The Interactive Fly

Genes involved in tissue and organ development

Fat body and the regulation of metabolism

Fat body development and maintenance of fat cell survival
Genetic control of the distinction between fat body and gonadal mesoderm
Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

Fat body regulation of metabolism, organism homeostasis, growth, and development
A nutrient sensor mechanism controls Drosophila growth
Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila
Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body
A Drosophila toolkit for HA-tagged proteins unveils a block in autophagy flux in the last instar larval fat body
Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster
A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan
Early life exercise training and inhibition of apoLpp mRNA expression to improve age-related arrhythmias and prolong the average lifespan in Drosophila melanogaster
Fat Quality Impacts the Effect of a High-Fat Diet on the Fatty Acid Profile, Life History Traits and Gene Expression in Drosophila melanogaster
MEF2 is an in vivo immune-metabolic switch
HP1a-mediated heterochromatin formation promotes antimicrobial responses against Pseudomonas aeruginosa infection
The control of lipid metabolism by mRNA splicing in Drosophila
CoA protects against the deleterious effects of caloric overload in Drosophila
Drosophila HNF4 directs a switch in lipid metabolism that supports the transition to adulthood
Thermal stress depletes energy reserves in Drosophila
Co-option of immune effectors by the hormonal signalling system triggering metamorphosis in Drosophila melanogaster
Decapentaplegic retards lipolysis during metamorphosis in Bombyx mori and Drosophila melanogaster
The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila
A salivary gland-secreted peptide regulates insect systemic growth
Fat body-derived Spz5 remotely facilitates tumor-suppressive cell competition through Toll-6-α-Spectrin axis-mediated Hippo activation
Anti-Tumor Effect of Turandot Proteins Induced via the JAK/STAT Pathway in the mxc Hematopoietic Tumor Mutant in Drosophila
Tumor Cytokine-Induced Hepatic Gluconeogenesis Contributes to Cancer Cachexia: Insights from Full Body Single Nuclei Sequencing
Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants
Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition
Wds-Mediated H3K4me3 Modification Regulates Lipid Synthesis and Transport in Drosophila
MicroRNA miR-263b-5p Regulates Developmental Growth and Cell Association by Suppressing Laminin A in Drosophila
Integrated Stress Response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation
Spenito-dependent metabolic sexual dimorphism intrinsic to fat storage cells
Integrating lipid metabolism, pheromone production and perception by Fruitless and Hepatocyte nuclear factor 4
Involvement of neuronal tachykinin-like receptor at 86C in Drosophila disc repair via regulation of kynurenine metabolism
MicroRNA miR-263b-5p Regulates Developmental Growth and Cell Association by Suppressing Laminin A in Drosophila

Fat body, fat storage, and physiology
High amylose starch consumption induces obesity in Drosophila melanogaster and metformin partially prevents accumulation of storage lipids and shortens lifespan of the insects
Lipoproteins in Drosophila melanogaster--assembly, function, and influence on tissue lipid composition
Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila
THADA regulates the organismal balance between energy storage and heat production
Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila
The exchangeable apolipoprotein Nplp2 sustains lipid flow and heat acclimation in Drosophila
The SR proteins SF2 and RBP1 regulate triglyceride storage in the fat body of Drosophila
Lactate production is a prioritised feature of adipocyte metabolism
Drosophila Snazarus regulates a lipid droplet population at plasma membrane-droplet contacts in adipocytes
FOXO-mediated repression of Dicer1 regulates metabolism, stress resistance, and longevity in Drosophila
Seipin is required for converting nascent to mature lipid droplets
Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism
Downregulation of Perilipin1 by the Immune Deficiency Pathway Leads to Lipid Droplet Reconfiguration and Adaptation to Bacterial Infection in Drosophila
Endoplasmic reticulum-associated protein degradation contributes to Toll innate immune defense in Drosophila melanogaster
Enteric Pathogens Modulate Metabolic Homeostasis in the Drosophila melanogaster host
TOR signaling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila
Gut-derived peptidoglycan remotely inhibits bacteria dependent activation of SREBP by Drosophila adipocytes
CG32803 is the fly homolog of LDAF1 and influences lipid storage in vivo
Acclimation temperature affects thermal reaction norms for energy reserves in Drosophila
Loss of Stearoyl-CoA Desaturase 1 leads to cardiac dysfunction and lipotoxicity
High fat diet induced abnormalities in metabolism, growth, behavior, and circadian clock in Drosophila melanogaster
The F-box gene Ppa promotes lipid storage in Drosophila
Recruitment of Peroxin 14 to lipid droplets affects lipid storage in Drosophila
NAD kinase sustains lipogenesis and mitochondrial metabolism through fatty acid synthesis
Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the immune deficiency signaling pathway
Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens
Basidiomycota species in Drosophila gut are associated with host fat metabolism

Fat body and insulin signaling
Growth-blocking peptides as nutrition-sensitive signals for insulin secretion and body size regulation
Drosophila lipin interacts with insulin and TOR signaling pathways in the control of growth and lipid metabolism
Sir2 acts through Hepatocyte Nuclear Factor 4 to maintain insulin signaling and metabolic homeostasis in Drosophila
dSir2 deficiency in the fatbody, but not muscles, affects systemic insulin signaling, fat mobilization and starvation survival in flies
Ecdysone-induced microRNA miR-276a-3p controls developmental growth by targeting the insulin-like receptor in Drosophila
Drosophila cytokine Unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion
The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults
A fat-tissue sensor couples growth to oxygen availability by remotely controlling insulin secretion
The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate
Differential metabolic sensitivity of insulin-like-response- and TORC1-dependent overgrowth in Drosophila fat cells
PPA1 Regulates Systemic Insulin Sensitivity by Maintaining Adipocyte Mitochondria Function as a Novel PPARgamma Target Gene
Edem1 activity in the fat body regulates insulin signalling and metabolic homeostasis in Drosophila
Adipose mitochondrial metabolism controls body growth by modulating systemic cytokine and insulin signaling
The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response
Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells
Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression
High fat diet-induced TGF-beta/Gbb signaling provokes insulin resistance through the tribbles expression
Domeless receptor loss in fat body tissue reverts insulin resistance induced by a high-sugar diet in Drosophila melanogaster
Meep, a Novel Regulator of Insulin Signaling, Supports Development and Insulin Sensitivity via Maintenance of Protein Homeostasis in Drosophila melanogaster =
A Novel Drosophila Model to Investigate Adipose tissue Macrophage Infiltration (ATM) and Obesity highlights the Therapeutic Potential of Attenuating Eiger/TNFα Signaling to Ameliorate Insulin Resistance and ATM
Remote control of insulin secretion by fat cells in Drosophila
Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes
Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor

Regulation of fat body function
Role and regulation of starvation-induced autophagy in the Drosophila fat body
Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila
Dual lipolytic control of body fat storage and mobilization in Drosophila
Flightless-I Controls Fat Storage in Drosophila
Lipid-gene regulatory network reveals coregulations of triacylglycerol with phosphatidylinositol/lysophosphatidylinositol and with hexosyl-ceramide
Mio/dChREBP coordinately increases fat mass by regulating lipid synthesis and feeding behavior in Drosophila
Systemic and mitochondrial effects of metabolic inflexibility induced by high fat diet in Drosophila melanogaster
Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition
Lipid droplet subset targeting of the Drosophila protein CG2254/dmLdsdh1
Salt-Inducible kinase 3 provides sugar tolerance by regulating NADPH/NADP+ redox balance
An autonomous metabolic role for Split ends
MRT, functioning with NURF complex, regulates lipid droplet size
NF-kappaB shapes metabolic adaptation by attenuating Foxo-mediated lipolysis in Drosophila
Dietary cysteine drives body fat loss via FMRFamide signaling in Drosophila and mouse
Galphaq, Ggamma1 and Plc21C control Drosophila body fat storage
The regulation of triglyceride storage by ornithine decarboxylase (Odc1) in Drosophila
The role of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb27C in regulating lipid storage in the Drosophila fat body
Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements
Tumor induction in Drosophila imaginal epithelia triggers modulation of fat body lipid droplets
Drosophila PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity
The heterogeneous nuclear ribonucleoprotein (hnRNP) Glorund functions in the Drosophila fat body to regulate lipid storage and transport
E2F/Dp inactivation in fat body cells triggers systemic metabolic changes
Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila
Drosophila STING protein has a role in lipid metabolism
STING controls energy stress-induced autophagy and energy metabolism via STX17
Histone acetyltransferase NAA40 modulates acetyl-CoA levels and lipid synthesis
A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones
Sima, a Drosophila homolog of HIF-1alpha, in fat body tissue inhibits larval body growth by inducing Tribbles gene expression
orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control
Cytoophidia coupling adipose architecture and metabolism
Microscopic and biochemical monitoring of endosomal trafficking and extracellular vesicle secretion in an endogenous in vivo model
Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting
Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease
The splicing factor 9G8 regulates the expression of NADPH-producing enzyme genes in Drosophila
Transportin-serine/arginine-rich (Tnpo-SR) proteins are necessary for proper lipid storage in the Drosophila fat body
Endoplasmic reticulum-associated protein degradation contributes to Toll innate immune defense in Drosophila melanogaster
The ESCRT-III Protein Chmp1 Regulates Lipid Storage in the Drosophila Fat Body/A>
Fear-of-intimacy-mediated zinc transport is required for Drosophila fat body endoreplication
FGF signaling promotes spreading of fat body precursors necessary for adult adipogenesis in Drosophila
The role of SR protein kinases in regulating lipid storage in the Drosophila fat body

Hormonal and neural regulation of fat body function
A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila
An obligatory role for neurotensin in high-fat-diet-induced obesity
Regulation of energy stores and feeding by neuronal and peripheral CREB activity in Drosophila
Identification and characterization of mushroom body neurons that regulate fat storage in Drosophila
Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila
Endocrine signals fine-tune daily activity patterns in Drosophila
Steroid hormone signaling is essential for pheromone production and oenocyte survival
Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae
Sex determination gene transformer regulates the male-female difference in Drosophila fat storage via the adipokinetic hormone pathway
Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation

Immunity and the fat body
Immune Control of Animal Growth in Homeostasis and Nutritional Stress in Drosophila
The mode of expression divergence in Drosophila fat body is infection-specific
The Nuclear Receptor Seven Up Regulates Genes Involved in Immunity and Xenobiotic Response in the Adult Drosophila Female Fat Body
Fat body cells are motile and actively migrate to wounds to drive repair and prevent infection
Inherent constraints on a polyfunctional tissue lead to a reproduction-immunity tradeoff

Remote functions of the fat body
Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila
Adipocyte metabolic pathways regulated by diet control the female germline stem cell lineage in Drosophila
Transforming growth factor beta/Activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression
Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia
Obesity-associated cardiac dysfunction in starvation-selected Drosophila melanogaster
Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila
A biological timer in the fat body comprised of Blimp-1, betaFTZ-F1 and Shade regulates pupation timing in Drosophila melanogasterDown-regulation of a cytokine secreted from peripheral fat bodies improves visual attention while reducing sleep in Drosophila
Kynurenine Metabolism in the Fat Body Non-autonomously Regulates Imaginal Disc Repair in Drosophila
Fat-body brummer lipase determines survival and cardiac function during starvation in Drosophila melanogaster
Tryptophan regulates Drosophila zinc stores

Genes expressed in fat body

Genetic control of the distinction between fat body and gonadal mesoderm

The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the visceral muscles are derived from a series of segmentally repeated primordia in the Drosophila mesoderm. This work describes the early development of the fat body and its relationship to the gonadal mesoderm, as well as the genetic control of the development of these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).

In each of parasegments 10-12 one of these primary dorsolateral regions generates somatic gonadal precursors instead of fat body. The balance between fat body and somatic gonadal fate in these serially homologous cell clusters is controlled by at least five genes. A model is suggested in which tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).

Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms

In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, larval nutrition was varied and effects on cell cycle initiation and maintenance were monitored in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. Ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition (Britton, 1998).

These results suggest that multiple pathways are involved in regulating the onset of cell proliferation in different tissue types in response to the global nutritional cue. Mitotic and endoreplicating cell cycles are regulated differently in response to the nutritional state: the endoreplicating tissues (ERTs) require continuous nutrition to cycle, whereas the mitotic cells cycle in a nutrition-independent manner once activated. In addition, the mechanism of cell cycle arrest in the two types of quiescent cells is different: quiescent ERTs can be driven into S-phase by ectopic expression of either of the G1/S regulators E2F or Cyclin E, while neither of these regulators can induce quiescent neuroblasts to enter S-phase.Conversely, quiescent neuroblasts but not quiescent ERTs are induced to reenter the cell cycle in response to a mitogen produced by the larval fat body (Britton, 1998).

The differential responses of the mitotic and endoreplicative cell cycles to nutrient withdrawal may provide an important mechanism for survival of the organism and reproduction in the face of food shortages in the wild. When nutrients become limiting, available resources can be dedicated to maintaining growth and proliferation in the mitotic tissues which are required to form the reproductive adult. Indeed, larvae are capable of pupating at a much smaller size than they normally do. A 'critical size' has been defined at which larvae are able to pupariate without further feeding. The small pupae which are formed by these larvae produce normal, fertile, but small adult flies (Britton, 1998).

Embryonic neuroblasts have an intrinsic program of cell proliferation. Each type of neuroblast has a specific identity, expresses unique and dynamic combinations of sublineage genes, and will give rise to a precise number and type of progeny before exiting the cell cycle. Interestingly, temporal control of sublineage gene expression in embryonic neuroblasts can be independent of cell cycle progression. Thus arresting a proliferating neuroblast in mid-lineage could lead to the desynchronization of sublineage gene expression and the loss of certain types of progeny, a result which could have disastrous consequences for the developing CNS (Britton, 1998).

In a food withdrawal experiment it was observed that many activated neuroblasts continued to proliferate for up to 7 days after food withdrawal, however a subset of them did not. This observation was most striking in the abdominal region of the VNC. The abdominal neuroblast lineages are much shorter than those of the majority of brain and thoracic neuroblasts, with a single abdominal neuroblast producing as few as four neurons during its postembryonic period of proliferation. Since the abdominal neuroblasts generally complete their entire larval program of proliferation in less than 2 days, it is not surprising that after 7 days of culture on sucrose the majority of these neuroblasts have exited the cell cycle. It is suspected that the reduction in labeled neuroblasts observed in all regions of the CNS over the course of this experiment is due to a subset of neuroblasts completing their intrinsic program of proliferation and exiting the cell cycle (Britton, 1998).

The insect fat body is the source of the majority of hemolymph proteins, including lipid binding proteins, juvenile hormone binding proteins and esterases, peptides which mediate the insect immune response, and vitellogenins involved in oocyte maturation in the adult female. The fat body is also responsible for synthesizing the stores of protein, lipid and glycogen which sustain the animal throughout metamorphosis. Ultrastructurally, the fat body shows a dramatic response to starvation. In Calpodes larvae, starvation leads to a rapid reorganization of the fat body including loss of mitochondria and rough endoplasmic reticulum (RER) by autophagy and depletion of stored metabolites. Refeeding induces mitochondrial divisions andincreases in RER content as well as the eventual replenishment of depleted stores. This study observed dramatic changes in the larval fat body in the course of starvation experiments, including a loss of tissue cohesion and changes in opacity. These changes probably reflect the alteration in composition the fat body cells undergo as stores of metabolites are mobilized to support proliferating mitotic tissues during starvation (Britton, 1998).

Previous studies have demonstrated that the adult female fat body is able to regulate yolk gene transcription in response to the nutritional environment. Interestingly, there is evidence that a component of the adult female abdomen is also capable of supporting the proliferation of larval tissues in a nutrition dependent manner. It has been demonstrated that the proliferation of imaginal disc fragments transplanted into the abdominal cavity of adult female hosts is dependent onnutrition. This study has found that when quiescent central nervous systems from starved larvae are transplanted into the abdomens of fed adult female hosts, larval neuroblasts reenter the cell cycle in what appears to be a normal spatiotemporal pattern. An appealing hypothesis is that production of the neuroblast mitogen in the fat body is regulated at the transcriptional level under the control of nutritional enhancers similar to those identified in the regions upstream of yolk protein genes. The ability of something in the adult female abdomen to activate proliferation in quiescent neuroblasts suggests that similar fat body-derived mitogens are produced in the larval and adult female fat bodies. This adult mitogen could have a role in controlling proliferation in the adult, perhaps functioning to regulate some oogenic process in response to the nutritional state. Indeed, oogenesis is inhibited in adult females fed on sucrose (Britton, 1998).

The dramatic response of the fat body to starvation, the demonstration that there is a mechanism for nutritional controlof transcription in adult female fat body, and the similar abilities of the adult female abdomen and the larval fat body to support nutrition-dependent cell cycle activation lend support to the proposal that the fat body is responsible for mediating the nutritional response in larval neuroblasts. The results of co-culture experiment demonstrate that the fat body supplies a diffusible factor which stimulates larval neuroblasts to enter the cell cycle (Britton, 1998).

A nutrient sensor mechanism controls Drosophila growth

Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. This study provides evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. A genetic screen for growth modifiers discovered slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. These results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism (Colombani, 2003).

In multicellular organisms, the control of growth depends on the integration of various genetic and environmental cues. Nutrient availability is one of the major environmental signals influencing growth and, as such, has dictated adaptative responses during evolution toward multicellularity. In particular, complex humoral responses ensure that growth and development are properly coordinated with nutritional conditions (Colombani, 2003).

In isolated cells, amino acid withdrawal leads to an immediate suppression of protein synthesis, suggesting that cells are protected by active sensing mechanims that block translation prior to depletion of internal amino acid stores. In many mammalian cell types, changes in amino acid diet affect the binding of the translation repressor 4EBP1 to initiation factor eIF4E and the activity of ribosomal protein S6 kinase (S6K). These two signaling events require the activity of TOR (target of rapamycin), a conserved kinase recently shown to participate in a nutrient-sensitive complex both in mammalian cells and in yeast. Mutations in the Drosophila TOR homolog (dTOR) results in cellular and physiological responses characteristic of amino acid deprivation and establish that TOR is cell autonomously required for growth in a multicellular organism. Furthermore, the TSC (tuberous sclerosis complex) tumor suppressor, consisting of a TSC1 and TSC2 heterodimer (TSC1/2), as well as the small GTPase Rheb participate to the regulation of TOR function. Overall, these data suggest that TSC, Rheb, TOR, and S6K participate in a conserved pathway that coordinates growth with nutrition in a cell-intrinsic manner (Colombani, 2003).

In multicellular organisms, humoral controls are believed to buffer variations in nutrient levels. However, little is known about how growth of individual cells is coordinated. In vertebrates, growth-promoting action of the growth hormone (GH) is mostly relayed to peripheral tissues through the production of IGF-I. Binding of IGF-I to its cognate receptor tyrosine kinase (IGF-IR) induces phosphorylation of insulin receptor substrates (IRS), which in turn activate a cascade of downstream effectors. These include phospho-inositide 3-kinase (PI3K), which generates the second messenger phosphatidylinositol-3,4,5-P3 (PIP3), and thereby activates the AKT/PKB kinase. Genetic manipulation of IGF-I, IGF-IR, PI3K, and AKT in mice modulates tissue growth in vivo thus demonstrating a requirement of the IGF pathway for growth. In Drosophila, both loss- and gain-of function studies have also exemplified the role of a conserved insulin/IGF signaling pathway in the control of growth. Ligands for the unique insulin receptor (Inr) constitute a family of seven peptides related to insulin, the Drosophila insulin-like peptides (Dilps). Remarkably, three dilp genes (dilp2, dilp3, and dilp5) are expressed in a cluster of seven median neurosecretory cells (m-NSCs) in the larval brain, suggesting that they have an endocrine function. Indeed, ablation of the seven dilp-expressing mNSCs in larvae induces a systemic growth defect (Colombani, 2003).

Both in flies and mice, mutations in IRS provoke growth retardation as well as female sterility similar to what is observed in starved animals. Moreover, PI3K activity in Drosophila larvae depends on the availability of proteins in the food. Overall, this supports the notion that the insulin/IGF pathway might coordinate tissue growth with nutritional conditions. However, upon amino acid withdrawal, neither PI3K nor AKT/PKB activities are downregulated in mammalian or insect cells in culture, suggesting that this pathway does not directly respond to nutrient shortage. Hence, an intermediate sensor mechanism must link nutrient availability to insulin/IGF signaling (Colombani, 2003).

An intriguing possibility is that specific organs could function as nutrient sensors and induce a nonautonomous modulation of insulin/IGF growth signaling in response to changes in nutrient levels. This study used a genetic approach in Drosophila to assess both the cellular and humoral responses to amino acid deprivation in the context of a developing organism. The insect fat body (FB) has important storage and humoral functions associated with nutrition, comparable to vertebrate liver and adipose tissue. During larval stages, the FB accumulates large stores of proteins, lipids, and carbohydrates, which are normally degraded by autophagy during metamorphosis in order to supply the developing tissues but can also be remobilized during larval life to compensate transitory nutrient shortage. In addition to its storage function, the FB also has endocrine activity and supports growth of imaginal disc explants and DNA replication of larval brains in coculture. This study demonstrates that the FB operates as a sensor for variations in nutrient levels and coordinates growth of peripheral tissues accordingly via a humoral mechanism (Colombani, 2003).

In the course of a P[UAS]-based overexpression screen for growth modifiers, a P[UAS]-insertion line (UY681) was found to cause growth retardation upon ectopic activation. Sequence analysis revealed that P(UY)681 is inserted in a predicted gene (CG11128) that encodes a putative protein showing strong homology with amino acid permeases of the cationic amino acid transporter (CAT) family. The P[UAS] element is inserted in the first intron of the CG11128 gene, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. To assess the function of this transporter, 3H-arginine uptake was measured in S2 cells. Results indicate that amino acid uptake is either enhanced by transfection of a CG11128 cDNA or suppressed by RNAi, indicating that the encoded protein presents CAT activity. In situ hybridization revealed basal levels of CG11128 expression in most larval tissues but much higher levels in the FB and the gut, two tissues involved in amino acid processing (Colombani, 2003).

By P element remobilization, an imprecise excision was obtained that deletes the sequences encoding the N-terminal half of the protein. 87% of homozygous mutant animals die during larval stages. The few viable adults emerged after a 2 day delay and were smaller and markedly slimmer than control animals. The associated gene was named slimfast (slif) and the excision allele slif1. Weight measurement indicated that homozygous slif1 adult males displayed a 16% mass reduction compared to control. Accordingly, adult wing size was reduced by 8% due to a reduction of both cell size and cell number. When the slif1 allele was in trans to Df(3L)Δ1AK, a deficiency covering the locus, larval lethality was slightly enhanced, suggesting that slif1 corresponds to a strong hypomorphic allele. The amino acid transporter function of slif, as well as the phenotypes observed upon reduction of slif function suggest that slif mutant animals might suffer amino acid deprivation. A major consequence of amino acid deprivation in larvae is the remobilization of nutrient stores in the FB, which typically results in aggregation of storage vesicles. Consistently, fusion of storage vesicles was observed in the FB of slif1 larvae and was indistinguishable from that observed in animals fed on protein-free media (Colombani, 2003).

GAL4 induction of P(UY)681 resulted in a growth-deficient phenotype similar to that of slif1 loss of function. The antisense orientation of P(UY)681 suggested that the growth defect following GAL4 induction was due to an RNAi effect. Indeed, Northern blot analysis revealed that ubiquitous GAL4-dependent activation of P(UY)681 using the daughterless-GAL4 (da-GAL4) driver strongly reduced slif mRNA levels. Only two of the three alternative first exons are potentially affected by the antisense RNA, possibly explaining the residual accumulation of slif mRNAs in da-GAL4; P(UY)681 animals. Most of these animals died at larval stage, similar to what was observed for slif1 mutants. Specific induction of P(UY)681in the wing disc using the MS1096-GAL4 driver provoked a reduction of the adult wing size, which could be either rescued by coactivation of a UAS-slif transgene or enhanced by reducing slif gene dosage with the heterozygous Df(3L)Δ1AK deficiency. Thus, GAL4-dependent activation of P(UY)681 reduces slif function and defines a conditional loss-of-function allele hereafter termed slifAnti (Colombani, 2003).

As expected, loss of slif function using the slifAnti allele also mimicked amino acid deprivation. Accordingly, ubiquitous slifAnti induction in growing larvae resulted in storage vesicle aggregation and strong reduction of global S6 kinase activity, similar to what was reported in animals raised on protein-free diet. Additionally, an increase in PEPCK1 gene transcription was observed, similar to the effect of amino acid withdrawal. In summary, this study has identified two loss-of-function alleles of the slif gene whose defects mimic physiological aspects of amino acid deprivation. Importantly, the conditional slifAnti allele provides a unique tool to mimic an amino acid deprivation in a tissue-specific manner (Colombani, 2003).

This study established that the FB is a sensor tissue for amino acid levels, as downregulation of the Slif amino acid transporter within the FB is sufficient to induce a general reduction in the rate of larval growth. In contrast, specific disruption of slif in imaginal discs, larval gut, or salivary glands did not induce a nonautonomous growth response, suggesting that these tissues do not participate in the systemic control of growth. The dilp-expressing median neurosecretory cells (m-NSCs) also affect growth control, since selective ablation of these cells in the larval brain induces an overall reduction of animal size. In response to complete sugar and protein starvation, the m-NSCs stop expressing dilp3 and dilp5 genes, suggesting that these neurons also sense nutrient levels. This study shows that the selective reduction of slif function in these cells has no obvious effect on tissue growth and animal development. This indicates that the seven dilp-expressing m-NSCs do not constitute a general amino acid sensor. In contrast, the role of m-NSCs in carbohydrate homeostasis and the observation that they stop expressing certain dilp genes when larvae are deprived of sugar rather suggests that these cells have a role in sensing carbohydrate levels (Colombani, 2003 and references therein).

This analysis also provides a framework in which to understand the phenotype of minidisc, a mutation in an amino acid transporter gene that exhibits nonautonomous growth defects in imaginal discs (Colombani, 2003).

In a number of model systems, both PI3K and TOR have been implicated in linking growth to nutritional status and, until recently, were considered as intermediates of a common regulatory pathway. In yeast, the TOR kinase is part of a cell-autonomous nutrient sensor, which controls protein synthesis, ribosome biogenesis, nutrient import, and autophagy. Genetic analysis in Drosophila indicates that dTOR is required for cell-intrinsic growth control. The results obtained using the slifAnti allele in the wing disc indicate that individual tissues have indeed the potential to respond to amino acid deprivation in a cell-autonomous manner. Nonetheless, this study also demonstrates that the TOR nutritional checkpoint participates in a systemic control of larval growth emanating from the FB. Within a developing organism, each cell may integrate these two distinct inputs regarding nutritional status, one originating from a systemically-acting FB sensor, and the other from TOR-dependent signaling in individual cells. One can further speculate that depending on the strength and duration of starvation, different in vivo nutritional checkpoints will be hierarchically recruited to protect the animal and that the systemic control might, in most physiological situations, override the cell-autonomous control. Indeed, as the data demonstrate, the FB sensor is sufficient to induce a general and coordinated response to starvation without calling individual cell-autonomous mechanisms into play (Colombani, 2003).

Several lines of evidence indicate that the PI3K pathway is not part of the sensor mechanism in FB cells. First, a sensor for PI3K activity in the FB is only marginally affected by amino acid deprivation in that tissue, indicating that the cell-autonomous response to amino acid starvation does not directly influence PI3K signaling. This is reminiscent of previous observations in mammalian cultured cells, showing that PI3K activity does not respond to variations in amino acid levels. Moreover, inhibition of PI3K signaling by dPTEN expression in the FB is not sufficient to trigger the sensing mechanism. Although, dPTEN overexpression causes a complete disappearance of the PI3K sensor accompanied by growth suppression of FB cells, the FB maintains a critical mass that allows for normal larval growth. In contrast, the regulatory subunit p60 whose overexpression potently inhibits PI3-kinase in flies has been shown to induce a systemic effect on larval growth when overexpressed in the FB using an Adh-Gal4 driver. This study found that a pumpless ppl-GAL4-directed expression of p60 also provokes a strong suppression of larval growth and a dramatic inhibition of FB development in young larvae. Thus, the systemic effect on growth observed upon p60 overexpression most likely results from a drastic reduction of FB mass, which then fails to support normal larval growth (Colombani, 2003).

These results further indicate that PI3K signaling is a remote target of the humoral message that originates from the FB in response to amino acid deprivation. This is in agreement with previous data showing that PI3K activity is downregulated by dietary amino acid deprivation and explains why global PI3-kinase inhibition mimics cellular and organismal effects of starvation. The existence of a humoral relay reconciles these in vivo studies with the absence of direct PI3K responsiveness to amino acid levels (Colombani, 2003).

The relative resistance of imaginal disc growth to the systemic control exerted by the FB correlates with maintenance of PI3K activity in these tissues. This is in agreement with previous observations that cells in the larval brain and in imaginal discs maintain a slow rate of proliferation under protein starvation, while larval endoreduplicating tissues (ERTs) arrest. This difference might be attributed to the basal levels of dilp2 expression observed in imaginal discs, allowing a moderate growth rate of these tissues through an autocrine/paracrine mechanism. It was recently shown that clonal induction of PI3K potently induces cell-autonomous growth response even in fasting larvae, indicating that some nutrients are still accessible to support cell growth within a fasted larva. The main function of a general sensor could be to preserve these limited nutrients for use by high priority tissues. In this context, local PI3K activation through an autocrine loop in imaginal tissues could favor the growth of prospective adult structures in adverse food conditions. Thus, the FB would have an active role in controlling the allocation of resources depending on nutritional status. In this respect, it is noteworthy that FB cells are relatively resistant to the FB-derived humoral signal, since the PI3K sensor is not drastically affected in the FB of ppl>slifAnti animals. Thereby, essential regulatory functions of the FB could be preserved even in severely restricted nutritional conditions (Colombani, 2003).

How does the FB signal to other tissues? This study suggests that a humoral signal relays information from the FB amino acid sensor and systemically inhibits PI3K signaling. In addition, this downregulation is not due to a direct inhibition of dilp expression by neurosecretory cells in the brain. Nevertheless, it cannot be ruled out that the secretion of these molecules is subjected to regulation in the mNSCs. Both in vivo and in insect cell culture, several imaginal discs growth factors (IDGF) secreted by the FB have been proposed to function synergistically with Dilp signaling to promote growth. However, this study did not find any modification of IDGF expression in the FB of larvae raised on water- or sugar-only diet, or upon FB induction of slifanti. In vertebrates, the different functions of the circulating IGF-I are modulated through its association with IGF-BPs and acid labile subunit (ALS). In particular, the formation of a ternary complex with ALS leads to a considerable extension of IGF-I half-life. The finding that a Drosophila ALS ortholog is expressed within the FB in an amino acid-dependent manner provides a new avenue to study the molecular mechanisms of nonautonomous growth control mediated by the FB (Colombani, 2003).

This study highlights the contribution that genetics can provide to unravel the mechanisms of physiological control. Using a genetic tool to mimic amino acid deprivation, it was demonstrated that nutrition systemically controls body size through an amino acid sensor operating in the FB. It is proposed that (1) in metazoans, a systemic nutritional sensor modulates the conserved TOR-signaling pathway, and (2) the response to sensor activation is relayed by a hormonal mechanism, which triggers an Inr/PI3K-dependent response in peripheral tissues (Colombani, 2003).

Growth-blocking peptides as nutrition-sensitive signals for insulin secretion and body size regulation

In Drosophila, the fat body, functionally equivalent to the mammalian liver and adipocytes, plays a central role in regulating systemic growth in response to nutrition. The fat body senses intracellular amino acids through Target of Rapamycin (TOR) signaling, and produces an unidentified humoral factor(s) to regulate insulin-like peptide (ILP) synthesis and/or secretion in the insulin-producing cells. This study found that two peptides, Growth-Blocking Peptide (GBP1) and CG11395 (GBP2), are produced in the fat body in response to amino acids and TOR signaling. Reducing the expression of GBP1 and GBP2 (GBPs) specifically in the fat body results in smaller body size due to reduced growth rate. In addition, GBPs were found to stimulate ILP secretion from the insulin-producing cells, either directly or indirectly, thereby increasing insulin and insulin-like growth factor signaling activity throughout the body. These findings fill an important gap in understanding of how the fat body transmits nutritional information to the insulin producing cells to control body size (Koyama, 2016).

Role and regulation of starvation-induced autophagy in the Drosophila fat body

In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).

Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).

In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).

The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).

Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).

Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).

The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).

How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).

In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).

Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).

Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila

Energy homeostasis, a fundamental property of all organisms, depends on the ability to control the storage and mobilization of fat, mainly triacylglycerols (TAG), in special organs such as mammalian adipose tissue or the fat body of flies. Malregulation of energy homeostasis underlies the pathogenesis of obesity in mammals including human. A screen was performed to identify nutritionally regulated genes that control energy storage in the Drosophila. The brummer (bmm) gene encodes the lipid storage droplet-associated TAG lipase Brummer, a homolog of adipocyte triglyceride lipase (ATGL). Food deprivation or chronic bmm overexpression depletes organismal fat stores in vivo, whereas loss of bmm activity causes obesity in flies. These study identifies a key factor of insect energy homeostasis control. Their evolutionary conservation suggests Brummer/ATGL family members to be implicated in human obesity and establishes a basis for modeling mechanistic and therapeutic aspects of this disease in the fly (Grönke, 2005).

Providing constant energy supply despite variability in food access and metabolic energy demand is a fundamental property of animals. Key to an individual's survival during food deprivation is the ability to mobilize stored energy resources accumulated during periods of excessive energy supply. In organisms as different as humans and the fruit fly Drosophila, energy-rich diet components are converted into glycogen and, to a larger extent, triacylglycerols (TAG), the storage forms of carbohydrate and fat, respectively. Storage fat is deposited in intracellular lipid droplets of specialized organs called the adipose tissue in mammals or the fat body in Drosophila. In mammals, adipose tissue cooperates with the digestive tract and the central nervous system to hardwire an integrated molecular communication network ensuring the lifelong integrity of an organism's energy homeostasis under varying environmental conditions. In the peripheral fat storage tissue, a regulated balance between lipogenesis and lipolysis is believed to continuously match acute energy needs by TAG mobilization and readjust organismal storage fat content to a genetically determined setpoint during periods of excessive energy supply. Chronic imbalance of energy storage control by the lack or malfunction of regulatory genes results in excessive fat accumulation and is causative to the obesity pandemics in human populations as well as to related phenotypes in rodent models (Grönke, 2005).

A key regulator of storage fat lipolysis in mammalian adipocytes is the hormone-sensitive lipase (HSL). Activated HSL interacts with perilipin at the lipid droplet membrane to eventually mobilize TAG. Acute HSL activation relies on posttranslational modification by protein kinase A (PKA) in response to hormonal β-adrenoceptor stimulation and subsequent activation of the cAMP second messenger signaling pathway. In addition, extensive fasting causes upregulation of mouse HSL mRNA and protein, supporting the enzyme's importance in acute and chronic TAG mobilization control. However, HSL knockout mice are viable and not obese, having substantial residual lipolytic activity. Accordingly, additional TAG lipases of the nutrin family, such as the most recently identified human adipose triglyceride lipase (ATGL), have been implicated in mammalian lipolysis. However, the in vivo relevance of this lipase family in fat storage control on the organismal level waits to be analyzed (Grönke, 2005 and references therein).

In insects, storage fat lipolysis is stimulated by adipokinetic hormone (AKH) in various species including the grasshopper Locusta migratoria, the tobacco hornworm Manduca sexta, and Drosophila, suggesting a general role in insect energy balance control. Like in mammalian TAG mobilization, AKH-stimulated lipolysis in the insect fat body relies on signaling via a G protein-coupled receptor (see Drosophila Gonadotropin-releasing hormone receptor/Adipokinetic hormone receptor), increase in intracellular cAMP, and activation of PKA. An insect TAG lipase, however, which makes the storage fat metabolically accessible for the energy-demanding target tissues, is currently unknown (Grönke, 2005).

Given the intriguing similarities in the regulatory mechanisms of TAG mobilization between mammals and insects, a genome-wide transcriptome profiling was performed in Drosophila to screen for nutritionally regulated and evolutionary conserved lipolysis effectors. This study presents the functional in vivo analysis of brummer, which encodes a TAG lipase of the nutrin family, whose lack causes obesity in the fly (Grönke, 2005).

To screen for nutritionally regulated genes, a genome-wide transcriptome analysis was performed, comparing gene expression of fed and food-deprived adult Drosophila flies. Sorting the total of 223 starvation-responsive genes according to their predicted function reveals that most of the starvation-induced genes are coding for metabolic enzymes (n = 44). In addition, genes coding for cytochromes (n = 10), metabolite transporters (n = 6), kinases (n = 5), and proteins involved in lipid metabolism (n = 7) are upregulated under starvation. Few metabolic enzymes (n = 7) are downregulated in response to starvation, whereas proteases and protease inhibitors form the largest group (n = 38). Nearly half of the starvation-induced metabolic enzymes are involved in carbohydrate catabolism, including key regulators like hexokinase (encoded by Hex-C), transketolase (CG8036), and phosphoglucomutase (Pgm) or enzymes involved in the breakdown of sugars like an α-Amylase (AmyD), a α-Glucosidase (CG11909), and six maltases (CG11669, CG8690, CG30359, CG30360, CG14934, CG14935). Protein degradation is reflected by the upregulation of genes involved in amino acid catabolism, including two aminotransferases encoded by got2 and spat, a phenylalanine-4-monooxygenase (henna), a 4-hydroxyphenylpyruvate-dioxygenase (CG11796), and a homogentisate-1,2-dioxygenase (hgo). The starvation-induced metabolic activation is further reflected by the transcriptional upregulation of five regulatory kinases or kinase subunits, which have all been implicated in energy homeostasis control. While the pyruvate dehydrogenase kinase encoded by pdk is critical for the regulation of oxidative glucose metabolism, the β subunit of the SNF1/AMP-activated protein kinase (AMPK) acts as a cellular energy sensor, and the cAMP-activated protein kinase A (PKA) promotes glycogen and TAG catabolism. The SNF4 γ subunit loechrig has been implicated in cholesterol homeostasis control. In addition, Lk6 kinase mutants have recently been described to have increased organismal TAG content, suggesting a function of the kinase in the control of organismal lipid storage (Grönke, 2005 and references therein).

Among the seven upregulated genes involved in lipid metabolism are genes encoding a putative TAG-lipase (CG5966), phospholipase A2 (CG1583), low-density lipoprotein receptor (LpR2), long chain fatty acid CoA ligase (CG9009), and carnitine-O-palmitoyltransferase (CPTI). Anabolic reactions of the lipid metabolism are repressed under starvation, as indicated by the transcriptional downregulation of a lipogenic 1-acylglycerol-3-P-O-acyltransferase (CG4753) and a long chain fatty acid elongase (CG6261). Moreover, the PAT domain containing lipid storage droplet-associated protein Lsd-1 and three TAG lipases are among the nine genes involved in lipid metabolism that are downregulated in response to starvation (Grönke, 2005).

Taken together, genome-wide transcriptome profiling of fed versus food-deprived flies displays various regulatory aspects of the metabolic starvation response in Drosophila, including carbohydrate, amino acid, and lipid catabolism. However, no function has been assigned to 25% of the 223 starvation-responsive genes. Among those, the gene CG5295 was found in region 70F5 on chromosome 3L, termed brummer (bmm), is upregulated upon starvation. The single bmm transcript, which encodes a 507 amino-acid-long protein (BMM) closely related to TTS-2/ATGL of mouse and human, is expressed during all ontogenetic stages of the fly. It is highly enriched in the energy storage tissue as well as the food-absorbing parts of the digestive tract, i.e., the larval midgut and gastric caeca. Quantitative Northern blot analysis confirms sustained transcriptional upregulation in response to food-deprivation and downregulation upon refeeding. The nutritional regulation and the patterns of bmm expression suggest that bmm participates in the control of energy homeostasis (Grönke, 2005).

BMM contains a patatin-like domain (PLD) including a serine hydrolase motif, originally described in plant acyl-hydrolases, and a so-called Brummer box (BB) of unknown function. The BB motif is found in a number of PLD-containing proteins, which are refered to as the Brummer/Nutrin subfamily. It includes the Anopheles BMM ortholog, a Drosophila paralogue called doppelgänger von brummer (dob; CG5560), the human proteins Adiponutrin, GS2-like, TTS-2/ATGL and GS2, Caenorhabditis elegans C05D11.7 and D1054.1 as well as Arabidopsis NP_174597 (Grönke, 2005).

PLD proteins are phospholipases in plants, human and Pseudomonas or TAG lipases, as recently shown for the human Brummer/Nutrin family members TTS-2.2/ATGL, GS2, and Adiponutrin. Recombinant BMM exhibits esterase activity on an esterified fatty acid (6,8-difluoro-4-methylumbelliferyl octanoate) as substrate but fails to catalyze the release of fatty acid from either the A2 position of a phospholipid (PAP), the glycosylphosphatidylinositol (GPI) membrane glycolipid membrane anchor of GPI-modified proteins (5′-nucleotidase, Gce1), or monoacylglycerol (MAG). However, it cleaves TAG in vitro, whereas the BMMS38A mutant, in which serine residue 38 of the catalytic center had been replaced by alanine, is enzymatically inactive. Thus, bmm as its mammalian homologs are candidates for nutritionally regulated in vivo effectors of TAG mobilization (Grönke, 2005).

To test whether bmm promotes fat mobilization in vivo, bmm loss-of-function mutant alleles (bmm1 and bmm2) were generated by mobilization of a transposable P element located in the first exon of bmm. Precise excision of the P element, as obtained with bmmrev, served as genetically matched control for phenotypic analysis. Embryos lacking both maternal and zygotic bmm activity are lethal, indicating that bmm carries an essential function. They develop pleiotropic degeneration phenotypes and have increased TAG levels in late embryogenesis. Embryonic lethality can be partially rescued by a paternally provided functional bmm gene and almost completely reverted by ubiquitous bmm expression from a cDNA-bearing transgene. Similar phenotypes and a reduced embryonic hatching rate have been reported for mutants of the perilipin-like fly gene Lsd-2. These results suggest that bmm fulfils a vital function in TAG mobilization during embryogenesis (Grönke, 2005).

Flies lacking only zygotic BMM lipase activity develop normally but show progressive obesity accumulating 17% (immature adults, <1 day old) to 101% (mature adults, 6 days old) more storage fat compared to control flies. Conversely, transgene-dependent bmm overexpression in fat body cells of fed flies, which mimics the effect of starvation-induced upregulation of bmm transcription, depletes the TAG content of immature and mature adults by 96% and 46%, respectively. These effects were not observed upon transgenic expression of the enzymatically inactive bmmS38A mutant, indicating that the TAG mobilization is caused by the lipase activity of BMM. bmm-dependent differences of organismal TAG content are also reflected by the lipid storage phenotype of fat body cells showing variously sized storage droplets in bmm1 mutant fat body cells and their reduction in size and number upon overexpression of the gene. The effect of BMM is specific for the fat-based aspect of energy storage, since the glycogen content is not affected in bmm mutant or bmm-overexpressing flies (Grönke, 2005).

Excessive fat storage in flies lacking bmm function reduces the median lifespan by only 10%. Acute TAG mobilization is impaired but not abolished in bmm mutants. While controls deplete their storage TAG during starvation, bmm mutants are able to consume 73% of their prestarvation fat content. Accordingly, food-deprived bmm mutants outlive controls by 56% on the expense of their increased prestarvation fat storage. The lipolytic activity present in bmm mutants allows fuelling their extended survival under food deprivation by metabolizing in total 65% more TAG than controls. Thus, as in mammals, mobilization of TAG storage in flies is controlled by more than one TAG lipase. Candidate effectors of bmm-independent TAG mobilization are the bmm paralogue dob and the genes CG5966 and CG11055, which code for a starvation-induced putative TAG lipase and a Drosophila HSL homolog, respectively (Grönke, 2005).

To possibly extend the functional similarity between mammalian and Drosophila TAG lipases, it was asked whether BMM localizes at the surface of lipid droplets. Transgenic flies expressing BMM:EGFP fusion protein variants in their fat body cells allow examination of BMM intracellular localization and lipolytic activity in vivo. Ubiquitous expression of BMM:EGFP or BMM reverts the obese phenotype of bmm mutant flies. Targeting of BMM:EGFP but not BMMS38A:EGFP expression to the fat body of otherwise wild-type flies depletes the organismal TAG storage and reduces both the number and size of lipid droplets in fat body cells. BMM:EGFP localizes at islands on the droplet surface, often at interdroplet contact sites). In contrast, nonfunctional BMMS38A:EGFP distributes homogenously over the droplet surfaces. The evolutionary conserved part of BMM including the Brummer box is sufficient to properly localize the protein on lipid droplets, likely to represent active sites of BMM-dependent TAG mobilization. Other BMM-related lipases, such as hamster desnutrin and human TTS-2.1/ATGL, also localize on lipid droplets, but their localization sequences are presently unknown (Grönke, 2005).

The results indicate that the surface of lipid droplets is an evolutionary conserved intracellular compartment boundary for organismal TAG storage control, as has been suggested for mammalian adipocytes where perilipin modulates activity of HSL and possibly non-HSL lipases such as ATGL. Lack of perilipin results in lean mice with increased lipolysis and reverses the obese phenotype of leptin receptor-deficient mutants. The perilipin-like LSD-2 of fly localizes to lipid droplets of fat cells and adjusts organismal TAG content in a dosage-dependent manner, suggesting that it functions as an evolutionary conserved modulator of lipolysis. In fact, bmm Lsd-2 double mutants have wild-type TAG levels, indicating that loss of Lsd-2 activity compensates for the lack of bmm. Conversely, combined overexpression of bmm and Lsd-2 in the fat body can partially revert the complementary phenotypes caused by the overexpression of each of the two genes. These data demonstrate that the lipid droplet-associated factors Brummer and LSD-2, which have opposite roles in organismal fat storage, act in an antagonistic manner (Grönke, 2005).

This first in vivo analysis of any insect lipase demonstrates a remarkable conservation of effectors controlling organismal fat storage in mammals and flies, emphasizing the value of Drosophila for research in energy homeostasis. On the basis of these results in the fly, it is speculated that mammalian members of the brummer/nutrin gene family like ATGL play an essential role in organismal fat mobilization and that malfunction of Brummer-homologous TAG lipases might contribute to mammalian obesity. Accordingly, stimulating Brummer-like lipase activity is a potential therapeutic approach to control TAG release from adipose tissue in obese patients, and lipase activators could be tested in the fly model (Grönke, 2005).

Dual lipolytic control of body fat storage and mobilization in Drosophila

Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. A Drosophila mutant was generated lacking the receptor (AKHR; FlyBase name -- Gonadotropin-releasing hormone receptor or GRHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ss-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. These data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis (Grönke, 2007).

Expression studies in a heterologous tissue culture system and in Xenopus oocytes identified AKH-responsive G protein-coupled receptors in Drosophila, such as the one encoded by the AKHR (or CG11325) gene. AKHR is expressed during all ontogenetic stages of the fly. It consists of seven exons, which encode a predicted protein of 443 amino acids. In late embryonic and larval stages, AKHR is expressed in the fat body. This finding is consistent with its predicted role as transmitter of the lipolytic AKH signal in this organ (Grönke, 2007).

In order to examine the effect of AKHR signaling on fat storage and mobilization in vivo, two different P element-insertion mutants were used, CG11188A1332 and AKHRG6244, which are located close to and within the AKHR gene, respectively. CG11188A1332 flies carrying the transposable element integration designated A1332 allow for the transcriptional activation of the adjacent AKHR gene. This ability was used for AKHR gain-of-function studies by overexpression of AKHR in the fat body of flies. Overexpression of AKHR in response to a fat body-specific Gal4 inducer causes dramatic reduction of organismal fat storage. This finding could be recapitulated by fat body-targeted AKHR expression from a cDNA-based upstream activation sequence (UAS)-driven AKHR transgene. These gain-of-function results suggest a critical in vivo role for AKHR in storage-lipid homeostasis of the adult fly (Grönke, 2007).

Flies of strain AKHRG6244, which carry a P element integration in the AKHR untranslated leader region, were used to generate the AKHR deletion mutants AKHR1 and AKHR2, as well as the genetically matched control AKHRrev, which possesses a functionally restored AKHR allele. As exemplified for embryonic and larval stages, AKHR1 mutants lack AKHR transcript. Ad libitum-fed flies without AKHR function are viable, fertile, and have a normal lifespan. However, such flies accumulate lipid storage droplets in the fat body and have 65%-127% more body fat than the controls. These results indicate that AKHR1 mutants develop an obese phenotype. The same result was obtained with AKHR2 and AKHR1/AKHR2 transheterozygous mutant flies, as well as with flies lacking the AKH-producing cells of the neuroendocrine system due to targeted ablation by the cell-directed activity of the proapoptotic gene reaper. Conversely, chronic overexpression of AKH provided by a fat body-targeted AKH transgene of otherwise wild-type flies largely depletes lipid storage droplets and organismal fat stores. However, the obese phenotype of AKHR mutants is unresponsive to AKH, indicating that AKHR is the only receptor transmitting the lipolytic signal induced by AKH in vivo. Collectively, these data demonstrate that AKH-dependent AKHR signaling is critical for the chronic lipid-storage homeostasis in ad libitum-fed flies (Grönke, 2007).

Studies on various insect species helped elucidate several components and mediators of the lipolytic AKH/AKHR signal transduction pathway. However, the identity of the TAG lipase(s) executing the AKH-induced fat mobilization program remained elusive. Besides the Drosophila homolog of the TG lipase from the tobacco hornworm Manduca sexta, the recently identified Brummer lipase, a homolog of the mammalian ATGL, is a candidate member of the AKH/AKHR pathway. This is based on the striking similarity between the phenotypes of AKHR and bmm mutants. Ad libitum-fed flies lacking either AKHR or bmm activity, store excessive fat. Both mutants show incomplete storage-fat mobilization (Grönke, 2005) and starvation resistance (Grönke, 2005) in response to food deprivation. Starvation resistance of these mutants might be caused by their increased metabolically accessible fat stores and/or changes in their energy expenditure due to locomotor activity reduction as described for flies with impaired AKH signaling. Despite the phenotypic similarities of their mutants, however, AKHR and bmm are members of two different fat-mobilization systems in vivo. Several lines of evidence support this conclusion. On one hand, AKH overexpression reduces the excessive TAG storage of bmm mutants, while on the other, bmm-induced fat mobilization can be executed in AKHR mutants. Thus, AKH/AKHR signaling is not a prerequisite for Brummer activity. Moreover, genetic epistasis experiments support this idea that AKHR and bmm belong to different control systems of lipocatabolism in vivo. Double-mutant analysis reveals that the obesity of AKHR and bmm single mutants is additive. Accordingly, AKHR bmm double-mutant flies store about four times as much body fat as control flies and accumulate excessive lipid droplets in their fat body cells (Grönke, 2007).

Thin layer chromatography (TLC) analysis was used to compare the storage-fat composition of AKHR and bmm single mutants with AKHR bmm double-mutant and control flies. Excessive body fat accumulation in AKHR bmm double mutants is on the one hand due to TAG, which is increased compared to AKHR and bmm single-mutant flies. Additionally, an uncharacterized class of TAG (TAGX) appears exclusively in AKHR bmm double mutants. In contrast to TAG, changes in diacylglycerol (DAG) content do not substantially contribute to the differences in body fat content in any of the analyzed genotypes. Taken together a quantitative increase and a qualitative change in the TAG composition account for the extreme obesity in AKHR bmm double-mutant flies (Grönke, 2007).

To address the in vivo response of AKHR bmm double mutants to induced energy-storage mobilization, flies were starved and their survival curve monitored. AKHR bmm double mutants die rapidly after food deprivation. In contrast to the starvation-resistant obese AKHR and bmm single mutants, the double mutants are not capable of mobilizing even part of their excessive fat stores. AKHR bmm double mutants do not, however, suffer from a general block of energy-storage mobilization because they can access and deplete their carbohydrate stores. These data demonstrate that energy homeostasis in AKHR bmm double-mutant flies is imbalanced by a severe and specific lipometabolism defect, which cannot be compensated in vivo (Grönke, 2007).

The nature of Brummer as a TAG lipase and AKHR as a transmitter of lipolytic AKH signaling suggests that the extreme storage-fat accumulation and starvation sensitivity of ad libitum-fed AKHR bmm double mutants is due to severe lipolysis dysfunction. To address this possibility in vitro, lipolysis rate measurements on fly fat body cell lysates and lysate fractions of control flies were performed. Results show that the cytosolic fraction of fat body cells contains the majority of basal and starvation-induced lipolytic activity against TAG, similar to the activity distribution in mammalian adipose tissue. Little basal and induced total TAG cleavage activity localizes to the lipid droplet fraction, whereas the pellet fraction including cellular membranes shows low basal, non-inducible TAG lipolysis. Lipolysis activity against DAG is similarly distributed between fat body cell fractions. However, in accordance with the function of DAG as major transport lipid in Drosophila, DAG lipolysis in fat body cells is not induced in response to starvation (Grönke, 2007).

Based on the lysate fraction analysis of control flies, cytosolic fat body cell extracts were used to assess the basal and starvation-induced lipolytic activity of mutant and control flies on TAG, DAG, and cholesterol oleate substrates. Whereas DAG and cholesterol oleate cleavage activity of fat body cells is comparable between all genotypes and physiological conditions tested, TAG lipolysis varies widely. Compared to control flies, basal TAG lipolysis of AKHR bmm double mutants is reduced by 80% and induced TAG cleavage is completely blocked, consistent with the flies' extreme obesity and their inability to mobilize storage fat. The impairment of basal lipolysis in the double mutants is largely due to the absence of bmm function, because it is also detectable in bmm single-mutant cells, whereas basal lipolysis in AKHR mutants is not reduced. Interestingly, bmm mutants mount a starvation-induced TAG lipolysis response after short-term (6 h), but not after extended (12 h), food deprivation. Conversely, AKHR mutant cells lack an early lipolysis response, but exhibit strong TAG cleavage activity after extended food deprivation. These data suggest that induced storage-fat mobilization in fly adipocytes relies on at least two lipolytic phases: an early, AKH/AKHR-dependent phase and a later, Brummer-dependent phase. Accordingly, it is speculated that the obesity of bmm and AKHR mutant flies is caused by different mechanisms: chronically low basal lipolysis in bmm mutants and, in AKHR mutants, lack of induced lipolysis during short-term starvation periods that is characteristic of organisms with discontinuous feeding behavior. It is acknowledged, however, that in vitro lipolysis assays on artificially emulsified substrates allow only a limited representation of the lipocatabolism in vivo, because lipid droplet-associated proteins modulate the lipolytic response in the insect fat body and mammalian tissue. Moreover, excessive fat accumulation in AKHR mutants may be in part due to increased lipogenesis because AKH signaling has been demonstrated to repress this process in various insects (Grönke, 2007).

Fat body cells of control flies (AKHRrev bmmrev) exhibit basal TAG lipolysis, which is doubled by starvation-induced lipolysis after 6 h or 12 h of food deprivation. bmm mutant cells have reduced basal lipolysis and lack induced lipolysis after 12 h starvation. AKHR mutant cells lack early (6 h) induced lipolysis, but show strong starvation-induced lipolysis after 12 h food deprivation. AKHR bmm double mutants have reduced basal lipolysis and lack starvation-induced lipolysis altogether (Grönke, 2007).

The finding of the dual lipolytic control in the fly raises the question of whether the two systems involved act independently of each other or whether one system responds to the impairment of the other. Modulation of transcription is an evolutionarily conserved regulatory mechanism of lipases from the ATGL/Brummer family. ATGL is transcriptionally up-regulated in fasting mice, as is bmm transcription in starving flies. Moreover bmm overexpression depletes lipid stores in the fat body of transgenic flies. Accordingly, bmm transcription was analyzed in response to modulation of AKH/AKHR signaling to assess a potential regulatory interaction between the two lipolytic systems. Compared to the moderate starvation-induced up-regulation of bmm in control flies, the gene is hyperstimulated in flies with impaired AKH signaling. As early as 6 h after food deprivation, bmm transcription is up-regulated by a factor of 2.5-3 in flies lacking the AKH-producing neuroendocrine cells (AKH-ZD) or in AKHR mutant flies. Conversely, chronic expression of AKH in the fat body suppresses bmm transcription. Bmm hyperstimulation in AKHR mutants is consistent with a subsequent strong increase of starvation-induced TAG lipolysis observed 12 h after food deprivation. Taken together, these data demonstrate an AKH/AKHR-independent activation mechanism of bmm and suggest the existence of compensatory regulation between bmm and the AKH/AKHR lipolytic systems, the mechanism of which is currently unknown (Grönke, 2007).

The results presented in this study provide in vivo evidence that the fly contains two induced lipolytic systems. One system confers AKH/AKHR-dependent lipolysis, a signaling pathway, which assures rapid fat mobilization by cAMP signaling and PKA activity. Drosophila's second lipolytic system involves the Brummer lipase, which is responsible for most of the basal and part of the induced lipolysis in fly fat body cells, likely via transcriptional regulation. Currently, it is unknown whether Brummer activity is post-translationally modulated by an α/β hydrolase domain-containing protein like the regulation of its mammalian homolog ATGL by CGI-58. Homology searches between mammalian and Drosophila genomes identify the CGI-58-related fly gene CG1882 and the putative Hsl homolog CG11055, providing additional support for the evolutionary conservation of fat-mobilization systems. However, differences in lipid transport physiology (i.e., DAG transport in Drosophila, and FFA in mammals) suggest a different substrate specificity or tissue distribution of fly Hsl compared to its mammalian relative (Grönke, 2007).

Future studies will not only unravel the crosstalk between the two Drosophila lipocatabolic systems, but also disclose the identity of additional genes involved in this process, such as the upstream regulators of bmm. This study substantiates the emerging picture of the evolutionary conservation between insect and mammalian fat-storage regulation and emphasizes the value of Drosophila as a powerful model system for the study of human lipometabolic disorders (Grönke, 2007).

Flightless-I Controls Fat Storage in Drosophila

Triglyceride homeostasis is a key process of normal development and is essential for the maintenance of energy metabolism. Dysregulation of this process leads to metabolic disorders such as obesity and hyperlipidemia. This study reports a novel function of the Drosophila flightless-I (fliI) gene in lipid metabolism. Drosophila fliI mutants were resistant to starvation and showed increased levels of triglycerides in the fat body and intestine, whereas fliI overexpression decreased triglyceride levels. These flies suffered from metabolic stress indicated by increased levels of trehalose in hemolymph and enhanced phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha). Moreover, upregulation of triglycerides via a knockdown of fliI was reversed by a knockdown of desat1 in the fat body of flies. These results indicate that fliI suppresses the expression of desat1, thereby inhibiting the development of obesity; fliI may, thus, serve as a novel therapeutic target in obesity and metabolic diseases (Park, 2018).

Remote control of insulin secretion by fat cells in Drosophila

Insulin-like peptides (ILPs) couple growth, metabolism, longevity, and fertility with changes in nutritional availability. In Drosophila, several ILPs called Dilps are produced by the brain insulin-producing cells (IPCs), from which they are released into the hemolymph and act systemically. In response to nutrient deprivation, brain Dilps are no longer secreted and accumulate in the IPCs. The larval fat body, a functional homolog of vertebrate liver and white fat, couples the level of circulating Dilps with dietary amino acid levels by remotely controlling Dilp release through a TOR/RAPTOR-dependent mechanism. Ex vivo tissue coculture was used to demonstrate that a humoral signal emitted by the fat body transits through the hemolymph and activates Dilp secretion in the IPCs. Thus, the availability of nutrients is remotely sensed in fat body cells and conveyed to the brain IPCs by a humoral signal controlling ILP release (Géminard, 2009).

Due to the lack of immunoassay, the study of the regulation of Dilp levels in Drosophila has been limited so far to the analysis of their expression level in response to nutritional conditions. This study presents evidence that the secretion of Dilp2 and Dilp5 as well as a GFP linked to a signal peptide (secGFP) is controlled by the nutritional status of the larva. The data also indicate that the IPCs have the specific ability to couple secretion with nutritional input. This suggests that all Dilps produced in the IPCs could be subjected to a common control on their secretion that could therefore override differences in their transcriptional regulation. It was further shown that the regulation of Dilp secretion plays a key role in controlling Dilp circulating levels and biological functions, since blocking neurosecretion in the IPCs led to growth and metabolic defects, and conversely, expression of Dilp2 in nonregulated neurosecretory cells is lethal upon starvation. Interestingly, previous reports suggest that Dilp release could also be controlled in the adult IPCs, raising the possibility that this type of regulation contributes to controlling metabolic homeostasis, reproduction, and aging during adult life (Géminard, 2009).

Dilp release is not activated by high-carbohydrate or high-fat diets, but rather depends on the level of amino acids and in particular on the presence of branched-chain amino acids like leucine and isoleucine. This finding is consistent with the described mechanism of TOR activation by leucine in mammalian cells (Avruch, 2009: Nicklin, 2009). In particular, it was recently shown that Rag GTPases can physically interact with mTORC1 and regulate its subcellular localization in response to L-leucine (Sancak, 2008). Interestingly, the present work indicates that amino acids do not directly signal to the IPCs, but rather they act on fat-body cells to control Dilp release. TOR signaling has been previously shown to relay the nutritional input in fat-body cells. Tor signaling is required for the remote control of Dilp secretion, since inhibition of Raptor-dependent TOR activity in fat cells provokes Dilp retention. Surprisingly, activation of TOR signaling in fat cells of underfed larvae is sufficient to induce Dilp release, indicating that TOR signaling is the major pathway relaying the nutrition signal from the fat body to the brain IPCs. In contrast, inhibition of PI3K activity in fat cells does not appear to influence Dilp secretion in the brain. This result is in line with previous in vivo data showing that reduction of PI3K levels in fat cells does not induce systemic growth defects. Altogether, this suggests that the nutritional signal is read by a TOR-dependent mechanism in fat cells, leading to the production of a secretion signal that is conveyed to the brain by the hemolymph (Géminard, 2009).

Ex vivo brain culture experiments demonstrate that hemolymph or dissected fat bodies from fed larvae constitute an efficient source for the Dilp secretion factor. This signal is absent in underfed animals, suggesting that it could be identified by comparative analysis of fed and underfed states. The nature of the secretion signal is unknown. It is produced and released in the hemolymph by fat cells, and its production relies on TORC1 function. Given the role of TORC1 in protein translation, one could envisage that the secretion factor is a protein or a peptide for which translation is limited by TORC1 activity and relies on amino acid input in fat-body cells. In mammals, fatty acids and other lipid molecules have the capacity to amplify glucose-stimulated insulin secretion in pancreatic β cells. The fly fat body carries important functions related to lipid metabolism, and a recent link has been established between TOR signaling and lipid metabolism in flies (Porstmann, 2008), leaving open the possibility that a TOR-dependent lipid-based signal could also operate in this regulation. Interestingly, carbohydrates do not appear to contribute to the regulation of insulin secretion by brain cells in flies. This finding is reminiscent of the absence of expression of the Sur1 ortholog in the IPCs and suggests that global carbohydrate levels are controlled by the glucagon-like AKH produced by the corpora cardiaca cells (Géminard, 2009).

These experiments demonstrate that Dilp secretion is linked to the polarization state of the IPC membrane, suggestive of a calcium-dependent granule exocytosis, like the one observed for insulin and many other neuropeptides. The nature of the upstream signal controlling membrane depolarization is not known. Recent data concerning the function of the nucleostemin gene ns3 in Drosophila suggest that a subset of serotonergic neurons in the larval brain act on the IPCs to control insulin secretion (Kaplan, 2008). Therefore, it remains to be known whether the IPCs or upstream serotonergic neurons constitute a direct target for the secretion signal. So far, no link has been established between the serotonergic stimulation of IPC function and the nutritional input (Géminard, 2009).

In 1998, J. Britton and B. Edgar presented experiments where starved brain and fed fat bodies were cocultured, allowing arrested brain neuroblasts to resume proliferation in the presence of nutrients (Britton and Edgar, 1998). From these experiments, the authors proposed that quiescent neuroblasts were induced to re-enter the cell cycle by a mitogenic factor emanating from the fed fat bodies. The present data extend these pioneer findings and suggest the possibility that the factor sent by the fed fat bodies is the secretion factor that triggers Dilp release from the IPCs, allowing neuroblasts to continue their growth and proliferation program through paracrine Dilp-dependent activation (Géminard, 2009).

In conclusion, this work combines genetic and physiology approaches on a model organism to decipher key physiological regulations and opens the route for a genetic study of the molecular mechanisms controlling insulin secretion in Drosophila (Géminard, 2009).

A neuronal relay mediates a nutrient responsive gut/fat body axis regulating energy homeostasis in adult Drosophila

The control of systemic metabolic homeostasis involves complex inter-tissue programs that coordinate energy production, storage, and consumption, to maintain organismal fitness upon environmental challenges. The mechanisms driving such programs are largely unknown. This study shows that enteroendocrine cells in the adult Drosophila intestine respond to nutrients by secreting the hormone Bursicon alpha, which signals via its neuronal receptor DLgr2/Rickets. Bursicon alpha/DLgr2 regulate energy metabolism through a neuronal relay leading to the restriction of glucagon-like, adipokinetic hormone (AKH) production by the corpora cardiaca and subsequent modulation of AKH receptor signaling within the adipose tissue. Impaired Bursicon alpha/DLgr2 signaling leads to exacerbated glucose oxidation and depletion of energy stores with consequent reduced organismal resistance to nutrient restrictive conditions. Altogether, this work reveals an intestinal/neuronal/adipose tissue inter-organ communication network that is essential to restrict the use of energy and that may provide insights into the physiopathology of endocrine-regulated metabolic homeostasis (Scopelliti, 2018).

Maintaining systemic energy homeostasis is crucial for the physiology of all living organisms. A balanced equilibrium between anabolism and catabolism involves tightly coordinated signaling networks and the communication between multiple organs. Excess nutrients are stored in the liver and adipose tissue as glycogen and lipids, respectively. In times of high energy demand or low nutrient availability, nutrients are mobilized from storage tissues. Understanding how organs communicate to maintain systemic energy homeostasis is of critical importance, as its failure can result in severe metabolic disorders with life-threatening consequences (Scopelliti, 2018).

The intestine is a key endocrine tissue and central regulator of systemic energy homeostasis. Enteroendocrine (ee) cells secrete multiple hormones in response to the nutritional status of the organism and orchestrate systemic metabolic adaptation across tissues. Recent work reveals greater than expected diversity, plasticity, and sensing functions of ee cells. Nevertheless, how ee cells respond to different environmental challenges and how they coordinate systemic responses is unclear. A better understanding of ee cell biology will directly impact understanding of intestinal physiopathology, the regulation of systemic metabolism, and metabolic disorders (Scopelliti, 2018).

Functional studies on inter-organ communication are often challenging in mammalian systems, due to their complex genetics and physiology. The adult Drosophila midgut has emerged as an invaluable model system to address key aspects of systemic physiology, host-pathogen interactions, stem cell biology and metabolism, among other things. As in its mammalian counterpart, the Drosophila adult intestinal epithelium displays multiple subtypes of ee cells with largely unknown functions. Recent work has demonstrated nutrient-sensing roles of ee cells (Scopelliti, 2018 and references therein).

The role of Bursicon/DLgr2 signaling has long been restricted to insect development, where the heterodimeric form of the hormone Bursicon, made by α and β subunits, is produced by a subset of neurons within the CNS during the late pupal stage and released systemically to activate its receptor DLgr2 in peripheral tissues to drive post-molting sclerotization of the cuticle and wing expansion. A recent study demonstrated a post-developmental activity for the α subunit of Bursicon (Bursα), which is produced by a subpopulation of ee cells in the posterior midgut, where it paracrinally activates DLgr2 in the visceral muscle (VM) to maintain homeostatic intestinal stem cell (ISC) quiescence (Scopelliti, 2014; Scopelliti, 2016; Scopelliti, 2018).

This study reports an unprecedented systemic role for Bursα regulating adult energy homeostasis. This work identifies a novel gut/fat body axis, where ee cells orchestrate organismal metabolic homeostasis. Bursα is systemically secreted by ee cells in response to nutrient availability and acts through DLgr2+ neurons to repress adipokinetic hormone (AKH)/AKH receptor (AKHR) signaling within the fat body/adipose tissue to restrict the use of energy stores. Impairment of systemic Bursα/DLgr2 signaling results in exacerbated oxidative metabolism, strong lipodystrophy, and organismal hypersensitivity to nutrient deprivation. This work reveals a central role for ee cells in sensing organismal nutritional status and maintaining systemic metabolic homeostasis through coordination of an intestinal/neuronal/adipose tissue-signaling network (Scopelliti, 2018).

This study shows that ee cells secrete Bursα in the presence of plentiful nutrients, while caloric deprivation reduces its systemic release and consequently results in hormone accumulation within ee cells. Interestingly, it was observed that conditions leading to the latter scenario are accompanied by reduced bursα transcription. The reasons underlying the inverse correlation between midgut bursα mRNA and protein levels are unclear and may represent part of a negative feedback mechanism for ultimate control of further protein production. A similar phenomenon is described during the regulation of the secretion of other endocrine hormones, such as DILPs (Scopelliti, 2018).

The results show that Bursα within ee cells is preferably regulated in response to dietary sugars. This is further supported by the function of Glut1 as at least one of the transmembrane sugar transporters connecting nutrient availability to Bursα signaling. Glut1 is the closest homolog of the mammalian regulator of ee incretin secretion SLC2A2, and it has been shown to positively regulate the secretion of peptide hormones in flies (Park, 2014). Whether Glut1 is a central sensor of dietary sugars and hormone secretion by ee cells remains to be addressed. However, it is likely that, in the face of challenges, such as starvation, multiple mechanisms of nutrient sensing and transport converge to allow a robust organismal adaptation to stressful environmental conditions (Scopelliti, 2018).

Reduction of systemic Bursα/DLgr2 signaling induces a complex metabolic phenotype, characterized by lipodystrophy and hypoglycemia, which is accompanied by hyperphagia. These phenotypes are not due to poor nutrient absorption or uptake by tissues or impaired synthesis of energy stores but are rather a consequence of increased catabolism. This is supported by a higher rate of glucose-derived 13C incorporation into TCA cycle intermediates, accompanied by increased mitochondrial respiration and body-heat production (Scopelliti, 2018).

While glucose tracing experiments help explain the hypoglycemic phenotype of Bursα/DLgr2-compromised animals even in the context of hyperphagia, they do not directly address the reduction in fat body triacylglycerides (TAGs). The latter would require 13C6-palmitate tracing for assessment of the rate of lipid oxidation and incorporation into the TCA cycle. This was precluded by overall poor uptake of 13C6-palmitate into adult animals even after prolonged periods of feeding. However, the depletion of fat body TAG stores in the presence of normal de novo lipid synthesis in Bursα/DLgr2-impaired animals strongly suggests that at least part of the increased rate of O2 consumption in those animals results from increased lipid breakdown via mitochondrial fatty acid oxidation. Consistently, increased O2 consumption rates and the thermogenic phenotype of Bursα/DLgr2-deficient animals are attenuated upon reduction of AKH/AKHR signaling. Finally, the functional role of Hormone-sensitive lipase (dHSL) in the fat body further supports the regulation of lipid breakdown by AKH/AKHR signaling as at least one of the key aspects mediating the role of Bursα/DLgr2 signaling in adult metabolic homeostasis (Scopelliti, 2018).

Previous work revealed that ee Bursα is required to maintain homeostatic ISC quiescence in the adult Drosophila midgut; that is, in the midgut of unchallenged and well-fed animals (Scopelliti, 2014, Scopelliti, 2016). Such a role of Bursα is mediated by local or short-range signaling through DLgr2 expressed within the midgut VM (Scopelliti, 2014). This study demonstrates a systemic role of Bursα that does not involve VM-derived DLgr2 but rather signals through its neuronal receptor. In that regard, the paracrine and endocrine functions of Bursα/DLgr2 are uncoupled. However, the regulation of ee-derived Bursα by nutrients is likely to affect local as well as systemic Bursα/DLgr2 signaling. Retention of Bursα within ee as observed in conditions of starvation may impair the hormone's signaling into the VM, which, in principle, would lead to ISC hyperproliferation (Scopelliti, 2014). In fact, under full nutrient conditions, genetic manipulations impairing systemic Bursα signaling, such as ee Glut1 knockdown or osbp overexpression, lead to ISC hyperproliferation comparable with that observed upon bursα knockdown (Scopelliti, 2014). This represents an apparent conundrum, as ISC proliferation is not the expected scenario in the context of starvation. However, starvation completely overcomes ISC proliferation in Bursα-impaired midguts. This is consistent with recent evidence showing that restrictive nutrient conditions, such as the absence of dietary methionine or its derivative S-adenosyl methionine, impair ISC proliferation in the adult fly midgut, even in the presence of activated mitogenic signaling pathways (Obata, 2018). Altogether, these data support a scenario in which starvation, while preventing systemic and local Bursα/DLgr2 signaling, would not result in induction of ISC proliferation as a side effect (Scopelliti, 2018).

Drosophila DLgr2 is the ortholog of mammalian LGR4, -5, and -6 with closer homology to LGR4. While LGR5 and 6 are stem cell markers in several tissues, such as small intestine and skin, LGR4 depicts broader expression patterns and physiological functions. LGR4, -5, and -6 are best known to enhance canonical Wnt signaling through binding to R-spondins. However, several lines of evidence support a more promiscuous binding affinity for LGR4, which can act as a canonical G-protein coupled receptor inducing iCa2+ and cyclic AMP signaling (Scopelliti, 2018).

Interestingly, an activating variant of LGR4 (A750T) is linked to obesity in humans, while the nonsense mutation c.376C>T (p.R126X) is associated with reduced body weight. Recent reports show that LGR4 homozygous mutant (LGR4m/m) mice display reduced adiposity and are resistant to diet- or leptin-induced obesity. These phenotypes appear to derive from increased energy expenditure through white-to-brown fat conversion and are independent of Wnt signaling. The tissue and molecular mechanisms mediating this metabolic role of LGR4 remain unclear. Therefore, the current paradigm may lead to a better understanding of LGR4's contribution to metabolic homeostasis and disease. Importantly, the results highlight the intestine and ee cells in particular as central orchestrators of metabolic homeostasis and potential targets for the treatment of metabolic dysfunctions (Scopelliti, 2018).

Bursicon is an insect-specific hormone. Therefore, direct mammalian translation of the signaling system presented in this study is unlikely. However, given the clear parallels between the metabolic functions of DLgr2 and LGR4, analysis of enteroendocrine cell-secreted factors in mammalian systems may reveal new and unexpected ligands for LGR4 (Scopelliti, 2018).

An obligatory role for neurotensin in high-fat-diet-induced obesity

Obesity and its associated comorbidities (for example, diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually and are among the most prevalent and challenging conditions confronting the medical profession. Neurotensin (NT; also known as NTS), a 13-amino-acid peptide predominantly localized in specialized enteroendocrine cells of the small intestine and released by fat ingestion, facilitates fatty acid translocation in rat intestine, and stimulates the growth of various cancers. The effects of NT are mediated through three known NT receptors (NTR1, 2 and 3; also known as NTSR1, 2, and NTSR3, respectively). Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality; however, a role for NT as a causative factor in these diseases is unknown. This study shows that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. It was further demonstrated that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates fatty acid absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3 (also known as sortilin). Consistent with the findings in mice, expression of NT in Drosophila midgut enteroendocrine cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, it was shown that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. These findings directly link NT with increased fat absorption and obesity and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment (Li, 2016).

High amylose starch consumption induces obesity in Drosophila melanogaster and metformin partially prevents accumulation of storage lipids and shortens lifespan of the insects

There are very few studies that have directly analyzed the effects of dietary intake of slowly digestible starches on metabolic parameters of animals. The present study examined the effects of slowly digestible starch with high amylose content (referred also as amylose starch) either alone, or in combination with metformin on the development, lifespan, and levels of glucose and storage lipids in the fruit fly Drosophila melanogaster. Consumption of amylose starch in concentrations 0.25-10% did not affect D. melanogaster development, whereas 20% starch delayed pupation and reduced the number of larvae that reached the pupal stage. Starch levels in larval food, but not in adult food, determined levels of triacylglycerides in eight-day-old adult flies. Rearing on diet with 20% starch led to shorter lifespan and a higher content of triacylglycerides in the bodies of adult flies as compared with the same parameters in flies fed on 4% starch diet. Food supplementation with 10mM metformin partly attenuated the negative effects of high starch concentrations on larval pupation and decreased triacylglyceride levels in adult flies fed on 20% starch. Long-term consumption of diets supplemented with metformin and starch decreased lifespan of the insects, compared with the diet supplemented with starch only. The data show that in Drosophila high starch consumption may induce a fat fly phenotype and metformin may partially prevent it (Abrat, 2018).

Lipid-gene regulatory network reveals coregulations of triacylglycerol with phosphatidylinositol/lysophosphatidylinositol and with hexosyl-ceramide

Lipid homeostasis is important for executing normal cellular functions and maintaining physiological conditions. The biophysical properties and intricate metabolic network of lipids underlie the coordinated regulation of different lipid species in lipid homeostasis. To reveal the homeostatic response among different lipids, this study systematically knocked down 40 lipid metabolism genes in Drosophila S2 cells by RNAi and profiled the lipidomic changes. Clustering analyses of lipids reveal that many pairs of genes acting in a sequential fashion or sharing the same substrate are tightly clustered. Through a lipid-gene regulatory network analysis, it was further found that a reduction of triacylglycerol (TAG) is associated with an increase of phosphatidylinositol (PI) and lysophosphatidylinositol (LPI) or a reduction of hexosyl-ceramide (HexCer) and hydroxylated hexosyl-ceramide (OH-HexCer). Importantly, negative coregulation between TAG and LPI/PI, and positive coregulation between TAG and HexCer, were also found in human Hela cells. Together, these results reveal coregulations of TAG with PI/LPI and with HexCer in lipid homeostasis (Wang, 2018).

Drosophila lipin interacts with insulin and TOR signaling pathways in the control of growth and lipid metabolism

Lipin proteins have key functions in lipid metabolism, acting as both phosphatidate phosphatases (PAPs) and nuclear regulators of gene expression. This study shows that the insulin and TORC1 pathways independently control functions of Drosophila dLipin. Reduced signaling through the insulin receptor strongly enhances defects caused by dLipin deficiency in fat body development, whereas reduced signaling through TORC1 leads to translocation of dLipin into the nucleus. Reduced expression of dLipin results in decreased signaling through the insulin receptor-controlled PI3K/Akt pathway and increased hemolymph sugar levels. Consistent with this, downregulation of dLipin in fat body cell clones causes a strong growth defect. The PAP, but not the nuclear activity of dLipin is required for normal insulin pathway activity. Reduction of other enzymes of the glycerol-3 phosphate pathway similarly affects insulin pathway activity, suggesting an effect mediated by one or more metabolites associated with the pathway. Together, these data show that dLipin is subject to intricate control by the insulin and TORC1 pathways and that the cellular status of dLipin impacts how fat body cells respond to signals relayed through the PI3K/Akt pathway (Schmitt, 2015).

Normal growth and the maintenance of a healthy body weight require a balance between food intake, energy expenditure and organismal energy stores. Two signaling pathways, the insulin pathway and the target of rapamycin (TOR) complex 1 (TORC1) pathway, play a critical role in this balancing process. Insulin or, in Drosophila, insulin-like peptides called Dilps are released into the circulatory system upon food consumption and stimulate cellular glucose uptake while promoting storage of surplus energy in the form of triacylglycerol (TAG or neutral fat). Nutrients, in particular amino acids, activate the TORC1 pathway, which stimulates protein synthesis leading to cellular and organismal growth. The two pathways are interconnected to allow crosstalk, but the extent and biological significance of crosstalk seems to be highly dependent on the physiological context and may be different in different animal groups. For instance, tuberous sclerosis protein TSC 2, which together with TSC1 inhibits TORC1 signaling, can be phosphorylated by Akt, the central kinase of the insulin pathway, in both mammals and Drosophila melanogaster. However, phosphorylation of TSC2 by Akt is not required for normal growth and development in Drosophila, whereas in mammalian cells Akt phosphorylation of TSC2 is required for normal TORC1 activity and the resulting activation of ribosomal protein kinase S6K1 (Schmitt, 2015).

Studies in mice have identified one of the three mammalian lipin paralogs, lipin 1, as a major downstream effector mediating effects of insulin and TORC1 signaling on lipid metabolism. In both Drosophila and mice, proteins of the lipin family function as key regulators of TAG storage and fat tissue development. Lipins execute their biological functions through two different biochemical activities, a phosphatidate phosphatase (PAP) activity that converts phosphatidic acid (PA) into diacylglycerol (DAG), and a transcriptional co-regulator activity, mediated by an LxxIL motif located in close proximity to the catalytic motif of the protein. The PAP activity of lipin constitutes an essential step in the glycerol-3 phosphate pathway that leads to the production to TAG, which is stored in specialized cells in the form of fat droplets (adipose tissue in mammals and fat body in insects). In addition, the product of the PAP activity of lipin, DAG, is a precursor for the synthesis of membrane phospholipids. As a transcriptional co-regulator, mammalian lipin 1 directly regulates the gene encoding nuclear receptor PPARγ, which regulates mitochondrial fatty acid β-oxidation, and the yeast lipin homolog has been shown to regulate genes required for membrane phospholipid synthesis (Schmitt, 2015 and references therein).

In cultured adipocytes, insulin stimulates phosphorylation of lipin 1 in a rapamycin- sensitive manner, suggesting that phosphorylation is mediated mTORC1. Phosphorylation by mTOR blocks nuclear entry of lipin 1 and, thus, access to target genes. Interestingly, non- phosphorylated lipin 1 that has migrated into the nucleus affects nuclear protein levels, but not mRNA levels, of the transcription factor SREBP1, which is a key regulator of genes involved in fatty acid and cholesterol synthesis. This effect requires the catalytic activity of lipin 1, suggesting that not all nuclear effects of the protein may result from a direct regulation of gene transcription. The lowering of nuclear SREBP protein abundance by lipin 1 counteracts the effects of Akt on lipid metabolism, which activates lipogenesis in a TORC1- dependent manner by activation of SREBP (Schmitt, 2015).

Lipins are not only subject to control by insulin and TORC1 signaling, they also have an effect on the insulin sensitivity of tissues. Lipin 1-deficient mice exhibit insulin resistance and elevated insulin levels, whereas over-expression in adipose tissue increases insulin sensitivity. Similarly, in humans, lipin 1 levels in adipose tissue are inversely correlated with glucose and insulin levels as well as insulin resistance. While these data indicate that adipose tissue expression of lipin 1 is an important determinant of insulin sensitivity, the underlying mechanism remains poorly understood. This study presents evidence that the only Drosophila lipin homolog, dLipin, cell- autonomously controls the sensitivity of the larval fat body to stimulation of the insulin/PI3K/Akt pathway. dLipin mutant larvae have increased hemolymph sugar levels, and larval fat body cells that are deficient of dLipin exhibit a severe growth defect. Loss-of-function and rescue experiments show that dLipin's PAP activity and an intact glycerol-3 phosphate pathway are required for normal insulin pathway activity in fat body cells. Similar to the control of lipin 1 in mammalian cells, the insulin/PI3K pathway controls functions of dLipin in fat tissue development and fat storage, and the TORC1 pathway controls nuclear translocation of dLipin. However, in an apparent contrast to regulation of lipin 1 in mammals, the current data suggest that the two pathways exert at least part of their effects on dLipin independent of one another (Schmitt, 2015).

The data indicated that normal growth of fat body cells depends on sufficient levels of dLipin. Interestingly, cytoplasmic growth seems to be more affected by lack of dLipin than endoreplicative growth, as indicated by an increased nucleocytoplasmic ratio. How does dLipin affect growth? Fat body cells of dLipin mutants and cells in which dLipin is downregulated by RNAi exhibit a striking lack of the second messenger PIP3 in the cell membrane, associated with reduced cellular levels of active Akt. These data indicate that dLipin has an influence on signaling through the canonical InR/PI3K/Akt pathway. PIP2 levels in the cell membrane were unchanged in dLipin-deficient fat body, indicating that lack of PIP3 was not caused by scarcity of the substrate of PI3K. Since RNAi knockdown of dLipin was sufficient to prevent an increase in cell growth induced by overexpression of a constitutively active form of the catalytic subunit of PI3K, Dp110, it seems that disruption of the InR/PI3K/Akt pathway occurs either at the level of PI3K or the PI3K antagonist PTEN (Schmitt, 2015).

The hemolymph of dLipin mutant larvae contains increased levels of sugars, a condition which may result from insulin resistance and/or decreased Dilp levels. The data strongly suggest that insulin resistance at least contributes to increased sugar levels for two reasons. First, reduction of dLipin specifically in the fat body reduces insulin responses in this tissue, but not in other tissues. This suggests that insulin (Dilp) levels are unaffected. Second, mosaic data show that lack of dLipin affects cell growth, which is controlled by the InR/PI3K/Akt pathway, in a cell-autonomous manner. Thus, individual cells that lack dLipin show impaired growth in an otherwise normal physiological background, further supporting the notion that lack of dLipin affects insulin (Dilp) sensitivity, but not insulin signaling itself. Consistent with the current observations in Drosophila, insulin resistance is one of the phenotypes exhibited by fld mice that lack lipin 1. Similar to mice, expression of lipin 1 in humans is positively correlated with insulin sensitivity of liver and adipose tissue. However, mechanisms that mediate effects of lipins on insulin sensitivity are not well understood. The current data show that dLipin's PAP activity is required for normal insulin sensitivity and that reduction of GPAT4 or AGPAT3, two other enzymes of the glycerol-3 phosphate pathway, has similar effects on membrane-associated PIP3 as reduction of dLipin. This suggests that the effect of dLipin on insulin sensitivity is mediated by intracellular changes in metabolites, e.g., TAGs or fatty acids, that are brought about by changed flux through the glycerol-3 phosphate pathway (Schmitt, 2015).

The data show that reduced activity of InR in dLipin-deficient fat body leads to a phenotype that strongly resembles the severe fat body phenotype of dLipin loss-of- function mutants. This observation strongly suggests that reduced signaling through InR further reduces the activity of dLipin. Since reduced activity of InR has no substantial impact on dLipin protein levels, a likely explanation for this effect is that the InR pathway controls the activity of dLipin through post-translational modification. This interpretation is supported by data showing that phosphorylation of dLipin in Drosophila S2 cells responds to insulin stimulation, and it is consistent with a substantial body of evidence showing that mammalian lipin 1 is regulated by phosphorylation in response to insulin signaling. This suggests that functions of the insulin signaling pathway in the regulation of lipins are evolutionarily conserved (Schmitt, 2015).

In contrast to reduced signaling through the InR/PI3K pathway, reduced signaling through TORC1 led to translocation of dLipin into the nucleus. A similar translocation into the cell nucleus has been observed for lipin 1 after loss of TORC1 in mammalian cells. Consistent with the role of TORC1 as a nutrient sensor, nuclear enrichment of dLipin is observed during starvation, and previous work has shown that the presence of dLipin is critical for survival during starvation. Together, these data suggest that both dLipin and lipin 1 have essential nuclear, gene-regulatory functions during starvation. What may be the genes controlled by nuclear lipins, and how do they control gene expression? In the mouse, it has been shown that lipin 1 can directly activate the gene encoding nuclear receptor PPARγ and that overexpression of lipin 1 leads to the activation of genes involved in fatty acid transport and β-oxidation, TCA cycle, and oxidative phosphorylation, including many target genes of PPARγ. At the same time, expression of genes involved in fatty acid and TAG synthesis is diminished (Finck, 2006). This suggests that lipins may directly regulate genes to promote the utilization of fat stores during starvation, although gene expression studies are necessary at physiological protein levels that distinguish between the effects of nuclear and cytoplasmic lipin to confirm this hypothesis. Chromatin immunoprecipitation experiments with both yeast and mammalian cells have shown that lipins associate with regulatory regions of target genes, suggesting that nuclear lipins act as transcriptional co-regulators. Interestingly, however, lipin 1 that has translocated into the nucleus can also influence gene expression through an unknown PAP-dependent mechanism that controls nuclear levels of the transcription factor SREBP, which positively controls genes required for sterol and fatty acid synthesis. This suggests that nuclear lipins may use alternate mechanisms to bring about changes in gene expression. It will be interesting to further investigate these mechanisms taking advantage of the large size and the polytene chromosomes of fat body cells in Drosophila (Schmitt, 2015).

Interestingly, robust nuclear translocation of dLipin was observed after reducing TORC1 activity, but no nuclear translocation of dLipin was seen when signaling through the insulin pathway was reduced, neither after moderate (InR DN) or severe reduction (p60). This suggests that the InR/PI3K pathway can control functions of dLipin independent of TORC1 in Drosophila. Two observations further support this proposition. First, reduction of dLipin affects cytoplasmic and endoreplicative growth differently when enhancing growth defects associated with diminished TORC1 activity, leading to an increase in the nucleocytoplasmic ratio. No such increase was observed after reduction of TORC1 alone, suggesting that enhancement of the growth defect is an additive effect that is caused by reduced PI3K/Akt signaling and not by further reduction of TORC1 activity. Second, whereas reduction of TORC1 in the fat body leads to a systemic growth defect, lack of dLipin in the fat body does not affect organismal growth and reduction of dLipin does not affect growth of animals that lack TOR (Schmitt, 2015).

Whereas the data do not indicate that InR/PI3K signaling has an effect on the intracellular distribution of dLipin, insulin stimulates cytoplasmic retention of lipin 1 in mammalian cells in a rapamycin-sensitive manner. This suggests that the effect is mediated by TORC1, which can also regulate lipin 1 in certain cells in a rapamycin-insensitive manner. However, it is noteworthy that lipin 1 contains at least 19 serine and threonine phosphorylation sites, and that some of these sites appear to be recognized by other kinases than TOR. In view of these findings, and considering that not all phosphorylations of lipin 1 stimulated by insulin are sensitive to rapamycin, it cannot be excluded that one or more other insulin-sensitive kinases contribute to the regulation of lipin 1 and other lipins. While data on the insulin and TORC1 regulation of lipin 1 were obtained with cultured cell lines, the current whole-animal data suggest that indeed an additional pathway may exist through which insulin regulates functions of lipins independent of TORC1. It is important to note that genetic studies in Drosophila have provided a number of examples indicating that the insulin and TORC1 pathways act independently of one another when studied in the context of specific tissues during normal development. For instance, activity of the ribosomal protein kinase S6K, which is a major target of TORC1 in both flies and mammals, is unaffected by mutations of insulin pathway components in Drosophila. Furthermore, insulin and TORC1 independently control different aspects of hormone production by the Drosophila ring gland. It will be interesting to see whether whole-animal studies in mammalian systems will reveal a similar, at least partial, independence of insulin and TORC1 signaling in the control of lipins. Specifically, future work will have to address in detail the functional importance of the many phosphorylation sites found in both mammalian and fly lipins, and identify all kinases involved, to determine the extent to which regulation is conserved between fly and mammalian lipins (Schmitt, 2015).

Sir2 acts through Hepatocyte Nuclear Factor 4 to maintain insulin signaling and metabolic homeostasis in Drosophila

SIRT1 is a member of the sirtuin family of NAD+-dependent deacetylases, which couple cellular metabolism to systemic physiology. This study shows that loss of the Drosophila SIRT1 homolog sir2 leads to the age-progressive onset of hyperglycemia, obesity, glucose intolerance, and insulin resistance. Tissue-specific functional studies show that Sir2 is both necessary and sufficient in the fat body to maintain glucose homeostasis and peripheral insulin sensitivity. This study reveals a major overlap with genes regulated by the nuclear receptor Hepatocyte Nuclear Factor 4 (HNF4). Drosophila HNF4 mutants display diabetic phenotypes similar to those of sir2 mutants, and protein levels for dHNF4 are reduced in sir2 mutant animals. Sir2 exerts these effects by deacetylating and stabilizing dHNF4 through protein interactions. Increasing dHNF4 expression in sir2 mutants is sufficient to rescue their insulin signaling defects, defining this nuclear receptor as an important downstream effector of Sir2 signaling. This study provides a genetic model for functional studies of phenotypes related to type 2 diabetes and establishes HNF4 as a critical downstream target by which Sir2 maintains metabolic health (Palu, 2016).

This study shows that sir2 mutants display a range of metabolic defects that parallel those seen in mouse Sirt1 mutants, including hyperglycemia, lipid accumulation, insulin resistance, and glucose intolerance. These results suggest that the fundamental metabolic functions of Sirt1 have been conserved through evolution and that further studies in Drosophila can be used to provide insights into its mammalian counterpart. An additional parallel with Sirt1 is seen in tissue-specific studies, where sir2 function is shown to be necessary and sufficient in the fat body to maintain insulin signaling and suppress hyperglycemia and obesity, analogous to the role of Sirt1 in the liver and white adipose. These results are also consistent with published studies of insulin sensitivity in Drosophila, which have shown that the fat body is the critical tissue that maintains glucose and lipid homeostasis through its ability to respond properly to insulin signaling (Palu, 2016).

These studies also define the dHNF4 nuclear receptor as a major target for Sir2 regulation. Consistent with this, dHNF4 mutants display a range of phenotypes that resemble those of sir2 mutants, including hyperglycemia, obesity, and glucose intolerance. As expected, these defects are more severe in dHNF4 loss-of-function mutants, consistent with sir2 mutants only resulting in a partial loss of dHNF4 protein. Sir2 interacts with dHNF4 and appears to stabilize this protein through deacetylation. This is an established mechanism for regulating protein stability, either through changes in target protein conformation that allow ubiquitin ligases to bind prior to proteasomal degradation, or through alternate pathways. Further studies, however, are required to determine if this is a direct protein-protein interaction or part of a higher order complex (Palu, 2016).

Although two papers have shown that mammalian Sirt1 can control HNF4A transcriptional activity through a protein complex, only one gene was identified as a downstream target of this regulation, PEPCK, leaving it unclear if this activity is of functional significance. The current study suggests that this regulatory connection is far more extensive. The observation that one third of the genes down-regulated in sir2 mutants are also down-regulated in dHNF4 mutants (including pepck), and most of the genes up-regulated in sir2 mutants are up-regulated in dHNF4 mutants, establishes this nuclear receptor as a major downstream target for Sir2 regulation. It will be interesting to determine if the extent of this regulatory connection has been conserved through evolution (Palu, 2016).

Despite this regulatory control, the over-expression of an HNF4 transgene was only able to partially restore the insulin signaling response and not the defects in carbohydrate homeostasis in sir2 mutants. This lack of complete rescue is not surprising, given that the Sirt1 family targets a large number of transcription factors, histones, and enzymes, providing multiple additional pathways for metabolic regulation. Moreover, the activity or target recognition of dHNF4 may be altered when it is hyperacetylated, in which case merely over-expressing this factor would not fully restore normal function. Future studies can examine more direct targets, both previously characterized and uncharacterized, for their functions in suppressing diabetes downstream of Sir2-dependent regulation (Palu, 2016).

Finally, sir2 mutants represent a new genetic model for studying the age-dependent onset of phenotypes related to type 2 diabetes. Newly-eclosed sir2 mutant adults are relatively healthy, with elevated levels of free glucose and glycogen but otherwise normal metabolic functions. Their health, however, progressively worsens with age, with two-week-old sir2 mutants displaying lipid accumulation, fasting hyperglycemia, and reduced insulin signaling accompanied by insulin resistance. This is followed by the onset of glucose intolerance by three weeks of age. Previous studies of type 2 diabetes in Drosophila have relied on dietary models using wild-type animals that are subjected to a high sugar diet. Although this is a valuable approach to better define the critical role of diet in diabetes onset, it is also clear that the likelihood of developing type 2 diabetes increases with age. The discovery that sir2 mutants display this pathophysiology provides an opportunity to exploit the power of Drosophila genetics to better define the mechanisms that lead to the stepwise onset of metabolic dysfunction associated with diabetes (Palu, 2016).

Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes

Metabolic adaptation to changing dietary conditions is critical to maintain homeostasis of the internal milieu. In metazoans, this adaptation is achieved by a combination of tissue-autonomous metabolic adjustments and endocrine signals that coordinate the mobilization, turnover, and storage of nutrients across tissues. To understand metabolic adaptation comprehensively, detailed insight into these tissue interactions is necessary. This study characterize the tissue-specific response to fasting in adult flies and identified an endocrine interaction between the fat body and liver-like oenocytes that regulates the mobilization of lipid stores. Using tissue-specific expression profiling, it was confirmed that oenocytes in adult flies play a central role in the metabolic adaptation to fasting. Furthermore, it was found that fat body-derived Drosophila insulin-like peptide 6 (dILP6) induces lipid uptake in oenocytes, promoting lipid turnover during fasting and increasing starvation tolerance of the animal. Selective activation of insulin/IGF signaling in oenocytes by a fat body-derived peptide represents a previously unidentified regulatory principle in the control of metabolic adaptation and starvation tolerance (Chatterjee, 2014).

Regulation of energy stores and feeding by neuronal and peripheral CREB activity in Drosophila

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), this stud examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot that functions as adipose tissue and the liver in flies, regulating energy balance, stress resistance and feeding behavior. It was found that disruption of CREB function in neurons reduces glycogen and lipid stores and increases sensitivity to starvation. Expression of DN-CREB in the fat body also reduces glycogen levels, while it does not affect starvation sensitivity, presumably due to increased lipid levels in these flies. Interestingly, blocking CREB activity in the fat body increased food intake. These flies do not show a significant change in overall body size, suggesting that disruption of CREB activity in the fat body causes an obese-like phenotype. Using a transgenic CRE-luciferase reporter, it was further demonstrated that disruption of the adipokinetic hormone receptor, which is functionally related to mammalian glucagon and beta-adrenergic signaling, in the fat body reduces CRE-mediated transcription in flies. This study demonstrates that CREB activity in either neuronal or peripheral tissues regulates energy balance in Drosophila, and that the key signaling pathway regulating CREB activity in peripheral tissue is evolutionarily conserved (Iijima, 2009).

This study provides in vivo evidence that both neuronal and peripheral CREB activities are involved in the regulation of energy balance in flies. Blocking CREB activity in neurons causes reductions in both glycogen and lipid stores and a higher sensitivity to starvation stress. In contrast, while disruption of CREB function in the fat body also reduces glycogen levels, it increases lipid stores, and does not affect starvation sensitivity. Since there was no significant change in overall body size in these flies, disruption of CREB activity in the fat body causes an obese-like phenotype. These results also indicate that CREB activity can both increase and reduce lipid stores in flies depending on its site of action. Recently, two distinct populations of Drosophila brain neurons that regulate fat deposition were identified in Drosophila (Al-Anzi, 2009). It will be interesting to determine in which neurons CREB functions to regulate energy metabolism in flies (Iijima, 2009).

In a recent study, TORC-mediated CREB activity in neurons was shown to positively regulate glycogen and lipid stores in flies. This is based on results showing that expression of TORC in neurons rescues the starvation sensitivity of TORC mutant flies. In addition, expression of TORC in neurons partially rescues the lower energy stores of these mutants. While supporting the conclusions of this study with respect to the role of neuronal CREB activity, the current results also provide evidence that CREB in the fat body plays roles in energy balance. Moreover, in contrast to the normal feeding behavior of a TORC mutant, it was found that blocking CREB activity in the fat body increases food intake. Thus, disruption of CREB functions has a broader impact on energy metabolism and feeding behavior than the loss of TORC. It is likely that not all CREB functions depend on TORC. In support of this, although a TORC null mutant is viable and fertile, CREB mutants are lethal (Iijima, 2009).

This study found that adipokinetic hormone (adipokinetic hormone/Adipokinetic hormone receptor) signaling in the fat body, which is thought to be functionally related to glucagon/glucogon receptor signaling in the mammalian liver, positively regulates CRE-mediated transcription. In the mammalian liver, CREB activates the gluconeogenic program following a glucagon stimulus. Recent studies reported that promoting AKH signaling in the fat body significantly reduces, while loss of AKHR function modestly increases, glycogen levels in flies, presumably through AKH/AKHR-mediated carbohydrate catabolism in the fat body (Gronke, 2007; Bharucha, 2008). However, this study found that blocking CREB activity in the fat body significantly reduces glycogen levels, which would seem to contradict the proposed role of AKH/AKHR in mediating carbohydrate catabolism in the fat body. One possibility is that CREB activity in the fat body regulates multiple aspects of glucose/glycogen metabolism in addition to the AKH/AKHR-mediated pathway, and that blocking all CREB functions in the fat body reduces total glycogen levels as a net effect. In fact, significant CREB activity was remaining in AKHR mutant flies, suggesting that other signaling pathways might contribute to the activation of CREB activity in the fat body. Further studies will be required to delineate the role of CREB activity in the fat body in carbohydrate metabolism and its relationship with the AKH signaling pathway (Iijima, 2009).

This study found that blocking CREB activity in the fat body increased lipid stores. AKH/AKHR is also thought to be functionally related to β-adrenergic signaling in mammalian adipose tissue, which activates protein kinase A (PKA) and stimulates lipolysis by phosphorylating hormone-sensitive lipase and perilipin. In Drosophila, the promotion of AKH signaling in the fat body reduces lipid levels, whereas loss of AKHR function has the opposite effect; this is partly ascribed to altered activity in lipocatabolic systems. In addition, AKH signaling has been shown to repress the lipogenesis pathway in various insects. Interestingly, blocking CREB activity in mammalian liver causes excessive fat accumulation, resulting in 'fatty liver' through overactivation of liposynthesis. Future analysis will unravel whether CREB activity in the fat body represses liposynthesis and/or promotes lipid catabolism under the control of AKH/AKHR signaling (Iijima, 2009).

In summary, these results demonstrate that CREB is involved in both central and peripheral regulation of energy balance and feeding behavior in Drosophila. Future studies of CREB in flies hold great promise for revealing the mechanisms underlying energy balance and feeding behavior. Such studies will likely contribute to understanding of human metabolic disorders (Iijima, 2009).

Drosophila cytokine Unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion

In Drosophila, the fat body (FB), a functional analog of the vertebrate adipose tissue, is the nutrient sensor that conveys the nutrient status to the insulin-producing cells (IPCs) in the fly brain to release Drosophila insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. This study identified Unpaired 2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the fed state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, it was found that human Leptin can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin (Rajan, 2012).

Previous studies have postulated the existence of secreted factors, produced by the FB, that stimulate systemic growth by stimulating cell proliferation and that the FB - the fly nutrient sensor - couples Dilp secretion from the brain IPCs depending on the nutritional status. This study shows that the JAK/STAT ligand Upd2, a type 1 cytokine signal, is involved in the inter-organ communication between the FB and the brain IPCs. Human Leptin can rescue the upd2 mutant phenotypes, implying that an invertebrate model system is suited to address questions pertaining to Leptin biology. (Rajan, 2012).

Upd proteins have secondary structures predicted to have α-helices similar to that of type I cytokines belonging to the IL-6 family, and sequence alignments suggest that they show some similarity to vertebrate Leptins. Among the three Upd ligands that activate the Dome receptor, only Upd2 plays a significant role in communicating the nutritional status from the FB. This is somewhat surprising as all three Upd proteins are secreted JAK/STAT pathway agonists and are able to activate the JAK/STAT pathway non-autonomously in-vivo. However, the signal sequences of the different Upds appear to confer them with different biophysical properties, as illustrated by tissue culture assays showing that, while Upd1 and Upd3 associate primarily with the extracellular matrix, Upd2 is easily detectable in the media. In addition, secretion assays showed that Upd2 is able to condition tissue culture media more potently than either Upd1 or Upd3. Altogether, these results suggest that Upd2 activates JAK/STAT signaling at greater distances than Upd1 or Upd3 (Rajan, 2012).

As evidenced by the growth and metabolic phenotypes of FB-specific knockdown, Upd2 seems to be required only in the FB but the reason for this tissue specificity is unclear. A previous study, which analyzed the Upd2 protein using a hidden Markov model, suggested that Upd2 is probably not secreted via the 'classical' Golgi-ER machinery because it lacks a signal peptide. In fact, other type I cytokines involved in inter-organ cross-talk also lack the signal peptide and are secreted by unconventional secretory pathways. Thus, one possible reason for the tissue specificity of Upd2 could be that the FB is the only tissue that can secrete this protein in an active form. Future work, contingent on the development of techniques and reagents to detect Upd2 in the fly hemolymph, will clarify this issue (Rajan, 2012).

The identification of Upd2 as a nutrient regulated signal from the FB that does not depend on AAs but is produced in response to dietary fats and sugars reveals that different nutrient-specific secreted factors exist in the fly. Interestingly, the upregulation of upd2 levels in FB knockdown of slif suggests the existence of a homeostatic feedback loop whereby Upd2, in the context of low protein, promotes utilization of fat and carbohydrate energy sources. High sugar diets in flies have been shown to trigger a lipogenesis program akin to high fat diets in mammals, suggesting that Upd2 is most likely downstream of signals that are produced by increased fat stores. This is a highly significant finding given that it questions a broadly prevailing view that one dominant secreted factor downstream of AAs governs nearly all aspects of systemic growth and metabolism in flies. The findings support the model that the fly FB secretes numerous factors that regulate systemic growth and metabolism downstream of various components of the fly diet (Rajan, 2012).

The results indicate that STAT activation in GABAergic neurons inhibits their firing. Previous work has implied that the GABA-B-receptors in Dilp neurons inhibit Dilp release. Given that these GABAergic neurons are pre-synaptic to the IPCs, it is proposed that activation of STAT in GABAergic neurons relieves the IPCs from repression, thus resulting in Dilp release. This is reminiscent to the observation that first order-neurons responding to adipose-derived Leptin are the inhibitory GABAergic neurons expressing LepRs. When LepRs are activated by Leptin they regulate Stat3 phosphorylation which, by an unknown mechanism, inhibits the firing of the GABAergic neurons. This in turn relieves the repression on a neuronal group called POMC (propiomelanocortin) neurons allowing them to fire. Thus, this circuit module is strikingly reminiscent to what is observe in the fly. (Rajan, 2012).

There are many outstanding questions yet to be resolved regarding the signaling mechanisms by which the JAK/STAT pathway regulates GABAergic neurons. The target(s) of the JAK/STAT pathway in regulating neuronal firing in mammalian GABAergic neurons remains to be identified. It has been suggested that Leptin activation of STAT signaling may be required for the long-term effects of Leptin’s action on energy homeostasis rather than for acute effects of Leptin, and that the acute effects of Leptin on the membrane potential of certain neuronal groups require activation of PI3-K signaling rather than STAT. However, the role of JAK/STAT versus PI3-K signaling in modulating the electrophysiology of the presynaptic GABAergic neurons is yet to be clarified , especially as previous studies were done on non-GABAergic neuronal groups. Altogether, further investigations into the role of JAK/STAT signaling in modulating neurotransmission in GABAergic neurons will be necessary to clarify how JAK/STAT signaling regulates their activities. Importantly, based on the similarity of the circuits and the conservation of the signaling pathways, studies in the fly are likely to provide insights relevant to mammalian neurophysiology. (Rajan, 2012).

The physiology of Leptin signaling in vertebrates is undoubtedly more complex and different from the physiology of flies. upd2- mutant flies are smaller and leaner whereas mutations in Leptin in mammals are associated with obesity. There is however some striking parallels. It was found that under starvation upd2 mRNA steady-state levels drop significantly, and there is a significant increase of upd2 mRNA expression under high fat diets. This is similar to the alteration in Leptin levels during starvation and high fat diets. Examination of the role of Leptin in the physiology of starvation, by providing mice with exogenous Leptin during periods of nutrient restriction, revealed that the primary physiological role of Leptin is to regulate the neuroendocrine system during starvation. Leptin reduced the reproductive capacity and increased stress hormone levels, which in turn increases the survival capacity of the organism under adverse nutrient conditions. Consistent with this, flies with ablated IPCs, which are unable to produce insulin, perform much better under starvation conditions and increased stress conditions. Given that the role of Upd2 is to promote insulin secretion, the reduction of Upd2 levels during starvation decreases Dilp secretion and increases the chances of survival under starvation (upd2- mutants are more starvation resistant than the wild-type controls). Hence, in this context, the primary physiological role of Upd2 and Leptin converge (Rajan, 2012).

dSir2 deficiency in the fatbody, but not muscles, affects systemic insulin signaling, fat mobilization and starvation survival in flies

Sir2 is an evolutionarily conserved NAD+ dependent protein. Although, SIRT1 has been implicated to be a key regulator of fat and glucose metabolism in mammals, the role of Sir2 in regulating organismal physiology, in invertebrates, is unclear. Drosophila has been used to study evolutionarily conserved nutrient sensing mechanisms, however, the molecular and metabolic pathways downstream to Sir2 (dSir2) are poorly understood. This study has knocked down endogenous dSir2 in a tissue specific manner using gene-switch gal4 drivers. Knockdown of dSir2 in the adult fatbody leads to deregulated fat metabolism involving altered expression of key metabolic genes. The results highlight the role of dSir2 in mobilizing fat reserves and demonstrate that its functions in the adult fatbody are crucial for starvation survival. Further, dSir2 knockdown in the fatbody affects dilp5 (insulin-like-peptide) expression, and mediates systemic effects of insulin signaling. This report delineates the functions of dSir2 in the fatbody and muscles with systemic consequences on fat metabolism and insulin signaling. In conclusion, these findings highlight the central role that fatbody dSir2 plays in linking metabolism to organismal physiology and its importance for survival (Banerjee, 2012).

This study reports that dSir2 is a critical factor that regulates metabolic homeostasis and mediates organismal physiology. Using genetic tools (inducible RNAi) that negate background effects, concrete results are provided that highlight the importance of endogenous dSir2 in the whole body, and in metabolically relevant tissues, such as fatbody and muscle. The findings point out the importance of nutrient signaling in eliciting dSir2-dependent molecular changes, which play an important role in tissue specific metabolic functions that affect systemic outputs in flies. By describing a metabolic phenotype in flies that lack dSir2, this study reiterates that Drosophila can be used to study sirtuin biology, but also highlight the evolutionary conservation of dSir2/SIRT1 functions in regulating organismal physiology (Banerjee, 2012)

Until now, the conservation of molecular mechanisms underlying Sir2 biology was poorly addressed in invertebrates. It is only in mammals that a functional interplay between metabolic flux, SIRT1 and its downstream molecular factors has been addressed, thus far (Longo, 2006; Canto, 2009; Finkel, 2009). Results from backcrossed dSir2 mutant and whole body dSir2 knockdown flies indicated that absence or down-regulation of dSir2 expression results in gross metabolic defects. Interestingly, it was observed that the effects on glucose levels were different in these two cases. The differences in glucose levels might reflect the systemic alterations in response to a complete absence of the protein in the case of mutants and down-regulation of expression in the case of knockdowns. It is interesting to note that studies in Sirt1+/-, liver specific Sirt1 knockout and knockdown micehave also yielded seemingly conflicting results. Specifically, with respect to glucose metabolism, these differences indicate that the manifestation of functions of Sir2/SIRT1 might be dependent upon the extent to which its expression is altered. Importantly, this underpins the need to further investigate the molecular interactions that bring about such varied phenotypes, in both mammals and flies (Banerjee, 2012).

It is important to note that consistent phenotypic, metabolic and molecular readouts were obtained with respect to fat metabolism in dSir2-mutant and -RNAi flies. A decrease (or absence) of dSir2 expression was found to result in increased fat storage in the fatbodies, as determined by oil red staining and biochemical analyses. This fat accumulation is due to altered expression of genes involved in fat metabolism. Importantly, it was shown that genes involved in fat breakdown are downregulated in the dSir2 knockdown flies, in addition to an upregulation of genes involved in fat synthesis. These findings are not only in accordance with the results obtained from dSir2 mutant larvae but also implicate dSir2 as a key player in fat metabolism in adult flies (Banerjee, 2012).

A role for dSir2 was uncovered in regulating systemic insulin signaling in flies. To investigate if the ability of dSir2 to mediate insulin signaling emanates from a specific tissue, dilp5 expression was assayed in fatbody and muscle specific dSir2RNAi flies. Interestingly, it was found that knocking down dSir2 only in the fatbody, but not muscles led to increased dilp5 expression, and mimicked dSir2 mutants and whole body dSir2RNAi flies. Specifically, this study addressed the role of dSir2 in the fatbody to mediate systemic effects on insulin signaling. Further investigations should help understand the dSir2-dependent molecular and physiological links between the fatbody and medial secretory neurons (MSNs). Very recently, hepatic SIRT1 was shown to mediate peripheral insulin signaling in mice. Importantly, the current findings underpin the importance of dSir2/SIRT1 in the homologous metabolic tissues, fatbody and liver, on systemic insulin signaling (Banerjee, 2012).

Efforts to link the molecular functions of dSir2 and organismal physiology led to the implication of dSir2 in starvation survival. dSir2 mutants and whole body dSir2RNAi flies succumb to starvation earlier than the controls and interestingly, are phenocopied by fatbody dSir2RNAi flies. Moreover it was shown that this is due to an inability to mobilize fat reserves from the fatbody, and a resultant of decreased expression of lipid breakdown genes, both under fed and starved conditions. The importance of dSir2 in the fatbody and fat mobilization is corroborated by an absence of deregulated fat metabolism in muscle specific dSir2RNAi flies. Further, a lack of starvation phenotype when dSir2 is knocked down from the muscles highlights the physiological relevance of fatbody (Banerjee, 2012).

In summary, this study has elucidated the significance of the functions of dSir2 in the fatbody in mediating central and peripheral effects on metabolic homeostasis and insulin signaling. Therefore, it is concluded that dSir2 is a key component that links dietary inputs with organismal physiology and survival. Most importantly, this study highlights the functions of dSir2 in the fatbody as a deterministic factor in governing fly physiology. This study delineates the functions of dSir2 in two metabolic tissues in affecting organismal survival. Metabolic homeostasis and the ability to utilize stored energy reserves are also crucial for mediating the effects of calorie restriction. These results, which emphasize the importance of dSir2 in maintaining homeostasis, reiterates its role in calorie restriction. Finally, this report highlights the need to further investigate the functions of dSir2, and should motivate future studies to understanding Sir2's interactions with other pathways and importance during aging (Banerjee, 2012).

Degradation of arouser by endosomal microautophagy is essential for adaptation to starvation in Drosophila

Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation, was identified and characterized. Hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. This analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, these data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser (Jacomin, 2021)

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans. The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis (Shi, 2022).

A Drosophila toolkit for HA-tagged proteins unveils a block in autophagy flux in the last instar larval fat body

For in vivo functional analysis of a protein of interest (POI), multiple transgenic strains with a POI that harbors different tags are needed but generation of these strains is still labor-intensive work. To overcome this, a versatile Drosophila toolkit was developed with a genetically encoded single-chain variable fragment for the HA epitope tag: 'HA Frankenbody'. This system allows various analyses of HA-tagged POI in live tissues by simply crossing an HA Frankenbody fly with an HA-tagged POI fly. Strikingly, the GFP-mCherry tandem fluorescent-tagged HA Frankenbody revealed a block in autophagic flux and an accumulation of enlarged autolysosomes in the last instar larval and prepupal fat body. Mechanistically, lysosomal activity was downregulated at this stage, and endocytosis, but not autophagy, was indispensable for the swelling of lysosomes. Furthermore, forced activation of lysosomes by fat body-targeted overexpression of Mitf, the single MiTF/TFE family gene in Drosophila, suppressed the lysosomal swelling and resulted in pupal lethality. Collectively, it is proposed that downregulated lysosomal function in the fat body plays a role in the metamorphosis of Drosophila (Murakawa, 2022).

Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster

Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. This report shows that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies, but lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Over-expression of wild type Atg1 in adult fly muscle or adipocytes reduces lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. These research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation (Bierlein, 2023).

A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan

In gonadal tissues, the Piwi-interacting (piRNA) pathway preserves genomic integrity by employing 23-29 nucleotide (nt) small RNAs complexed with argonaute proteins to suppress parasitic mobile sequences of DNA called transposable elements (TEs). Although recent evidence suggests that the piRNA pathway may be present in select somatic cells outside the gonads, the role of a non-gonadal somatic piRNA pathway is not well characterized. This study reports a functional somatic piRNA pathway in the adult Drosophila fat body including the presence of the piRNA effector protein Piwi and canonical 23-29 nt long TE-mapping piRNAs. The piwi mutants exhibit depletion of fat body piRNAs, increased TE mobilization, increased levels of DNA damage and reduced lipid stores. These mutants are starvation sensitive, immunologically compromised and short-lived, all phenotypes associated with compromised fat body function. These findings demonstrate the presence of a functional non-gonadal somatic piRNA pathway in the adult fat body that affects normal metabolism and overall organismal health (Jones, 2016).

This study has shown evidence for a fully functional piRNA pathway in a non-gonadal somatic tissue, the adult fly fat body, that is likely to be necessary for proper tissue function and overall organismal health. These results demonstrate that the adult fat body piRNA pathway exhibits canonical characteristics found in gonadal somatic cells, and its activity likely positively affects the function of a tissue important to metabolic homeostasis and physiological health. Although it has not been possible to entirely rule out a contribution of the gonadal piRNA pathway to fat body function, many of the phenotypes observed are opposite to those typically seen in animals with compromised gonadal tissue function and therefore likely represent the effect of a loss of the fat body piRNA pathway. For example, the shortened lifespan and reduced lipid stores in piRNA pathway mutants demonstrate that the piRNA pathway is essential in the health and functioning of non-gonadal somatic tissues, as reduction or ablation of gonadal function in flies often extends lifespan and increases lipid stores rather than decreasing lifespan and fat storage. Recent studies in wild-type flies have also demonstrated an important link between TE activity and longevity, and these studies demonstrating partial rescue of the shortened lifespan in flamenco mutants upon administration of a reverse transcription inhibitor further support this association (Jones, 2016).

Interest in a function for the piRNA pathway in the soma has increased recently as new roles for this pathway are being illuminated. The piRNA pathway's association with tissues that maintain a degree of immortalization similar to that exhibited in the germline is of particular interest. For example, the somatic stem cell niches of Hydra maintain an active piRNA pathway that represses TEs, possibly contributing to this organism's remarkably long lifespan. These studies, together with the current findings, suggest that the presence of a piRNA pathway in normal somatic tissues may offer an additional cellular defence against TE reactivation and possible somatic genomic damage. The finding of a role for the piRNA pathway in preserving metabolic homeostasis and the overall health of the fly suggests the potential importance of the piRNA pathway in other somatic tissues. Finally, interventions specifically augmenting the piRNA pathway may provide significant benefits to maintaining genomic integrity, tissue function and healthy lifespan (Jones, 2016).

Early life exercise training and inhibition of apoLpp mRNA expression to improve age-related arrhythmias and prolong the average lifespan in Drosophila melanogaster

Cardiovascular disease (CVD) places a heavy burden on older patients and the global healthcare system. A large body of evidence suggests that exercise training is essential in preventing and treating cardiovascular disease, but the underlying mechanisms are not well understood. This study used the Drosophila melanogaster animal model to study the effects of early-life exercise training (Exercise) on the aging heart and lifespan. It was found in flies that age-induced arrhythmias are conserved across different genetic backgrounds. The fat body is the primary source of circulating lipoproteins in flies. Inhibition of fat body apoLpp (Drosophila apoB homolog) demonstrated that low expression of apoLpp reduced the development of arrhythmias in aged flies but did not affect average lifespan. At the same time, exercise can also reduce the expression of apoLpp mRNA in aged flies and have a protective effect on the heart, which is similar to the inhibition of apoLpp mRNA. Although treatment of UAS-apoLpp(RNAi) and exercise alone had no significant effect on lifespan, the combination of UAS-apoLpp(RNAi) and exercise extended the average lifespan of flies. Therefore, it is concluded that UAS-apoLpp(RNAi) and exercise are sufficient to resist age-induced arrhythmias, which may be related to the decreased expression of apoLpp mRNA, and that UAS-apoLpp(RNAi) and exercise have a combined effect on prolonging the average lifespan (Ding, 2022).

Fat Quality Impacts the Effect of a High-Fat Diet on the Fatty Acid Profile, Life History Traits and Gene Expression in Drosophila melanogaster

Feeding a high-fat diet (HFD) has been shown to alter phenotypic and metabolic parameters in Drosophila melanogaster. However, the impact of fat quantity and quality remains uncertain. Butterfat (BF) was first used as an example to investigate the effects of increasing dietary fat content (3-12%) on male and female fruit flies. Although body weight and body composition were not altered by any BF concentration, health parameters, such as lifespan, fecundity and larval development, were negatively affected in a dose-dependent manner. When fruit flies were fed various 12% HFDs (BF, sunflower oil, olive oil, linseed oil, fish oil), their fatty acid profiles shifted according to the dietary fat qualities. Moreover, fat quality was found to determine the effect size of the response to an HFD for traits, such as lifespan, climbing activity, or fertility. Consistently, a highly fat quality-specific transcriptional response to three exemplary HFD qualities was also found with a small overlap of only 30 differentially expressed genes associated with the immune/stress response and fatty acid metabolism. In conclusion, these data indicate that not only the fat content but also the fat quality is a crucial factor in terms of life-history traits when applying an HFD in D. melanogaster (Eickelberg, 2023).

MEF2 is an in vivo immune-metabolic switch

Infections disturb metabolic homeostasis in many contexts, but the underlying connections are not completely understood. To address this, paired genetic and computational screens were used in Drosophila to identify transcriptional regulators of immunity and pathology and their associated target genes and physiologies. It was shown that Mef2 is required in the fat body for anabolic function and the immune response. Using genetic and biochemical approaches, it was found that MEF2 is phosphorylated at a conserved site in healthy flies and promotes expression of lipogenic and glycogenic enzymes. Upon infection, this phosphorylation is lost, and the activity of MEF2 changes-MEF2 now associates with the TATA binding protein to bind a distinct TATA box sequence and promote antimicrobial peptide expression. The loss of phosphorylated MEF2 contributes to loss of anabolic enzyme expression in Gram-negative bacterial infection. MEF2 is thus a critical transcriptional switch in the adult fat body between metabolism and immunity (Clark, 2013).

This study identified Mef2 as a factor critical for energy storage and the inducible immune response in the Drosophila fat body. Many infection-induced antimicrobial peptides depend on Mef2 for normal expression. In consequence, flies lacking Mef2 activity in the fat body are severely immunocompromised against a variety of infections. Mef2 sites are also associated with genes encoding key enzymes of anabolism, and Mef2 is required for normal expression of these genes; consequently, flies lacking Mef2 function in the fat body exhibit striking reductions in the total levels of triglyceride and glycogen. These two groups of target genes are counterregulated during infection; the anabolic targets of Mef2 are reduced in expression when antimicrobial peptides are induced. Fat body MEF2 was shown to exist in two states with distinct physiological activities. In uninfected animals, MEF2 is phosphorylated at T20 and can promote the expression of its metabolic targets. In infected animals, T20 is dephosphorylated, and MEF2 associates with the TATA-binding protein to bind a compound MEF2-TATA sequence found in the core promoters of antimicrobial peptides. The loss of T20-phosphorylated MEF2 promotes the loss of anabolic transcripts in flies with Gram-negative bacterial infection. These data, taken together, suggest that the central role of MEF2 in promoting fat body anabolism and immune activity reflects a switch between distinct transcriptional states regulated, at least in part, by differential affinity for TBP determined by T20 phosphorylation (Clark, 2013).

The signaling mechanisms regulating T20 phosphorylation and MEF2-TBP association are clearly of critical importance. The ability of p70 S6K to phosphorylate this residue is congruent with the ability of S6K to enhance anabolism and repress catab- olism in response to nutrient signals (Laplante, 2012). However, others have shown T20 phosphorylation by PKA, suggesting that T20 phosphorylation may be regulated by more than one pathway in vivo. The role of TAK1 may be similarly complex. TAK1 is required for formation of the MEF2-TBP complex upon Gram-negative infection, but this effect may be indirect. For example, reduced S6K phosphorylation after infection may result from insulin resistance driven by TAK1 via JNK. TAK1- dependent JNK activation is required for normal AMP induction in vivo, but it remains possible that some novel pathway is the critical connection between TAK1 and MEF2-TBP complex formation (Clark, 2013).

In mammals, in addition to hematopoietic roles, Mef2c regulates B cell proliferation upon antigen stimulation, and Mef2d regulates IL2 and IL10 in T cells. The possibility that Mef2 family proteins might be important direct activators of innate responses has not previously been examined. This study shows that Mef2 is a core transcriptional component of the innate immune response of the adult fly. Equally, vertebrate Mef2 family proteins are critical regulators of muscle metabolism, activated by physical activity to promote expression of PGC-1a and the glucose transporter Glut4. Glut4 regulation by MEF2 is known in adipose tissue as well as in muscle 1998); it is an intriguing possibility that MEF2 is as important a regulator of adipose metabolism in vertebrates as it has been show to be in flies (Clark, 2013).

Infection-induced metabolic disruption leading to cachexia is present in vertebrates as well as in insects, most notoriously in Gram-negative sepsis and persistent bacterial infections such as tuberculosis. The current data suggest that wasting seen after infection may be due, in part, to the requirement for MEF2 to serve different transcriptional functions in different conditions; the MEF2 immune-metabolic transcriptional switch may be a mechanistic constraint that forces the fly into metabolic pathophysiology in contexts of persistent immune activation. Alternatively, the loss of MEF2-driven anabolic transcripts due to infection may be productive, either by altering systemic energy usage or by increasing the production or release of one or more antimicrobial metabolites. Recent work has highlighted a distinction between 'resistance' type immune mechanisms, in which the host attempts to eradicate an invading organism, and 'tolerance' type mechanisms, in which the host response is oriented toward reducing the damage done by infection. The distinct metabolic and immune requirements for MEF2, combined with the obligation on the part of the host to raise some measure of resistance to systemic infection, may limit the achievable level of tolerance in persistent infections (Clark, 2013).

HP1a-mediated heterochromatin formation promotes antimicrobial responses against Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is a Gram-negative bacterium that causes severe infectious disease in diverse host organisms, including humans. Effective therapeutic options for P. aeruginosa infection are limited due to increasing multidrug resistance and it is therefore critical to understand the regulation of host innate immune responses to guide development of effective therapeutic options. The epigenetic mechanisms by which hosts regulate their antimicrobial responses against P. aeruginosa infection remain unclear. This study used Drosophila melanogaster to investigate the role of heterochromatin protein 1a (HP1a), a key epigenetic regulator, and its mediation of heterochromatin formation in antimicrobial responses against PA14, a highly virulent P. aeruginosa strain. Animals with decreased heterochromatin levels showed less resistance to P. aeruginosa infection. In contrast, flies with increased heterochromatin formation, either in the whole organism or specifically in the fat body-an organ important in humoral immune response-showed greater resistance to P. aeruginosa infection, as demonstrated by increased host survival and reduced bacterial load. Increased heterochromatin formation in the fat body promoted the antimicrobial responses via upregulation of fat body immune deficiency (imd) pathway-mediated antimicrobial peptides (AMPs) before and in the middle stage of P. aeruginosa infection. The fat body AMPs were required to elicit HP1a-mediated antimicrobial responses against P. aeruginosa infection. Moreover, the levels of heterochromatin in the fat body were downregulated in the early stage, but upregulated in the middle stage, of P. aeruginosa infection. These data indicate that HP1a-mediated heterochromatin formation in the fat body promotes antimicrobial responses by epigenetically upregulating AMPs of the imd pathway. This study provides novel molecular, cellular, and organismal insights into new epigenetic strategies targeting heterochromatin that have the potential to combat P. aeruginosa infection (Wu, 2022).

Mio/dChREBP coordinately increases fat mass by regulating lipid synthesis and feeding behavior in Drosophila

During nutrient excess, triglycerides are synthesized and stored to provide energy during times of famine. The presence of high glucose leads to the activation of carbohydrate response element binding protein (">ChREBP), a transcription factor that induces the expression of a number of glycolytic and lipogenic enzymes. ChREBP is expressed in major metabolic tissues and while there is a basic understanding of ChREBP function in liver, in vivo genetic systems to study the function of ChREBP in other tissues are lacking. This study characterized the role of the Drosophila homolog of ChREBP, Mondo (also known as Mio), in controlling fat accumulation in larvae and adult flies. In Mio mutants, high sugar-induced lipogenic enzyme mRNA expression is blunted and lowering Mio levels specifically in the fat body using RNA interference leads to a lean phenotype. A lean phenotype is also observed when the gene bigmax, the fly homolog of ChREBP's binding partner Mlx, is decreased in the larval fat body. Interestingly, depleting Mio in the fat body results in decreased feeding providing a potential cause of the lowered triglycerides observed in these animals. However, Mio does not seem to function as a general regulator of hunger-induced behaviors as decreasing fat body Mio levels has no effect on sleep under fed or starved conditions. Together, these data implicate a role for Mio in controlling fat accumulation in Drosophila and suggests that it may act as a nutrient sensor in the fat body to coordinate feeding behavior with nutrient availability (Sassu, 2012).

After a meal, the oxidation of sugars and fats provides energy for basic cellular functions. Excess calories that are not used for energy are stored mainly as glycogen and triglycerides. This response was selected for throughout evolution as a means of preparing an organism for times of scarce food sources. However, in today’s western society where food is abundant and readily available, this ability of an animal to store surplus nutrients leads to excess fat accumulation and metabolic diseases such as obesity and diabetes. This effect is pronounced after a prolonged high sugar diet as these conditions lead to an acute increase in the activity of enzymes necessary for fat synthesis, the chronic production of lipogenic enzymes, and the concurrent synthesis and storage of fats (Sassu, 2012).

A key regulator of this chronic response to high sugar intake and fat storage in mammals is the transcription factor, carbohydrate response element binding protein (ChREBP). In response to high glucose conditions, ChREBP translocates into the nucleus, where it activates the expression of pyruvate kinase and many lipogenic enzymes, including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and ATP citrate lyase (ATPCL), ultimately leading to increased fat accumulation (Benhamed, 2012; Postic, 2007). In order to fully activate transcription, ChREBP must heterodimerize with another transcription factor called Max-like protein X (Mlx) (Stoeckman, 2004). ChREBP is expressed most highly in liver and adipose tissue, but significant expression is also observed in skeletal muscle, the intestine and kidney. Conversely, Mlx has a relatively ubiquitous expression pattern. While there is a basic understanding of ChREBP function in the liver, in vivo systems to study tissue-specific functions of ChREBP are lacking (Sassu, 2012).

Drosophila is an excellent model system for investigating the tissue-specific control of metabolism. Flies have specialized organ systems for nutrient uptake, storage, and metabolism that are functionally analogous to mammalian systems. The Drosophila midgut is the site of both digestion and nutrient uptake, while the fat body of the fly is the site of glycogen and triglyceride storage. Many important metabolic genes and pathways in mammals are also highly conserved in flies (see table in Baker, 2007) and these genes can be manipulated easily using the genetic tools available in the Drosophila system, allowing the information identified to be applied to mammal (Sassu, 2012).

The Drosophila genome contains a ChREBP-like gene named Mlx interactor (Mio) (also known as Mondo) and an Mlx-like gene called bigmax. However, very little functional data exists for these gene products. Mio and bigmax are expressed throughout embryogenesis and are enriched in the fat body and malpighian tubules. Mio mRNA levels are also increased when larvae are fed 20% sucrose, a condition that also leads to increased expression of fat synthesis enzymes, suggesting a role for Mio in regulating high-sugar induced lipogenic gene expression. Therefore, it was hypothesized that Mio and bigmax are involved in regulating fat storage in the fly. This study found that the induction of lipogenic enzyme expression in response to high sugar is blocked in Mio mutant animals. Consistent with this finding, knocking down Mio and bigmax in the fly fat body leads to decreased fat storage. Further, overall food consumption is also blunted when Mio expression is decreased, providing a potential explanation for the observed lean phenotype. These data identify a novel role for Mio in the fat body to regulate the storage of triglycerides and overall food consumption and further supports the use of Drosophila as a model system for understanding tissue-specific control of metabolism and behavior (Sassu, 2012).

This study has shown that decreasing Mio levels specifically in the fat body leads to lower triglycerides in larvae and adult flies. This suggests that Mio plays a role in lipid accumulation in these animals and supports the hypothesis that Mio acts to regulate fat storage in Drosophila. These findings are in agreement with data from a study where ChREBP knockout mice were found to have lower triglyceride levels in their adipose tissue compared to wild type control mice. ChREBP acts with the myc-family transcription factor Mlx to activate transcription. This study has shown that decreasing the expression of the Drosophila homolog of Mlx, bigmax, in the larval fat body results in decreased triglycerides, suggesting that Mio and bigmax may act together to control fat metabolism. Further biochemical analysis of these two proteins is necessary to determine whether they bind to each other in order to activate the transcription of target genes (Sassu, 2012).

Previous studies have shown that a high sugar diet leads to changes in expression of multiple genes involved in fat synthesis. The factors involved in up-regulating the transcription of these genes are, however, unknown. Mio is a likely regulator of the transcription of some of these lipogenic genes as its mammalian homolog is regulated in response to high sugar. Data presented in this study showing that high sugar-induced lipogenic gene expression is blunted in Mio mutants provides support for this hypothesis. However, the targets described in this study are probably only a small subset of Mio-regulated genes, and further experimentation is necessary in order to identify the full complement of genes that are regulated by Mio (Sassu, 2012).

The Drosophila fat body has been implicated in the regulation of both feeding and sleep. This study shows that decreased levels of Mio in the fat body lead to decreased food consumption, but not altered sleep. Mio knockdown flies were tested for sleep during fed and starved states and did not differ from wildtype under either condition. These findings raise the possibility that Mio selectively acts to regulate feeding behavior. Testing Mio-deficient flies in additional hunger-dependent assays such as appetitive memory and sucrose-yeast food choice would address this question (Sassu, 2012).

The decreased feeding in Mio knockdown flies could be responsible for the decreased fat per cell and overall lower triglyceride levels observed in these Mio mutants. One question from this study that remains unanswered is how Mio functions in the fat body to control feeding. The findings that Mio expression in the fat body is necessary for normal feeding suggests that Mio acts in the fat body as a sensor capable of detecting the status of the body’s energy reserves and conveying that information to the brain to control feeding patterns accordingly. An inherent ability of the fat body to release peptides into the hemolymph of the fly could explain this proposed communication between these organs. It is possible that Mio may activate the transcription of a factor secreted from the fat body that acts as the messenger to the brain. In order to identify whether such a Mio-responsive factor exists, the full complement of Mio target genes needs to be identified (Sassu, 2012).

It is also possible that the fat body may be communicating with the brain through a direct neuronal connection. In mammals, white adipose tissue is directly innervated by the sympathetic nervous system. This connection is thought to be a major stimulus for initiating the mobilization of lipid stores. A similar connection between the fat body and the brain may be present in Drosophila, but evidence supporting this claim is lacking. In summary, the data presented in this study shows that Mio, the Drosophila homolog of mammalian ChREBP, functions in the fat body to promote both lipid storage as well as feeding behavior. These data provide support for Mio acting as a nutrient sensor in the Drosophila fat body to coordinate metabolism and behavior in response to changes in nutrient abundance. This study also describes a genetic system for identifying and understanding the genes and mechanisms involved in controlling feeding and metabolism under high sugar conditions (Sassu, 2012).

The control of lipid metabolism by mRNA splicing in Drosophila

The storage of lipids is an evolutionarily conserved process that is important for the survival of organisms during shifts in nutrient availability. Triglycerides are stored in lipid droplets, but the mechanisms of how lipids are stored in these structures are poorly understood. Previous in vitro RNAi screens have implicated several components of the spliceosome in controlling lipid droplet formation and storage, but the in vivo relevance of these phenotypes is unclear. In this study, specific members of the splicing machinery were identified that are necessary for normal triglyceride storage in the Drosophila fat body. Decreasing the expression of the splicing factors U1-70K, U2AF38, U2AF50 in the fat body resulted in decreased triglyceride levels. Interestingly, while decreasing the SR protein 9G8 in the larval fat body yielded a similar triglyceride phenotype, its knockdown in the adult fat body resulted in a substantial increase in lipid stores. This increase in fat storage is due in part to altered splicing of the gene for the beta-oxidation enzyme CPT1, producing an isoform with less enzymatic activity. Together, these data indicate a role for mRNA splicing in regulating lipid storage in Drosophila and provide a link between the regulation of gene expression and lipid homeostasis (Gingras, 2014)

Lipoproteins in Drosophila melanogaster--assembly, function, and influence on tissue lipid composition

Interorgan lipid transport occurs via lipoproteins, and altered lipoprotein levels correlate with metabolic disease. However, precisely how lipoproteins affect tissue lipid composition has not been comprehensively analyzed. This study identified the major lipoproteins of Drosophila melanogaster; genetics and mass spectrometry were used to study their assembly, interorgan trafficking, and influence on tissue lipids. The apoB-family lipoprotein Lipophorin (Lpp) is the major hemolymph lipid carrier. It is produced as a phospholipid-rich particle by the fat body, and its secretion requires Microsomal Triglyceride Transfer Protein (MTP). Lpp acquires sterols and most diacylglycerol (DAG) at the gut via Lipid Transfer Particle (LTP), another fat body-derived apoB-family lipoprotein. The gut, like the fat body, is a lipogenic organ, incorporating both de novo-synthesized and dietary fatty acids into DAG for export. This study identified distinct requirements for LTP and Lpp-dependent lipid mobilization in contributing to the neutral and polar lipid composition of the brain and wing imaginal disc. These studies define major routes of interorgan lipid transport in Drosophila and uncover surprising tissue-specific differences in lipoprotein lipid utilization (Palm, 2012).

The major inter-organ lipid transport routes in Drosophila are executed by a single lipoprotein, Lpp, which is scaffolded by the apoB homologue apoLpp. Its major polar lipid constituents are long-chain PE and sterols, and its major neutral lipid is medium-chain DAG. Lpp lipidation takes place in two consecutive steps, which require distinct lipid transfer proteins, MTP and LTP, and take place in different organs: fat body and gut. ApoLpp is translated and lipidated in the fat body by an MTP-dependent mechanism, resulting in the formation of dense Lpp particles rich in PE. These are recruited to the gut, where they are further loaded with DAG and sterols through the activity of LTP. Thus, although Lpp originates in the fat body, it is loaded both with fat body and gut lipids (Palm, 2012).

Lipidation of mammalian apoB, like that of Drosophila apoLpp, proceeds in two distinct steps, formation of primordial phospholipid-rich lipoprotein particles, and subsequently acquisition of bulk neutral lipid. However, this process occurs entirely in the secretory pathway of producing cells. MTP has been proposed to be required both for initial transfer of phospholipids, and for the recruitment of TAG to the ER lumen for incorporation into lipoproteins. Interestingly, Drosophila MTP has been shown to promote the secretion of apoB-containing lipoproteins from COS cells, and to transfer phospholipids, but not TAG, between liposomes. This suggested that MTP acquired the ability to transfer TAG in the vertebrate lineage. Experiments described in this study show that Drosophila MTP is required for the production of the two Drosophila apoB-family lipoproteins Lpp and LTP in vivo; they further show that MTP is insufficient to load Lpp with normal quantities of DAG, the major neutral lipid of Lpp. These data support the idea that MTP originally evolved to promote the assembly of phospholipid-rich apoB-family lipoproteins (Palm, 2012).

The novel Drosophila apoB-family lipoprotein LTP shares many properties with the Lipid Transfer Particle purified from the hemolymph of several insects, including Manduca and Locusta. The scaffolding proteins of Drosophila LTP, apoLTPI and apoLTPII, are generated from a single precursor, apoLTP. Orthologous apoB-family proteins of other insects are therefore plausible candidates for the scaffolding proteins of their LTP particles. Insect LTPs were shown to contain a third, small protein subunit, apoLTPIII. Biochemical experiments do not address whether Drosophila LTP might contain an apoLTPIII subunit, because LTP is of such low abundance that silver staining barely detects the much larger apoLTPI. Sequence analysis of apoLTP does not suggest the existence of a protease cleavage site that could give rise to a protein of the size of apoLTPIII, and neither apoLTPI nor apoLTPII antibodies detect an additional protein of this size. Thus, if apoLTPIII exists in Drosophila, it is not likely to be derived from the apoLTP precursor (Palm, 2012).

The function of LTP as a lipid transfer protein rather than a carrier of bulk hemolymph lipid uncovers surprising evolutionary plasticity of the apoB lipoprotein family. Insect LTPs have been studied in vitro in a wide range of systems. In different contexts, they have been shown to facilitate the exchange of DAG and phospholipids between Lpp and fat body or gut, and even between insect and human lipoproteins of different densities. Studies of feeding Drosophila larvae indicate that only a subset of the lipid transfer activities of LTP may be relevant under specific metabolic conditions in vivo. LTP moves DAG and sterols from the larval gut onto Lpp. However, it does not facilitate significant net transfer of fat body lipids to Lpp. Consistent with this, radiolabeling experiments showed that the rate of DAG transfer from larval Manduca fat body to Lpp exceeds the rate of the reverse process. This may reflect a dominance of nutritional lipid uptake and fat storage in feeding larvae (Palm, 2012).

Although no Drosophila HDL-like lipoprotein was identified, it is noted that LTP and Lpp share some functional features with mammalian HDL, despite being scaffolded by unrelated apolipoproteins. Together, Lpp and LTP mediate efflux of sterols from the gut to circulation. Conceivably, other tissues that recruited both lipoproteins might efflux sterol for reverse transport (Palm, 2012).

While it is clear that dietary lipids do contribute to Lpp DAG, the gut does not directly incorporate dietary fatty acids into DAG destined for export. The long-chain fatty acids that predominate in the diet strikingly differ from the medium-chain fatty acids in Lpp DAG. A possible explanation is that the gut remodels dietary fatty acids, conceivably via limited β-oxidation. Interestingly, the gut is also a lipogenic organ and a significant fraction of the medium-chain fatty acids found in Lpp DAG derives from de novo fatty acid synthesis in this organ. In more primitive animals, such as Caenorhabditis elegans, lipid uptake, storage and lipogenesis all occur in the gut. More complex animals, including Drosophila, have developed separate organ systems for lipid storage and lipogenesis. However, the data show that this separation of functions is not absolute in the fly. Rather, other nutrients such as amino acids or sugars might be partially converted to lipid by the gut, instead of being transported intact into circulation. It would be interesting to ask what circumstances favor this conversion. Intriguingly, de novo lipogenesis has been observed in the mammalian gut, especially under conditions of insulin resistance, and has been proposed to contribute to the postprandial dyslipidemia observed in this state. Drosophila may be a useful model to explore this problem (Palm, 2012).

Gut and fat body differ in how they respond to blockage of lipid export to Lpp. Enterocytes vastly and rapidly expand their normally moderate stores of medium-chain DAG and TAG. This occurs even in the absence of dietary lipids, when exported lipids are derived from endogenous fatty acid synthesis. Thus, the gut has a flexible capacity for lipid storage. In contrast, the larval fat body maintains its neutral lipid stores within tight limits. When lipoprotein transport is blocked, endogenous lipid synthesis from other dietary components may suffice to build the large TAG stores of this organ. Furthermore, even though the fat body normally supplies the entire animal with large amounts of lipoproteins, TAG stores hardly increase when Lpp is not produced. Homeostatic mechanisms must maintain fat body TAG levels. In this way, the fat body differs from the gut, which accumulates fat when lipoprotein export is blocked, similar to mammalian gut and liver (Palm, 2012).

Peripheral tissues cannot maintain normal TAG levels in the absence of Lpp. The wing disc depends on Lpp for a large fraction of its fat stores. Interestingly, this work indicates that lipid delivery from the fat body and gut differently contributes to wing disc neutral lipids. TAG species containing medium-chain fatty acids depends on LTP and Lpp-mediated DAG mobilization from the gut. TAG species containing long-chain fatty acids also depend on Lpp-mediated lipid delivery, but are less affected by a blockage of DAG export from the gut. As Lpp is produced in the fat body, this suggests that long-chain TAG in wing discs may be derived from lipids supplied by the fat body. The most abundant source of long-chain fatty acids in Lpp is PE, which raises the possibility that wing discs use Lpp phospholipids to build cellular fat stores. Consistent with this, cultured murine hepatocytes convert a significant fraction of LDL or HDL-derived PC to TAG, although the in vivo relevance of this pathway remains to be explored. However, Lpp still contains reduced amounts of medium-chain DAG when LTP-mediated lipid loading is impaired. Thus, long-chain fatty acids in wing disc TAG might also derive from elongation of medium-chain fatty acids. Interestingly, although medium-chain DAG is the most abundant lipid transported through circulation, tissues store only minor amounts of neutral lipid containing medium-chain fatty acids. This would be consistent with the idea that tissues either elongate these fatty acids or subject them to β-oxidation (Palm, 2012).

The brain also requires Lpp-mediated lipid delivery to build its TAG stores. Interestingly, the brain stores normal levels of TAG when gut lipid mobilization is inhibited. While this does not exclude the possibility that the brain may directly acquire lipids from the gut under normal conditions, it indicates that TAG levels in this organ are more resistant to fluctuations in nutritional conditions than those in the wing disc (Palm, 2012).

In addition to providing fatty acids for neutral lipid storage, lipoproteins also influence the phospholipid composition of wing disc and gut: Lpp knock-down specifically reduces those PE species that are most abundant in Lpp. This suggests that Lpp might directly deliver PE to the cellular membranes of wing disc and gut. It further raises the possibility that phospholipid synthesis in other tissues could have organism-wide effects on membrane lipid composition. Since PE-rich Lpp particles are assembled in the fat body, this tissue is a likely source of these lipids. However, the brain does not depend on Lpp to maintain its normal membrane phospholipid composition. Furthermore, previous work suggested that the brain is more resistant to sterol depletion than other tissues. In general, these data indicate that the lipid composition of the brain is more tightly and autonomously controlled than that of other tissues (Palm, 2012).

In mammals, cellular lipid synthesis and lipid supply from circulation are coordinated through the SREBP pathway. Since Drosophila SREBP is regulated by PE instead of sterols, it will be interesting to explore whether altered PE levels in Lpp-deprived wing discs might activate SREBP signaling and increase lipid synthesis or lipoprotein uptake. If true, coordination of cellular lipid synthesis with lipid supply through lipoproteins is an evolutionarily conserved function of the SREBP pathway (Palm, 2012).

Lipoproteins transport large amounts of lipids through circulation - including many of the polar and neutral lipid species present in cells. These data indicate that in Drosophila, individual organs utilize lipoprotein-derived lipids not only for fat storage but also for membrane homeostasis. ApoB-deficient human patients, and patients with dyslipidemia suffer from various abnormalities in peripheral tissues. The data suggest that it may be worthwhile to examine how these perturbations alter the membrane lipid composition of affected tissues (Palm, 2012).

Adipocyte amino acid sensing controls adult germline stem cell number via the amino acid response pathway and independently of Target of Rapamycin signaling in Drosophila

How adipocytes contribute to the physiological control of stem cells is a critical question towards understanding the link between obesity and multiple diseases, including cancers. Previous studies have revealed that adult stem cells are influenced by whole-body physiology through multiple diet-dependent factors. For example, nutrient-dependent pathways acting within the Drosophila ovary control the number and proliferation of germline stem cells (GSCs). The potential role of nutrient sensing by adipocytes in modulating stem cells in other organs, however, remains largely unexplored. This study report that amino acid sensing by adult adipocytes specifically modulates the maintenance of GSCs through a Target of Rapamycin-independent mechanism. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response pathway within adipocytes, causing increased rates of GSC loss. These studies reveal a new step in adipocyte-stem cell crosstalk (Armstrong, 2014).

Stem cell lineages are inextricably linked to whole-body physiology and nutrient availability in multiple organisms. For example, diet influences wound healing, hematopoietic transplants and cancer risk in humans, and evidence ranging from human epidemiological to model organism experimental data suggests that diet-dependent pathways impact a variety of adult stem cells. As intact living organisms vary their dietary input, multiple tissues and organs sense and respond to diet; however, knowledge of how inter-organ communication contributes to the dietary control of adult stem cells remains limited (Armstrong, 2014).

The obesity epidemic has brought to light the crucial importance of normal adipocyte function in maintaining a healthy physiology. Adipocytes are highly sensitive to diet and produce long-range factors with key roles in metabolism, reproduction and other physiological processes. Conversely, dysfunctional adipocytes underlie the link between obesity and several diseases, including cancers. Whether sensing of dietary inputs by adipocytes leads to specific effects on adult stem cells in other organs, however, remains largely unexplored (Armstrong, 2014).

Drosophila female germline stem cells (GSCs) sense and respond to diet through complex endocrine mechanisms. Two or three GSCs reside within a well-defined niche in the germarium, the anterior region of the ovariole. Each asymmetric GSC division yields another GSC and a cystoblast that forms a 16-cell cyst, which is enveloped by follicle cells to generate a follicle that develops through oogenesis to form a mature oocyte. On a yeast-rich diet, GSCs and their progeny grow and proliferate faster than on a yeast-free diet, and this response is mediated by diet-dependent factors that act on or within the ovary. For example, optimal levels of Target of Rapamycin (TOR) activity likely controlled by circulating amino acids are intrinsically required in GSCs for their proliferation and maintenance. Insulin-like peptides produced by median neurosecretory cells in the brain act directly on GSCs to modulate how fast they proliferate to generate new cystoblasts. In parallel, insulin-like peptides act directly on cap cells, the major cellular components of the niche, to control GSC maintenance via two mechanisms. Insulin-like peptides promote the response of cap cells to Notch ligands, which are required for proper cap cell numbers, and also GSC-cap cell attachment via E-cadherin. These previous studies, however, did not address whether or how nutrient sensing by adipocytes influences the dietary response of GSCs and their descendants (Armstrong, 2014 and references therein).

Drosophila adipocytes, together with hepatocyte-like oenocytes, compose the fat body, a nutrient-sensing organ with endocrine roles. In the larval fat body, TOR activation downstream of amino acid sensing results in the production of unknown factors that modulate overall growth of the organism. In both the larval and adult fat body, sensing of sugars and lipids leads to the production of a leptin-like cytokine, Unpaired 2 (Upd2), which controls the secretion of brain insulin-like peptides. This study reports that partially inhibiting amino acid transport in adult adipocytes results in a specific reduction in the number of ovarian GSCs and that, surprisingly, this effect is independent of TOR signaling. Instead, reduced amino acid levels and the consequent increase in uncoupled tRNAs trigger activation of the GCN2-dependent amino acid response (AAR) pathway within adipocytes, causing increased rates of GSC loss. These results indicate that amino acid sensing by adipocytes through a TOR-independent mechanism is communicated to GSCs to control their maintenance, thereby contributing to their response to diet. These findings bring to light the importance of elucidating how adipocytes contribute to the regulation of various adult stem cell types by diet, and how these mechanisms might be adversely affected in obese individuals (Armstrong, 2014).

Adipocyte metabolic pathways regulated by diet control the female germline stem cell lineage in Drosophila

Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. This study conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-coA production are upregulated, promoting a shift of glucose metabolism towards macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems (Matsuoka, 2017).

Transforming growth factor beta/Activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression

Organisms need to assess their nutritional state and adapt their digestive capacity to the demands for various nutrients. Modulation of digestive enzyme production represents a rational step to regulate nutriment uptake. However, the role of digestion in nutrient homeostasis has been largely neglected. This study analyzed the mechanism underlying glucose repression of digestive enzymes in the adult Drosophila midgut. Glucose represses the expression of many carbohydrases and lipases. The data reveal that the consumption of nutritious sugars stimulates the secretion of the transforming growth factor β (TGF-β) ligand, Dawdle, from the fat body. Dawdle then acts via circulation to activate TGF-β/Activin signaling in the midgut, culminating in the repression of digestive enzymes that are highly expressed during starvation. Thus, this study not only identifies a mechanism that couples sugar sensing with digestive enzyme expression but points to an important role of TGF-β/Activin signaling in sugar metabolism (Chng, 2004).

Digestive enzymes expression is subjected to complex regulation. However, apart from the regulation of magro (lipase) by the nutrient-sensitive DHR96 and dFOXO (Karpac, 2013). It is noteworthy that an arbitrary threshold for RNA-seq analysis has rejected several genes whose repression was more subtle. For this, it has been have independently verified Amy-p, Amy-d, CG9466, CG9468, and CG6283 to be repressed by glucose through qRT-PCR. Thus, the actual repertoire of carbohydrases and lipases affected by glucose could be potentially larger (Karpac, 2013).

To date, little is known about the contribution of digestion on sugar homeostasis. It seems likely that glucose repression of carbohydrases and lipases is aimed at reducing the amount of sugars and lipids that are available for absorption. Consistent with this view, glucose transmembrane transporters were also found among genes that were downregulated by dietary glucose. A high-sugar diet in Drosophila is associated with dire consequences such as hyperglycemia, insulin resistance, and increased fat accumulation. Thus, reducing both carbohydrases and lipases expression may restrict the nutritional load available for absorption into the circulation when carbohydrate stores in the organism are sufficient and fats are accumulating. In accordance with this, early postprandial glucose level was elevated in the hemolymph when TGF-β/Activin pathway function was compromised in the midgut, a condition associated with elevated digestive enzymes expression. However, when the levels of TAG, glycogen, glucose, and trehalose were monitored after 2 weeks on a high-sugar diet, no significant differences were observed between flies whereby Smad2 or Babo were knocked down in the midgut and control. Sugar homeostasis is a tightly regulated process involving multiple tissues. One possibility would be that the postprandial increase in glucose was counteracted by early acting satiety response when hemolymph glucose level passed a certain threshold, thus limiting the net amount of glucose entering the circulation. Clearly, the role of glucose repression in sugar homeostasis and metabolism warrants additional research. An understanding of how the repertoire of digestive enzymes respond to other nutriments in the diet will provide insights into how an organism may rebalance its diet after ingestion and improve understanding of nutrients homeostasis (Karpac, 2013).

In this study, it was also shown that digestive enzyme repression is induced only by nutritious carbohydrates in the diet. Arabinose, a sweet-tasting sugar with no nutritional value, and L-glucose, another nonutilizable sugar did not suppress amylase and maltase expression. Hence, postprandial activation of gustatory receptors in the gut are considered to be an unlikely mechanism for glucose repression of digestive enzymes. Instead, all these are suggestive of an underlying sugar-sensing mechanism to ensure that carbohydrate digestive capacity toward utilizable carbohydrate sources are not comprised until nutritional sufficiency is attained (Karpac, 2013).

In Drosophila, sugar homeostasis is often associated with the AKH and insulin signaling, whereas insulin signaling is also modulated by proteins and amino acids in the diet. Recently, it has been shown that Daw expression is modulated by insulin signaling, and Daw was identified as a target of dFOXO (Bai, 2013), raising the possibility that glucose repression may be similarly affected by insulin signaling. Surprisingly, disrupting both AKH and insulin signaling did not compromise glucose repression. Instead, this study identified a key role for TGF-β/Activin signaling in this process. Whereas Daw expression may be modulated by insulin signaling, the results clearly showed that glucose repression is mediated through an insulin-independent mechanism. More recently, Ghosh (2014) has demonstrated that Daw is required for insulin secretion, suggesting that the TGF-β/Activin pathway may function upstream of the insulin signaling. It is also noteworthy that, whereas compromising insulin signaling is known to raise circulating sugar levels, this did not affect the ability of flies to repress digestive enzymes in response to dietary glucose. One possible explanation is that Daw expression in response to glucose is dependent on the nutritional state perceived cell autonomously by the fat body cells. Thus, if nutrient sensing in these cells is not compromised, Daw induction and glucose repression can be achieved. Future research should clarify the mechanism underlying Daw induction by nutritious sugar and define the possible interactions between TGF-β/Activin and other sugar-sensing mechanisms (Karpac, 2013).

The TGF-β/Activin pathway in Drosophila has been previously studied in the context of larval brain development, neuronal remodeling, wing disc development, and, more recently, aging and pH homeostasis. This study addresses the physiological function of the TGF-β/Activin pathway in the adult midgut. When the TGF-β/Activin signaling was disrupted in the adult midgut, glucose repression was abolished. Conversely, increasing TGF-β/Activin signaling in the midgut, through the overexpression of the constitutive active form of Babo or Smad2, was sufficient to repress both amylase and maltase expression. Furthermore, glucose repression is mediated by the TGF-β ligand Daw, produced and secreted from the fat body, a metabolic tissue functionally analogous to the mammalian liver and adipose tissue. Thus, this study uncovers a physiological role for the TGF-β/Activin pathway in adapting carbohydrate and lipase digestion in response to the nutritional state of the organism. Because many features of digestion and absorption are conserved between flies and mammals, it will be of interest to investigate the role of TGF-β/Activin pathway in mammalian digestion (Karpac, 2013).

Recent studies have attributed a role for Daw in aging and pH homeostasis, two processes tightly linked to metabolism. Thus, it is likely that Daw induced from the fat body in response to carbohydrate in the diet will induce a more global response instead of a local response, affecting only digestive enzyme expression. As such, Daw may act as a central mediator for glucose homeostasis by regulating sugar level in the circulation. When there are sufficient carbohydrates in the diet, Daw expression restricts the expression of carbohydrase and glucose transporters. Concurrently, at the postabsorption level, Daw in the circulation may act directly or indirectly (via insulin signaling) to maintain circulating sugar level. A broader role for Daw in sugar homeostasis is reinforced by the findings that Daw mutant larvae were more sensitive to a high-sugar diet. Similarly, this study found overexpression of Daw, but not Myo, Mav, or Actβ, renders flies sensitive to sugar starvation. Along this line, in C. elegans, the TGF-β signaling is reported to be elevated and required in neurons for satiety. There were also several observations that hyperglycemia is linked to increased TGF-β activity in mammals. Hence, the role of TGF-β/Activin signaling in sugar homeostasis requires further investigation in Drosophila and other organisms (Karpac, 2013).

In conclusion, this study revealed a remarkable resilience in the regulation of carbohydrate and lipid-acting enzymes expression to ensure that digestive capacity in the midgut is not compromised before certain metabolic criteria in the fat body is attained. The study also unraveled a role of the TGF-β/Activin-signaling pathway in the adult Drosophila midgut, which has not been appreciated. It reinforced the notion that the gut is not a passive tube for nutriment flow. Rather, it dynamically modulates digestive enzyme expression in response to the organism’s nutritional state through endocrine signals derived from other metabolic tissues (Karpac, 2013).

Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia

Aging of immune organs, termed as immunosenescence, is suspected to promote systemic inflammation and age-associated disease. The cause of immunosenescence and how it promotes disease, however, has remained unclear. This study reports that the Drosophila fat body, a major immune organ, undergoes immunosenescence and mounts strong systemic inflammation that leads to deregulation of immune deficiency (IMD) signaling in the midgut of old animals. Inflamed old fat bodies secrete circulating peptidoglycan recognition proteins that repress IMD activity in the midgut, thereby promoting gut hyperplasia. Further, fat body immunosenecence is caused by age-associated lamin-B reduction specifically in fat body cells, which then contributes to heterochromatin loss and derepression of genes involved in immune responses. As lamin-associated heterochromatin domains are enriched for genes involved in immune response in both Drosophila and mammalian cells, these findings may provide insights into the cause and consequence of immunosenescence during mammalian aging (Chen, 2014).

By analyzing gene expression changes upon aging in fat bodies and midguts, it was shown that an increase of immune response in the fat body is accompanied by a striking reduction in the midgut. Specifically, it was demonstrate that the age-associated increase in Immune deficiency (IMD) signaling in fat bodies leads to reduction of IMD activity in the midgut, which in turn contributes to midgut hyperplasia. This fat body to midgut effect requires peptidoglycan recognition proteins (PGRPs) secreted from fat body cells and is mediated by both bacteria dependent and independent pathways. Therefore, fat body aging contributes to systemic inflammation, which contributes to the disruption of gut homeostasis. Importantly, it was shown that the age-associated lamin-B loss in fat body cells causes the derepression of a large number of immune responsive genes, thereby resulting in fat body-based systemic inflammation (Chen, 2014).

B-type lamins have long been suggested to have a role in maintaining heterochromatin and gene repression. Consistently, this study's global analyses of fat body depleted of lamin-B revealed a loss of heterochromatin and derepression of a large number of immune responsive genes. This is further supported by ChIP-qPCR analyses of H3K9me3 on specific IMD regulators. Recent studies in different cell types show that tethering genes to nuclear lamins do not always lead to their repression. Deleting B-type lamins or all lamins in mouse ES cells or trophectdoderm cells does not result in derepression of all genes in LADs. In light of these studies, it is suggested that the transcriptional repression function of lamin-B could be gene and cell type dependent. Interestingly, GO analyses revealed a significant enrichment of immune responsive genes in Lamin-associated domains (LADs) in four different mammalian cell types and Drosophila Kc cells. Since the large-scale pattern of LADs is conserved in different cell types in mammals, it is possible that the immune-responsive genes are also enriched in LADs in the fly fat body cells. Supporting this notion, the IKKγ, key, which is one of the two derepressed IMD regulators and was found to exhibit H3K9me3 reduction and gene activation, is localized to LADs in Kc cells. It is speculated that lamin-B might play an evolutionarily conserved role in repressing a subset of inflammatory genes in certain tissues, such as the immune organs, in the absence of infection or injury. Consistently, senescence-associated lamin-B1 loss in mammalian fibroblasts is correlated with senescence-associated secretory phenotype senescence-associated secretory phenotype (SASP). Although the in vivo relevance of fibroblast SASP in chronic inflammation and aging-associated diseases in mammals remains to be established, the findings in Drosophila provide insights and impetus to investigate the role of lamins in immunosenescence and systemic inflammation in mammals (Chen, 2014).

Lamin-B gradually decreases in fat body cells of aging flies, whereas lamin-C amount remains the same. Since it has been recently shown that the assembly of an even and dense nuclear lamina is dependent on the total lamin concentration, the age-associated appearance of lamin-B and lamin-C gaps around the nuclear periphery of fat body cells is likely caused by the drop of the lamin-B level. How aging triggers lamin-B loss is unknown, but it appears to be posttranscriptional, because lamin-B transcripts in fat bodies remain unchanged upon aging. Interestingly, among the tissues examined, no changes of lamin-B and lamin-C proteins were found in cells in the heart tube, oenocytes, or gut epithelia in old flies. Therefore, the age-associated lamin-B loss does not occur in all cell types in vivo. A systematic survey to establish the cell/tissue types that undergo age-associated reduction of lamins in both flies and mammals should provide clues to the cause of loss. Deciphering how advanced age leads to lamin loss should open the door to further investigate the cellular mechanism that contributes to chronic systemic inflammation and how it in turn promotes age-associated diseases in humans (Chen, 2014).

Old Drosophila gut is known to exhibit increased microbial load, which would cause increased stress response and activation of tissue repair, thereby leading to midgut hyperplasia. Systemic inflammation caused by lamin-B loss in fat body leads to repression of local midgut IMD signaling. The upregulation of targets of IMD in the aged whole gut has been recently reported, while a downregulation of target genes was observed in the current analyses of the midgut. However, the previous study found a similar upregulation of the genes when performing RNA-seq of the whole gut (Chen, 2014).

These studies reveal an involvement of bacteria in the repression of midgut IMD signaling by the PGRPs secreted from the fat body. How PGRPs from the fat body repress midgut IMD is still unknown. One possibility is that the body cavity bacteria contribute to the maintenance of midgut IMD activity, and the increased circulating PGRPs limit these bacteria. The circulating PGRPs may also reduce midgut IMD activity indirectly by affecting other tissues. The evidence suggests that lamin-B loss could also contribute to midgut hyperplasia independent of the IMD pathway. While it will be important to further address these possibilities, the findings have revealed a fat body mediated inflammatory pathway that can lead to reduced migut IMD, increased gut microbial accumulation, and midgut hyperplasia upon aging (Chen, 2014).

Interestingly, microbiota changes also occur in aging human intestine and have been linked to altered intestinal inflammatory states and diseases. Although, much effort has been devoted to understand how local changes in aging mammalian intestines affect gut microbial community, the cause remains unclear. The findings in Drosophila reveal the importance of understanding the impact of immunosenescence and systemic inflammation on gut microbial homeostasis. Indeed, if increased circulating inflammatory cytokines perturb the ability of local intestine epithelium and the gut-associated lymphoid tissue to maintain a balanced microbial community, the unfavorable microbiota in the old intestine would cause chronic stress response and tissue repair, thereby leading to uncontrolled cell growth as observed in age-associated cancers (Chen, 2014).

Obesity-associated cardiac dysfunction in starvation-selected Drosophila melanogaster

There is a clear link between obesity and cardiovascular disease, but the complexity of this interaction in mammals makes it difficult to study. Among the animal models used to investigate obesity-associated diseases, Drosophila melanogaster has emerged as an important platform of discovery. In the laboratory, Drosophila can be made obese through lipogenic diets, genetic manipulations and adaptation to evolutionary stress. While dietary and genetic changes that cause obesity in flies have been demonstrated to induce heart dysfunction, there have been no reports investigating how obesity affects the heart in laboratory-evolved populations. This paper studied replicated populations of Drosophila that had been selected for starvation resistance for over 65 generations. These populations evolved characteristics that closely resemble hallmarks of metabolic syndrome in mammals. Starvation-selected Drosophila have dilated hearts with impaired contractility. This phenotype appears to be correlated with large fat deposits along the dorsal cuticle, which alter the anatomical position of the heart. A strong relationship was demonstrated between fat storage and heart dysfunction, as dilation and reduced contractility can be rescued through prolonged fasting. Unlike other Drosophila obesity models, the starvation-selected lines do not exhibit excessive lipid deposition within the myocardium and rather store excess triglycerides in large lipid droplets within the fat body. These findings provide a new model to investigate obesity-associated heart dysfunction (Hardy, 2015).

CoA protects against the deleterious effects of caloric overload in Drosophila

A Drosophila model of type 2 diabetes was developed in which high sugar (HS) feeding leads to insulin resistance. In this model, adipose triglyceride storage is protective against fatty acid toxicity and diabetes. Initial biochemical and gene expression studies suggested that deficiency in acetyl-CoA might underlie reduced triglyceride synthesis in animals during chronic HS feeding. Focusing on the Drosophila fat body, which is specialized for triglyceride storage and lipolysis, a series of experiments was undertaken to test the hypothesis that CoA could protect against the deleterious effects of caloric overload. Quantitative metabolomics revealed a reduction in substrate availability for CoA synthesis in the face of an HS diet. Further reducing CoA synthetic capacity by expressing fat body-specific RNAi targeting pantothenate kinase (fumble) or phosphopantothenoylcysteine decarboxylase (PPCS) exacerbated HS-diet-induced accumulation of free fatty acids. Dietary supplementation with pantothenic acid (vitamin B5, a precursor of CoA) ameliorated HS-diet-induced free fatty acid accumulation and hyperglycemia while increasing triglyceride synthesis. Taken together, these data support a model where free CoA is required to support fatty acid esterification and to protect against the toxicity of HS diets (Musselman, 2016).

Previous studies have shown a reduced capacity for TG synthesis in obesity that is accompanied by increases in FFAs, ceramides, and DAG, all potential mediators of lipotoxicity. Still, it remains unknown what mechanisms limit the ability of animals to store excess carbons from dietary sugar as TG. In this study, a dramatic upregulation in the expression of CoA synthetic enzymes was observed, prompting a closer look at these steps of the pathway. The CoA pool is known to be limiting for several metabolic processes, including the TCA cycle, ketogenesis, lipogenesis, and mitochondrial fatty acid import and β-oxidation. Although all of these pathways were not investigated, data support a model where CoA is limiting in the face of caloric excess, reducing animal fitness by contributing to metabolic lipotoxicity (Musselman, 2016 and references therein).

The Drosophila gut may be an important source of pantothenate. The fly gut is known to harbor commensal bacteria that regulate nutritional status and might help to provide pantothenate, as has been demonstrated in mammals. Measurable quantities of this nutrient in isolated guts were observed, although no change in pantetheine or pantothenate levels was observed upon HS feeding. Increased gut expression of genes predicted to encode the pantetheine hydrolase vanin-like and the pantothenate transporter, CG10444, may represent an attempt of the gut to compensate for inadequate CoA levels and suggests a concerted systemic effort to provide this nutrient to the FB (Musselman, 2016 and references therein).

One open question is: what metabolites indicate an increased requirement for pantothenate in peripheral tissues? The carnitine-acyl carnitine system is one way in which free CoA pools are maintained in cells. Serum acyl-carnitine concentrations reflect an excess of intracellular acyl groups, increasing when fatty acid oxidation is defective in the presence of increased FFAs. It follows that these acyl-carnitines might accumulate when metabolic flux is reduced during insulin resistance. Increased long-chain carnitine esters have been observed in the serum, liver, muscle, and urine of individuals with obesity and T2D, although reduced levels of long-chain acyl-carnitines have also been associated with metabolic syndrome and T2D. Rodent models of obesity and T2D also accumulate acyl-carnitines. In Drosophila, acyl-carnitines decline with age, along with obesity. Perhaps circulating acyl-carnitines signal a demand for CoA to enable proper fatty acid esterification into TG in the FB and adipose. Data from this study support a model where CoA bioavailability enables metabolic flexibility and channeling of the endocrine fatty acid pool (Musselman, 2016 and references therein).

Another potential rate-limiting substrate for CoA synthesis in the face of caloric overload is cysteine, although data suggest that cysteine is not limiting in the context of caloric overload. Cysteine supplementation alone slightly reduces fitness on HS diets and does not rescue HS phenotypes. Metabolite analysis shows that cysteine levels are slightly elevated in HS-fed FBs compared with controls. Further increasing cysteine levels could adversely affect redox status in the FB, impairing cellular processes and masking any benefit to lipogenesis. It is interesting to note that some studies have shown a benefit for cysteine supplementation in T2D. It is presumable that a number of metabolites have the potential to become rate-limiting under different physiological conditions. Nonetheless, data from this study support a substrate-limited model where increasing the production of CoA benefits animal health in the face of a HS diet (Musselman, 2016 and references therein).

PA is available over-the-counter as calcium pantothenate in vitamin B5 supplements. In another study, pantothenate supplementation was shown to promote CoA-dependent keto­genesis and improve liver function in an animal model of nonalcoholic fatty liver disease. This study proposes that vitamin B5 represents a potential therapy for insulin resistance resulting from overnutrition. Although pantothenate supplementation would be expected to increase adiposity, a significant benefit can be expected in terms of metabolic health. PA’s low cost and toxicity profile make it an especially attractive target for future clinical studies (Musselman, 2016 and references therein).

Tissue nonautonomous effects of fat body methionine metabolism on imaginal disc repair in Drosophila

Regulatory mechanisms for tissue repair and regeneration within damaged tissue have been extensively studied. However, the systemic regulation of tissue repair remains poorly understood. To elucidate tissue nonautonomous control of repair process, it is essential to induce local damage, independent of genetic manipulations in uninjured parts of the body. This study developed a system in Drosophila for spatiotemporal tissue injury using a temperature-sensitive form of diphtheria toxin A domain driven by the Q system to study factors contributing to imaginal disc repair. Using this technique, it was demonstrated that methionine metabolism in the fat body, a counterpart of mammalian liver and adipose tissue, supports the repair processes of wing discs. Local injury to wing discs decreases methionine and S-adenosylmethionine, whereas it increases S-adenosylhomocysteine in the fat body. Fat body-specific genetic manipulation of methionine metabolism results in defective disc repair but does not affect normal wing development. These data indicate the contribution of tissue interactions to tissue repair in Drosophila, as local damage to wing discs influences fat body metabolism, and proper control of methionine metabolism in the fat body, in turn, affects wing regeneration (Kashio, 2016).

A biological timer in the fat body comprised of Blimp-1, betaFTZ-F1 and Shade regulates pupation timing in Drosophila melanogaster

During the development of multicellular organisms, many events occur with precise timing. In Drosophila, pupation occurs about 12 hours after puparium formation, and its timing is believed to be determined by the release of a steroid hormone, ecdysone (E), from the prothoracic gland. This study demonstrates that the ecdysone-20-monooxygenase, Shade, determines the pupation timing by converting E to 20-hydroxyecdysone (20E) in the fat body, which is the organ that senses nutritional status. The timing of shade expression is determined by its transcriptional activator βFTZ-F1. The βFTZ-F1 gene is activated after a decline in the expression of its transcriptional repressor Blimp-1, which is temporally expressed around puparium formation in response to a high titer of 20E. The expression level and stability of Blimp-1 is critical for the precise timing of pupation. Thus, it is proposed that Blimp-1 molecules function as sands in an hourglass for this precise developmental timer system. Furthermore, the data suggest a biological advantage results from both the use of a transcriptional repressor for the time determination, and association of developmental timing with nutritional status of the organism (Akagi, 2016).

The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults

Although mutations in HNF4A were identified as the cause of Maturity Onset Diabetes of the Young 1 (MODY1) two decades ago, the mechanisms by which this nuclear receptor regulates glucose homeostasis remain unclear. This study reports that loss of Drosophila HNF4 recapitulates hallmark symptoms of MODY1, including adult-onset hyperglycemia, glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS). These defects are linked to a role for dHNF4 in promoting mitochondrial function as well as the expression of Hex-C, a homolog of the MODY2 gene Glucokinase. dHNF4 is required in the fat body and insulin-producing cells to maintain glucose homeostasis by supporting a developmental switch toward oxidative phosphorylation and GSIS at the transition to adulthood. These findings establish an animal model for MODY1 and define a developmental reprogramming of metabolism to support the energetic needs of the mature animal (Barry, 2016).

The association of MODY subtypes with mutations in specific genes provides a framework for understanding the monogenic heritability of this disorder as well as the roles of the corresponding pathways in systemic glucose homeostasis. This paper investigated the long-known association between HNF4A mutations and MODY1 by characterizing a whole-animal mutant that recapitulates the key symptoms associated with this disorder. Drosophila HNF4 is shown to be required for both GSIS and glucose clearance in adults, acting in distinct tissues and multiple pathways to maintain glucose homeostasis. Evidence is provided that dHNF4 promotes mitochondrial OXPHOS by regulating nuclear and mitochondrial gene expression. Finally, the expression of dHNF4 and its target genes is shown to be dramatically induced at the onset of adulthood, contributing to a developmental switch toward GSIS and oxidative metabolism at this stage in development. These results provide insights into the molecular basis of MODY1, expand understanding of the close coupling between development and metabolism, and establish the adult stage of Drosophila as an accurate context for genetic studies of GSIS, glucose clearance, and diabetes (Barry, 2016).

Drosophila HNF4 mutants display late-onset hyperglycemia accompanied by sensitivity to dietary carbohydrates, glucose intolerance, and defects in GSIS - hallmarks of MODY1. These defects arise from roles for dHNF4 in multiple tissues, including a requirement in the IPCs for GSIS and a role in the fat body for glucose clearance. The regulation of GSIS by dHNF4 is consistent with the long-known central contribution of pancreatic β-cells to the pathophysiology of MODY1 (Fajans and Bell, 2011). Similarly, several MODY-associated genes, including GCK, HNF1A and HNF1B, are important for maintaining normal hepatic function. These distinct tissue-specific contributions to glycemic control may explain why single-tissue Hnf4A mutants in mice do not fully recapitulate MODY1 phenotypes and predict that a combined deficiency for the receptor in both the liver and pancreatic β-cells of adults would produce a more accurate model of this disorder (Barry, 2016).

This study used metabolomics, RNA-seq, and ChIP-seq to provide initial insights into the molecular mechanisms by which dHNF4 exerts its effects on systemic metabolism. These studies revealed several downstream pathways, each of which is associated with maintaining homeostasis and, when disrupted, can contribute to diabetes. These include genes identified in previous study of dHNF4 in larvae that act in lipid metabolism and fatty acid β-oxidation, analogous to the role of Hnf4A in the mouse liver to maintain normal levels of stored and circulating lipids (Hayhurst, 2001; Palanker, 2009). Extensive studies have linked defects in lipid metabolism with impaired β-cell function and peripheral glucose uptake and clearance, suggesting that these pathways contribute to the diabetic phenotypes of dHNF4 mutants. An example of this is pudgy, which is expressed at reduced levels in dHNF4 mutants and encodes an acyl-CoA synthetase that is required for fatty acid oxidation (Xu, 2012). Interestingly, pudgy mutants have elevated triglycerides, reduced glycogen, and increased circulating sugars, similar to dHNF4 mutants, suggesting that this gene is a critical downstream target of the receptor. It is important to note, however, that the metabolomic, RNA-seq, and ChIP-seq studies were conducted on extracts from whole animals rather than individual tissues. As a result, some of the findings may reflect compensatory responses between tissues, and some tissue-specific changes in gene expression or metabolite levels may not be detected by the current approach. Further studies using samples from dissected tissues would likely provide a more complete understanding of the mechanisms by which dHNF4 maintains systemic physiology (Barry, 2016).

Notably, the Drosophila GCK homolog encoded by Hex-C is expressed at reduced levels in dHNF4 mutants. The central role of GCK in glucose sensing by pancreatic β-cells as well as glucose clearance by the liver places it as an important regulator of systemic glycemic control. Functional data supports these associations by showing that Hex-C is required in the fat body for proper circulating glucose levels, analogous to the role of GCK in mammalian liver. Unlike mice lacking GCK in the β-cells, no effect is seen on glucose homeostasis when Hex-C is targeted by RNAi in the IPCs. This is possibly due to the presence of a second GCK homolog in Drosophila, Hex-A, which could act alone or redundantly with Hex-C to mediate glucose sensing by the IPCs. In mammals, GCK expression is differentially regulated between hepatocytes and β-cells through the use of two distinct promoters, and studies in rats have demonstrated a direct role for HNF4A in promoting GCK expression in the liver. These findings suggest that this relationship has been conserved through evolution. In addition, the association between GCK mutations and MODY2 raise the interesting possibility that defects in liver GCK activity may contribute to the pathophysiology of both MODY1 and MODY2 (Barry, 2016).

Interestingly, gene ontology analysis indicates that the up-regulated genes in dHNF4 mutants correspond to the innate immune response pathways in Drosophila. This response parallels that seen in mice lacking Hnf4A function in enterocytes, which display intestinal inflammation accompanied by increased sensitivity to DSS-induced colitis and increased permeability of the intestinal epithelium, similar to humans with inflammatory bowel disease. Disruption of Hnf4A expression in Caco-2 cells using shRNA resulted in changes in the expression of genes that act in oxidative stress responses, detoxification pathways, and inflammatory responses, similar to the effect seen in dHNF4 mutants. Moreover, mutations in human HNF4A are associated with chronic intestinal inflammation, irritable bowel disease, ulcerative colitis, and Crohn's disease, suggesting that these functions are conserved through evolution. Taken together, these results support the hypothesis that dHNF4 plays an important role in suppressing an inflammatory response in the intestine. Future studies are required to test this hypothesis in Drosophila. In addition, further work is required to better define the regulatory functions of HNF4 that are shared between Drosophila and mammals. Although the current work suggests that key activities for this receptor have been conserved in flies and mammals, corresponding to the roles of HNF4 in the IPCs (β-cells) for GSIS, fat body (liver) for lipid metabolism and glucose clearance, and intestine to suppress inflammation, there are likely to be divergent roles as well. One example of this is the embryonic lethality of Hnf4A mutant mice, which is clearly distinct from the early adult lethality reported here for dHNF4 mutants. Further studies are required to dissect the degree to which the regulatory functions of this receptor have been conserved through evolution (Barry, 2016).

It is also important to note that mammalian Hnf4A plays a role in hepatocyte differentiation and proliferation in addition to its roles in metabolism. This raises the possibility that early developmental roles for dHNF4 could impact the phenotypes reported in this study in adults. Indeed, all of the current studies involve zygotic dHNF4 null mutants that lack function throughout development. In an effort to address this possibility and distinguish developmental from adult-specific functions, a conditional dHNF4 mutant allele is currently being constructed using CRISPR/Cas9 technology. Future studies using this mutation should allow conducting a detailed phenotypic analysis of this receptor at different stages of Drosophila development (Barry, 2016).

It is also interesting to speculate that the current functional studies of dHNF4 uncover more widespread roles for MODY-associated genes in glycemic control, in addition to the link with MODY2 described in this study. HNF1A and HNF1B, which are associated with MODY3 and MODY5, respectively, act together with HNF4A in an autoregulatory circuit in an overlapping set of tissues, with HNF4A proposed to be the most upstream regulator of this circuit. The observation that Drosophila do not have identifiable homologs for HNF1A and HNF1B raises the interesting possibility that dHNF4 alone replaces this autoregulatory circuit in more primitive organisms. The related phenotype of these disorders is further emphasized by cases of MODY3 that are caused by mutation of an HNF4A binding site within the HNF1A promoter. Consistent with this link, MODY1, MODY3 and MODY5 display similar features of disease complication and progression, and studies of HNF1A and HNF4A in INS-1 cells have implicated roles for these transcription factors in promoting mitochondrial metabolism in β-cells. In line with this, mitochondrial diabetes is clearly age progressive, as are MODY1, 3, and 5, but not MODY2, which represents a more mild form of this disorder. Furthermore, the severity and progression of MODY3 is significantly enhanced when patients carry an additional mutation in either HNF4A or mtDNA. Overall, these observations are consistent with the well-established multifactorial nature of diabetes, with multiple distinct metabolic insults contributing to disease onset (Barry, 2016).

RNA-seq analysis supports a role for dHNF4 in coordinating mitochondrial and nuclear gene expression. This is represented by the reduced expression of transcripts encoded by the mitochondrial genome, along with effects on nuclear-encoded genes that act in mitochondria. In addition, ChIP-seq revealed that several of the nuclear-encoded genes are direct targets of the receptor. Mitochondrial defects have well-established links to diabetes-onset, with mutations in mtDNA causing maternally-inherited diabetes and mitochondrial OXPHOS playing a central role in both GSIS and peripheral glucose clearance. Consistent with this, functional studies indicate that dHNF4 is required to maintain normal mitochondrial function and that defects in this process contribute to the diabetic phenotypes in dHNF4 mutants (Barry, 2016).

It is important to note that the number of direct targets for dHNF4 in the nucleus is difficult to predict with the current dataset. A relatively low signal-to-noise ratio in ChIP-seq experiment allowed identification of only 37 nuclear-encoded genes as high confidence targets by fitting the criteria of proximal dHNF4 binding along with reduced expression in dHNF4 mutants. Future ChIP-seq studies will allow expansion of this dataset to gain a more comprehensive understanding of the scope of the dHNF4 regulatory circuit and may also reveal tissue-restricted targets that are more difficult to detect. Nonetheless, almost all of the genes identified as direct targets for dHNF4 regulation correspond to genes involved in mitochondrial metabolism, including the TCA cycle, OXPHOS, and lipid catabolism, demonstrating that this receptor has a direct impact on these critical downstream pathways that influence glucose homeostasis (Barry, 2016).

An unexpected and significant discovery in these studies is that dHNF4 is required for mitochondrial gene expression and function. Several lines of evidence support the model that dHNF4 exerts this effect through direct regulation of mitochondrial transcription, although a number of additional experiments are required to draw firm conclusions on this regulatory connection. First, most of the 13 protein-coding genes in mtDNA are underexpressed in dHNF4 mutants. RNA-seq studies have been conducted of Drosophila nuclear transcription factor mutants and similar effects on mitochondrial gene expression have not been reported previously. Second, dHNF4 protein is abundantly bound to the control region of the mitochondrial genome, representing the fifth strongest enrichment peak in the ChIP-seq dataset. Although the promoters in Drosophila mtDNA have not yet been identified, the site bound by dHNF4 corresponds to a predicted promoter region for Drosophila mitochondrial transcription and coincides with the location of the major divergent promoters in human mtDNA. It is unlikely that the abundance of mtDNA relative to nuclear DNA had an effect on the ChIP-seq peak calling because the MACS2 platform used for this analysis accounts for local differences in read depth across the genome (including the abundance of mtDNA). In addition, although the D-loop in mtDNA has been proposed to contribute to possible false-positive ChIP-seq peaks in mammalian studies, the D-loop structure is not present in Drosophila mtDNA. Nonetheless, additional experiments are required before it can be concluded that this apparent binding is of regulatory significance for mitochondrial function. Third, the effects on mitochondrial gene expression do not appear to be due to reduced mitochondrial number in dHNF4 mutants. This is consistent with the normal expression of mt:Cyt-b in dHNF4 mutants, which has a predicted upstream promoter that drives expression of the mt:Cyt-b and mt:ND6 operon (although mt:ND6 RNA could not be detected in northern blot studies). Fourth, immunostaining for dHNF4 shows cytoplasmic protein that overlaps with the mitochondrial marker ATP5A, in addition to its expected nuclear localization. Some of the cytoplasmic staining, however, clearly fails to overlap with the mitochondrial marker, making it difficult to draw firm conclusions from this experiment. Multiple efforts to expand on this question biochemically with subcellular fractionation studies have been complicated by abundant background proteins that co-migrate with the receptor in mitochondrial extracts. New reagents are currently being developed to detect the relatively low levels of endogenous dHNF4 protein in mitochondria, including use of the CRISPR/Cas9 system for the addition of specific epitope tags to the endogenous dHNF4 locus. Finally, multiple hallmarks of mitochondrial dysfunction were observed, including elevated pyruvate and lactate, specific alterations in TCA cycle metabolites, reduced mitochondrial membrane potential, reduced levels of ATP, and fragmented mitochondrial morphology. These phenotypes are consistent with the reduced expression of key genes involved in mitochondrial OXPHOS, and studies showing that decreased mitochondrial membrane potential and ATP production are commonly associated with mitochondrial fragmentation (Barry, 2016).

Although unexpected, the proposal that dHNF4 may directly regulate mitochondrial gene expression is not unprecedented. A number of nuclear transcription factors have been localized to mitochondria, including ATFS-1, MEF2D, CREB, p53, STAT3, along with several nuclear receptors, including the estrogen receptor, glucocorticoid receptor, and the p43 isoform of the thyroid hormone receptor. The significance of these observations, however, remains largely unclear, with few studies demonstrating regulatory functions within mitochondria. In addition, these factors lack a canonical mitochondrial localization signal at their amino-terminus, leaving it unclear how they achieve their subcellular distribution. In contrast, one of the five mRNA isoforms encoded by dHNF4, dHNF4-B, encodes a predicted mitochondrial localization signal in its 5'-specific exon, providing a molecular mechanism to explain the targeting of this nuclear receptor to this organelle. Efforts are currently underway to conduct a detailed functional analysis of dHNF4-B by using the CRISPR/Cas9 system to delete its unique 5' exon, as well as establishing transgenic lines that express a tagged version of dHNF4-B under UAS control. Future studies using these reagents, along with available dHNF4 mutants, should allow dissection of the nuclear and mitochondrial functions of this nuclear receptor and their respective contributions to systemic physiology (Barry, 2016).

Finally, it is interesting to speculate whether the role for dHNF4 in mitochondria is conserved in mammals. A few papers have described the regulation of nuclear-encoded mitochondrial genes by HNF4A. In addition, several studies have detected cytoplasmic Hnf4A by immunohistochemistry in tissue sections, including in postnatal pancreatic islets and hepatocytes. Moreover, the regulation of nuclear/cytoplasmic shuttling of HNF4A has been studied in cultured cells. The evolutionary conservation of the physiological functions of HNF4A, from flies to mammals, combined with these prior studies, argue that more effort should be directed at defining the subcellular distribution of HNF4A protein and its potential roles within mitochondria. Taken together with these studies in Drosophila, this work could provide new directions for understanding HNF4 function and MODY1 (Barry, 2016).

Physiological studies by George Newport in 1836 noted that holometabolous insects reduce their respiration during metamorphosis leading to a characteristic 'U-shaped curve' in oxygen consumption. Subsequent classical experiments in Lepidoptera, Bombyx, Rhodnius and Calliphora showed that this reduction in mitochondrial respiration during metamorphosis and dramatic rise in early adults is seen in multiple insect species, including Drosophila. Consistent with this, the activity of oxidative enzyme systems and the levels of ATP also follow a 'U-shaped curve' during development as the animal transitions from a non-feeding pupa to a motile and reproductively active adult fly. Although first described over 150 years ago, the regulation of this developmental increase in mitochondrial activity has remained undefined. This study shows that this temporal switch is dependent, at least in part, on the dHNF4 nuclear receptor. The levels of dHNF4 expression increase dramatically at the onset of adulthood, accompanied by the expression of downstream genes that act in glucose homeostasis and mitochondrial OXPHOS. This coordinate transcriptional switch is reduced in dHNF4 mutants, indicating that the receptor plays a key role in this transition. Importantly, the timing of this program correlates with the onset of dHNF4 mutant phenotypes in young adults, including sugar-dependent lethality, hyperglycemia, and defects in glucose-stimulated insulin secretion, indicating that the upregulation of dHNF4 expression in adults is of functional significance. It should also be noted, however, that dHNF4 target genes are still induced at the onset of adulthood in dHNF4 mutants, albeit at lower levels, indicating that other regulators contribute to this switch in metabolic state. Nonetheless, the timing of the induction of dHNF4 and its target genes in early adults, and its role in promoting OXPHOS, suggest that this receptor contributes to the end of the 'U-shaped curve' and directs a systemic transcriptional switch that establishes an optimized metabolic state to support the energetic demands of adult life (Barry, 2016).

Interestingly, a similar metabolic transition towards OXPHOS was recently described in Drosophila neuroblast differentiation, mediated by another nuclear receptor, EcR. Although this occurs during early stages of pupal development, prior to the dHNF4-mediated transition at the onset of adulthood, the genes involved in this switch show a high degree of overlap with dHNF4 target genes that act in mitochondria, including ETFB, components of Complex IV, pyruvate carboxylase, and members of the α-ketoglutarate dehydrogenase complex. This raises the possibility that dHNF4 may contribute to this change in neuroblast metabolic state and play a more general role in supporting tissue differentiation by promoting OXPHOS (Barry, 2016).

Only one other developmentally coordinated switch in systemic metabolic state has been reported in Drosophila and, intriguingly, it is also regulated by a nuclear receptor. Drosophila Estrogen-Related Receptor (dERR) acts in mid-embryogenesis to directly induce genes that function in biosynthetic pathways related to the Warburg effect, by which cancer cells use glucose to support rapid proliferation (Tennessen, 2011; Tennessen, 2014b). This switch toward aerobic glycolysis favors lactate production and flux through biosynthetic pathways over mitochondrial OXPHOS, supporting the ~200-fold increase in mass that occurs during larval development. Taken together with the current work on dHNF4, these studies define a role for nuclear receptors in directing temporal switches in metabolic state that meet the changing physiological needs of different stages in development. Further studies should allow better definition of these regulatory pathways as well as determine how broadly nuclear receptors exert this role in coupling developmental progression with systemic metabolism (Barry, 2016).

Although little is known about the links between development and metabolism, it is likely that coordinated switches in metabolic state are not unique to Drosophila, but rather occur in all higher organisms in order to meet the distinct metabolic needs of an animal as it progresses through its life cycle. Indeed, a developmental switch towards OXPHOS in coordination with the cessation of growth and differentiation appears to be a conserved feature of animal development. Moreover, as has been shown for cardiac hypertrophy, a failure to coordinate metabolic state with developmental context can have an important influence on human disease (Barry, 2016).

In addition to promoting a transition toward systemic oxidative metabolism in adult flies, dHNF4 also contributes to a switch in IPC physiology that supports GSIS. dHNF4 is not expressed in larval IPCs, but is specifically induced in these cells at adulthood. Similarly, the fly homologs of the mammalian ATP-sensitive potassium channel subunits, Sur1 and Kir6, which link OXPHOS and ATP production to GSIS, are not expressed in the larval IPCs but are expressed during the adult stage. They also appear to be active at this stage as cultured IPCs from adult flies undergo calcium influx and membrane depolarization upon exposure to glucose or the anti-diabetic sulfonylurea drug glibenclamide. In addition, reduction of the mitochondrial membrane potential in adult IPCs by ectopic expression of an uncoupling protein is sufficient to reduce IPC calcium influx, elevate whole-animal glucose levels, and reduce peripheral insulin signaling. This switch in IPC physiology is paralleled by a change in the nutritional signals that trigger DILP release. Amino acids, and not glucose, stimulate DILP2 secretion by larval IPCs. Rather, glucose is sensed by the corpora cardiaca in larvae, a distinct organ that secretes adipokinetic hormone, which acts like glucagon to maintain carbohydrate homeostasis during larval stages. Interestingly, this can have an indirect effect on the larval IPCs, triggering DILP3 secretion in response to dietary carbohydrates (Kim, 2015). Adult IPCs, however, are responsive to glucose for DILP2 release (Park, 2014). In addition, dHNF4 mutants on a normal diet maintain euglycemia during larval and early pupal stages, but display hyperglycemia at the onset of adulthood, paralleling their lethal phase on a normal diet. Taken together, these observations support the model that the IPCs change their physiological state during the larval-to-adult transition and that dHNF4 contributes to this transition toward glucose-stimulated insulin secretion. The observation that glucose is a major circulating sugar in adults, but not larvae, combined with its ability to stimulate DILP2 secretion from adult IPCs, establishes this stage as an experimental context for genetic studies of glucose homeostasis, GSIS, and diabetes. Functional characterization of these pathways in adult Drosophila will allow he power of model organism genetics to be harnessed to better understand the regulation of glucose homeostasis and the factors that contribute to diabetes (Barry, 2016).

Drosophila HNF4 directs a switch in lipid metabolism that supports the transition to adulthood

Animals must adjust their metabolism as they progress through development in order to meet the needs of each stage in the life cycle. This study shows that the dHNF4 nuclear receptor acts at the onset of Drosophila adulthood to direct an essential switch in lipid metabolism. Lipid stores are consumed shortly after metamorphosis but contribute little to energy metabolism. Rather, dHNF4 directs their conversion to very long chain fatty acids and hydrocarbons, which waterproof the animal to preserve fluid homeostasis. Similarly, HNF4alpha is required in mouse hepatocytes for the expression of fatty acid elongases that contribute to a waterproof epidermis, suggesting that this pathway is conserved through evolution. This developmental switch in Drosophila lipid metabolism promotes lifespan and desiccation resistance in adults and suppresses hallmarks of diabetes, including elevated glucose levels and intolerance to dietary sugars. These studies establish dHNF4 as a regulator of the adult metabolic state (Storelli, 2018).

Drosophila breeds and feeds on rotting fruits, which are ephemeral ecosystems. Thus, while the Drosophila larva lives inside a semi-liquid nutritive substrate and feeds extensively to support its rapid increase in mass, the mature adult fly emerges into an unpredictable environment where it uses flight for dispersal and the colonization of more favorable niches. Similar to all insects, the large surface-to-volume ratio of Drosophila adults makes them vulnerable to dehydration. In addition, the metabolic demands of flight require a high rate of gas exchange via the respiratory system, increasing water loss. This study shows that the successful transition from a stationary and protected pupa to a motile adult that can survive in a dry environment is dependent on the dHNF4 nuclear receptor, which acts in the oenocytes to direct a transcriptional program that supports the rapid production of very long-chain fatty acids (VLCFAs) and cuticular hydrocarbons. This activity is consistent with earlier studies, which have demonstrated an important role for oenocytes in VLCFA/hydrocarbon synthesis and desiccation resistance, and places dHNF4 as a central transcriptional regulator for oenocyte function. This study shows, however, that whole-body and hemolymph TAG levels drop to low levels between emergence and day 3 of adulthood, on either a sugar-free diet or a rich 15% sugar diet. The lipids contained in larval adipose cells thus appear to be consumed independently of nutritional input, suggesting that they contribute to non-energetic functions. Moreover, control adults can survive for weeks in the absence of stored lipids, further indicating that they provide a relatively minor source of energy at this stage in development (Storelli, 2018).

It is proposed that lipids in the newly emerged adult are shunted toward a purpose that is more profound than energy production at this stage in development -- the formation of a waterproof cuticle -- and that this occurs in a dHNF4-dependent manner. Stored lipid levels are unaffected in newly emerged dHNF4 mutant adults relative to controls. Lipids, however, accumulate rapidly after this stage in oenocytes, in the presence or absence of nutrients, representing a developmental role for dHNF4 in maintaining lipid homeostasis in this cell type. The timing of lipid accumulation in the oenocytes of starved and fed dHNF4 mutants coincides with the timing of lipid loss and larval adipose cell death. Genetic studies demonstrate that dHNF4 is specifically required in oenocytes to suppress developmental steatosis. This appears to be due to its role in inducing the expression of genes involved in VLCFA elongation. Oenocyte-specific silencing of KAR or ACC recapitulates the developmental steatotic phenotype, while silencing genes that encode downstream or upstream steps in the hydrocarbon biosynthetic pathway has no effect on the lipid levels in oenocytes. Further studies are needed to determine the molecular mechanisms that link VLCFA elongation with oenocyte lipid homeostasis. Finally, adults normally emerge with low hydrocarbon levels that increase dramatically over 3 days, even in the absence of food, in agreement with the hypothesis that the larval adipose cells provide the primary source of precursors for hydrocarbon production. These results are consistent with previous reports, which have shown that dietary manipulation during larval life affects the blend of hydrocarbons in young adult. Consistent with this, oenocyte-specific dHNF4 RNAi in mature adults was shown to lead to a similar degree of lipid accumulation as that seen upon inducing RNAi at emergence. Further studies are required to address this continuing role for dHNF4 in mature adults. In addition, the mechanisms that partition stored lipids toward VLCFA/hydrocarbon production or energy production remain to be determined. Finally, the key functions described for dHNF4 in oenocytes are shared between males and females, indicating that they are not sex specific (Storelli, 2018).

Studies of starvation resistance indicate that newly emerged Drosophila adults can tolerate the absence of nutrients for at least a week. During the non-feeding period of metamorphosis, pupae consume about half of their glycogen and TAG stores and most of the trehalose gathered during the larval stages. In spite of this depletion of energy reserves, sufficient nutrients persist to allow the newly emerged adult to survive and disperse in their new environment. Under hydrated conditions, control lines emerge with the ability to survive from 1 to 3 weeks in the absence of nutrients. It is suggested that these conditions provide insufficient humidity to accurately distinguish the effects of dehydration from the effects of nutrient depletion. Without moisture or food, Drosophila die in less than a day, with a median lifespan of 13 hr. Thus, as is well known in mammals, sufficient hydration is critical for assessing the ability of animals to properly mobilize stored nutrients for survival during starvation (Storelli, 2018).

Changes in metabolism must accompany each stage in development in order to allow normal progression through the life cycle. Although little is known about how development and metabolism are coupled, nuclear receptors appear to play a central role in this process. Consistent with this, a previous study of dHNF4 showed that it is highly up-regulated at the end of metamorphosis as the fly begins its adult life, coordinately inducing genes involved in glucose homeostasis and oxidative phosphorylation (Barry, 2016). Functional studies showed that dHNF4 acts at this stage in the insulin-producing cells to maintain glucose-stimulated insulin secretion and acts in the fat body to promote glucose clearance. This study expands upon these activities for dHNF4 at the onset of adulthood to include an essential role in supporting VLCFA and hydrocarbon production in the oenocytes to allow adult survival and dispersion (Storelli, 2018).

Key genes involved in VLCFA/hydrocarbon production are induced at the onset of adulthood in control adults and reduced in expression in dHNF4 mutants, including KAR, Cpr, Cyp4g1, and several genes encoding predicted elongases. In addition, dHNF4 transcriptional activity can be activated by LCFAs and VLCFAs, which appear to act as ligands for this nuclear receptor. This is consistent with studies of mammalian HNF4α, which have identified LCFAs as ligands that can trigger the conformational changes required for coactivator recruitment. Thus, the free fatty acids generated by lipolysis from the larval adipose cells could act as ligands for dHNF4 in oenocytes, driving the transcription of genes in the VLCFA/hydrocarbon pathway. The resulting VLCFAs might further activate dHNF4, providing a feed-forward loop to enhance VLCFA/hydrocarbon production. These levels of regulation could ensure that free fatty acids are rapidly and efficiently converted into VLCFAs and hydrocarbons to provide an effective waterproof barrier for the young adult (Storelli, 2018).

Simple carbohydrates are required for optimal Drosophila longevity, yet dHNF4 and VLCFA/hydrocarbon biosynthesis are required in oenocytes to provide tolerance to these nutrients. In addition, increased humidity is sufficient to rescue sugar toxicity and hyperglycemia in dHNF4 mutants, suggesting that defects in fluid homeostasis are contributing to these phenotypes. Defects in fluid homeostasis could occur in dHNF4 mutants because of impaired waterproofing of the cuticle or the trachea. Parvy and colleagues demonstrated that, besides providing precursors for hydrocarbon synthesis, VLCFA metabolism in oenocytes is required to remotely waterproof the respiratory system in larvae. Notably, disrupting VLCFA production in oenocytes via ACC, KAR, or FASNCG17374 RNAi in this cell type results in defects in tracheal waterproofing and larval lethality. Lethality between larval and pupal stages was observed when these RNAi constructs were driven with a constitutive oenocyte-specific GAL4 driver. However, defects in tracheal air filling or lethality are not seen in dHNF4 mutant larvae or in animals with constitutive dHNF4 RNAi expression in oenocytes, suggesting that there are no significant effects on tracheal waterproofing in these animals. In addition, altering cuticular hydrocarbon production while leaving VLCFA synthesis intact (by silencing Cyp4g1 in oenocytes) induces sugar toxicity and defects in glucose homeostasis. Thus it is postulated that reduced hydrocarbon production by oenocytes and altered cuticular waterproofing play central roles in the physiological defects observed in dHNF4 mutants (Storelli, 2018).

Understanding of diabetes in humans provides possible models to explain the physiological mechanisms that link dHNF4 activity in oenocytes to fluid and carbohydrate homeostasis. In mammals, excess blood glucose cannot be effectively reabsorbed by the kidney and is excreted with urine. One possibility is that dHNF4 mutants provide a sensitized genetic context for the development of a hyperosmolar hyperglycemic state in Drosophila. Increased transepidermal water loss could reduce hemolymph volume, contributing to the development of hyperglycemia on a sugar-containing diet. This hypothesis could explain the elevated glucose levels observed in dHNF4 mutants and the normalization of glucose levels in high humidity. Further studies are required to test this possibility and dissect the mechanisms that couple dHNF4 function in oenocytes to systemic carbohydrate homeostasis (Storelli, 2018).

A number of studies have focused on characterizing the signaling pathways that govern glucose sensing and metabolism in Drosophila. These studies have provided evidence for glucose-induced cellular damage via oxidative stress or the formation of advanced glycation end-products. In spite of these efforts, however, more remains to be learned about the mechanisms underlying dietary sugar toxicity. This work suggests unexpected roles for oenocyte lipid metabolism and hydration status in regulating glucose homeostasis and suppressing sugar toxicity in Drosophila (Storelli, 2018).

Interestingly, the role of VLCFAs in reducing trans-epidermal water loss is conserved through evolution. Similarly, mouse mutants for Elovl1 and Elovl4 are born normally but die shortly thereafter from acute dehydration, similar to dHNF4 mutant adults. This lethality is accompanied by reduced epithelial barrier function, defects in the lamellar structure of newborn skin, reduced levels of VLCFAs, and the absence of key acylceramides that contribute to skin hydrophobicity. Genetic studies of the gene encoding ELOVL3, which elongates C20 fatty acids to C22 and C24, revealed similar functions. Elovl3 mutant mice have disrupted skin and hair morphology resulting in increased trans-epidermal water loss. Taken together, these results indicate that VLCFA production is critical for proper lipid metabolism and survival after birth in mouse, revealing a perinatal transition that parallels the role for VLCFAs and hydrocarbons in newly emerged Drosophila adults. Interestingly, these studies also link Elovl gene expression to HNF4α function in mice. Elovl3, Elovl5, and KAR are all expressed at reduced levels in HNF4α mutant hepatocytes, indicating that this regulatory link is conserved through evolution. Further studies are required to determine if HNF4α exerts a similar role in mammalian skin and if its activity is required for epithelial barrier function and desiccation resistance (Storelli, 2018).

Steroid hormone signaling is essential for pheromone production and oenocyte survival

Many of the lipids found on the cuticles of insects function as pheromones and communicate information about age, sex, and reproductive status. To identify genes that control cuticular lipid production in Drosophila, a RNA interference screen was performed and Direct Analysis in Real Time and gas chromatography mass spectrometry were performed to quantify changes in the chemical profiles. Twelve putative genes were identified whereby transcriptional silencing led to significant differences in cuticular lipid production. Amongst them, a gene was identified that was named spidey (CG1444), which encodes a putative steroid dehydrogenase that has sex- and age-dependent effects on viability, pheromone production, and oenocyte survival. Transcriptional silencing or overexpression of spidey during embryonic development results in pupal lethality and significant changes in levels of the ecdysone metabolite 20-hydroxyecdysonic acid and 20-hydroxyecdysone. In contrast, inhibiting gene expression only during adulthood resulted in a striking loss of oenocyte cells and a concomitant reduction of cuticular hydrocarbons, desiccation resistance, and lifespan. Oenocyte loss and cuticular lipid levels were partially rescued by 20-hydroxyecdysone supplementation. Taken together, these results identify a novel regulator of pheromone synthesis and reveal that ecdysteroid signaling is essential for the maintenance of cuticular lipids and oenocytes throughout adulthood (Chiang, 2016).

Seipin is required for converting nascent to mature lipid droplets

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, this study shows that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. These observations identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs (Wang, 2016).

Downregulation of Perilipin1 by the Immune Deficiency Pathway Leads to Lipid Droplet Reconfiguration and Adaptation to Bacterial Infection in Drosophila

Lipid droplets (LDs), the highly dynamic intracellular organelles, are critical for lipid metabolism. Dynamic alterations in the configurations and functions of LDs during innate immune responses to bacterial infections and the underlying mechanisms, however, remain largely unknown. This study traced the time-course morphology of LDs in fat bodies of Drosophila after transient bacterial infection. Detailed analysis shows that perilipin1 (plin1), a core gene involved in the regulation of LDs, is suppressed by the immune deficiency signaling, one major innate immune pathway in Drosophila. During immune activation, downregulated plin1 promotes the enlargement of LDs, which in turn alleviates immune reaction-associated reactive oxygen species stress. Thus, the growth of LDs is likely an active adaptation to maintain redox homeostasis in response to immune deficiency activation. Therefore, this study provides evidence that plin1 serves as a modulator on LDs' reconfiguration in regulating infection-induced pathogenesis, and plin1 might be a potential therapeutic target for coordinating inflammation resolution and lipid metabolism (Wang, 2021).

Enteric Pathogens Modulate Metabolic Homeostasis in the Drosophila melanogaster host

On daily basis, living beings work out an armistice with their microbial flora and a scuffle with invading pathogens to maintain a normal state of health. Although producing virulence factors and escaping the host's immune machinery are the paramount tools used by pathogens in their 'arm race' against the host, this study provides insight into another facet of pathogenic embitterment by presenting evidence of the ability of enteric pathogens to exhibit pathogenicity through modulating metabolic homeostasis in Drosophila melanogaster. Escherichia coli and Shigella sonnei orally infected flies exhibit lipid droplet deprivation from the fat body, irregular accumulation of lipid droplets in the midgut, and significant elevation of systemic glucose and triglyceride levels. These detected metabolic alterations in infected flies could be attributed to differential regulation of peptide hormones known to be crucial for lipid metabolism and insulin signaling. Gaining a proper understanding of infection-induced alterations succeeds in curbing the pathogenesis of enteric diseases and sets the stage for promising therapeutic approaches to pursue infection-induced metabolic disorders (Najjar, 2022).

TOR signaling is required for host lipid metabolic remodelling and survival following enteric infection in Drosophila

When infected by enteric pathogenic bacteria, animals need to initiate local and whole-body defence strategies. While most attention has focused on the role innate immune anti-bacterial responses, less is known about how changes in host metabolism contribute to host defence. Using Drosophila as a model system, this study identified induction of intestinal target-of-rapamycin (TOR) kinase signaling as a key adaptive metabolic response to enteric infection. Enteric infection induces both local and systemic induction of TOR independently of the IMD innate immune pathway, and TOR functions together with IMD signaling to promote infection survival. These protective effects of TOR signaling are associated with re-modelling of host lipid metabolism. Thus, TOR is required to limit excessive infection-mediated wasting of host lipid stores by promoting an increase in the levels of gut- and fat body-expressed lipid synthesis genes. These data supports a model in which induction of TOR represents a host tolerance response to counteract infection-mediated lipid wasting in order to promote survival (Deshpande, 2022).

Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, this study set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. Differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, this study has dissected relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. This work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders (Abuhattum, 2022).

Basidiomycota species in Drosophila gut are associated with host fat metabolism

The importance of bacterial microbiota on host metabolism and obesity risk is well documented. However, the role of fungal microbiota on host storage metabolite pools is largely unexplored. This study investigated the role of microbiota on D. melanogaster fat metabolism, and examine interrelatedness between fungal and bacterial microbiota, and major metabolic pools. Fungal and bacterial microbiota profiles, fat, glycogen, and trehalose metabolic pools are measured in a context of genetic variation represented by whole genome sequenced inbred Drosophila Genetic Reference Panel (DGRP) samples. Increasing Basidiomycota, Acetobacter persici, Acetobacter pomorum, and Lactobacillus brevis levels correlated with decreasing triglyceride levels. Host genes and biological pathways, identified via genome-wide scans, associated with Basidiomycota and triglyceride levels were different suggesting the effect of Basidiomycota on fat metabolism is independent of host biological pathways that control fungal microbiota or host fat metabolism. Although triglyceride, glycogen and trehalose levels were highly correlated, microorganisms' effect on triglyceride pool were independent of glycogen and trehalose levels. Multivariate analyses suggested positive interactions between Basidiomycota, A. persici, and L. brevis that collectively correlated negatively with fat and glycogen pools. In conclusion, fungal microbiota can be a major player in host fat metabolism. Interactions between fungal and bacterial microbiota may exert substantial control over host storage metabolite pools and influence obesity risk (Bozkurt, 2023).

Gut-derived peptidoglycan remotely inhibits bacteria dependent activation of SREBP by Drosophila adipocytes
Bacteria that colonize eukaryotic gut have profound influences on the physiology of their host. In Drosophila, many of these effects are mediated by adipocytes that combine immune and metabolic functions. Enteric infection with some bacteria species was shown to triggers the activation of the SREBP lipogenic protein in surrounding enterocytes but also in remote fat body cells and in ovaries, an effect that requires insulin signaling. By activating the NF-κB pathway, the cell wall peptidoglycan produced by the same gut bacteria remotely, and cell-autonomously, represses SREBP activation in adipocytes. It was shown that by reducing the level of peptidoglycan, the gut born PGRP-LB amidase balances host immune and metabolic responses of the fat body to gut-associated bacteria. In the absence of such modulation, uncontrolled immune pathway activation prevents SREBP activation and lipid production by the fat body (Charroux, 2022).

CG32803 is the fly homolog of LDAF1 and influences lipid storage in vivo

The Seipin protein is a conserved key component in the biogenesis of lipid droplets (LDs). Recently, a cooperation between human Seipin and the Lipid droplet assembly factor 1 (LDAF1) was described. LDAF1 physically interacts with Seipin and the holocomplex safeguards regular LD biogenesis. The function of LDAF1 proteins outside mammals is less clear. In yeast, the lipid droplet organization (LDO) proteins, which also cooperate with Seipin, are the putative homologs of LDAF1. While certain functional aspects are shared between the LDO and mammalian LDAF1 proteins, the relationship between the proteins is under debate. This study identified the Drosophila melanogaster protein CG32803, which was re-named to dmLDAF1, as an insect member of this protein family. dmLDAF1 decorates LDs in cultured cells and in vivo and the protein is linked to the fly and mouse Seipin proteins. Altering the dmLDAF1 abundance affects LD size, number and overall lipid storage amounts. These results suggest that the LDAF1 proteins thus fulfill an evolutionarily conserved function in the biogenesis and biology of LDs (Chartschenko, 2020).

Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism

Seipin, the gene that causes Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), is important for adipocyte differentiation and lipid homeostasis. Previous studies in Drosophila revealed that Seipin promotes ER calcium homeostasis through the Ca(2+)-ATPase SERCA, but little is known about the events downstream of perturbed ER calcium homeostasis that lead to decreased lipid storage in Drosophila dSeipin mutants. This study shows that glycolytic metabolites accumulate and the downstream mitochondrial TCA cycle is impaired in dSeipin mutants. The impaired TCA cycle further leads to a decreased level of citrate, a critical component of lipogenesis. Mechanistically, Seipin/SERCA-mediated ER calcium homeostasis is important for maintaining mitochondrial calcium homeostasis. Reduced mitochondrial calcium in dSeipin mutants affects the TCA cycle and mitochondrial function. The lipid storage defects in dSeipin mutant fat cells can be rescued by replenishing mitochondrial calcium or by restoring the level of citrate through genetic manipulations or supplementation with exogenous metabolites. Together, these results reveal that Seipin promotes adipose tissue lipid storage via calcium-dependent mitochondrial metabolism (Ding, 2018).

Impaired lipid metabolism is associated with an imbalance in energy homeostasis and many other disorders. Excessive lipid storage results in obesity, while a lack of adipose tissue leads to lipodystrophy. Clinical investigations reveal that obesity and lipodystrophy share some common secondary effects, especially non-alcoholic fatty liver disease and severe insulin resistance. Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2/CGL2) is one of the most severe lipodystrophy diseases. Patients with BSCL2 manifest almost total loss of adipose tissue as well as fatty liver, insulin resistance, and myohypertrophy. BSCL2 results from mutation of the Seipin gene, which is highly conserved from yeast to human (Ding, 2018).

To study the function of Seipin, genetic models were established in different organisms, including yeast, fly, and mouse, and in human cells. As a transmembrane protein residing in the endoplasmic reticulum (ER) and in the vicinity of lipid droplet (LD) budding sites, Seipin has been shown to be involved in LD formation, phospholipid metabolism, lipolysis, and ER calcium homeostasis. As a result of the functional studies in these models, several factors that interact with Seipin protein were identified, such as the phosphatidic acid phosphatase lipin, 14-3-3β, and glycerol-3-phosphate acyltransferase (GPAT). Drosophila Seipin (dSeipin) functions tissue autonomously in preventing ectopic lipid accumulation in salivary gland (a non-adipose tissue) and in promoting lipid storage in fat tissue (Tian, 2011). The non-adipose tissue phenotype is likely attributed to the increased level of phosphatidic acid (PA) generated by elevated GPAT activity (Pagac, 2016). In adipose tissue Seipin interacts with the ER Ca2+-ATPase SERCA, whose activity is reduced in dSeipin mutants, leading to reduced ER calcium levels. Further genetic analysis suggested that the perturbed level of intracellular calcium contributes to the lipodystrophy. However, it is not known how the depleted ER calcium pool causes decreased lipid storage (Ding, 2018).

Besides the ER, mitochondria are another important intracellular calcium reservoir. Mitochondrial calcium is mainly derived from the ER through the IP3R channel. IP3R not only releases calcium from the ER into the cytosol, but also provides sufficient Ca2+ at mitochondrion-associated ER membranes (MAMs) for activation of the mitochondrial calcium uniporter. The mitochondrial Ca2+ level varies greatly in different cell types and can be modulated by influx and efflux channel proteins, such as MCU and NCLX, a mitochondrial Na+/Ca2+ exchanger. A proper mitochondrial Ca2+ level is implicated in mitochondrial integrity and function. Mitochondrial calcium is needed to support the activity of the mitochondrial matrix dehydrogenases in the TCA cycle. TCA cycle intermediates are used for the synthesis of important compounds, including glucose, amino acids, and fatty acids. Acetyl-CoA, as the basic building block of fatty acids, is generally derived from glycolysis, the TCA cycle, and fatty acid β-oxidation. In mammalian adipocytes, acetyl-CoA derived from the TCA cycle intermediate citrate is crucial for de novo lipid biosynthesis, which contributes significantly to lipid storage (Ding, 2018 and references therein).

This study used multiple comparative omics to analyze the proteomic, transcriptomic, and metabolic differences between larval fat cells of dSeipin mutants and wild type. The results reveal an impairment in channeling glycolytic metabolites to mitochondrial metabolism in dSeipin mutant fat cells, and scarcity of mitochondrial Ca2+, are the causative factors of this metabolic dysregulation. Evidence is provided showing that dSeipin lipodystrophy is rescued by restoring mitochondrial calcium or replenishing citrate. It is proposed that the low ER Ca2+ level in dSeipin mutants cannot maintain a sufficiently high mitochondrial Ca2+ concentration to support the TCA reactions. This in turn leads to reduced lipogenesis in dSeipin mutants (Ding, 2018).

Seipin promotes fat tissue lipid storage via calcium-dependent mitochondrial metabolism. Defective ER calcium homeostasis in dSeipin mutants is associated with reduced mitochondrial calcium and impaired mitochondrial function, such as low production of TCA cycle metabolites. Restoring mitochondrial calcium levels or replenishing citrate, a key TCA cycle product and also an important precursor of lipogenesis, rescues the lipid storage defects in dSeipin mutant fat cells (Ding, 2018).

This study investigated the underlying causes of Seipin-dependent lipodystrophy by integrating multiple omic analyses, including RNA-seq, quantitative proteomics, and metabolomic analysis. Compared to previous studies based on genetics and traditional cellular phenotypic analysis, these combinatory omic approaches provide an unprecedented spectrum of molecular phenotypes, which not only add new information but also pinpoint logical directions for further investigations (Ding, 2018).

Omics analyses, in particular lipidomic analysis, have been utilized to investigate the underlying mechanisms in several previous Seipin studies and led to the finding that PA is elevated in several Seipin mutant models. In this study, based on genetic rescuing assays and quantitative proteomics analysis, it was initially proposed that downregulated glycolysis is the cause of lipodystrophy. However, both the RNA-seq results and metabolomic data argue against this possibility and suggest a new mechanism. Despite reduced levels of glycolytic enzymes, transcription of the corresponding genes is not affected, and glycolytic metabolites, in particular pyruvate, are increased in dSeipin mutants compared to wild type. Metabolomic data further show that citrate and isocitrate, which are the products of the first two steps of the mitochondrial TCA cycle, are dramatically decreased in dSeipin mutants, suggesting a defective metabolic flow downstream of pyruvate. These results lead to a new possibility that the lipid storage defects in dSeipin mutants are caused by a defective TCA cycle and this is indeed supported by the metabolic flux analysis. These findings further suggest the involvement of mitochondria. In line with this, the previous discovery that fatty acid β-oxidation is elevated in dSeipin mutant fat cells may reflect compensation for the reduced TCA cycle and lipogenesis. This possibility is supported by the results of genetic and citrate-supplement rescue experiments and by citrate measurements (Ding, 2018).

It is known that glycolytic enzymes and metabolites are regulated by a metabolic feedback loop, which may complicate the explanation of genetic interactions. The current findings highlight that although genetic analysis and rescue results provide important clues, multiple lines of evidence are critical for unraveling complex intracellular pathways. In this case, the combination of omic results and genetic analysis led to the finding that mitochondrial metabolism is important in Seipin-associated lipodystrophy (Ding, 2018).

Mitochondria are hubs in key cellular metabolic processes, including the TCA cycle, ATP production, and amino acid catabolism. Mitochondria also play a central role in lipid homeostasis by controlling two seemingly opposite metabolic pathways, lipid biosynthesis, and fatty acid breakdown. Therefore, impairment of mitochondrial function in different tissues may lead to different, even opposite, phenotypes in lipid storage. In tissues where lipid biosynthesis is the major pathway, defective mitochondria might result in reduced lipid storage, whereas in tissues where fatty acid oxidation prevails, the same defect might lead to increased lipid storage. Reduced lipid storage in dSeipin mutants suggests the former case. The reduced level of citrate and other TCA cycle products in dSeipin mutants suggests an impairment of mitochondrial function. The reduction of OCR and ATP production, the decreased Rhod-2 staining, and the aberrant enrichment of mitochondria within autophagosomes all further support this notion. Interestingly, in mouse brown adipose tissue, Seipin mutation increases mitochondrial respiration along with normal MitoTracker labeling (Zhou, 2016). The discrepancies suggest that Seipin may have cell type-specific functions. Unlike white adipose tissue, which favors lipid storage/biosynthesis, brown adipose tissue is prone to fatty acid breakdown (Ding, 2018).

The link between mitochondria and Seipin was concealed in several previous studies. GPATs, which are recently reported Seipin-interacting proteins, participate in many mitochondrial processes. For example, mitochondria from brown adipocytes that are deficient in GPAT4 exhibit high oxidative levels, and mitochondrial GPAT is required for mitochondrial dynamics. PA, which is elevated in Seipin mutants, is required for mitochondrial morphology and function. Similarly, mitochondrial impairments were also observed in various lipodystrophic conditions. Downregulation of mitochondrial transcription and altered mitochondrial function were indicated in type III congenital generalized lipodystrophy. Multiple mitochondrial metabolic processes are altered in mice with lipodystrophy caused by Zmpste24 mutation. HIV patients treated with anti-retroviral therapy manifest partial lipodystrophy and impaired mitochondria in adipocytes. Moreover, mitochondrial dysfunction in adipose tissue triggers lipodystrophy and systemic disorders in mice. Therefore, the contribution of mitochondrial dysfunction to the cause or development of lipodystrophic conditions warrants further examination (Ding, 2018).

It has been previously reported that dSeipin/SERCA-mediated ER calcium homeostasis is critical for lipid storage (Bi, 2014). Consistent with this, transcripts encoding calcium signaling factors are enriched in the genes that are differentially expressed between dSeipin mutants and wild type. Mitochondrial calcium is transported from the ER through the ER-resident channel IP3R. The reduction of mitochondrial calcium in dSeipin mutant fat cells suggests that the decreased ER calcium leads to an insufficient level of mitochondrial calcium. Importantly, RNAi of a putative Drosophila mitochondrial calcium efflux channel (NCLX/CG18660) not only restores the mitochondrial calcium level but also rescues the lipid storage defects in dSeipin mutants, indicating that mitochondrial calcium is key for dSeipin-mediated lipid storage. This explains the previous finding that the lipid storage defects in dSeipin mutants are rescued by RNAi of RyR, which is not required for ER-mitochondrion calcium transport, but not by RNAi of IP3R (Ding, 2018).

Cellular calcium has been linked to lipid storage and related diseases in recent studies. Comprehensive genetic screening in Drosophila showed that ER calcium-related proteins are key regulators of lipid storage. In particular, SERCA, as the sole ER calcium influx channel and an interacting partner of Seipin, has been repeatedly implicated in lipid metabolism. Dysfunctional lipid metabolism can disrupt ER calcium homeostasis by inhibiting SERCA and further disturbing systemic glucose homeostasis. Increased SERCA expression was shown to have dramatic anti-diabetic benefits in mouse models. In a genomewide association study, SERCA was been found to be associated with obesity. In addition, cellular calcium influx is important for transcriptional programming of lipid metabolism, including lipolysis in mice. The current study further elucidates that ER calcium and mitochondrial calcium are important for cellular lipid homeostasis. It also provides a new insight into the pathogenic mechanism of congenital lipodystrophy (Ding, 2018).

Since Seipin mutations lead to opposite effects on lipid storage in adipose tissue (lipodystrophy) and non-adipose tissues (ectopic lipid storage), numerous studies have been carried out to understand the underlying mechanisms. In Seipin mutants, elevated GPAT activity leads to an increased level of PA. This may cause the formation of supersized lipid droplets in non-adipose cells because of the fusogenic property of PA in lipid leaflets, and may also lead to adipogenesis defects due to the potential role of PA as an inhibitor of preadipocyte differentiation. The Seipin-mediated lipid storage phenotype is further complicated by the role of Seipin in lipid droplet formation, which is mainly studied in unicellular eukaryotic yeast or in cultured cells from multicellular eukaryotic organisms. Seipin has been found in the ER-LD contact sites, which are considered as essential subcellular foci for LD formation/maturation. Moreover, in mammalian adipose tissue, the role of Seipin in lipogenesis or lipolysis may also be masked by the defect in early adipogenesis (Ding, 2018).

How can previous findings in different model organisms and different cell types be reconciled? Seipin has been characterized as a tissue-autonomous lipid modulator. It is likely that Seipin participates in lipid metabolism via distinct mechanisms in different tissues. Alternatively, the metabolic processes that involve Seipin may have different outcomes in different tissues. For example, mitochondria have a different impact on lipid metabolism in different tissues: In non-fat cells, mitochondria mainly direct energy mobilization, whereas in fat cells, mitochondria mainly lead anabolism. The molecular role of Seipin and the phenotypic outcomes in Seipin mutants may rely on specific cellular and developmental contexts (Ding, 2018).

Acclimation temperature affects thermal reaction norms for energy reserves in Drosophila

Organisms have evolved various physiological mechanisms to cope with unfavourable environmental conditions. The ability to tolerate non-optimal thermal conditions can be substantially improved by acclimation. This study examined how an early-life acclimation to different temperatures (19 °C, 25 °C and 29 °C) influences thermal reaction norms for energy stores in Drosophila adults. The results show that acclimation temperature has a significant effect on the amount of stored fat and glycogen (and their relative changes) and the optimal temperature for their accumulation. Individuals acclimated to 19 °C had, on average, more energy reserves than flies that were initially maintained at 25 °C or 29 °C. In addition, acclimation caused a shift in optimal temperature for energy stores towards acclimation temperature. Significant population differences in this response were detected. The effect of acclimation on the optimal temperature for energy stores was more pronounced in flies from the temperate climate zone (Slovakia) than in individuals from the tropical zone (India). Overall, it was found that the acclimation effect was stronger after acclimation to low (19 °C) than to high (29 °C) temperature. The observed sensitivity of thermal reaction norms for energy reserves to acclimation temperature can have important consequences for surviving periods of food scarcity, especially at suboptimal temperatures (Klepsatel, 2020).

Drosophila PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity

PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine and endocrine mechanisms. This study investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). Genetic approaches and single-nuclei sequencing were combined to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. This study found that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. These findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells (Ghosh, 2020).

The presence in vertebrates of multiple PDGF/VEGF signaling ligands and cognate receptors makes it difficult to assess their roles in inter-organ communication. Additionally, understanding the tissue-specific roles of these molecules, while circumventing the critical role they play in regulating tissue vascularization, is equally challenging in vertebrate models. This study investigated the tissue-specific roles of the ancestral PDGF/VEGF-like factors and the single PDGF/VEGF-receptor in Drosophila in lipid homeostasis. The results demonstrate that in adult flies the PDGF/VEGF like factor, Pvf1, is a muscle-derived signaling molecule (myokine) that suppresses systemic lipid synthesis by signaling to the Drosophila hepatocyte-like cells/oenocytes (Ghosh, 2020).

The Drosophila larval and adult adipose tissues have distinct developmental origins. The larval adipose tissue undergo drastic morphological changes during metamorphosis and dissociate into individual large spherical cells. These free-floating adipose cells persist to the young adult stage where they play a crucial role in protecting the animal from starvation and desiccation. These larval adipose tissue cells are ultimately removed via cell death. Adult-specific adipose tissue cells develop during the pupal stage from yet unknown progenitor cells and have very little lipid stores in newly eclosed flies. Over the period of next 3-5 days the adult adipose tissue builds up its lipid reserves through feeding and de-novo lipid synthesis. However, at the end of the lipid build-up phase, the rate of lipid synthesis must be suppressed to avoid over-loading of the adipose tissue and prevent lipid toxicity. The data suggest that muscle Pvf1 signaling suppresses lipid synthesis at the end of the adult adipose tissue lipid build-up phase. Pvf1 production in the adult muscles peaks around the time when adult adipose tissue lipid stores reach steady state capacity. In turn, muscle-derived Pvf1 suppresses lipid synthesis and lipid incorporation by activating TOR signaling in the oenocytes (Ghosh, 2020).

This study reveals that Pvf1 is abundant in the tubular muscles of the Drosophila leg and abdomen. In these striated muscles, the protein localizes between individual myofibrils and is particularly enriched at the M and Z bands. Drosophila musculature can be broadly categorized into two groups, the fibrillar muscles and the tubular muscles, with distinct morphological and physiological characteristics. Drosophila IFMs of the thorax belong to the fibrillar muscle group and are stretch-activated, oxidative, slow twitch muscles that are similar to vertebrate cardiac muscles. By contrast, Drosophila leg muscles and abdominal muscles belong to the tubular muscle group. These muscles are striated, Ca2+ activated, and glycolytic in nature. The tubular muscles are structurally and functionally closer to vertebrate skeletal muscles. Expression of Pvf1 in the tubular muscles of the Drosophila leg may reflect a potentially conserved role of this molecule as a skeletal-muscle-derived myokine. The fact that most of the myokines in vertebrates were identified in striated skeletal muscles supports this possibility . Moreover, vertebrate VEGF ligands, VEGF-A and VEGF-B, have also been shown to be stored and released from skeletal muscles (Ghosh, 2020).

Interestingly, in vertebrates, the expression and release of VEGF ligands are regulated by muscle activity. In mice, expression of VEGF-B in the skeletal muscles is regulated by PGC1-α, one of the key downstream effectors of muscle activity. Additionally, expression of VEGF-B is upregulated in both mouse and human skeletal muscles in response to muscle activity. Similarly, expression of VEGF-A is induced by muscle contraction. No effect of muscle activity on the expression levels of pvf1 was observed in the Drosophila muscles. Whether muscle activity regulates release of Pvf1 primarily could not be demonstrated due to the difficulty in collecting adequate amounts of hemolymph from the adult males. However, the localization of Pvf1 to the M/Z bands suggests a potential role for muscle activity in Pvf1 release. The M and Z bands of skeletal muscles are important centers for sensing muscle stress and strain. These protein-dense regions of the muscle house a number of proteins that can act as mechano-sensors and mediate signaling events including translocation of selected transcription factors to the nucleus. Pvf1, therefore, is ideally located to be able to sense muscle contraction and be released in response to muscle activity. Further work, contingent on the development of new tools and techniques, will be necessary to measure Pvf1 release into the hemolymph and study the regulation of this release by exercise (Ghosh, 2020).

Previous work has shown that Pvf1 released from gut tumors generated by activation of the oncogene yorkie leads to wasting of Drosophila muscle and adipose tissue (Song, 2019). Adipose tissue wasting in these animals is characterized by increased lipolysis and release of free fatty acids (FFAs) in circulation. However, no role was observed of Pvf signaling in regulating lipolysis in the adipose tissue of healthy well-fed flies without tumors. Loss of PvR signaling in the adipose tissue did not have any effect on lipid content. Additionally, over-expressing Pvf1 in the muscle did not lead to the bloating phenotype commonly seen in cachectic animals with gut tumors. It is concluded that Pvf1 affects wasting of the adipose tissue only in the context of gut tumors and that the effect could involve the following mechanisms: (1) the gut tumor releases pathologically high levels of Pvf1 into circulation leading to ectopic activation of PvR signaling in the adipose tissue, and, that such high levels of Pvf1 are not released by the muscle (even when pvf1 is over-expressed in the muscle); (2) Pvf1 causes adipose tissue wasting in the context of other signals that emanate from the gut tumor that are not available in flies that do not have tumors (Ghosh, 2020).

Only oenocyte-specific loss of PvR signaling phenocopies the obesity phenotype caused by muscle-specific loss of Pvf1, indicating that muscle-Pvf1 primarily signals to the oenocytes to regulate systemic lipid content. Additionally, muscle-specific loss of Pvf1, as well as oenocyte-specific loss of PvR and its downstream effector TOR, leads to an increase in the rate of lipid synthesis. These observations indicate a role for the Drosophila oenocytes in lipid synthesis and lipid accumulation in the adipose tissue. Oenocytes have been implicated in lipid metabolism previously and these cells are known to express a diverse set of lipid metabolizing genes including but not limited to fatty acid synthases, fatty acid desaturases, fatty acid elongases, fatty acid β-oxydation enzymes and lipophorin receptors. Functionally, the oenocytes are proposed to mediate a number of lipid metabolism processes. Oenocytes tend to accumulate lipids during starvation (presumably for the purpose of processing lipids for transport to other organs and generation of ketone bodies) and are necessary for starvation induced mobilization of lipids from the adipose tissue. This role is similar to the function of the liver in clearing FFAs from circulation during starvation for the purpose of ketone body generation, and redistribution of FFAs to other organs by converting them to TAG and packaging into very-low density lipoproteins. However, a [1-14C]-oleate chase assay did not show any effect of oenocyte-specific loss of PvR/TOR signaling on the rate of lipid utilization, indicating that this pathway does not affect oenocyte-dependent lipid mobilization (Ghosh, 2020).

Oenocytes also play a crucial role in the production of very-long-chain fatty acids (VLCFAs) needed for waterproofing of the cuticle (Storelli, 2019). Results of a starvation resistance assay indicate that loss of the muscle-to-oenocyte Pvf1 signaling axis does not affect waterproofing of the adult cuticle. Storelli (2019) recently showed that the lethality observed in traditionally used starvation assays is largely caused by desiccation unless the assay is performed under saturated humidity conditions. Since a starvation assay was performed under 60% relative humidity (i.e. non-saturated levels), it is likely that desiccation played a partial role in causing starvation-induced lethality. Any defects in waterproofing of the adult cuticle would have led to reduced starvation resistance. However, both muscle-specific loss of Pvf1 and oenocyte-specific loss of PvR led to increased starvation resistance suggesting normal waterproofing in these animals. The increased starvation resistance in these animals is likely the result of these animals having higher stored lipid content that helps them to survive longer without food (Ghosh, 2020).

Insect oenocytes were originally believed to be lipid synthesizing cells because they contain wax-like granules. These cells express a large number of lipid-synthesizing genes and the abundance of smooth endoplasmic reticulum further suggest a role for this organ in lipid synthesis and transport. However, evidence for potential involvement of the oenocytes in regulating lipid synthesis and lipid deposition in the adipose tissue has been lacking. The fact that two of the three fatty acid synthases (fasn2 and fasn3) encoded by the Drosophila genome are expressed specifically in adult oenocytes suggests a potential role for these cells in lipid synthesis. The observation that oenocyte-specific loss of PvR and its downstream effector TOR leads to increased lipid synthesis and increased lipid content of the adipose tissue strongly supports this possibility. The data further suggests that involvement of the oenocytes in mediating lipid synthesis is more pronounced in newly eclosed adults when the adipose tissue needs to actively build up its lipid stores. In later stages of life, the lipid synthetic role of the oenocytes is repressed by the muscle-to-oenocyte Pvf1 signaling axis. This observation also raises the question of whether FFAs made in the oenocytes can be transported to the adipose tissue for storage. This possibility was tested by over-expressing the lipogenic genes fasn1 and fasn3, which regulate the rate limiting steps of de-novo lipid synthesis, in the oenocytes. It was found that excess lipids made in the oenocytes do end up in the adipose tissue of the animal leading to increased lipid droplet size in the adipose tissue. Taken together, these results provide evidence for the role of Drosophila oenocytes in lipid synthesis and storage of neutral lipids in the adipose tissue of the animal. Interestingly, the vertebrate liver is also one of the primary sites for de-novo lipid synthesis and lipids synthesized in the liver can be transported to the adipose tissue for the purpose of storage. Hence, the fundamental role of the oenocytes and the mammalian liver converge with respect to their involvement in lipid synthesis (Ghosh, 2020).

Oenocyte-specific loss of the components of the Pi3K/Akt1/TOR signaling pathway was observed to lead to increased lipid synthesis. The increased rate of lipid synthesis in flies lacking TOR signaling in the oenocytes is paradoxical to current knowledge of how TOR signaling affects expression of lipid synthesis genes. In both vertebrates and flies, TOR signaling is known to facilitate lipid synthesis by inducing the expression of key lipid synthesis genes such as acetyl CoA-carboxylase and fatty acid synthase via activation of SREBP-1 proteins. Therefore this study checked how oenocyte-specific loss of TOR signaling affects expression of oenocyte-specific fatty acid synthases (fasn2 and fasn3) and oenocyte non-specific fatty acid synthesis genes (fasn1 and acc). Oenocyte-specific loss of TOR strongly down-regulated only fasn2 and fasn3, while the expression of adipose tissue specific fasn1 and acc did not change, indicating that TOR signaling is required for the expression of lipogenic genes in the oenocytes. An increase in lipid synthesis in response to loss of TOR in the oenocytes is quite intriguing and the mechanism remains to be addressed. The increase in lipid synthesis is thought not to happen in the oenocytes since loss of TOR signaling rather reduces expression of lipogenic genes in the oenocytes. The increase in lipid synthesis could happen either as a result of compensatory upregulation of lipid synthesis in the adipose tissue or due to disruption of an as yet unknown role of the oenocytes in lipid synthesis that hinges on TOR signaling. The fact that the expression levels of fasn1 and acc does not change significantly in animals lacking TOR signaling in the oenocytes indicates that compensatory upregulation of lipid synthesis, if present, does not happen through transcriptional upregulation of lipid synthesis genes in the adipose tissue. It is still possible, however, that the increase in lipid synthesis is caused by post-translational modifications of the enzymes. Alternatively, loss of TOR in the oenocyte could affect tissue distribution of lipids or impair clearing of dietary lipids via formation of cuticular hydrocarbons. Understanding the tissue specific alterations in gene expression and changes in the phosphorylation states of key lipogenic proteins in the adipose tissue of animals lacking TOR signaling in oenocytes could shed more light on the mechanisms involved (Ghosh, 2020).

Interestingly, the data suggests that the Drosophila InR does not play a role in activating TOR signaling in the oenocytes. While loss of TOR signaling in the oenocytes leads to obesity, loss of InR signaling does not. Additionally, loss of oenocyte specific InR signaling did not have any effect on p4EBP levels in oenocytes. Moreover, InR signaling and TOR signaling also diverge in their roles in regulating the size of oenocytes. While loss of InR signaling leads to a significant reduction in the size of oenocytes, loss of TOR does not. Further suggesting that TOR does not act downstream of InR in oenocytes. Rather, the data suggests that in wildtype well-fed flies TOR signaling in oenocytes is activated by the Pvf receptor. Interestingly, insulin dependent activation of TOR is not universal. For instance, in the specialized cells of non-obese mouse liver, InR does not play any role in activation of TOR and downstream activation of SREBP-1c (Ghosh, 2020).

Drosophila larval oenocytes are known to accumulate lipids in response to starvation. It has also been showed that starving adult females for 36 hr is capable of inducing lipid accumulation in the oenocytes and that this response is dependent of InR signaling. Since TOR signaling is a known metabolic regulator, one alternate hypothesis that could explain some of the data is that loss of PvR/TOR signaling leads to a starvation like response specifically in the oenocyte leading to InR-dependent accumulation of lipid droplets. To address this possibility, single nuclei sequencing of the adult male abdominal cuticle (and the tissues residing within) derived from oenots>tsc1,tsc2 flies. The animals were raised under identical experimental conditions as control animals. Then the two snRNA-seq data sets were re-analyzed after correcting for batch effects using harmony. The resulting UMAP plots for both genotypes look similar to the original UMAP plot for the control flies and identifies all the clusters reported (see Differential snRNA-seq of the abdominal cuticle upon oenocyte-specific loss of TOR). The percentage of nuclei that constitute each of the major clusters remained similar in both genotypes and the top marker genes for each of the clusters did not change. The oenocyte-specific gene expression profiles from both data sets were subsequently converted to pseudobulk expression for the genes that were detected. This allowed comparison of the expression profiles of the oenocytes from control animals and animals lacking TOR signaling in oenocytes. The effect of losing TOR on the expression of the 47 genes that had been reported to be up-regulated in oenocytes in response to starvation was specifically looked at. Thirty-six of these genes were detected by single nuclei sequencing analysis, however, none of them changed significantly. Based on this observation, it is concluded that loss of TOR signaling most likely does not mount a starvation like response in the oenocytes (Ghosh, 2020).

Serum levels of VEGF-A is high in obese individuals and drops rapidly in response to bariatric surgery, suggesting a role for VEGF-A in obesity. However, evidence on whether VEGF-A or other VEGFs are deleterious vs beneficial in the context of the pathophysiology of obesity is unclear. Adipose tissue-specific over-expression of both VEGF-B and VEGF-A has been shown to improve adipose tissue vascularization, reduce hypoxia, induce browning of fat, increase thermogenesis, and protect against obesity. At the same time, blocking VEGF-A signaling in the adipose tissue of genetically obese mice leads to reduction of body weight gain, improvement in insulin sensitivity, and a decrease in adipose tissue inflammation. Moreover, systemic inhibition of VEGF-A or VEGF-B signaling by injecting neutralizing monoclonal antibodies have also shown remarkable effects in improving insulin sensitivity in the muscle, adipose tissue, and the liver of high-fat diet-induced mouse models of obesity and diabetes. Although the evidence on the roles of VEGF/PDGF signaling ligands in obesity and insulin resistance is well established, the mechanisms clearly are quite complex and are often context dependent. Consequently, a wider look at various tissue specific roles of PDGF/VEGF signaling will be necessary to comprehensively understand the roles of PDGF/VEGF signaling in lipid metabolism. The current work demonstrates an evolutionarily conserved role for PDGF/VEGF signaling in lipid metabolism and a non-endothelial cell dependent role of the signaling pathway in lipid synthesis. Additionally, these findings suggest an atypical tissue-specific role of TOR signaling in suppressing lipid synthesis at the level of the whole organism. Further studies will be required to determine whether vertebrate VEGF/PDGF and TOR signaling exerts similar roles either in the vertebrate liver or in other specialized organ (Ghosh, 2020).

This study made use of snRNA-Seq technology to identify expression of Pvr precisely in certain tissues in the complex abdominal region, which harbors several metabolically active tissues including adipose tissues, oenocytes, and muscle in Drosophila. As yet, there is no systematic study of a complete transcriptomics resource of each of these tissues considering the difficulty in dissecting and segregating these tissues for downstream sequencing. Thus, this study also provides a rich resource of gene expression profiles, paving way for a systems-level understanding of each of these tissues in Drosophila (Ghosh, 2020).

Loss of Stearoyl-CoA Desaturase 1 leads to cardiac dysfunction and lipotoxicity

Diets high in carbohydrates are associated with type 2 diabetes and its comorbidities, including hyperglycemia, hyperlipidemia, obesity, hepatic steatosis and cardiovascular disease. This study used a high-sugar diet to study the pathophysiology of diet-induced metabolic disease in Drosophila melanogaster. High-sugar diets produce hyperglycemia, obesity, insulin resistance, and cardiomyopathy in flies along with ectopic accumulation of toxic lipids, or lipotoxicity. Stearoyl-CoA desaturase 1 is an enzyme that contributes to long-chain fatty acid metabolism by introducing a double bond into the acyl chain. Knockdown of stearoyl-CoA desaturase 1 in the fat body reduced lipogenesis and exacerbated pathophysiology in flies reared on high-sugar diets. These flies exhibited dyslipidemia and growth deficiency in addition to defects in cardiac and gut function. The lipidome of these flies was assessed using tandem mass spectrometry to provide insight into the relationship between potentially lipotoxic species and type 2 diabetes-like pathophysiology. Oleic acid supplementation is able to rescue a variety of phenotypes produced by stearoyl-CoA desaturase 1 RNAi, including fly weight, triglyceride storage, gut development, and cardiac failure. Taken together, these data suggest a protective role for monounsaturated fatty acids in diet-induced metabolic disease phenotypes (Tuthill, 2021).

The F-box gene Ppa promotes lipid storage in Drosophila. The F-box gene Ppa promotes lipid storage in Drosophila

Lipid is one of the important components of living organisms. The precise regulation and homeostasis maintenance of lipid metabolism are essential to human health. The ubiquitination pathway regulates lipid metabolism by degrading lipid-related proteins. Ppa encodes an F-box protein, which is a member of the SCF ubiquitination complex. Previous studies reported that Ppa regulated the body segmentation and the correct localization of centromere histones, while its function in lipid metabolism has not been reported. In this study, Drosophila melanogaster was used to explore the function of Ppa in lipid storage. The subcellular localization of PPA was detected by fusion with green fluorescent protein. The deletion mutant of Ppa was constructed via CRISPR/Cas9 technology. The morphological changes of lipid droplets in deletion mutants and Ppa overexpression flies were analyzed by BODIPY 493/503 or Nile red staining. Further, Ppa was overexpressed in the deletion mutant to verify its function. The results showed that PPA-GFP fusion protein were localized in the nuclei of salivary gland and fat body. Compared with the control flies, the lipid droplets in Ppa deletion mutants became smaller, and overexpression of Ppa exhibited larger lipid droplets. Overexpression of Ppa in the deletion mutant could restore the lipid droplets to normal state. In summary, this study demonstrated that Ppa could promote lipid storage in Drosophila (Yang, 2021).

Recruitment of Peroxin 14 to lipid droplets affects lipid storage in Drosophila

Both peroxisomes and lipid droplets regulate cellular lipid homeostasis. Direct inter-organellar contacts as well as novel roles for proteins associated with peroxisome or lipid droplets occur when cells are induced to liberate fatty acids from lipid droplets. This study has shown a non-canonical role for a subset of peroxisome-assembly [Peroxin (Pex)] proteins in this process in Drosophila. Transmembrane proteins Pex3, Pex13 and Pex14 were observed to surround newly formed lipid droplets. Trafficking of Pex14 to lipid droplets was enhanced by loss of Pex19, which directs insertion of transmembrane proteins like Pex14 into the peroxisome bilayer membrane. Accumulation of Pex14 around lipid droplets did not induce changes to peroxisome size or number, and co-recruitment of the remaining Peroxins was not needed to assemble peroxisomes observed. Increasing the relative level of Pex14 surrounding lipid droplets affected the recruitment of Hsl lipase. Fat body-specific reduction of these lipid droplet-associated Peroxins caused a unique effect on larval fat body development and affected their survival on lipid-enriched or minimal diets. This revealed a heretofore unknown function for a subset of Pex proteins in regulating lipid storage (Ueda, 2022).

NAD kinase sustains lipogenesis and mitochondrial metabolism through fatty acid synthesis

Lipid storage in fat tissue is important for energy homeostasis and cellular functions. Through RNAi screening in Drosophila fat body, this study found that knockdown of a Drosophila NAD kinase (NADK), which phosphorylates NAD to synthesize NADP de novo, causes lipid storage defects. NADK sustains lipogenesis by maintaining the pool of NADPH. Promoting NADPH production rescues the lipid storage defect in the fat body of NADK RNAi animals. Furthermore, NADK and fatty acid synthase 1 (FASN1) regulate mitochondrial mass and function by altering the levels of acetyl-CoA and fatty acids. Reducing the level of acetyl-CoA or increasing the synthesis of cardiolipin (CL), a mitochondrion-specific phospholipid, partially rescues the mitochondrial defects of NADK RNAi. Therefore, NADK- and FASN1-mediated fatty acid synthesis coordinates lipid storage and mitochondrial function (Xu, 2021).

Lipid homeostasis is important for human health, and its dysregulation is tightly associated with many metabolic diseases, such as type 2 diabetes, hepatic steatosis, cardiovascular disease, and cancer. Cellular lipid homeostasis is regulated by the opposing actions of lipid accumulation, including lipid uptake, de novo lipogenesis and lipid storage, and lipid mobilization, such as lipolysis, lipid oxidation, and lipid efflux. Excess lipid storage or insufficient lipid storage causes obesity or lipodystrophy, respectively (Xu, 2021).

Acetyl-CoA carboxylase (ACC) and FASN mediate fatty acid synthesis from acetyl-CoA during de novo lipogenesis. The fatty acids are then esterified for storage as neutral lipids such as triglycerides (TAGs). The lipid droplet, an organelle with a neutral lipid core and a phospholipid monolayer, is the hub for lipid storage. Understanding of the regulation of lipid storage and lipid droplet dynamics has significantly advanced in recent years. Many processes, including neutral lipid synthesis and degradation, composition of phospholipids, lipid droplet biogenesis and fusion, calcium homeostasis, and lipophagy, together determine lipid storage. Nevertheless, the mechanisms regulating lipid storage and lipid droplet dynamics in vivo are not completely clear (Xu, 2021).

To reduce lipid storage, TAG is mobilized through cytosolic lipolysis to release fatty acids, which are subsequently broken down, mainly in mitochondria, into acetyl-CoA units by lipid oxidation. Therefore, defective mitochondria often lead to lipid accumulation. For example, inhibition of β-oxidation in mitochondria causes lipid accumulation in Drosophila brain. Interestingly, besides conducting fatty acid oxidation, mitochondria also provide substrates and energy for de novo fatty acid synthesis. Both the acetyl-CoA and ATP required by fatty acid synthesis are derived from mitochondria. Impairment of mitochondrial function affects lipogenesis and lipid droplet accumulation. Therefore, impairment of mitochondrial function probably has a context-dependent effect on lipid storage (Xu, 2021).

Conversely, dysregulation of lipid storage also affects mitochondrial function. In the heart, cytoplasmic adipose TAG lipase (ATGL), which hydrolyzes TAG from lipid droplets, affects lipid storage and mitochondrial biogenesis and oxidative metabolism. Similarly, in islet β cells, ATGL knockdown impairs mitochondrial respiration and ATP production, and a PPARδ agonist rescues these mitochondrial defects (Xu, 2021).

Mechanistically, ATGL-mediated lipid droplet lipolysis induces the expression of genes involved in mitochondrial oxidation and respiration by activating the master regulators PPARα/PPARγ and PGC-1α. These studies pinpoint a close relationship between the mitochondrion and the lipid droplet, despite the compartmentalized features of lipid storage and lipid breakdown. Several metabolites, including acetyl-CoA and fatty acids, appear to mediate the two-way communication between these two organelles. De novo lipogenesis is tightly associated with acetyl-CoA and fatty acids. However, despite a few reports showing that lipogenesis inhibitors cause various mitochondrial dysfunctions in cancer, the question of whether and how de novo lipogenesis affects mitochondrial function has not been properly addressed (Xu, 2021).

Through an RNAi screen in Drosophila, this study found that CG6145, a cytosolic NAD kinase (NADK), affects lipid storage in fat body by providing NADPH, an essential reductant in lipogenesis. NADK RNAi causes similar de novo lipogenesis defects as FASN1 RNAi. More importantly, both NADK RNAi and FASN1 RNAi larvae exhibit reduced mitochondrial content. Finally, it was revealed that de novo fatty acid synthesis regulates mitochondrial mass, at least partially, by controlling PGC-1α acetylation and cardiolipin (CL) synthesis (Xu, 2021).

This study shows that NADK affects lipid storage and mitochondrial metabolism in Drosophila. NADK is essential for generating NADP and NADPH, the latter of which is important for de novo fatty acid synthesis. Besides lipid storage, NADK-mediated fatty acid synthesis also contributes to mitochondrial function, possibly through two different mechanisms: one is through acetyl-CoA and PGC-1α acetylation, and the other is through synthesis of the mitochondrion-specific phospholipid CL (Xu, 2021).

Despite the obvious requirement for NADPH in de novo fatty acid synthesis and other metabolic reactions, knowledge about the physiological function and impact of NADK on metabolic homeostasis in different organisms and tissues is limited. This study demonstrated the importance of NADK in animal lipid storage in vivo. NADK determines the level of NADP(H). Increasing NADPH availability rescues the defects in NADK RNAi, which confirms that NADPH is a key determinant of lipid storage. In support of this idea, NADPH-producing enzymes, such as G6PD and ME, promote lipid production in oleaginous microbes. The expression and activities of these enzymes are also correlated with lipid storage in mammals. These observations suggest that NADK and the level of NADPH are previously unappreciated regulators of organismal lipid storage. Interestingly, insulin, which promotes the synthesis and storage of lipids, activates NADK by Akt-mediated phosphorylation, which suggests that NADK may respond to physiological conditions to regulate lipid storage (Xu, 2021).

Besides lipogenesis, this stufy found that NADK also influences mitochondrial metabolism. The amounts of mitochondria and lipid droplets are decreased in both NADK RNAi and FASN1 RNAi, raising the possibility that these two closely linked organelles are co-regulated. Mitochondria regulate lipid metabolism by providing energy and substrates for lipogenesis and a site for fatty acid degradation. Lipid droplets, acting as an important organelle of lipid metabolism, also regulate mitochondrial function. Interestingly, elevating lipolysis by ATGL overexpression reduces the amount of lipid droplets, but it increases mitochondrial content, which suggests that reduced lipid storage per se is not the cause of the reduced mitochondrial mass in both NADK RNAi and FASN1 RNAi. Previous studies have shown that ATGL-mediated lipolysis promotes mitochondrial metabolism and biogenesis through activation of PPARs or Sirt1/PGC-1α. NADK RNAi and FASN1 RNAi exert a stronger effect on mitochondrial function than on lipolysis, which might be attributed to the severe decline in the level of fatty acids. Interestingly, PGC-1α acetylation mediates the regulation of mitochondrial function by both lipolysis and lipogenesis. Therefore, de novo fatty acid synthesis regulates the dynamics of both lipid droplets and mitochondria (Xu, 2021).

Fatty-acid-dependent activation of PPARs and Sirt1 is rather specific. The ligands of PPARs are primarily unsaturated and long-chain fatty acids, while Sirt1 is activated by monounsaturated fatty acids within a restricted range of concentrations. This study found that RNAi of the fat-body-specific PGC-1α homolog srl only moderately reduced mitochondrial mass, in contrast to the strong effect of NADK and FASN1 RNAi. In addition, knockdown of PPAR homologs in fat body caused no obvious mitochondrial phenotype. Therefore, it is likely that fatty acids also regulate mitochondrial function through other mechanism(s). In addition, the rescue of NADK RNAi and FASN1 RNAi by different exogenously supplied fatty acids (including saturated, monounsaturated, and odd-chain fatty acids) and by BMM overexpression suggests a general mechanism with limited or low fatty acid selectivity (Xu, 2021).

Phospholipid synthesis, which affects mitochondrial function in many ways, also requires fatty acids. CL is a mitochondrion-specific phospholipid and is important for almost every aspect of mitochondrial integrity, including crista organization, mitochondrial protein import, and assembly. It is a rather unique phospholipid, harboring four fatty acyl chains, and it undergoes remodeling, which makes it sensitive to the availability and composition of fatty acids. Importantly, the rescue of mitochondrial defects in NADK RNAi and FASN1 RNAi by several genetic manipulations to increase CL production suggests that decreased CL synthesis contributes to the mitochondrial phenotype in NADK RNAi and FASN1 RNAi. The mitochondrial morphology in NADK RNAi and FASN1 RNAi is not completely identical with CLS RNAi. In addition, the rescue effect of CLS overexpression is not comparable with fatty acid supplementation. These observations suggest that fatty acids might also regulate mitochondria through other mechanisms (Xu, 2021).

De novo fatty acid synthesis is important for many biological processes. For example, the activity of fatty acid synthesis is stimulated in some cancer cells or proliferating stem cells. Its inhibition suppresses cell proliferation and survival. It is generally thought that fatty acid synthesis mainly affects these processes by providing structural and signaling lipids\, and limited attention has been paid to the causative role of mitochondrial dysfunction, which is also important for cancer progression and stem cell homeostasis. For example, inhibition of PGC-1α or OXPHOS suppresses cancer cell survival and metastasis under oxidative or bioenergetic stress conditions. In addition, mitochondrial mass is associated with prostate cancer progression. Inhibition of mitochondrial biogenesis was identified as a therapeutic strategy for acute myeloid leukemia. Although OXPHOS activity is restricted in many cancer cells, mitochondrial content, dynamics, and metabolic activity are important for tumorigenesis and stem cell homeostasis (Xu, 2021).

Considering the findings of this study, it is possible that fatty acid synthesis-regulated mitochondrial function may be critical for cancer cell growth and stem cell differentiation. For example, fatty acid and lipid synthesis promote hepatocellular carcinoma development, accompanied by increased CL levels and OXPHOS activity. Inhibition of FASN or ACC reduces mitochondrial oxygen consumption, changes mitochondrial morphology, and affects the levels of mitochondrial proteins and metabolites in cancer and stem cells (Xu, 2021).

Both NADK and FASN are considered as potential targets for cancer therapy because of their lipogenic and other functions. NADK and FASN act as important regulators of lipid storage by restricting the capacity of fatty acid synthesis. This study showed that NADK- and FASN1-mediated fatty acid synthesis regulates mitochondrial function, probably by altering the levels of acetyl-CoA and CL. More physiological functions and molecular mechanisms of NADK and fatty acid synthesis may be revealed through the fatty-acid-mitochondrion link (Xu, 2021).

This study has demonstrated that increased PGC-1 acetylation and reduced CL synthesis are responsible for mitochondrial phenotype in NADK RNAi and FASN1 RNAi. However, reduced acetyl-CoA level and CLS overexpression only partially rescued mitochondrial phenotype. Exogenous fatty acid supplement completely restored mitochondrial mass in NADK RNAi and FASN1 RNAi, suggesting that fatty acid synthesis might regulate mitochondrial mass via other mechanisms as well. In addition, these studies were conducted in fat cells, which are specialized for lipid storage. It remains to be determined whether these findings apply to other cell types (Xu, 2021).

Enteric bacterial infection in Drosophila induces whole-body alterations in metabolic gene expression independently of the immune deficiency signaling pathway

When infected by intestinal pathogenic bacteria, animals initiate both local and systemic defence responses. These responses are required to reduce pathogen burden and also to alter host physiology and behavior to promote infection tolerance, and they are often mediated through alterations in host gene expression. This study used transcriptome profiling to examine gene expression changes induced by enteric infection with the Gram-negative bacteria Pseudomonas entomophila in adult female Drosophila. Infection was found to induce a strong upregulation of metabolic gene expression, including gut and fat body-enriched genes involved in lipid transport, lipolysis, and beta-oxidation, as well as glucose and amino acid metabolism genes. Furthermore, the classic innate immune deficiency (Imd)/Relish/NF-KappaB pathway were not required for, and in some cases limits, these infection-mediated increases in metabolic gene expression. Enteric infection with Pseudomonas entomophila downregulates the expression of many transcription factors and cell-cell signaling molecules, particularly those previously shown to be involved in gut-to-brain and neuronal signaling. Moreover, as with the metabolic genes, these changes occurred largely independent of the Imd pathway. Together, this study identifies many metabolic, signaling, and transcription factor gene expression changes that may contribute to organismal physiological and behavioral responses to enteric pathogen infection (Deshpande, 2022).

Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor

Animals adapt their growth rate and body size to available nutrients by a general modulation of insulin-insulin-like growth factor signaling. In Drosophila, dietary amino acids promote the release in the hemolymph of brain insulin-like peptides (Dilps), which in turn activate systemic organ growth. Dilp secretion by insulin-producing cells involves a relay through unknown cytokines produced by fat cells. This study identified Methuselah (Mth) as a secretin-incretin receptor subfamily member required in the insulin-producing cells for proper nutrient coupling. Using genetic and ex vivo organ culture experiments, it was shown that the Mth ligand Stunted is a circulating insulinotropic peptide produced by fat cells. Therefore, Sun and Mth define a new cross-organ circuitry that modulates physiological insulin levels in response to nutrients (Delanoue, 2016).

Environmental cues, such as dietary products, alter animal physiology by acting on developmental and metabolic parameters like growth, longevity, feeding, and energy storage or expenditure. The systemic action of this control suggests that intermediate sensor tissues evaluate dietary nutrients and trigger hormonal responses. Previous work in Drosophila melanogaster established that a specific organ called the fat body translates nutritional information into systemic growth-promoting signals. The leptinlike Janus kinase-signal transducers and activators of transcription (JAK-STAT) ligand unpaired 2 and the CCHamid2 peptide are produced by fat cells in response to both sugar and fat and trigger a metabolic response. Dietary amino acids activate TORC1 signaling in fat cells and induce the production of relay signals that promote the release of insulin-like peptides (Dilps) by brain insulin-producing cells (IPCs). Two fat-derived peptides (GBP1 and GBP2) activate insulin secretion in response to a protein diet, although their receptor and neural targets remain uncharacterized. To identify critical components of this organ crosstalk, a genetic screen was conducted in Drosophila larvae. The gene methuselah (mth), which encodes a heterotrimeric GTP-binding protein (G protein)-coupled receptor belonging to the subfamily of the secretin-incretin receptor subfamily came out as a strong hit. Impairing mth function in the IPCs reduces larval body growth, whereas silencing mth in a distinct set of neurons or in the larval fat body had no impact on pupal volume. Larvae in which expression of the mth gene is reduced by RNA interference (RNAi), specifically in the IPCs (hereafter, dilp2>mth-Ri), present an accumulation of Dilp2 and Dilp5 in the IPCs, whereas dilp2 gene expression remains unchanged, a phenotype previously described as impaired Dilp secretion. Indeed, forced depolarization of the IPCs rescues pupal volume and Dilp2 accumulation upon IPC-specific mth depletion. Therefore, Mth is required for Dilps secretion and larval body growth (Delanoue, 2016).

Two peptides encoded by the stunted (sun) gene, SunA and SunB, serve as bona fide ligands for Mth and activate a Mth-dependent intracellular calcium response. Silencing sun in fat cells, but no other larval tissue, of well-fed larvae mimics the mth loss-of-function phenotype with no effect on the developmental timing. Conversely, overexpression of sun in the larval fat body (lpp>sun) partially rescues the systemic growth inhibition observed upon feeding larvae a diet low in amino acids or upon 'genetic starvation' [silencing of the slimfast (slif) gene in fat cells. This growth rescue is abolished in mth1 homozygous mutants. This shows that Sun requires Mth to control growth. However, sun overexpression has no effect in animals fed a normal diet. A modification of sun expression does not prevent fat body cells from responding to amino acid deprivation as seen by the level of TORC1 signaling, general morphology, and lipid droplet accumulation but affects the ability of larvae to resist to starvation (Delanoue, 2016).

Dilp2-containing secretion granules accumulate in the IPCs following starvation and are rapidly released upon refeeding. Mth is required in the IPCs to promote Dilp secretion after refeeding, and forced membrane depolarization of IPCs using a bacterial sodium channel (dilp2>NaChBac) is dominant over the blockade of Dilp2 secretion in dilp2>mth-Ri animals. This dominance indicates that Mth acts upstream of the secretion machinery. In addition, Dilp2 secretion after refeeding is abrogated in lpp>sun-Ri animals, and overexpression of sun in fat cells prevents Dilp2 accumulation upon starvation. Altogether, these findings indicate that Mth and its ligand Sun are two components of the systemic nutrient response controlling Dilp secretion (Delanoue, 2016).

Hemolymph from fed animals triggers Dilp2 secretion when applied to brains dissected from starved larvae. This insulinotropic activity requires the function of Mth in the IPCs and the production of Sun by fat body cells. Conversely, overexpressing sun in the fat body (lpp>sun) is sufficient to restore insulinotropic activity to the hemolymph of starved larvae. A 2-hour incubation with a synthetic peptide corresponding to the Sun isoform A (Sun-A) is also sufficient to induce Dilp secretion from starved brains. A similar effect is observed with an N-terminal fragment of Sun (N-SUN) that contains the Mth-binding domain but not with a C-terminal fragment (C-SUN) that does not bind Mth. The insulinotropic effect of N-SUN is no longer observed in brains from larvae of the mth allele, mth1 . This absence of effect indicates that N-SUN action requires Mth in the brain. In addition, preincubation of control hemolymph with antiserum containing Sun antibodies specifically suppresses its insulinotropic function. These results indicate that Sun is both sufficient and necessary for insulinotropic activity in the hemolymph of protein-fed animals (Delanoue, 2016).

To directly quantify the amount of circulating Sun protein, Western blot experiments wee performed on hemolymph using antibodies against Sun. A 6-kD band was detected in hemolymph collected from fed larvae, and size was confirmed using Schneider 2 (S2) cell extracts. The band intensity was reduced upon sun knockdown in fat body cells but not in gut cells. Therefore, circulating Sun peptide appears to be mostly contributed by fat cells, as suggested by functional experiments. The levels of circulating Sun are strongly reduced upon starvation. In line with this, sun transcripts are drastically reduced after 4 hours of protein starvation and start increasing after 1 hour of refeeding, whereas expression of the sun homolog CG31477 is not modified. sun transcription is not affected by blocking TORC1, the main sensor for amino acids in fat body cells. However, adipose-specific TORC1 inhibition induces a dramatic reduction of circulating Sun, indicating that TORC1 signaling controls Sun peptide translation or secretion from fat cells. PGC1-Spargel is a transcription activator, the expression of which relies on nutritional input. PGC1 was found to be required for sun transcription, and fat body silencing of PGC1 and sun induce identical larval phenotypes. Although PGC1 expression is strongly suppressed upon starvation, blocking TORC1 activity in fat cells does not reduce PGC1 expression. Conversely, knocking down PGC1 does not inhibit TORC1 activity. This finding suggests that PGC1 and TORC1 act in parallel. Therefore, Sun production by fat cells in response to nutrition is controlled at two distinct levels by PGC1 and TORC1 (Delanoue, 2016).

The Sun peptide is identical to the ε subunit of the mitochondrial F1F0-adenosine triphosphatase (F1F0-ATPase) synthase (complex V). Indeed, both endogenous Sun and Sun labeled with a hemagglutinin tag (Sun-HA) colocalize with mitochondrial markers in fat cells , and the Sun peptide cofractionates with mitochondrial complex V in blue native polyacrylamide gel electrophoresis. In addition, silencing sun in fat cells decreases mitochondrial Sun staining and the amounts of adenosine triphosphate (ATP). However, recent evidence indicates that an ectopic (ecto) form of the F1F0-ATP synthase is found associated with the plasma membrane in mammalian and insect cells. In addition, coupling factor 6, a subunit of complex V, is found in the plasma. Therefore, Stunted could participate in two separate functions carried by distinct molecular pools. To address this possibility, a modified form of Stunted carrying a green fluorescent protein (GFP) tag at its N terminus (GFP-Sun), next to the mitochondria-targeting signal (MTS), was used. When expressed in fat cells, GFP-Sun does not localize to the mitochondria, contrarily to a Sun peptide tagged at its C-terminal end (Sun-GFP). This suggests that addition of the N-terminal tag interferes with the MTS and prevents mitochondrial transport of Sun. However, both GFP-Sun and Sun-GFP are found in the hemolymph and rescue pupal size and Dilp2 accumulation in larvae fed a low-amino acid diet as efficiently as wild-type Sun (wt-Sun) and do so in a mth-dependent manner. This indicates that the growth-promoting function of Sun requires its secretion but not its mitochondrial localization and suggests the existence of one pool of Sun peptide located in the mitochondria devoted to F1F0-ATP synthase activity and ATP production and another pool released in the hemolymph for coupling nutrient and growth control. In this line, although fat body levels of Sun are decreased upon starvation, its mitochondrial localization is not reduced. This finding indicates that starvation affects a nonmitochondrial pool of Sun. In support of this, starved fat bodies contain normal levels of ATP and lactate, indicating that mitochondrial oxidative phosphorylation is preserved in fat cells in poor nutrient conditions. Last, other subunits from complex V (ATP5a) or complex I (NdufS3) were not detected in circulating hemolymph. Therefore, the release of Sun in the hemolymph relies on a specific mechanism (Delanoue, 2016).

In conclusion, this study has provided evidence for a molecular cross-talk between fat cells and brain IPCs involving the ligand Stunted and its receptor Methuselah. Stunted is a moonlighting peptide present both in the mitochondria as part of the F1F0-ATP synthase complex and as an insulinotropic ligand circulating in the hemolymph. The mechanism of Stunted release remains to be clarified. The beta subunit of the ectopic form of F1F0-ATP synthase is a receptor for lipoproteins, which serve as cargos for proteins and peptides. In addition, Drosophila lipid transfer particle-containing lipoproteins were shown to act on the larval brain to control systemic insulin signaling in response to nutrition. This suggests that Sun could be loaded on lipoproteins for its transport. Given the role of insulin-insulin-like growth factor (IGF) signaling in aging, the current findings could help in understanding the role of Sun/Mth in aging adult flies (Delanoue, 2016).

The same genetic screen previously identified the fly tumor necrosis factor α Eiger (Egr) as an adipokine necessary for long-term adaptation to protein starvation, and recent work pointed to other adipose factors, illustrating the key role of the larval fat body in orchestrating nutrient response. The multiplicity of adipose factors and their possible redundancy could explain the relatively mild starvation-like phenotype obtained after removal of only one of them. Overall, these findings suggest a model whereby partially redundant fat-derived signals account for differential response to positive and negative valence of various diet components, as well as acute versus long-term adaptive responses (Delanoue, 2016).

Thermal stress depletes energy reserves in Drosophila

Understanding how environmental temperature affects metabolic and physiological functions is of crucial importance to assess the impacts of climate change on organisms. This study used different laboratory strains and a wild-caught population of the fruit fly Drosophila melanogaster to examine the effect of temperature on the body energy reserves of an ectothermic organism. Permanent ambient temperature elevation or transient thermal stress was shown to cause significant depletion of body fat stores. Surprisingly, transient thermal stress induces a lasting "memory effect" on body fat storage, which also reduces survivorship of the flies upon food deprivation later after stress exposure. Functional analyses revealed that an intact heat-shock response is essential to protect flies from temperature-dependent body fat decline. Moreover, it was found that the temperature-dependent body fat reduction is caused at least in part by apoptosis of fat body cells, which might irreversibly compromise the fat storage capacity of the flies. Altogether, these results provide evidence that thermal stress has a significant negative impact on organismal energy reserves, which in turn might affect individual fitness (Klepsatel, 2016).

A fat-tissue sensor couples growth to oxygen availability by remotely controlling insulin secretion

Organisms adapt their metabolism and growth to the availability of nutrients and oxygen, which are essential for development, yet the mechanisms by which this adaptation occurs are not fully understood. This study describes an RNAi-based body-size screen in Drosophila to identify such mechanisms. Among the strongest hits is the fibroblast growth factor receptor homolog breathless necessary for proper development of the tracheal airway system. Breathless deficiency results in tissue hypoxia, sensed primarily in this context by the fat tissue through HIF-1a prolyl hydroxylase (Hph). The fat relays its hypoxic status through release of one or more HIF-1a-dependent humoral factors that inhibit insulin secretion from the brain, thereby restricting systemic growth. Independently of HIF-1a, Hph is also required for nutrient-dependent Target-of-rapamycin (Tor) activation. These findings show that the fat tissue acts as the primary sensor of nutrient and oxygen levels, directing adaptation of organismal metabolism and growth to environmental conditions (Texada, 2019).

This report identifies one tissue in particular, the fat body, which senses internal oxygen levels and regulates growth rate accordingly. The data show that, as an adaptive response to oxygen limitation, the fat tissue releases into the circulation one or more factors that inhibit the secretion of insulin from the brain to reduce systemic growth. The ability of oxygen to reduce systemic body growth through downregulation of insulin signaling requires Hph-dependent HIF-1a/Sima activity in the fat tissue. Furthermore, hypoxia and AA deprivation both reduce Hph activity in the fat tissue, and this reduction leads to suppression of Tor signaling, independently of HIF-1a/Sima. This is consistent with a requirement of Hph for cell growth. In other contexts, Sima is known to regulate Tor-pathway activity via the protein Scylla/REDD1. However, this pathway does not appear to be responsible for the effects of hypoxia on Tor activity observed in this study, as sima mutation does not block hypoxia- or starvation-induced Tor suppression. Likewise, Tor suppression is not necessary for the systemic growth reduction induced by hypoxia. The data suggest that Hph is involved in AA sensing, in addition to its well-described role in oxygen sensing, and that HIF-1a is not involved in this process. Together, this suggests that AA and oxygen sensing converge through Hph in the fat body to modulate systemic growth in response to environmental conditions (Texada, 2019).

Many of the known Drosophila adipokines that affect insulin secretion from the IPCs are regulated by Tor-pathway activity in the fat body, including CCHa-2, Egr, FIT, GBP and GBP2, and Sun. The finding that AA availability regulates Hph activity, and that Hph activity modulates Tor signaling, thus places Hph upstream of these known factors, in addition to the separate Sima-dependent and Tor-independent humoral factor(s) that modulate insulin secretion under hypoxia. Several routes by which AA availability regulates Tor have been investigated and Hph may modulate some of these and not others, thereby allowing for different responses to AA starvation and hypoxia. Indeed, the current work shows that HIF-1a/Sima is required for the growth-suppressive effect of hypoxia, but not for growth responses to varied dietary AA input, although Hph is involved in both. The mechanisms by which Hph activity, which simultaneously requires AAs and oxygen, allows or promotes Tor signaling is an interesting topic to investigate in future studies, as is the identity of the Tor-independent humoral factor(s) downstream of Sima (Texada, 2019).

The results show that hypoxia or loss of fat-body Hph activity Sima dependently represses Dilp3 and Dilp5 transcription, while having little or no suppressive effect on Dilp2 expression. This suggests that specific transcriptional regulation of Dilp3 and Dilp5 is an important component of the response to hypoxia. Consistent with this observation, previous studies have shown that the transcription of Dilp2, -3, and -5 are independently regulated. Nutrient deprivation reduces expression of Dilp3 and -5, while having no effect on Dilp2 expression, similar to the effects of exposure to hypoxia. This is consistent with the finding that both AA deprivation and hypoxia suppress Hph activity, although the downstream pathways involved appear likely to be different, as at least some aspects of nutrient deprivation are relayed through the Tor pathway, whereas the hypoxia-specific signal(s) shown here is not. This observed transcriptional response could conceivably arise secondarily to DILP-release inhibition via autocrine feedback regulation that operates in both Drosophila and mammals. However, lower secretion of DILPs, which were observed under hypoxic conditions, generally feeds back to induce an increase in the expression of Dilp3 and -5 rather than the decrease that was observed. Therefore, the hypoxia-induced alterations of Dilp3 and -5 expression appear to be specific transcriptional responses rather than feedback effects. Thus, beyond the identity of the fat-body factor involved, the mechanisms operating in the IPCs by which it regulates insulin-like gene expression and peptide release will be important to study in future experiments (Texada, 2019).

In mammals, several adipokines regulate β-cell function and insulin secretion, including Leptin, which conveys information about fat storage and is a functional analog of the Drosophila fat-derived cytokine Unpaired. Interestingly, strong increases were observed in fat-body LD size induced by Tor inhibition downstream of AA starvation, hypoxia, or Hph mutation, indicating a change in lipid metabolism within the fat tissue. Although this phenotype is Tor-dependent and not upstream of the particular HIF-1a-dependent factor described above, it is possible that additional signals related to lipid metabolism may be released by the fat body in response to hypoxia or starvation, such as a lipid-binding protein or even a lipid per se. For example, the mammalian fatty acid-binding protein 4 is an insulin-modulating adipokine that is influenced by obesogenic conditions that lead to adipose tissue hypoxia, and orthologous proteins are encoded by the Drosophila genome (Texada, 2019).

Most organisms stop growing after reaching a genetically predetermined species-characteristic size. Although insight from genetic studies in Drosophila into the mechanisms that regulate body growth with regard to nutrition helps to explain how organisms modulate their growth rate according to nutritional conditions, a mechanism that allows organisms to assess their size and stop their growth when they have reached an optimum has remained elusive. However, recent evidence suggests that body size in insects may be determined by a mechanism that involves oxygen sensing, and oxygen availability is known to place limits on insect body size. According to this recent insight, the limited growth ability of the tracheal system during development may limit overall body size via downstream oxygen sensing. The size of the tracheal system is established at the beginning of each developmental stage and remains largely fixed, aside from terminal branching, as the body grows until it eventually reaches the limit of the system's ability to deliver oxygen. This allows the body to assess its size by sensing internal oxygen concentrations and to terminate growth at a characteristic size that is determined by the size of the tracheal system. An RNAi screen shows that the FGF receptor Btl, which is a key factor essential for tracheal growth during development, is a main determinant of body size. Indeed, btl was a stronger hit than known size-governing genes. The data therefore support the notion that the tracheal system and oxygen sensing may be part of a size-assessment mechanism (Texada, 2019).

Oxygen homeostasis also requires the coordination of growth between the tissues that consume oxygen and those that deliver it. The development of the oxygen delivery system is therefore oxygen sensitive in both mammals and Drosophila. In mammals, local tissue hypoxia promotes angiogenesis via induction of many pro-angiogenic factors, including FGF. In Drosophila, tissue hypoxia induces expression of the FGF-like ligand Bnl, leading to branching of the tracheal airway tubes toward oxygen-deficient areas. This study shows that this mechanism operates independently of insulin, as reduced insulin signaling in the trachea has no effect on overall body growth. This system therefore allows an adaptive response to low oxygen by reducing overall body growth via suppression of insulin signaling, while promoting hypoxia-induced FGF-dependent tracheal growth to increase oxygen delivery (Texada, 2019).

Cell and tissue hypoxia are also observed in human conditions of obesity and cancer. The insect fat body performs the functions of mammalian fat and liver tissues. Accordingly, perturbation of systemic insulin signaling by adipose and hepatic tissue hypoxia is also observed in mammalian systems. In mammals, obesity induces hypoxia within adipose tissue due to the rarefaction of vascularization of this tissue, leading to the release of inflammatory mediators and other adipokines that are associated with the pathophysiology of obesity-related metabolic disorders including diabetes. Although loss of normal β-cell activity is considered a main factor in diabetes, the mechanism by which tissue hypoxia affects insulin secretion is poorly understood. The finding of one or more hypoxia-induced fat-body-derived insulinostatic factors may lead to insights into the role of adipose-tissue hypoxia in obesity and its impact on diabetes. Furthermore, a link is shown between oxygen and AA availability in the adipose tissue through Hph-dependent regulation of the Tor pathway, linking these pathways in a common metabolic response to oxygen limitation and nutrient scarcity (Texada, 2019).

Obesity also causes physical and hormonal changes that affect breathing patterns, leading to apnea and thus intermittent episodes of systemic hypoxia. These hypoxic periods can induce changes in the liver, leading to fatty liver disease and dyslipidemia. The alterations to fat-body lipid metabolism observed in this study may thus be relevant to human health as well. Furthermore, hypoxia-induced programs play important roles in tumor formation. During cancer development, tumor cells undergo a metabolic reprogramming, the so-called Warburg effect, in which their metabolism shifts from oxidative phosphorylation to glycolysis, and activation of HIF-1a is believed to play a key role in this shift. As the hypoxia-sensing mechanism and the insulin-signaling system are conserved between flies and mammals, understanding the effects of hypoxia on the fat body could thus provide insight into many human disease states. It will be of interest to study whether tissue hypoxia also inhibits Tor-pathway activity in mammalian adipocytes (Texada, 2019).

In conclusion, this study unravels a mechanism that allows organisms to adapt their metabolism and growth to environments with low oxygen. Hypoxia activates a fat-tissue oxygen sensor that remotely controls the secretion of insulin from the brain by inter-organ communication. This involves the inhibition of Hph activity, leading to the activation of a HIF-1a-dependent genetic program within the fat tissue, which then secretes one or more humoral signals that alter insulin-gene expression and repress insulin secretion, thereby slowing growth. AA scarcity, like oxygen deficiency, is shown to inhibit Hph activity, and the activity of Hph, but not of HIF-1a, is required for Tor activity in the fat body. Thus, in addition to its role in regulating the as-yet unidentified fat-body hypoxia signal via HIF-1a, Hph connects both oxygen and AA levels to the Tor pathway through an unknown HIF-1a-independent mechanism. Given the conservation of oxygen-sensing and growth-regulatory systems, and the influence of oxygen on growth between Drosophila and mammals, a similar adaption response may operate in mammals via adipose tissue oxygen sensing to maintain homeostasis (Texada, 2019).

Immune Control of Animal Growth in Homeostasis and Nutritional Stress in Drosophila

A large body of research implicates the brain and fat body (liver equivalent) as central players in coordinating growth and nutritional homeostasis in multicellular animals. In this regard, an underlying connection between immune cells and growth is also evident, although mechanistic understanding of this cross-talk is scarce. This study explored the importance of innate immune cells in animal growth during homeostasis and in conditions of nutrient stress. Drosophila larvae lacking blood cells eclose as small adults and show signs of insulin insensitivity. Moreover, when exposed to dietary stress of a high-sucrose diet (HSD), these animals are further growth retarded than normally seen in regular animals raised on HSD. In contrast, larvae carrying increased number of activated macrophage-like plasmatocytes show no defects in adult growth when raised on HSD and grow to sizes almost comparable with that seen with regular diet. These observations imply a central role for immune cell activity in growth control. Mechanistically, these findings reveal a surprising influence of immune cells on balancing fat body inflammation and insulin signaling under conditions of homeostasis and nutrient overload as a means to coordinate systemic metabolism and adult growth. This work integrates both the cellular and humoral arm of the innate immune system in organismal growth homeostasis, the implications of which may be broadly conserved across mammalian systems as well (Preethi, 2020).

The mode of expression divergence in Drosophila fat body is infection-specific

Transcription is controlled by interactions of cis-acting DNA elements with diffusible trans-acting factors. Changes in cis or trans factors can drive expression divergence within and between species, and their relative prevalence can reveal the evolutionary history and pressures that drive expression variation. Previous work delineating the mode of expression divergence in animals has largely used whole body expression measurements in one condition. Since cis-acting elements often drive expression in a subset of cell types or conditions, these measurements may not capture the complete contribution of cis-acting changes. This study quantified the mode of expression divergence in the Drosophila fat body, the primary immune organ, in several conditions, using two geographically distinct lines of D. melanogaster and their F1 hybrids. Expression was measured in the absence of infection and in infections with Gram-negative S. marcescens or Gram-positive E. faecalis bacteria, which trigger the two primary signaling pathways in the Drosophila innate immune response. The mode of expression divergence strongly depends on the condition, with trans-acting effects dominating in response to Gram-positive infection and cis-acting effects dominating in Gram-negative and pre-infection conditions. Expression divergence in several receptor proteins may underlie the infection-specific trans effects. Before infection, when the fat body has a metabolic role, there are many compensatory effects, changes in cis and trans that counteract each other to maintain expression levels. This work demonstrates that within a single tissue, the mode of expression divergence varies between conditions and suggests that these differences reflect the diverse evolutionary histories of host-pathogen interactions (Ramirez-Corona, 2021).

The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. This study found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels were found of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, this study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding understanding of the comprehensive regulatory mechanisms of final body size in animals (Yuan, 2020).

This study has discovered that in the insect fat body, 20E activates AMPK in two ways: By up-regulating the mRNA levels of all three AMPK subunits and by inducing energy stress to activate AMPK (Yuan, 2020).

The transcription levels of all three AMPK subunits, the protein level of AMPKα, and the phosphorylation level of AMPKα were all elevated in the Bombyx fat body at ∼4M and in the Drosophila fat body during pupariation, showing developmental profiles that were consistent with those of 20E signaling. Both the gain-of-function and loss-of-function experiments further demonstrate that AMPK is transcriptionally activated by 20E signaling. According to preliminary data, 20E-EcR-USP does not directly induce the expression of the AMPK-PP2A subunit genes, and further studies should be performed to investigate the detailed mechanisms whereby the 20E-triggered transcriptional cascade is involved in this transcriptional activation (Yuan, 2020).

20E is well known to act through the insect larval central nervous system (CNS) to induce wandering behavior and escape from food. Moreover, 20E slowly reduces insect feeding behavior and, thus, food consumption. Nevertheless, both the induction of wandering behavior and the reduction of feeding behavior can cause energy stress, such as sugar starvation, which ultimately increases the cellular AMP/ATP ratio, leading to the activation of AMPK. According to the Bombyx fat body results at ~4M, such a poor nutrition status promoted AMPK activity and inhibited IIS. Altogether, 20E slowly induces a sugar starvation-like condition to activate AMPK in the fat body by modulating CNS-controlled feeding behavior and wandering behavior in insects (Yuan, 2020).

IIS is an anabolic pathway, while AMPK accounts for catabolism, thus it naturally exists a mutual inhibition between IIS and AMPK. AMPK and PP2A might affect each other, and the AMPK-PP2A axis has been documented in mammalian cells. This study confirmed that the AMPK-PP2A axis exists in the Drosophila fat body, linking the antagonism of IIS by 20E (Yuan, 2020).

In addition to the dephosphorylation of AKT by PP2A, PP2A also dephosphorylates S6K, playing a key role in the attenuation of IIS and its downstream TORC1 activity. These studies determined that PP2A not only dephosphorylates AKT and inhibits TORC1 activity but also dephosphorylates InR and inactivates PI3K, showing that PP2A inhibits IIS starting from the dephosphorylation of InR. It is hypothesized that PP2A might dephosphorylate InR, PI3K, and AKT and, thus, inhibit IIS in an integrative manner (Yuan, 2020).

Finally, this study demonstrated that 20E activates the AMPK-PP2A axis to antagonize IIS in the insect fat body. After blocking either AMPK or PP2A, 20E no longer antagonizes IIS in the fat body. In summary, the AMPK-PP2A axis in the insect fat body is activated by 20E to antagonize IIS (Yuan, 2020).

Considering the similar regulatory functions in the antagonism of IIS by 20E, the possible relationship between miR-8/Ush and AMPK-PP2A was examined. Via bioinformatics prediction, it was found that miR-8 does not target AMPK or PP2A transcripts. Meanwhile, preliminary data showed that overexpression of AMPKCA or PP2ACA did not affect Ush expression in the fat body. Thus, it is supposed that AMPK-PP2A should function in parallel with miR-8 in the antagonism of IIS by 20E. It is concluded that the AMPK-PP2A axis is a crucial, but not a unique, pathway linking 20E to IIS (Yuan, 2020).

Previous studies and the current results together indicate that, similar to the inhibition of IIS in the larval fat body, activation of the fat body AMPK-PP2A axis reduces food consumption and thus restricts growth rate and body size in Drosophila. In other words, the AMPK-PP2A axis and IIS in the fat body play opposite developmental roles in regulating the larval growth rate and body size, and one crucial reason should be the modulation of feeding behavior and thus food consumption (Yuan, 2020).

The insect fat body, which is analogous to the mammalian liver, functions as an energy reservoir and nutrient sensor to regulate developmental timing. Fat body-derived amino acid signals, which involve Slimfast (the amino acid transporter) and TORC1 signaling, reactivate quiescent neuroblasts and finally control larval growth by regulating the synthesis and release of insulin/IGF. In addition to amino acid-dependent signals, certain other growth-promoting factors, such as CCHamide-2 and Unpaired 2, secreted from the fat body also affect the brain to remotely control insulin/IGF secretion in Drosophila. Moreover, IIS acts as the center of energy and nutrition response and positively regulates the larval growth rate partially by inhibiting autophagy in the Drosophila fat body. In contrast, 20E negatively regulates the larval growth rate by impeding IIS in the Drosophila fat body. Interestingly, preliminary results suggest that the AMPK-PP2A axis had little effect on fat body autophagy during normal feeding conditions and that TORC1 in the fat body plays little role in regulating the larval growth rate (Yuan, 2020).

It is likely that activation of the AMPK-PP2A axis and the inhibition of IIS in the fat body might affect the nutritional and endocrinal functions of this tissue. These changes in the fat body should cause the reduction of food consumption, resulting in the restriction of growth rate and body size. Investigating the detailed molecular mechanisms of how food consumption and its related feeding behavior and wandering behavior are regulated by hormonal and nutritional signals in the fat body might open a new window for understanding the regulatory mechanisms of final body size in insects. In future, it is worthwhile to examine whether the CNS. as well as neuropeptides and neurotransmitters, are involved in this regulation. Taking these data together, a model is proposed in which 20E antagonizes IIS by activating the AMPK-PP2A axis in the fat body to restrict the larval growth rate in insects. This study expands understanding of the comprehensive regulatory mechanisms underlying final body size determination in animals (Yuan, 2020).

Differential metabolic sensitivity of insulin-like-response- and TORC1-dependent overgrowth in Drosophila fat cells

Glycolysis and fatty acid (FA) synthesis directs the production of energy-carrying molecules and building blocks necessary to support cell growth, although the absolute requirement of these metabolic pathways must be deeply investigated. This study used Drosophila genetics and focused on the TOR (Target of Rapamycin) signaling network that controls cell growth and homeostasis. In mammals, mTOR (mechanistic-TOR) is present in two distinct complexes, mTORC1 and mTORC2; the former directly responds to amino acids and energy levels, whereas the latter sustains insulin-like-peptide (Ilp) response. The TORC1 and Ilp signaling branches can be independently modulated in most Drosophila tissues. This study shows that TORC1 and Ilp-dependent overgrowth can operate independently in fat cells and that ubiquitous over-activation of TORC1 or Ilp signaling affects basal metabolism, supporting the use of Drosophila as a powerful model to study the link between growth and metabolism. Cell-autonomous restriction of glycolysis or FA synthesis in fat cells was shown to retrain overgrowth dependent on Ilp signaling but not TORC1 signaling. Additionally, the mutation of FASN (Fatty acid synthase) results in a drop in TORC1 but not Ilp signaling, whereas, at the cell-autonomous level, this mutation affects none of these signals in fat cells. These findings thus reveal differential metabolic sensitivity of TORC1- and Ilp-dependent growth and suggest that cell-autonomous metabolic defects might elicit local compensatory pathways. Conversely, enzyme knockdown in the whole organism results in animal death. Importantly, this study weakens the use of single inhibitors to fight mTOR-related diseases and strengthens the use of drug combination and selective tissue-targeting (Devilliers, 2021).

Co-option of immune effectors by the hormonal signalling system triggering metamorphosis in Drosophila melanogaster

Insect metamorphosis is triggered by the production, secretion and degradation of 20-hydroxyecdysone (ecdysone). In addition to its role in developmental regulation, increasing evidence suggests that ecdysone is involved in innate immunity processes, such as phagocytosis and the induction of antimicrobial peptide (AMP) production. AMP regulation includes systemic responses as well as local responses at surface epithelia that contact with the external environment. At pupariation, Drosophila melanogaster increases dramatically the expression of three AMP genes, drosomycin (drs), drosomycin-like 2 (drsl2) and drosomycin-like 5 (drsl5). The systemic action of drs at pupariation is dependent on ecdysone signalling in the fat body and operates via the ecdysone downstream target, Broad. In parallel, ecdysone also regulates local responses, specifically through the activation of drsl2 expression in the gut. Finally, the relevance of this ecdysone dependent AMP expression for the control of bacterial load was confirmed by showing that flies lacking drs expression in the fat body have higher bacterial persistence over metamorphosis. In contrast, local responses may be redundant with the systemic effect of drs since reduction of ecdysone signalling or of drsl2 expression has no measurable negative effect on bacterial load control in the pupa. Together, these data emphasize the importance of the association between ecdysone signalling and immunity using in vivo studies and establish a new role for ecdysone at pupariation, which impacts developmental success by regulating the immune system in a stage-dependent manner. It is speculated that this co-option of immune effectors by the hormonal system may constitute an anticipatory mechanism to control bacterial numbers in the pupa, at the core of metamorphosis evolution (Nunes, 2021).

Decapentaplegic retards lipolysis during metamorphosis in Bombyx mori and Drosophila melanogaster

Insect morphogen Decapentaplegic (Dpp) functions as one of the key extracellular ligands of the Bone Morphogenetic Protein (BMP) signaling pathway. Previous studies in insects mainly focused on the roles of Dpp during embryonic development and the formation of adult wings. This study demonstrated a new role for Dpp in retarding lipolysis during metamorphosis in both Bombyx mori and Drosophila melanogaster. CRISPR/Cas9-mediated mutation of Bombyx dpp causes pupal lethality, induces an excessive and premature breakdown of lipids in the fat body, and upregulates the expressions of several lipolytic enzyme genes, including brummer (bmm), lipase 3 (lip3), and hormone-sensitive lipase (hsl), and lipid storage droplet 1 (lsd1), a lipid droplets (LD)-associated protein gene. Further investigation in Drosophila reveals that salivary gland-specific knockdown of the dpp gene and fat body-specific knockdown of Mad involved in Dpp signaling phenocopy the effects of Bombyx dpp mutation on pupal development and lipolysis. Taken together, these data indicate that the Dpp-mediated BMP signaling in the fat body maintains lipid homeostasis by retarding lipolysis, which is necessary for pupa-adult transition during insect metamorphosis (Qian, 2023).

The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila

Animals can sense internal nutrients, such as amino acids/proteins, and are able to modify their developmental programs in accordance with their nutrient status. In the fruit fly, Drosophila melanogaster, amino acid/protein is sensed by the fat body, an insect adipose tissue, through a nutrient sensor, target of rapamycin (TOR) complex 1 (TORC1). TORC1 promotes the secretion of various peptide hormones from the fat body in an amino acid/protein-dependent manner. Fat-body-derived peptide hormones stimulate the release of insulin-like peptides, which are essential growth-promoting anabolic hormones, from neuroendocrine cells called insulin-producing cells (IPCs). Although the importance of TORC1 and the fat body-IPC axis has been elucidated, the mechanism by which TORC1 regulates the expression of insulinotropic signal peptides remains unclear. This study shows that an evolutionarily conserved molecular chaperone, heat shock protein 90 (Hsp90), promotes the expression of insulinotropic signal peptides. Fat-body-selective Hsp90 knockdown caused the transcriptional downregulation of insulinotropic signal peptides. IPC activity and systemic growth were also impaired in fat-body-selective Hsp90 knockdown animals. Furthermore, Hsp90 expression depended on protein/amino acid availability and TORC1 signaling. These results strongly suggest that Hsp90 serves as a nutrient-responsive gene that upregulates the fat body-IPC axis and systemic growth. It is proposed that Hsp90 is induced in a nutrient-dependent manner to support anabolic metabolism during the juvenile growth period (Ohhara, 2021).

PPA1 Regulates Systemic Insulin Sensitivity by Maintaining Adipocyte Mitochondria Function as a Novel PPARgamma Target Gene

Downregulation of mitochondrial function in adipose tissue is considered as one important driver for the development of obesity-associated metabolic disorders. Inorganic Pyrophosphatase 1 (PPA1) is an enzyme catalyzes the hydrolysis of PPi to Pi, and is required for anabolism to take place in cells. Although alternation of PPA1 has been related to some diseases, the importance of PPA1 in metabolic syndromes has never been discussed before. This study found that global PPA1 knockout mice (PPA1(+/-)) showed impaired glucose tolerance and severe insulin resistance under HFD feeding. In addition, impaired adipose tissue development and ectopic lipid accumulation were also observed. Conversely, overexpression of PPA1 in adipose tissue by AAV injection can partly reverse the metabolic disorders in PPA1(+/-) mice, suggesting that impaired adipose tissue function is responsible for the metabolic disorders observed in PPA1(+/-) mice. Mechanistic studies revealed that PPA1 acted as a PPARγ (E75 and E78) target gene to maintain mitochondrial function in adipocytes. Furthermore, specific knockdown of PPA1 in fat body of Drosophila led to impaired mitochondria morphology, decreased lipid storage, and made Drosophila more sensitive to starvation. In conclusion, these finding findings demonstrated the importance of PPA1 in maintaining adipose tissue function and whole body metabolic homeostasis (Yin, 2021).

Edem1 activity in the fat body regulates insulin signalling and metabolic homeostasis in Drosophila

In Drosophila, nutrient status is sensed by the fat body, a functional homolog of mammalian liver and white adipocytes. The fat body conveys nutrient information to insulin-producing cells through humoral factors which regulate Drosophila insulin-like peptide levels and insulin signalling. Insulin signalling has pleiotropic functions, which include the management of growth and metabolic pathways. This paper reports that Edem1 (endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1), an endoplasmic reticulum-resident protein involved in protein quality control, acts in the fat body to regulate insulin signalling and thereby the metabolic status in Drosophila Edem1 limits the fat body-derived Drosophila tumor necrosis factor-α Eiger activity on insulin-producing cells and maintains systemic insulin signalling in fed conditions. During food deprivation, edem1 gene expression levels drop, which aids in the reduction of systemic insulin signalling crucial for survival. Overall, this study demonstrates that Edem1 plays a vital role in helping the organism to endure a fluctuating nutrient environment by managing insulin signalling and metabolic homeostasis (Pathak, 2021).

A salivary gland-secreted peptide regulates insect systemic growth

Insect salivary glands have been shown to function in pupal attachment and food lubrication by secreting factors into the lumen via an exocrine way. This study found in Drosophila that a salivary gland-derived secreted factor (Sgsf) peptide regulates systemic growth via an endocrine way. Sgsf is specifically expressed in salivary glands and secreted into the hemolymph. Sgsf knockout or salivary gland-specific Sgsf knockdown decrease the size of both the body and organs, phenocopying the effects of genetic ablation of salivary glands, while salivary gland-specific Sgsf overexpression increases their size. Sgsf promotes systemic growth by modulating the secretion of the insulin-like peptide Dilp2 from the brain insulin-producing cells (IPCs) and affecting mechanistic target of rapamycin (mTOR) signaling in the fat body. Altogether, this study demonstrates that Sgsf mediates the roles of salivary glands in Drosophila systemic growth, establishing an endocrine function of salivary glands (Li, 2022).

Fat body-derived Spz5 remotely facilitates tumor-suppressive cell competition through Toll-6-α-Spectrin axis-mediated Hippo activation

Tumor-suppressive cell competition is an evolutionarily conserved process that selectively removes precancerous cells to maintain tissue homeostasis. Using the polarity-deficiency-induced cell competition model in Drosophila, this study identify Toll-6, a Toll-like receptor family member, as a driver of tension-mediated cell competition through α-Spectrin (α-Spec)-Yorkie (Yki) cascade. Toll-6 aggregates along the boundary between wild-type and polarity-deficient clones, where Toll-6 physically interacts with the cytoskeleton network protein α-Spec to increase mechanical tension, resulting in actomyosin-dependent Hippo pathway activation and the elimination of scrib mutant cells. Furthermore, this study show that Spz5 secreted from fat body, the key innate organ in fly, facilitates the elimination of scrib clones by binding to Toll-6. These findings uncover mechanisms by which fat bodies remotely regulate tumor-suppressive cell competition of polarity-deficient tumors through inter-organ crosstalk and identified the Toll-6-α-Spec axis as an essential guardian that prevents tumorigenesis via tension-mediated cell elimination (Kong, 2022).

Epithelial cells possess intrinsic mechanisms to outcompete and eliminate early precancerous cells to maintain homeostasis. For instance, in a mouse model of esophageal carcinogenesis, the majority of newly developed tumor clones are eliminated through cell competition by adjacent normal epithelium. Similarly, surveillance mechanisms also exist in Drosophila epithelium to actively remove oncogenic clones composed of polarity-deficient cells. Genetic studies in flies have uncovered numerous mechanisms that regulate tumor-suppressive cell competition, including c-Jun-N-terminal kinase (JNK) signaling activation-mediated cell elimination, direct cell-cell interaction, secreted factors from epithelial cells, and inter-organ crosstalk between insulin-producing cells and precancerous cell-bearing discs (Kong, 2022).

Initially identified in Drosophila, the Hippo pathway is an evolutionarily conserved signaling cascade that plays crucial roles in various physiological and pathological contexts, ranging from tumor progression and embryogenesis to stem cell renewal and immune surveillance. Apart from its well-established roles in controlling cell proliferation and cell death, numerous studies have proved that the Hippo pathway also functions as a key mechanotransducer to sense mechanical changes in the microenvironment. Despite the identification of multiple essential mechanosensitive signaling molecules including RAP2, MAP4K, Agrin, and Spectrin, it remains poorly understood how mechanical stimuli are transmitted from plasma membrane localized receptors to activate Hippo signaling cascade-mediated cellular responses, especially in intact tissues. This study, through a genetic screen in Drosophila, uncovered a regulatory mechanism whereby mechanical tension drives tumor-suppressive cell competition though the Hippo pathway. The genetic and biochemistry data uncovered Toll-6 as an essential regulator of Hippo signaling and further identified α-Spec as an essential downstream component that regulates cell competition via tension-mediated actomyosin activation. Moreover, this study further demonstrated that inter-organ communication is critical for the removal of precancerous cells at a systemic level and discovered fat body (FB)-derived Spz5 as a crucial ligand (Kong, 2022).

This study demonstrated that polarity-deficient oncogenic clones are eliminated through tension-dependent cell competition and has identified Toll-6 as a key membrane receptor that physically interacts and acts through α-Spec to activate the Hippo pathway. It has long been recognized that both extrinsic cues such as ligands and intrinsic factors such as stiffness and cell-cell contact-mediated mechanical cues can determine cell fate and affect cell proliferation, yet relatively little is known about how the cytoskeleton system contributes to the elimination of precancerous cells during cell competition in vivo. The data show that both &alpha-Spec and Rho1, two essential cytoskeleton regulators, accumulate and facilitate the elimination of scrib clones. In addition, α-Spec as a crucial linker that bridges Toll-6 activation-induced tensional changes to cytosolic Hippo pathway activation. Interestingly, studies in the mammalian system showed that RhoA (human Rho1 ortholog) is responsible for mechanical force-induced cell extrusion. Thus, a similar tension-mediated cell-elimination mechanism might exist in the mammalian system to actively remove unfitted precancerous cells (Kong, 2022).

TLRs play critical roles in the innate immune response. The Drosophila genome encodes nine TLRs, of which only Toll (Tl/Toll-1) has a clear function in innate immunity. Interestingly, a paradoxical role of Tl in regulating cell competition has been reported. Activation of Tl in polarity-deficient clones suppresses the elimination of losers, whereas in the Myc-induced cell competition model, increased Tl activity accelerates the elimination of losers. Apart from Tl, Toll-2, Toll-3, Toll-7, Toll-8, and Toll-9 have been implicated in regulating cell competition in different contexts, while Toll-4 and Toll-5 have little effect. It is noteworthy that none of above studies has investigated the role of Toll-6 in cell competition. The current data not only reveal Toll-6 as a crucial regulator of tumor-suppressive cell competition but also show how the mechanical tension-mediated Hippo cascade is initiated from the cell membrane through the Toll-6-&alpha-Spec axis. Notably, this study found that Toll-6 was not required for Myc-induced cell competition. Given that TLRs are highly conserved in vertebrates and the elimination of scrib-depleted cells also exists in the mammalian system, further experiments are necessary to determine whether analogous mechanisms exist in mammals and humans to regulate mechanical tension-induced, Hippo pathway-mediated tumor-suppressive cell competition (Kong, 2022).

Inter-organ communication is essential for proper development and homeostasis maintenance of multicellular organisms under both physiological and pathological conditions. The tumor progression process is also shaped by the interactions between tumor and other organs, including the immune system. Recent studies in Drosophila have provided insightful understanding of the complex crosstalk between organs during tumorigenesis. The FB is the major immune organ of Drosophila, and it has been shown that intestinal tumor progression or abdominal tumor transplantation promotes the wasting behavior of FBs. The current findings that the transcription of spz5 is increased in the FB from scrib clone bearing larvae to facilitate tumor-suppressive cell competition may provide an in vivo mechanistic understanding of the inter-organ communications between FBs and remotely colonized precancerous clones. Together, the ism) around the boundary between losers and winners, which recruits α-Spec and provokes Hippo pathway-dependent elimination of scrib−/− clones. Meanwhile, the presence of scrib−/− loser cells in the eye disc will trigger a systemic effect on the distal organs, including FBs, which results in the transcription upregulation and secretion of Spz5, in turn forming a feedforward loop to reinforce the tumor-suppressive cell competition by binding to Toll-6 (Kong, 2022).

Although biochemical and genetic data demonstrate that Toll-6 physically interacts with α-Spec and that α-Spec is required for the elimination of scrib clones, these experiments were unable to explain the molecular mechanisms by which Toll-6 recruits α-Spec and initiates the downstream signaling transduction. Another limitation is that because the substantial analysis relies heavily on genetics to infer mechanism, enough rigorous biochemistry data was not included to prove how the binding of Toll-6 with α-Spec triggers Hippo signaling activation. Additionally, this study found that FB-derived Spz5 is essential for the elimination of scrib clones through inter-organ communications, but it is not understood completely how the spz5 mRNA level is upregulated systematically in the FBs of larvae that bear scrib mutant clones. Future work will be required to dissect the transcriptome changes of FBs upon precancerous clone induction in distal organs. Finally, this study showed that Spz5 acts through Toll-6 to regulate cell competition, and it is known that Spz5 can bind to other TLRs to regulate both cell death and survival through a three-tier mechanism (Foldi, 2017), suggesting that Spz5 can trigger intracellular signal transduction through ligand receptor binding. Nonetheless, a question that remains unsolved is why a signaling network that relays cell mechanical properties (Toll-6-α-Spec axis) should be regulated by a chemical ligand/receptor interaction; it would be interesting to further explore the underlying mechanisms (Kong, 2022).

Anti-Tumor Effect of Turandot Proteins Induced via the JAK/STAT Pathway in the mxc Hematopoietic Tumor Mutant in Drosophila

Several antimicrobial peptides supress the growth of lymph gland (LG) tumors in Drosophila multi sex comb (mxc) mutant larvae. The activity of another family of polypeptides, called Turandots, is also induced via the JAK/STAT pathway after bacterial infection; however, their influence on Drosophila tumors remains unclear. The JAK/STAT pathway was activated in LG tumors, fat body, and circulating hemocytes of mutant larvae. The mRNA levels of Turandot (Tot) genes increased markedly in the mutant fat body and declined upon silencing Stat92E in the fat body, indicating the involvement of the JAK/STAT pathway. Furthermore, significantly enhanced tumor growth upon a fat-body-specific silencing of the mRNAs demonstrated the antitumor effects of these proteins. The proteins were found to be incorporated into small vesicles in mutant circulating hemocytes (as previously reported for several antimicrobial peptides) but not normal cells. In addition, more hemocytes containing these proteins were found to be associated with tumors. The mutant LGs contained activated effector caspases, and a fat-body-specific silencing of Tots inhibited apoptosis and increased the number of mitotic cells in the LG, thereby suggesting that the proteins inhibited tumor cell proliferation. Thus, Tot proteins possibly exhibit antitumor effects via the induction of apoptosis and inhibition of cell proliferation (Kinoshita, 2023).

Tumor Cytokine-Induced Hepatic Gluconeogenesis Contributes to Cancer Cachexia: Insights from Full Body Single Nuclei Sequencing

A primary cause of death in cancer patients is cachexia, a wasting syndrome attributed to tumor-induced metabolic dysregulation. Despite the major impact of cachexia on the treatment, quality of life, and survival of cancer patients, relatively little is known about the underlying pathogenic mechanisms. Hyperglycemia detected in glucose tolerance test is one of the earliest metabolic abnormalities observed in cancer patients; however, the pathogenesis by which tumors influence blood sugar levels remains poorly understood. In this study, utilizing a Drosophila model, it was demonstrated that the tumor secreted interleukin-like cytokine Upd3 induces fat body expression of Pepck1 and Pdk, two key regulatory enzymes of gluconeogenesis, contributing to hyperglycemia. These data further indicate a conserved regulation of these genes by IL-6/JAK STAT signaling in mouse models. Importantly, in both fly and mouse cancer cachexia models, elevated gluconeogenesis gene levels are associated with poor prognosis. Altogether, this study uncovers a conserved role of Upd3/IL-6/JAK-STAT signaling in inducing tumor-associated hyperglycemia, which provides insights into the pathogenesis of IL-6 signaling in cancer cachexia (Liu, 2023).

Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae

The feeding of pests is one of the important reasons for losses of agricultural crop yield. This study aimed to reveal how juvenile hormone participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. Larvae of O. furnacalis exhibit a daily circadian rhythm on feeding, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a juvenile hormone (JH) active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by the brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide to O. furnacalis that controls feeding and metabolism. The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control (Yu, 2022).

Adipose mitochondrial metabolism controls body growth by modulating systemic cytokine and insulin signaling

Animals must adapt their growth to fluctuations in nutrient availability to ensure proper development. These adaptations often rely on specific nutrient-sensing tissues that control whole-body physiology through inter-organ communication. While the signaling mechanisms that underlie this communication are well studied, the contributions of metabolic alterations in nutrient-sensing tissues are less clear. This study show how the reprogramming of adipose mitochondria controls whole-body growth in Drosophila larvae. Dietary nutrients alter fat-body mitochondrial morphology to lower their bioenergetic activity, leading to rewiring of fat-body glucose metabolism. Strikingly, it was found that genetic reduction of mitochondrial bioenergetics just in the fat body is sufficient to accelerate body growth and development. These growth effects are caused by inhibition of the fat-derived secreted peptides ImpL2 and tumor necrosis factor alpha (TNF-α)/Eiger, leading to enhanced systemic insulin signaling. This work reveals how reprogramming of mitochondrial metabolism in one nutrient-sensing tissue can couple nutrient availability to whole-body growth (Sriskanthadevan-Pirahas, 2022).

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. This study found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, it will be used to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic (Keliinui, 2022).

The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response

Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. This study shows that Eiger, the Drosophila TNF-alpha, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-alpha converting enzyme (TACE) family protein CG7908 is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, this study has identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-alpha that mediates adaptive response to nutrient deprivation (Agrawal, 2016).

This study shows that the fly TNF Eiger functions as a metabolic hormone produced by the fat body in response to chronic protein deprivation. Elevated circulating levels of human TNF-α and TNFR are also observed in malnutrition conditions and in catabolic states associated with cachexia induced by sepsis and cancer. Moreover, this study presents evidence that the molecular mechanism leading to decreased insulin expression by TNF-α is conserved in mammalian β cells. Therefore, activation of TNF signaling could be an evolutionary conserved response to undernutrient imbalance, recently highjacked as an adaptive response to continuous nutritional surplus. Drosophila Eiger has so far mostly been implicated in local, autonomous responses, activating JNK signaling in the cells or tissues that express it. In recent studies, however, it has been postulated that Egr can diffuse outside of its expression domain in a paracrine manner. The current data now demonstrate that Egr circulates in the hemolymph and acts remotely, allowing crossorgan communication. This raises questions relative to the mode of transport of Eiger in the hemolymph and its specificity of action on remote target tissues. Indeed, although overexpression of Egr in fat cells leads to a strong increase in Egr levels in the hemolymph, flies show no obvious defects, suggesting that secreted Egr has limited access to peripheral tissues while in the hemolymph. Previous studies have shown that human and mouse TNF-α efficiently cross the blood-brain barrier (BBB) after i.v. injection and are detected in the cerebrospinal fluid in a process requiring the presence of TNF receptors in glial cells. This study shows that hemolymph Eiger can penetrate the brain and access to the insulin-producing cells. It will be important to evaluate in future experiments the mechanisms by which Egr travels in the hemolymph and across the larval BBB (Agrawal, 2016).

This study shows that an important aspect of Egr secretion relies on its shedding from the membrane by the convertase enzyme TACE. Drosophila TACE was recently shown to be required for Egr function in a tumor model using activated ras (Chabu, 2014). This study shows that transcription of TACE, but not egr, is induced in fat cells under low-protein diet (LPD) and that adipose TACE activity is critical for metabolic adaptation to low protein. Moreover, a genetic link was identified between TACE expression and TORC1, the main amino acid sensor in fat cells. Interestingly, REPTOR and REPTOR-BP, two transcription factors that are responsible for most of the transcription response to TORC1 inhibition, are not required for TACE expression, suggesting that an alternative mechanism is required for TACE induction in response to TORC1 inhibition following exposure to LPD. Vertebrate TACE/ADAM17 acts on a small number of cytokines and growth factors including TNF-α and several membrane receptors (Menghini, 2013). Mice deficient in TACE function are lean and resistant to high-fat-diet-induced obesity and diabetes type 2, a range of phenotypes that could be linked to TNF-α shedding defects. In mammals, TACE activity is controlled through balanced expression of the Tissue Inhibitor of Metalloprotease 3 (TIMP3). Although a TIMP3 homolog exists in Drosophila, there is no indication that it participates in modulating Drosophila TACE activity in addition to TACE transcriptional activation observed in LPD (Agrawal, 2016).

Circulating Eiger produced by adipose cells remotely acts on the brain neurosecretory cells that produce insulin (IPCs), leading to general body growth inhibition. This correlates with specific expression of the TNF receptor Grindelwald in the IPCs. Indeed, knocking down Grnd in these neurons mimics the effect of knocking down Egr in the fat body. No effect is observed upon knocking down the other fly TNFR Wengen in IPCs, indicating that Grnd mediates Egr metabolic action and that specific targeting of TNF signaling to the IPCs is the consequence of localized Grnd expression. As a consequence of Grnd activation, JNK signaling is elevated in the IPCs of animals raised on a LPD. TNF signaling is not required in the IPCs for the retention of Dilp2 observed upon acute protein starvation. By contrast, activation of JNK in larval IPCs leads to reduced expression of Dilp2 and Dilp5, two major circulating Dilps. Strikingly, this is reminiscent of the role described for JNK in vertebrate pancreatic β cells. Indeed, JNK inhibitors increase insulin expression in Langerhans islets from obese mice, suggesting that JNK represses expression of the insulin gene. These results are also in line with the present finding that TNF-α inhibits INS1 and INS2 gene transcription from Min6 cells and mouse islets (Agrawal, 2016).

In conclusion, this work unravels an anciently conserved mechanism by which TNF signaling mediates direct response to low nutrient through a fat-brain crossorgan communication leading to the modulation of growth. Other signals contributing to reduction of insulin signaling in condition of nutrient shortage have recently been identified in Drosophila. Systemic Hedgehog is produced by gut cells in response to low nutrients and targets both fat cells and ecdysone-producing cells to adapt larval growth to nutrient shortage. A hormone called Limostatin produced by the corpora cardiaca in low-glucose condition acts directly on adult insulin-producing cells to block insulin secretion. These findings together with the present work indicate that a variety of signals allow the integration of different physiological contexts for the proper control of insulin production (Agrawal, 2016).

Reduction of nucleolar NOC1 accumulates pre-rRNAs and induces Xrp1 affecting growth and resulting in cell competition

NOC1 is a nucleolar protein necessary in yeast for both transport and maturation of ribosomal subunits. This study shows that Drosophila NOC1 is necessary for rRNAs maturation and for a correct animal development. Its ubiquitous downregulation results in a dramatic decrease in polysome level and of protein synthesis. NOC1 expression in multiple organs, such as the prothoracic gland and the fat body, is necessary for their proper functioning. Reduction of NOC1 in epithelial cells from the imaginal discs results in clones that die by apoptosis, an event that is partially rescued in a M/+ background, suggesting that reduction of NOC1 induces the cells to become less fit and to acquire a loser state. NOC1 downregulation activates the pro-apoptotic eiger-JNK pathway and leads to an increase of Xrp1 that results in Dilp8 upregulation. These data underline NOC1 as an essential gene in ribosome biogenesis and highlight its novel functions in the control of growth and cell competition (Destefanis, 2022).

This study has shown that the Drosophila homologs of yeast NOC1, NOC2 and NOC3 are required for animal development and their ubiquitous reduction results in growth impairment and larval lethality. Ubiquitous overexpression of NOC1 is also detrimental but at the pupal stage, a phenotype that is rescued by co-expression of NOC1-RNAi, which allows the animals to develop to small adults. These data suggest that NOC1 expression must be tightly regulated, as either its reduction or overexpression may be detrimental for the cells. As demonstrated in yeast, the function of Drosophila NOC1 is not redundant with the other NOC proteins, and its overexpression does not compensate for the loss of NOC2 and NOC3. The reason for this behavior might be because NOC proteins function as heterodimers (NOC1-NOC2 and NOC2-NOC3) that are necessary for proper control of rRNA processing and the assembling of the 60S ribosomal subunits. Indeed, it has been demonstrated in yeast that the NOC1-NOC2 complex regulates the activity of ribosomal RNA protein-5 (Rpr5), which controls rRNA cleavage at the internal transcribed spacers ITS1 and ITS2 sequences to ensure the stoichiometric maturation of the 40S and 60S ribosomal subunits. This function is likely to be conserved also in flies. In fact, the current results show that reduction of NOC1 induces the accumulation of the intermediate ITS1 and ITS2 immature forms of rRNAs. Moreover, a reduction was observed in the relative abundance of 18S and 28S rRNAs, suggesting that NOC1 is also required in flies for proper rRNA processing and ribosome maturation. In line with this hypothesis, this study demonstrated that NOC1 reduction results in a strong decrease in ribosome abundance and assembling, which is also accompanied by a strong reduction of the 80S and the polysomes. In addition, a mild accumulation was observed of the 40S and 60S subunits, suggesting that the mature 80S ribosome might be unstable in NOC1-RNAi animals and that a small percentage of the ribosome disassembles into the two subunits, leading to the observed increase. In addition, given that NOC1 was identified as a predicted transcription factor, and because reduction of NOC1 results in a robust decrease in global protein synthesis, it cannot be excluded that specific factors involved in the 80S assembling are reduced or missing in NOC1-RNAi animals (Destefanis, 2022).

Analysis of protein-protein interaction using STRING indicates that CG7838/NOC1 might act in a complex with other nucleolar proteins. Indeed, NOC1 was shown to colocalize in the nucleolus with fibrillarin. Moreover, NOC1 overexpression also results in the formation of large round nuclear structures, which are significantly reduced when its expression is decreased by NOC1-RNAi . Interestingly, similar structures have been shown for CEBPz, the human homolog of NOC1, as visible in images from 'The Human Protein Atlas'. CEBPz (also called CBF2 and CTF2; OMIM-612828) is a transcription factor member of the CAAT-binding protein family, which are involved in Hsp70 complex activation and are upregulated in tumors, particularly in cells from patients with acute myeloid leukemia (AML). As NOC1 also has the conserved CBP domain, this suggests that it might also act as a transcription factor, a hypothesis corroborated by data in Drosophila (CHIP-Seq and genetic screens) that demonstrates how its expression is associated to promoter regions of genes with a function in the regulation of nucleolar activity and of ribosomal proteins. This observation is important as it opens up the possibility that NOC1 can control ribosome biogenesis through alternative mechanisms in addition to its control over rRNA transport and maturation. Moreover, this function might be conserved for CEBPz, because in a bioinformatic analysis nucleolar components and ribosomal proteins were identified as being upregulated in liver and breast tumors with an overexpression of CEBPz. Interestingly, misexpression of some of these targets, like Rpl5 and Rpl35a, have been associated with ribosomopathies, suggesting that mutations in CEBPz could contribute to tumorigenesis in these genetic diseases (Destefanis, 2022).

To better characterize NOC1 functions in vivo, its expression was modified in organs that are relevant for Drosophila physiology, such as the prothoracic gland (PG), the FB and the wing imaginal discs (Destefanis, 2022).

Although the overexpression of NOC1 in the PG does not affect development, its reduction significantly decreased ecdysone production, as shown by E74b mRNA levels. This reduction is significant both at 5 and at 12 days AEL, and occurs concomitantly with the reduction of the PG size. Consequently, NOC1-RNAi animals are developmentally delayed and do not undergo pupariation but rather continue to wander until they die at ~20 days AEL. These animals feed constantly and increase their size, accumulating fats and sugars in the FB cells, which augment their size. Previous work described the presence of hemocytes (macrophage-like cells) infiltrating the FB of these animals, a condition accompanied with an increase in JNK signaling and reactive oxygen species (ROS), likely released by the fat cells under stress conditions. Interestingly, this intercellular event recapitulates the chronic low-grade inflammation, or adipocyte tissue macrophage (ATM), a pathology associated with adipose tissue in obese people that represents the consequence of impaired lipid metabolism (Destefanis, 2022).

Reduction of NOC1, NOC2 or NOC3 in the FB results in smaller and fewer cells, whereas reduction of NOC1 in the whole organ inhibits animal development. The FB regulates animal growth by sensing amino acids concentrations in the hemolymph and remotely controlling the release of DILP2, DILP3 and DILP5 from the IPCs. The FB also stores the nutrients (fats and sugars) that are necessary during the catabolic process of autophagy to allow animals to survive metamorphosis. When nutrients are limited, larvae delay their development to accumulate fats and sugars until reaching their critical size, which ensures they can progress through metamorphosis. NOC1 downregulation in the FB alters its ability to store nutrients, and larvae proceed poorly through development. In addition, these animals show DILP2 accumulation in the IPCs even in normal feeding conditions, indicating that the remote signals responsible for DILP release are greatly reduced, phenocopying animals in starvation or with reduced levels of MYC in fat cells. Interestingly, it was also observed that Cg-NOC1-RNAi animals accumulate an abnormal amount of fats in non-metabolic organs, such as gut, brain and imaginal discs. This finding suggests that these animals are subjected to inter-organ dyslipidemia, a mechanism of lipid transport activated when the FB function is impaired, which triggers non-autonomous signals to induce other organs to store fats. Interestingly, this condition recapitulates dyslipidemia in humans, where the compromised adipose tissue releases lipoproteins of the APO family, inducing fat accumulation in organs. Notably, a similar condition has also been described in flies for mutations in members of the APOE family, outlining how the mechanisms controlling the inter-organ fat metabolism are conserved among species (Destefanis, 2022).

NOC1 depletion in clones analyzed in the wing imaginal discs triggers their elimination by apoptosis. This event is partially rescued when clones are induced in the hypomorphic background of the Minute(3)66D/+ mutation. These cells also upregulate the pro-apoptotic gene Xrp1, recently shown to be responsible for controlling translation and indirectly cell competition upon proteotoxic stress. Reduction of NOC1 in the wing imaginal disc prolongs larval development with upregulation of DILP8 normally induced by cellular damage and apoptosis. The fact that NOC1-RNAi cells upregulate, in addition to Xrp1, eiger, another pro-apoptotic gene and member of the TNFα family, and activate the JNK pathway, suggests that different mechanisms are converging in these cells to induce apoptosis and DILP8 upregulation. Genetic epistasis experiments were performed to define the relationship between Eiger signaling in NOC1-RNAi cells and how this is linked to Xrp1 transcriptional upregulation in response to nucleolar stress and DILP8 upregulation. This analysis showed that reduction of Eiger did not significantly affect DILP8 expression induced upon NOC1 downregulation. Owing to the embryonic lethality induced by the simultaneous reduction of NOC1 and Xrp1 in cells of the wing imaginal discs, using both rotund and nubbin promoters, the contribute of Eiger to Xrp1 and DILP8 transcriptional regulation upon NOC1-RNAi was analyzed. These data indicate that DILP8 upregulation was not significantly affected by the reduction of Eiger seen upon NOC1 reduction, confirming the data in vivo with DILP8-GFP. In addition, it is predicted that Xrp1 acts independently of Eiger, since Xrp1 mRNA upregulation is not rescued in imaginal discs from NOC1-RNAi; eiger-RNAi animals, pointing out to a more upstream role for Xrp1 in controlling the stress response following reduction of NOC1; the function of Eiger remains to be determined (Destefanis, 2022).

In conclusion, the data corroborate the role of NOC1 in mechanisms that induce proteotoxic stress adding NOC1 as a novel component that links defects in protein synthesis with cell competition. This study also showed the relevance of NOC1 in promoting nucleolar stress and apoptosis, both leading cause of tumor formation. The data support a potential role for the human homolog CEBPz in the context of tumorigenesis. Indeed, mutations in CEBPz are described in >1.5% of tumors of epithelial origins, suggesting that it might have a role in contributing to the signals that trigger proteotoxic stress associated to tumorigenesis. CEBPz was also found, together with the METTL3-METTL14 methyltransferase complex, to control cellular growth and to have a role in the regulation of H3K9m3 histone methylation in response to sonication-resistant heterochromatin (srHC), highlighting it as a moonlighting protein for RNA and heterochromatin modifications (Destefanis, 2022).

Wds-Mediated H3K4me3 Modification Regulates Lipid Synthesis and Transport in Drosophila

Lipid homeostasis is essential for insect growth and development. The complex of proteins associated with Set 1 (COMPASS)-catalyzed Histone 3 lysine 4 trimethylation (H3K4me3) epigenetically activates gene transcription and is involved in various biological processes, but the role and molecular mechanism of H3K4me3 modification in lipid homeostasis remains largely unknown. The present study showed in Drosophila that fat body-specific knockdown of will die slowly (Wds) as one of the COMPASS complex components caused a decrease in lipid droplet (LD) size and triglyceride (TG) levels. Mechanistically, Wds-mediated H3K4me3 modification in the fat body targeted several lipogenic genes involved in lipid synthesis and the Lpp gene associated with lipid transport to promote their expressions; the transcription factor heat shock factor (Hsf) could interact with Wds to modulate H3K4me3 modification within the promoters of these targets; and fat body-specific knockdown of Hsf phenocopied the effects of Wds knockdown on lipid homeostasis in the fat body. Moreover, fat body-specific knockdown of Wds or Hsf reduced high-fat diet (HFD)-induced oversized LDs and high TG levels. Altogether, this study reveals that Wds-mediated H3K4me3 modification is required for lipid homeostasis during Drosophila development and provides novel insights into the epigenetic regulation of insect lipid metabolism (Zhao, 2023).

MicroRNA miR-263b-5p Regulates Developmental Growth and Cell Association by Suppressing Laminin A in Drosophila

Basement membranes (BMs) play important roles under various physiological conditions in animals, including ecdysozoans. During development, BMs undergo alterations through diverse intrinsic and extrinsic regulatory mechanisms; however, the full complement of pathways controlling these changes remain unclear. This study found that fat body-overexpression of Drosophila miR-263b, which is highly expressed during the larval-to-pupal transition, resulted in a decrease in the overall size of the larval fat body, and ultimately, in a severe growth defect accompanied by a reduction in cell proliferation and cell size. Interestingly, it was further observed that a large proportion of the larval fat body cells were prematurely disassociated from each other. Moreover, evidence is presented that miR-263b-5p suppresses the main component of BMs, Laminin A (LanA). Through experiments using RNA interference (RNAi) of LanA, it was found that its depletion phenocopied the effects in miR-263b-overexpressing flies. Overall, these findings suggest a potential role for miR-263b in developmental growth and cell association by suppressing LanA expression in the Drosophila fat body (Kim, 2023).

Integrated Stress Response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation

Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. This study used Drosophila melanogaster as a model to establish the Integrated Stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor which instructs oogenesis. Atf4 was shown to regulate the lipase Brummer to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient sensing role for Atf4. This study also discovered that Atf4 promotes secretion of a fat body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, it is posited that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction: directly, by impacting yolk lipoprotein production and follicle maturation, and systemically, by regulating ovulation (Grmai, 2023).

Spenito-dependent metabolic sexual dimorphism intrinsic to fat storage cells

Metabolism in males and females is distinct. Differences are usually linked to sexual reproduction, with circulating signals (e.g. hormones) playing major roles. By contrast, sex differences prior to sexual maturity and intrinsic to individual metabolic tissues are less understood. 'This study analyzed Drosophila melanogaster larvae and find that males store more fat than females, the opposite of the sexual dimorphism in adults. Metabolic differences are intrinsic to the major fat storage tissue, including many differences in the expression of metabolic genes. Previous work identified fat storage roles for Spenito (Nito), a conserved RNA-binding protein and regulator of sex determination. Nito knockdown specifically in the fat storage tissue abolished fat differences between males and females. Nito is required for sex-specific expression of the master regulator of sex determination, Sex-lethal (Sxl). "Feminization" of fat storage cells via tissue-specific overexpression of a Sxl target gene made larvae lean, reduced the fat differences between males and females, and induced female-like metabolic gene expression. Altogether, this study supports a model in which Nito autonomously controls sexual dimorphisms and differential expression of metabolic genes in fat cells in part through its regulation of the sex determination pathway (Diaz, 2023).

Integrating lipid metabolism, pheromone production and perception by Fruitless and Hepatocyte nuclear factor 4

Sexual attraction and perception, governed by separate genetic circuits in different organs, are crucial for mating and reproductive success, yet the mechanisms of how these two aspects are integrated remain unclear. In Drosophila, the male-specific isoform of Fruitless (Fru), Fru (M), is known as a master neuro-regulator of innate courtship behavior to control perception of sex pheromones in sensory neurons. This study shows that the non-sex specific Fru isoform (Fru (COM)) is necessary for pheromone biosynthesis in hepatocyte-like oenocytes for sexual attraction. Loss of Fru (COM) in oenocytes resulted in adults with reduced levels of the cuticular hydrocarbons (CHCs), including sex pheromones; adults showed altered sexual attraction and reduced cuticular hydrophobicity. Hepatocyte nuclear factor 4 (Hnf4) was identified as a key target of Fru (COM) in directing fatty acid conversion to hydrocarbons in adult oenocytes. fru- and Hnf4 -depletion disrupts lipid homeostasis, resulting in a novel sex-dimorphic CHC profile, which differs from doublesex - and transformer -dependent sexual dimorphism of the CHC profile. Thus, Fru couples pheromone perception and production in separate organs for precise coordination of chemosensory communication that ensures efficient mating behavior (Sun, 2023).

Involvement of neuronal tachykinin-like receptor at 86C in Drosophila disc repair via regulation of kynurenine metabolism

Neurons contribute to the regeneration of projected tissues; however, it remains unclear whether they are involved in the non-innervated tissue regeneration. This study shows that a neuronal tachykinin-like receptor at 86C (TkR86C) is required for the repair of non-innervated wing discs in Drosophila. Using a genetic tissue repair system in Drosophila larvae, genetic screening was performed for G protein-coupled receptors to search for signal mediatory systems for remote tissue repair. An evolutionarily conserved neuroinflammatory receptor, TkR86C, was identified as the candidate receptor. Neuron-specific knockdown of TkR86C impaired disc repair without affecting normal development. The humoral metabolites of the kynurenine (Kyn) pathway regulated in the fat body were investigated because of their role as tissue repair-mediating factors. Neuronal knockdown of TkR86C hampered injury-dependent changes in the expression of vermillion in the fat body and humoral Kyn metabolites. These data indicate the involvement of TkR86C neurons upstream of Kyn metabolism in non-autonomous tissue regeneration (Kashio, 2023)

MicroRNA miR-263b-5p Regulates Developmental Growth and Cell Association by Suppressing Laminin A in Drosophila

Basement membranes (BMs) play important roles under various physiological conditions in animals, including ecdysozoans. During development, BMs undergo alterations through diverse intrinsic and extrinsic regulatory mechanisms; however, the full complement of pathways controlling these changes remain unclear. This study found that fat body-overexpression of Drosophila miR-263b, which is highly expressed during the larval-to-pupal transition, resulted in a decrease in the overall size of the larval fat body, and ultimately, in a severe growth defect accompanied by a reduction in cell proliferation and cell size. Interestingly, it was further observed that a large proportion of the larval fat body cells were prematurely disassociated from each other. Moreover, evidence is presented that miR-263b-5p suppresses the main component of BMs, Laminin A (LanA). Through experiments using RNA interference (RNAi) of LanA, it was found that its depletion phenocopied the effects in miR-263b-overexpressing flies. Overall, these findings suggest a potential role for miR-263b in developmental growth and cell association by suppressing LanA expression in the Drosophila fat body (Kim, 2023).

Ecdysone-induced microRNA miR-276a-3p controls developmental growth by targeting the insulin-like receptor in Drosophila

Animal growth is controlled by a variety of external and internal factors during development. The steroid hormone ecdysone plays a critical role in insect development by regulating the expression of various genes. In this study, it was found that fat body-specific expression of miR-276a, an ecdysone-responsive microRNA (miRNA), led to a decrease in the total mass of the larval fat body, resulting in significant growth reduction in Drosophila. Changes in miR-276a expression also affected the proliferation of Drosophila S2 cells. Furthermore, it was found that the insulin-like receptor (InR) is a biologically relevant target gene regulated by miR-276a-3p. In addition, its was found that miR-276a-3p is upregulated by the canonical ecdysone signalling pathway involving the ecdysone receptor and broad complex. A reduction in cell proliferation caused by ecdysone was compromised by blocking miR-276a-3p activity. Thus, these results suggest that miR-276a-3p is involved in ecdysone-mediated growth reduction by controlling InR expression in the insulin signalling pathway (Lee, 2023)

High fat diet-induced TGF-beta/Gbb signaling provokes insulin resistance through the tribbles expression

Hyperglycemia, hyperlipidemia, and insulin resistance are hallmarks of obesity-induced type 2 diabetes, which is often caused by a high-fat diet (HFD). However, the molecular mechanisms underlying HFD-induced insulin resistance have not been elucidated in detail. This study established a Drosophila model to investigate the molecular mechanisms of HFD-induced diabetes. HFD model flies recapitulate mammalian diabetic phenotypes including elevated triglyceride and circulating glucose levels, as well as insulin resistance. Expression of glass bottom boat (gbb), a Drosophila homolog of mammalian transforming growth factor-β (TGF-β), is elevated under HFD conditions. Furthermore, overexpression of gbb in the fat body produced obese and insulin-resistant phenotypes similar to those of HFD-fed flies, whereas inhibition of Gbb signaling significantly ameliorated HFD-induced metabolic phenotypes. tribbles, a negative regulator of AKT, is a target gene of Gbb signaling in the fat body. Overexpression of tribbles in flies in the fat body phenocopied the metabolic defects associated with HFD conditions or Gbb overexpression, whereas tribbles knockdown rescued these metabolic phenotypes. These results indicate that HFD-induced TGF-β/Gbb signaling provokes insulin resistance by increasing tribbles expression (Hong, 2016).

Abnormally high fat mass is a major risk factor for the development of diabetes. Previous studies emphasize that excess adiposity results in abnormal production of cytokines, growth factors, and hormones, which in turn causes secondary diseases like insulin resistance. This study has demonstrated that HFD-induced obesity triggered TGF-β signaling, which downregulates insulin signaling in the fat body. This study also demonstrated the role of tribbles, a novel target of TGF-β/Gbb signaling, in the development of insulin resistance (Hong, 2016).

Drosophila models were used in several recent studies of diet-induced obesity, insulin resistance, hyperglycemia, and hyperinsulinemia. In Drosophila larvae, a high-sugar diet induces type 2 diabetic phenotypes including hyperglycemia, high TG, and insulin resistance. Likewise, in adult flies, HFD feeding also induces high TG and altered glucose metabolism, and in mammals it causes cardiac dysfunctions like diabetic cardiomyopathy. This study has established a Drosophila model of obesity-induced insulin resistance, which has remarkable parallels with the mammalian system, and used it to observe and investigate the development of insulin resistance under chronic over-nutrition conditions. In addition, to study the Drosophila insulin-resistance phenotype in detail, this study has developed an ex vivo culture system (Hong, 2016).

When adult flies were fed a HFD, their short- and long-term metabolic responses were different: for example, expression and secretion of Dilp2 was increased by short-term HFD but decreased by long-term HFD. Insulin signaling, which was assayed by monitoring pAKT activation and expression of the dFOXO target genes d4E-BP and dInR, was activated in short-term but not long-term HFD, whereas TG and trehalose/glucose levels in hemolymph were increased by long-term HFD. Because these pathological phenotypes in flies were very similar to the phenotypes associated with insulin-resistant diabetes in mammals, it is concluded that HFD adult flies can be used as a model of type 2 diabetes (Hong, 2016).

In addition to increasing TG levels, HFD feeding in flies increased the expression of gbb. In mice, inhibition of TGF-β signaling by knockout of Smad3 protects against diet-induced obesity and diabetes. Inhibition of TGF-β signaling may improve adipose function and reverse the effects of obesity on insulin resistance. The TGF-β/Smad3 signaling also plays a key role in adipogenesis. However, it remains unclear how TGF-β signaling is related to the onset of diet-induced obesity and diabetes. This study examined the effects of Drosophila TGF-β family ligands on obesity. Of the genes that were tested, only gbb was upregulated by HFD. Gab regulates lipid metabolism and controls energy homeostasis by responding to nutrient levels (Ballard, 2010); consequently, gbb mutants have extremely low levels of fat in the fat body, resembling a nutrient-deprived phenotype (Ballard, 2010). On the contrary, gbb overexpression increased the TG level, mimicking the effects of nutrient-rich conditions. These data suggest that TGF-β/Gbb signaling is involved in HFD-induced obesity. Indeed, overexpression of gbb in the fat body phenocopied the TG and trehalose/glucose levels in flies fed a HFD. However, Dilp2 expression was increased by gbb overexpression in the fat body, consistent with the effects of short-term but not long-term HFD (Hong, 2016).

Focused was placed on three negative regulators of insulin signaling, PTP1b, PTEN, and tribbles 3 (TRB3), which are involved in insulin resistance in obese mammals. tribbles was upregulated in gbb-overexpressing cells and flies. In mammals, Tribbles encodes an evolutionarily conserved kinase that plays multiple roles in development, tissue homeostasis, and metabolism. A mammalian Tribbles homolog, Tribbles homolog 3 (TRB3), is highly expressed in liver tissue under fasting and diabetic conditions, and inhibits insulin signaling by direct binding to Akt and blocking phosphorylation-dependent Akt activation. Indeed, the expression level of TRB3 is elevated in patients with type 2 diabetes and animal models of this disease. In the systemic sclerosis model, TGF-β signaling can induce mammalian TRB3 and activates TGF-β signaling-mediated fibrosi. Recent work showed that Drosophila tribbles, like mammalian TRB3, inhibits insulin-mediated growth by blocking Akt activation. In this study, tribbles expression was increased in HFD conditions in both mice and flies, as well as in TGF-β-treated human liver cells. tribbles knockdown rescued the diabetic phenotypes caused by HFD, consistent with previous findings in mammals. In addition, tribbles knockdown rescued the diabetic phenotypes caused by gbb overexpression. These data strongly suggest that the evolutionarily conserved tribbles gene is a novel downstream target of Gbb signaling, and that tribbles knockdown rescues diabetic phenotypes in flies. Therefore, future studies should seek to elucidate TGF-β-Trb3 signaling and its functions in mammalian adipocytes; the resultant findings could suggest new strategies for preventing type 2 diabetes (Hong, 2016).

In summary, This study established a Drosophila insulin-resistance model and demonstrated that Gbb signaling in the fat body plays a critical role in obesity-mediated insulin resistance by regulating tribbles expression. These results provide insights regarding the function of Gbb/TGF-β signaling in metabolic disease, and suggest that this pathway represents a promising therapeutic target for treatment of obesity and diabetes (Hong, 2016).

Domeless receptor loss in fat body tissue reverts insulin resistance induced by a high-sugar diet in Drosophila melanogaster

Insulin resistance is a hallmark of type 2 diabetes resulting from the confluence of several factors, including genetic susceptibility, inflammation, and diet. Under this pathophysiological condition, the dysfunction of the adipose tissue triggered by the excess caloric supply promotes the loss of sensitivity to insulin at the local and peripheral level, a process in which different signaling pathways are involved that are part of the metabolic response to the diet. Besides, the dysregulation of insulin signaling is strongly associated with inflammatory processes in which the JAK/STAT pathway plays a central role. To better understand the role of JAK/STAT signaling in the development of insulin resistance, Drosophila melanogaster was used as a type 2 diabetes model generated by the consumption of a high-sugar diet (HSD). In this model, the effects were studied of inhibiting the expression of the JAK/STAT pathway receptor Domeless, in fat body, on adipose metabolism and glycemic control. The results show that the Domeless receptor loss in fat body cells reverses both hyperglycemia and the increase in the expression of the insulin resistance marker Nlaz, observed in larvae fed a high sugar diet. This effect is consistent with a significant reduction in Dilp2 mRNA expression and an increase in body weight compared to wild-type flies fed high sugar diets. Additionally, the loss of Domeless reduced the accumulation of triglycerides in the fat body cells of larvae fed HSD and also significantly increased the lifespan of adult flies. Taken together, the results show that the loss of Domeless in the fat body reverses at least in part the dysmetabolism induced by a high sugar diet in a Drosophila type 2 diabetes model (Lourido, 2021).

Systemic and mitochondrial effects of metabolic inflexibility induced by high fat diet in Drosophila melanogaster

Metabolic inflexibility is a condition that occurs following a nutritional stress which causes blunted fuel switching at the mitochondrial level in response to hormonal and cellular signalling. Linked to obesity and obesity related disorders, chronic exposure to a high fat diet (HFD) in animal models has been extensively used to induce metabolic inflexibility and investigate the development of various metabolic diseases. However, many questions concerning the systemic and mitochondrial responses to metabolic inflexibility remain. This study investigated the global and mitochondrial variations following a 10-day exposure to a HFD in adult Drosophila melanogaster. The results show that following 10-day exposure to the HFD, mitochondrial respiration rates measured in isolated mitochondria at the level of complex I were decreased. This was associated with increased contributions of non-classical providers of electrons to the electron transport system (ETS) such as the proline dehydrogenase (ProDH) and the mitochondrial glycerol-3-phosphate dehydrogenase (mtG3PDH) alleviating complex I dysfunctions, as well as with increased ROS production per molecule of oxygen consumed. These results also show an accumulation of metabolites from multiple different metabolic pathways in whole adult Drosophila and a drastic shift in the lipid profile which translated into decreased proportion of saturated and monounsaturated fatty acids combined with an increased proportion of polyunsaturated fatty acids. Thus, these results demonstrate the various responses to the HFD treatment in adult Drosophila melanogaster that are hallmarks of the development of metabolic inflexibility and reinforce this organism as a suitable model for the study of metabolic disorders (Cormier, 2021)

Identification and characterization of mushroom body neurons that regulate fat storage in Drosophila

Two neuronal populations, c673a and Fru-GAL4, regulate fat storage in fruit flies. Both populations partially overlap with a structure in the insect brain known as the mushroom body (MB), which plays a critical role in memory formation. This overlap prompted an examination of whether the MB is also involved in fat storage homeostasis. Using a variety of transgenic agents, the neural activity of different portions of the MB and associated neurons were selectively manipulated to decipher their roles in fat storage regulation. The data show that silencing of MB neurons that project into the &alpha:'β' lobes decreases de novo fatty acid synthesis and causes leanness, while sustained hyperactivation of the same neurons causes overfeeding and produces obesity. The &alpha:'β' neurons oppose and dominate the fat regulating functions of the c673a and Fru-GAL4 neurons. It was also shown that MB neurons that project into the γ lobe also regulate fat storage, probably because they are a subset of the Fru neurons. It was posible to identify input and output neurons whose activity affects fat storage, feeding, and metabolism. The activity of cholinergic output neurons that innervating the β'2 compartment (MBON-β'2mp and MBON-γ5β'2a) regulates food consumption, while glutamatergic output neurons innervating α' compartments (MBON-γ2α'1 and MBON-α'2) control fat metabolism. This study has identified a new fat storage regulating center, the α'β' lobes of the MB. The study also delineated the neuronal circuits involved in the actions of the α'β' lobes, and showed that food intake and fat metabolism are controlled by separate sets of postsynaptic neurons that are segregated into different output pathways (Al-Anzi, 2018).

Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila

Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. This study introduced RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. Organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, this study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases (Xu, 2019).

Much like mammals, flies have an energy storage tissue called fat body, which functions similar to liver and white adipose tissue. Importantly, the majority of extra energy in mammals and flies is deposited as triacylglycerols (TAGs) in adipose tissue intracellular organelles called Lipid Droplets (LDs). Notably, a number of lipid metabolism effectors and regulators has been found to be conserved from fly to man. One of these regulatory pathways is the store-operated calcium entry (SOCE), a major determinant of the versatile second messenger intracellular Ca2+ (iCa2+). One core component of SOCE is the endoplasmic reticulum (ER) calcium sensor Stromal interaction molecule (Xu, 2019).

In non-excitable cells, the canonical G protein-coupled receptor (GPCR) signaling generates inositol 1,4,5- trisphosphate (IP3) in the cytosol, which stimulates a first wave of Ca2+ release from ER stores to the cytoplasm by activating the calcium channel-receptor IP3R. Stim/STIM1 senses the ER calcium depletion and re-localizes to ER-plasma membrane junctions, where it interacts with plasma membrane calcium channel proteins (called olf186-F in flies and Calcium release-activated calcium channel protein 1 (ORAI1) in mammals) to form active Ca2+ release-activated Ca2+ channels (CRACs), thereby ultimately facilitating the extracellular calcium entry. With this iCa2+ increase, SOCE regulates a plethora of cellular processes over a wide temporal spectrum. Previously work used acute knockdown of Stim in the fat body to identify SOCE as an adiposity regulator in flies. A recent study confirmed that also mammalian STIM1 regulates lipid metabolism in liver and muscle . Moreover, the plasma membrane translocation of STIM1 required for normal SOCE is impaired in hepatocytes of obese mice, which causes further metabolic dysfunction. While fat accumulation in response to acute SOCE interference is well established, the adverse consequences of chronic iCa2+ malfunction are unknown as Stim/STIM loss-of-function is lethal in mice and in flies, while extended tissue-specific Stim knockdown using a temperature-controlled expression system suffers from technical limitations (Xu, 2019).

Therefore, in this study a novel strategy was developed called short-hairpin or double-stranded RNAi Pulse Induction (RiPI) to achieve tissue-specific, chronic knockdown of genes of interest exemplified by Stim in adult fly fat body. The siRNAs generated by a short-term RiPI are long-lasting, which chronically reduces the expression of Stim mRNA in adult fly fat tissue and consequently leads to progressive severe obesity in flies. Evidence is provided that chronic knockdown of Stim not only causes the tissue-autonomous dysfunction of lipid mobilization in response to Adipokinetic hormone (Akh) and reduced insulin signaling in fly fat body, but also remotely controls hyperphagia. The results suggest that chronic dysfunction of Stim/SOCE in the energy storage tissue can dramatically modulate the systemic regulatory network, which drives the organismal energy imbalance (Xu, 2019).

This study presents in vivo evidence for chronic targeted gene knockdown following RiPI in the adult Drosophila fat body. A short pulse induction of shRNA targeting the AkhR gene generates persisting siRNAs, which causes significant down-regulation of AkhR for at least 10 days. Persistence of RNAi has been associated with RNA-dependent RNA polymerase (RdRP)-mediated siRNA amplification in C. elegans and in human cells. In Drosophila, however, it remains controversial whether the genome encodes a functional RdRP. Therefore, slow degradation of the transgene-derived siRNAs might confer the chronic gene knockdown. In fact, RNAi effector double-stranded siRNAs (21nt and 24nt) are more stable than the 18nt double-stranded RNAs in the human cytosolic extract. Moreover, in human HEK293T cells, the anti-sense strand of siRNA is more resistant to intracellular nucleases compared to the sense strand of the siRNA duplex, which is likely due to the incorporation of anti-sense siRNAs into the activated RNA induced silencing complex (RISC). Therefore, the involvement of RISC might allow the slow degradation of siRNAs in adult Drosophila fat body cells. The slow decline of the siRNA level is apparently sufficient to chronically knockdown the endogenous gene expression of AkhR and Stim, which causes progressive body fat increase. This mode of action is further supported by the fact that pulsed overexpression of RNAi resistant Stim-mRNA only transiently rescues the fat content increase due to Stim-TRiPI. Consistently, long-term gene silencing (at least 11 days) is also observed in adult flies after injection of low concentrations of dsRNAs. Similarly, in an EGFP-transgenic mouse model, the inhibition of the reporter expression lasts as long as two months after siRNA injection. In summary, this study shows that in vivo RiPI generates long-lasting RNAi, which allows chronic knockdown of target genes in a tissue-specific manner. It is proposed that RiPI is a versatile tool to study causative relationships and temporal sequences in inter-organ communication processes (Xu, 2019).

Using RiPI, this study has established a Drosophila obesity model based on chronic, adipose tissue-directed knockdown of Stim, which shares remarkable similarity to characteristics of human obesity. First, the visibly enlarged abdomen of the obese flies corresponds to increased waist circumference, which gains importance as meaningful parameter to assess android adiposity. Similarly, body fat accumulation causes significant weight gain, another readout to quantify obesity in rodents and human. Second, the excessive fat accumulation correlates with climbing deficits of the obese flies, with physical fitness reduction being another hallmark of human adiposity. Moreover, obese Stim-TRiPI flies have reduced life span, which is reminiscent of the higher mortality rates in human obesity patients. Third, this study demonstrates that early-onset hyperphagia drives the positive energy balance in Stim-TRiPI flies. Consistently, increased food intake is the major driver of human obesity. Hyperphagia is linked to increased dietary glucose conversion into storage fat in obese Stim-TRiPI flies. Notably, increased food intake and elevated glucose conversion into storage lipids has also been reported after silencing obesity blocking neurons in the fly central brain. With hyperphagia being an important contributor, obesity development in Stim-RiPI flies is not monocausal. It is noteworthy that the rise in fat storage in Stim-DRiPI substantially exceeds the food intake increase. Moreover, matching the food intake of Stim-TRiPI On and Off flies still results in body fat accumulation. Importantly, there is a significantly reduced metabolic rate of Stim-DRiPI flies. Finally, the observed hyperglycemia at day 10, physical fitness reduction at day 24 and shortened life span of Stim-TRiPI On flies are associated with obesity development, similar to type 2 diabetes (T2D), exercise intolerance and mortality, which are also highly correlated with human obesity. In summary, chronic knockdown of Stim in the adult fat body causes fly obesity by a number of physiological factors culminating in organismal energy imbalance similar to mammalian adiposity (Xu, 2019).

This study highlights the critical roles played by Stim in interaction with Akh/AkhR signaling and insulin signaling in the fly fat body tissue. Reduced expression of Mdh1 and Gprk2 suggests impaired Akh/AkhR signaling in the fat body of Stim-TRiPI flies. Mammalian MDH1 has been linked to glycolysis in cells with mitochondrial dysfunction, obese Stim-TRiPI On flies display normal glycogen storage and mobilization during starvation. Similar findings are also observed in AkhA, AkhAP, and AkhR1 mutant fly larvae and adult flies, albeit their capability to mobilize glycogen is weakly impaired. A possible explanation is that flies employ corazonin, a starvation-responsive pathway complementary to Akh, to utilize glycogen. In addition to storage glycogen, the reduced expression of genes involved in lipolysis predicts an impairment of starvation-induced storage lipid mobilization. Indeed, obese Stim-TRiPI flies display an abnormal lipid mobilization profile under starvation and die with residual fat resources. Similarly, impaired lipid mobilization is also observed in flies with loss-of-function mutation in the TAG lipase gene bmm or in flies lacking either InsP3R or AkhR. Consistently, loss-of-function of STIM1/2 in mammalian cells, also impairs lipolysis via down-regulation of cAMP. Moreover, decreased catecholamine-stimulated lipolysis has been identified in human obese individuals. Collectively, thw results show that fat body tissue of obese Stim-RiPI On flies is resistant in response to Akh signaling, which drives the obesity development (Xu, 2019).

Moreover, this study supports the possibility to model T2D in adult flies. Obese Stim-TRiPI flies show reduced expression of the glucose clearance gene Hex-C, whose mammalian homolog was also suppressed in T2D patients. Besides, evidence is provided to support that obese Stim-TRiPI flies have hyperglycemia, impairment of insulin signaling in fat body tissue, and larger lipid droplets. Similar features were also described in fly larvae reared on high sugar diet36,87, which resemble mammalian insulin resistance88. Regarding unchanged circulating dIlp-2 level in obese Stim-DRiPI flies, insulin-like peptide secretion might be interfered by the knockdown of Stim in the insulin producing cells of Stim-DRiPI flies mediated by ubiquitous driver daGS, more investigation on circulating insulin levels of obese Stim-DRiPI flies by specific driver needs to be done in future. Interestingly, the indicators of insulin signaling impairment mentioned above occur at later stage of Stim-RiPI obesity development, and accordingly are possibly the consequence of Stim-TRiPI On mediated-fat gain, which also supports the concept that obesity compromises insulin signaling (Xu, 2019).

Apart from the specific role of the fat body in storage lipid handling and glucose clearance, this study shows that chronic Knockdown of Stim in this organ remotely promotes Akh secretion from the fly CC neuroendocrine cells, which leads to hyperphagia. RNAseq and gene expression analysis indicate a list of genes encoding candidate hormone or secreted proteins. Among them, CCHa2, daw, and Lst has been shown to function as hormones to regulate insulin-like peptide secretion. In addition, CCHa2, daw, Lst are also regulated by Akh overexpression in opposite direction. Whether differential expression of these genes mentioned above mediate the (mis)communication between the fat body and the CC cells is currently unknown. Nevertheless, the communication between the fat body and the CC cells is essential for the food intake increase as well as further obesity development induced by long-term knockdown of Stim. Interestingly, a study provided evidence that muscle tissue in flies communicates with the CC cells to control Akh secretion via the myokine Unpaired2 (Upd2). Upd2 had been previously shown to act as adipokine, which signals the fed state from the fat body. Unlike mammalian leptin, Upd2 remotely acts on insulin-producing cells in the central brain to regulate insulin secretion but not food intake. Recently, Akh mRNA expression was shown to be regulated by a gut-neuronal relay via midgut-secreted peptide Buriscon α in response to nutrients. Given the fact that the transcription of Akh is unaffected in Stim-RiPI On flies, identification of the adipokine, which regulates the Akh release directly or indirectly to affect food intake in the Stim-RiPI fly obesity model requires future research efforts (Xu, 2019).

In conclusion, this work introduces RNAi Pulse Induction as a novel in vivo paradigm for chronic, tissue-specific gene interference. RiPI makes essential genes accessible to long-term functional analysis in the adult fly, as exemplified in this study by establishing a Drosophila obesity model caused by chronic knockdown of Stim in the adult fat body. Moreover, this study reveals, that the fat body integrates the tissue-autonomous and the systemic branches of Akh signalling: by regulation of lipid mobilization via SOCE in the fat body, and possibly by remote-control of Akh secretion from the CC cells. Recently, the evolutionarily conserved role of SOCE in controlling energy metabolism has attracted the interest of mammalian studies. While Akh is structurally not conserved to humans, there is a growing number of remotely-controlled orexigenic peptide hormones in mammals with asprosin being one of the latest additions. Collectively, these findings in the fly add further evidence to the existence of conserved regulatory principles in animal energy homeostasis control emanating from SOCE signalling in fat storage tissues (Xu, 2019).

Endocrine signals fine-tune daily activity patterns in Drosophila

Animals need to balance competitive behaviors to maintain internal homeostasis. The underlying mechanisms are complex but typically involve neuroendocrine signaling. Using Drosophila, this study systematically manipulated signaling between energy-mobilizing endocrine cells producing adipokinetic hormone (AKH), octopaminergic neurons, and the energy-storing fat body to assess whether this neuroendocrine axis involved in starvation-induced hyperactivity also balances activity levels under ad libitum access to food. The results suggest that AKH signals via two divergent pathways that are mutually competitive in terms of activity and rest. AKH increases activity via the octopaminergic system during the day, while it prevents high activity levels during the night by signaling to the fat body. This regulation involves feedback signaling from octopaminergic neurons to AKH-producing cells (APCs). APCs are known to integrate a multitude of metabolic and endocrine signals. The results add a new facet to the versatile regulatory functions of APCs by showing that their output contributes to shape the daily activity pattern under ad libitum access to food (Pauls, 2021).

MRT, functioning with NURF complex, regulates lipid droplet size

Lipid droplets (LDs) are highly dynamic organelles that store neutral lipids. Through a gene overexpression screen in the Drosophila larval fat body, this study has identified that MRT, an Myb/switching-defective protein 3 (Swi3), Adaptor 2 (Ada2), Nuclear receptor co-repressor (N-CoR), Transcription factor (TF)IIIB (SANT)-like DNA-binding domain-containing protein, regulates LD size and lipid storage. MRT directly interacts with, and is functionally dependent on, the PZG and NURF chromatin-remodeling complex components. MRT binds to the promoter of plin1, the gene encoding the LD-resident protein perilipin, and inhibits the transcription of plin1. In vitro LD coalescence assays suggest that mrt overexpression or loss of plin1 function facilitates LD coalescence. These findings suggest that MRT functions together with chromatin-remodeling factors to regulate LD size, likely through the transcriptional repression of plin1 (Yao, 2018).

Lipid droplets (LDs) are widely distributed and highly evolutionarily conserved organelles that comprise a phospholipid monolayer, a neutral lipid core, and numerous LD-associated proteins. The size of LDs varies greatly in different kinds of cells and even in the same cell type under different physiological conditions. Adipocyte hypertrophy, characterized by increased LD size, has been proved to be the major mechanism that induces the expansion of adipose tissue in obese individuals (Yao, 2018).

The size of LDs is regulated by several distinct processes such as targeted delivery of neutral lipids from the endoplasmic reticulum (ER) to LDs via LD-ER bridges, local triglyceride (TAG) synthesis by LDs, atypical LD fusion, and LD coalescence. Numerous key factors in these processes, including proteins and phospholipids, have been identified. Perilipin1 (PLIN1), the first protein that was identified on LDs, is important for fat mobilization and regulates lipolysis in both mammals and flies. PLIN1 is post-translationally phosphorylated by protein kinase A (PKA) after β-adrenergic stimulation, which is essential for its function in regulating lipolysis. In mice, PLIN1 also mediates atypical fusion of LDs by activating FSP27. Transcriptionally, the Plin1 gene is activated by peroxisome proliferator-activated receptor-γ (PPARγ) and suppressed by liver X receptor-α (LXR-α). Whole-genome loss-of-function screens and functional studies in Caenorhabditis elegans and Drosophila have also identified many genes involved in lipid storage and LD size regulation. These findings have made significant contributions to understanding the mechanism of LD size regulation. However, the mechanisms that regulate and functionally integrate the activity of these genes under different physiological and pathological conditions need to be further explored (Yao, 2018).

Chromatin remodeling is an essential process for transcriptional regulation. The chromatin structure is rearranged to allow transcriptional regulatory proteins to access DNA that is wrapped around histones. Chromatin remodeling and nucleosome occupancy changes are associated with adipocyte differentiation and lipid homeostasis. In addition, histone modification changes are important for adipocyte differentiation, lipogenesis, and hepatic steatosis. However, the specific roles of chromatin remodeling in LD size regulation have not yet been fully explored (Yao, 2018).

Through a genetic gain-of-function screen in Drosophila, this study identified an LD size regulator, MRT, which promotes LD coalescence. The regulatory effect of MRT on LD size requires the MRT-interacting proteins PZG and components of the nucleosome remodeling factor (NURF) complex. Together, these findings reveal that an MRT-PZG-NURF axis regulates the size of LDs (Yao, 2018).

The LD coalescence process regulated by MRT and PLIN1 displays some interesting features. First, LDs were isolated from Drosophila larval fat bodies, and the LDs were kept in PBS buffer without adding ATP. Therefore, the LD coalescence process does not rely on an external energy supply. Second, unlike in the gradual LD fusion process induced by proteins, lipid transfer between LDs was not noticed during their contact time in most cases, and coalescence happened instantaneously. Third, the paired LDs sometimes separated, indicating that the LD-LD contact is not as stable as the protein-mediated LD-LD contact. Lastly, coalescence does not obviously depend on LD size or the size ratio of the LD pair. All of these features are quite consistent with the traits of LD coalescence triggered by membrane instability (Yao, 2018).

The elevated LD coalescence rate may result from increased levels of phosphatidic acid (PA) or phosphatidylethanolamine (PE) and reduced levels of phosphatidylcholine (PC). mrt overexpression reduces the PC content in the fat body, which is consistent with the decreased mRNA levels of ck and Cct1 that are important for PC synthesis. However, overexpression of Cct1 does not suppress the large LD phenotype caused by mrt overexpression. This suggests that either the reduced level of Cct1 is not the main reason for the formation of large LDs or that other factors are also required. Along the same lines, it remains to be addressed whether there is a link between Drosophila PLIN1 and the levels of PA or PC (Yao, 2018).

The transcriptional regulation of lipid metabolism, including adipogenesis, lipogenesis, and fatty acid oxidation, is being intensively studied. Compared to other aspects of lipid metabolism, the mechanisms for transcriptional regulation of LD size have been reported in only a few cases. PPARs and LXR-α transcriptionally regulate the expression of perilipin genes. In C. elegans, DAF-12 (homolog of vitamin D receptor [VDR]/LXR) promotes the thermosensitive formation of supersized LDs. In Drosophila, loss of function of TATA-binding protein [TBP]-related factor 2 (TRF2) significantly increases LD size by affecting the transcription of genes related to peroxisomal fatty acid β-oxidation. Interestingly, PZG has also been identified in the TRF2-containing complex. It appears to be contradictory that two complexes, TRF2-PZG and MRT-PZG, which share the same component, have opposite effects on LD size. It is possible that PZG negatively regulates TRF2 in LD size regulation. Alternatively, because PZG has TRF2-dependent and TRF2-independent functions, it is also possible that PZG is not required for TRF2-dependent LD size regulation or that the TRF2-PZG and MRT-PZG complexes have totally different targets in LD size regulation (Yao, 2018).

As the determinants of the accessibility of transcription factors to their target promoters, chromatin-remodeling factors also regulate lipid metabolism. Both ATP-dependent chromatin-remodeling factors and histone modification factors contribute to lipid homeostasis in mammals. As subunits of the SWI/SNF complexes, BAF60a and BAF60c control hepatic lipid metabolism by activating the transcription of fatty acid oxidation genes or lipogenic genes in response to different nutrient conditions and hormone signals. Other studies have shown that histone deacetylases, including HDAC1 and HDAC3, are implicated in regulating adipocyte differentiation, lipogenesis, and hepatic steatosis (Yao, 2018).

This study showed that MRT and its partner proteins the PZG and NURF complex components regulate LD size. MRT, with the help of the PZG and NURF complex, likely represses the transcription of its downstream targets to regulate LD size. plin1 is probably one of many MRT target genes, and further genomic and transcriptomic approaches may provide a global view of MRT-mediated transcription regulation. The relatively mild loss-of-function phenotype of mrt indicates that MRT, together with chromatin-remodeling complexes, likely modulates or balances the accessibility of promoters of LD size regulators, such as plin1. Moreover, the NURF complex belongs to the ISWI chromatin-remodeling family, and besides ISWI, there are three other ATP-dependent chromatin-remodeling families: SWI/SNF, CHD, and INO80. It remains to be determined whether other chromatin-remodeling complexes participate in LD size regulation and how they are functionally related to the MRT-PZG-NURF axis (Yao, 2018).

The current findings suggest that a balance between 'open' and 'closed' chromatin states is important for the transcriptional regulation of key LD size regulators. The MRT-PZG-NURF axis is involved in the regulation of key factors such as PLIN1 through the 'closed' state. Identifying the regulators of the 'open' state will ultimately reveal the full picture of transcriptional regulation of LD size regulation at the chromatin level (Yao, 2018).

NF-kappaB shapes metabolic adaptation by attenuating Foxo-mediated lipolysis in Drosophila

Metabolic and innate immune signaling pathways have co-evolved to elicit coordinated responses. However, dissecting the integration of these ancient signaling mechanisms remains a challenge. Using Drosophila, this study uncovered a role for the innate immune transcription factor nuclear factor kappaB (NF-kappaB)/Relish in governing lipid metabolism during metabolic adaptation to fasting. Relish in fat bodies was found to be required to restrain fasting-induced lipolysis, and thus conserve cellular triglyceride levels during metabolic adaptation, through specific repression of ATGL/Brummer lipase gene expression in adipose tissue (fat body). Fasting-induced changes in Brummer expression and, consequently, triglyceride metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, a critical metabolic transcription factor. Relish limits Foxo function by influencing fasting-dependent histone deacetylation and subsequent chromatin modifications within the Bmm locus. These results highlight that the antagonism of Relish and Foxo functions are crucial in the regulation of lipid metabolism during metabolic adaptation, which may further influence the coordination of innate immune-metabolic responses (Molaei, 2019).

In order to explore mechanistic connections between NF-κB and various metabolic control networks, lipid homeostasis was assessed in Drosophila lacking functional Relish (utilizing the relE20 allele) independent of pathogenic infection. Relish is similar to mammalian p100/p105 NF-κB proteins and contains a Rel-homology domain, as well as ankyrin repeats (found in mammalian inhibitory IκBs). During ad libitum feeding, NF-κB/Rel mutant adult female flies (relE20/relE20) have significantly less organismal TAGs than genetically matched controls (either OreR or relE20 / + heterozygote flies, 7 days old post eclosion). However, these changes in TAG correlated with decreases in acute and chronic feeding and can be rescued by high-calorie (sugar) diets, suggesting that steady-state differences in lipid homeostasis are potentially driven by changes in feeding behavior. Assaying the major fat storage tissues, this study also found that TAG-level reduction in mutant animals correlates with strong, but variable, decreases in neutral lipid content in fat body and/or adipose but not in the intestine (Molaei, 2019).

Since ad libitum effects on lipid homeostasis appear to correspond with feeding deficits (i.e., they are potentially indirect), changes in fat metabolism were assayed in Relish mutant animals during metabolic adaptation to fasting. relE20/relE20 mutant flies were sensitive to starvation. Furthermore, Relish-deficient animals displayed accelerated decreases in organismal TAG levels during acute fasting (at time points before significant death occurred), while controls flies showed little to no change at the same time points (always comparing within sibling genotypes). These changes in TAG levels correlate with a strong reduction of stored neutral lipids and/or lipid droplets in carcass fat body, suggesting that there is enhanced or accelerated lipid breakdown during metabolic adaptation in these animals (Molaei, 2019).

The insect fat body acts as a key sensor to link nutrient status and energy expenditure and, as such, is the major lipid depository (mainly TAGs) that combines energy storage, de novo synthesis, and breakdown functions of vertebrate adipose and hepatic tissues. This tissue is also essential for Toll- and Relish-mediated innate immune responses to bacterial infection. Critically, the fat body is integral to properly balance lipid catabolism and anabolism in order to modulate organismal energy homeostasis (through lipid supply to other tissues) in response to metabolic or dietary adaptation. Expression of full-length Relish in the fat body (CGGal4>UAS-Rel) can rescue reduced starvation survival rates and the accelerated loss of lipid storage in relE20/relE20 mutant flies during fasting. These data suggest that Relish function in the fat body is required to acutely maintain lipid homeostasis throughout the course of metabolic adaptation (Molaei, 2019).

To further confirm an autonomous and potentially direct role for Relish in the regulation of fasting-mediated changes in lipid metabolism, Relish was specifically in the fat body (using multiple, independent RNAi lines: named UAS-RelRNAi KK and GD). Attenuating Relish in the fat body of female flies (CGGal4>UAS-RelRNAi) leads to starvation sensitivity, as well as accelerated loss of organismal TAG levels and fat body lipid storage in response to fasting; additional RNAi control experiments can be found in Figures S1F-S1J. As expected, fasting-induced changes in fat body lipid storage occur before significant decreases in total TAG levels of whole animals are observed. Phenotypes were confirmed with an independent fat body driver (PplGal4; Figures S2A-S2C), and similar results were found utilizing males but not when utilizing another immune cell (hemocyte) driver (HmlGal4). Conversely, over-expressing full-length Relish (CGGal4>UAS-Rel) or a constitutively active N-terminal fragment (CGGal4>UAS-Rel.68) in the fat body significantly limits fasting-mediated decreases in lipids compared to controls (Molaei, 2019).

Furthermore, attenuation of upstream components of the Relish signaling pathway phenocopies these Relish loss-of-function effects on lipid metabolism during metabolic adaption. Relish is governed by conserved regulators TAK1 and the IKK (IκB Kinase) signalosome (which consists of homologs of both IKKβ [Drosophila Ird5] and IKKgamma [Drosophila Kenny (key)]), while the apical caspase DREDD is required for the proteolytic cleavage of the IkB domain, allowing for nuclear translocation. Inhibiting Kenny or DREDD in the fat body of female flies (CGGal4>UAS-DREDDRNAior KeyRNAi) leads to starvation sensitivity, as well as accelerated loss of organismal TAG levels and fat body lipid storage in response to fasting (compared to control flies (CGGal4>w1118). Similarly, attenuating upstream receptors usually required for NF-κB/Relish activation (PGRP family members PGRP-LC [trans-membrane] or PGRP-LE ) also leads to decreased lipid storage in the fat body after starvation, suggesting that at least part of the canonical innate immune pathway is required for these metabolic phenotypes (Molaei, 2019).

Taken together, these data show that Relish can autonomously regulate lipid metabolism in the fat body during metabolic adaptation and suggest that Relish may direct specific metabolic responses to control the breakdown of TAGs (Molaei, 2019).

Properly balancing energy homeostasis in response to metabolic adaptation depends on the ability to coordinate storage, breakdown, and mobilization of lipids, primarily TAG. This coordination requires precise control of metabolic response networks, including changes in metabolic gene expression. To determine potential mechanisms by which the Relish transcription factor could direct cellular TAG metabolism during fasting, transcriptional changes of various metabolic genes related to lipid catabolism or anabolism were measured in Relish-deficient animals. Specifically, the lipase Brummer (Bmm) was identified as being regulated by Relish. Bmm is the Drosophila homolog of mammalian adipose TAG lipase (ATGL), an enzyme that is critical for lipolysis. Bmm plays an essential and conserved role in TAG breakdown and, subsequently, fatty acid mobilization, from lipid droplets in fat storage tissues during metabolic adaptation. In control flies, bmm transcription is mildly induced during acute fasting, but in relE20/relE20 mutant flies, bmm expression is strongly up-regulated (from whole flies). These Relish-dependent changes in bmm transcription appear unique, as Relish deficiency does not impact fasting-induced changes in other lipases such as Drosophila hormone-sensitive lipase (dHSL), Drosophila lipase 4 (dlip4), or CG5966. Similar results were found in dissected fat body with specific attenuation of NF-κB/Relish in the same tissue (CGGal4>UAS-RelRNAi KK). These results suggest that Relish function is required to repress or limit Bmm expression in response to metabolic adaptation and subsequently restrain TAG breakdown (Molaei, 2019).

To correlate this difference in gene expression to differences in lipolysis, an assay was employed to measure dynamic changes in lipid content based on the incorporation of radiolabeled glucose (14C-glucose) into lipids during fatty acid synthesis in vivo. After acute feeding (1.5 h) of a diet containing 14C-glucose, Relish-mutant flies show drastic changes in glucose incorporation (synthesis) that is likely due to changes in feeding behavior. 16 h of feeding minimized these differences in synthesis, and subsequent analysis of newly synthesized 14C-labeled lipids during fasting showed an increased rate of breakdown in relE20/relE20mutant flies (47% in mutants compared to 20% in controls). This change in the rate of breakdown correlated with increases in free fatty acids. Finally, genetically attenuating Bmm lipase in the fat body (CGGal4>UAS-BmmRNAi) can rescue the accelerated loss of TAGs in relE20/relE20 mutant flies during fasting (Molaei, 2019).

These data collectively reveal that Relish function is required to limit fasting-induced Bmm gene expression and subsequently restrain TAG lipolysis during metabolic adaptation (Molaei, 2019).

Following these results, the mechanism by which the Relish transcription factor can context-dependently attenuate Bmm expression was explored. Utilizing Cis-element OVERrepresentation (Clover) software, this study identified conserved NF-κB DNA binding motifs (κB sequence sites identified as GGG R N YYYYY) throughout the first intron of the Bmm locus. To assess binding, a previously characterized Relish antibody was used to perform chromatin immunoprecipitation (ChIP)-qPCR experiments. Relish binding in fed or fasted wild-type flies is significantly enriched (compared to immunoprecipitations using serum controls) at binding motif(s) approximately 1 kb downstream from the transcriptional start site. This putative Bmm regulatory region was cloned upstream of RFP (red fluorescent protein) in order to generate in vivo expression reporters. While the unaltered region only slightly influenced RFP reporter activity in fed or fasted conditions, eliminating the Relish binding site leads to minimal enhanced reporter activity under fed conditions and strong increases in RFP activity during fasting (primarily in the fat body of the carcass and head). Thus, this Relish-binding site within the first Bmm intron acts as an important regulatory region to limit induced gene expression (Molaei, 2019).

Relish binding at this region is similar in fed and fasted states. No evidence was found of classical Relish transcriptional activation function during acute fasting. First, innate immune target gene expression (Drosomycin and Diptericin) and Relish DNA binding to innate immune gene promoters (Diptericin) were not changed during fasting. Second, metabolic adaptation did not significantly alter nuclear localization of Relish in the fat body. Thus, in order to explore how Relish limits or represses fasting-induced Bmm expression, despite its constitutive binding to DNA and distinct from its transcriptional activation function, histone and/or chromatin changes were assessed in Relish-deficient flies. Histone deacetylases (HDACs) have been shown to accumulate in the nucleus during metabolic adaptation, influencing gene expression in a fasting-dependent manner through chromatin regulation and transcription factor deacetylation. Furthermore, previous studies have linked interactions of NF-κB transcription factors and HDACs with NF-κB-dependent transcriptional repression. It was thus hypothesized that Relish might repress Bmm gene expression through influencing histone modifications during fasting, when histone modifiers (such as HDACs) in the nucleus are elevated. Using ChIP-qPCR, histone 3 lysine 9 acetylation (H3K9ac, a post-translational modification generally associated with transcriptional activation) was monitored at this Bmm regulatory region in Relish-deficient animals and controls. During feeding, there is no change in H3K9ac enrichment at this locus between genotypes. However, during fasting relE20/relE20 mutant flies display a significant enrichment (compared to controls) of H3K9ac at the site of Relish binding, indicative of promoter or enhancer activation. Analysis of modEncode ChIP sequencing databases associated with histone modifications (in adult female flies) also revealed that this site is generally enriched for other modifications linked to gene expression regulation (such as H3K27ac, H3K4me3, and H3K4me1), further indicating that this locus is an important regulatory region. Additionally, inhibiting a single HDAC in the fat body (Rpd3 [Drosophila HDAC1]; CGGal4>UAS-Rpd3RNAi) can drive small, but significant, increases in fasting-induced Bmm transcription (from whole flies; Figure 3D) and accelerate fat body lipid usage (Molaei, 2019).

Taken together, these data show that Relish can bind to a putative regulatory region within the Bmm locus during both feeding and fasting. In response to fasting, the presence of Relish can influence fasting-dependent histone acetylation and chromatin changes that are consistent with transcriptional repression (Molaei, 2019).

The unique ability of Relish to limit or repress fasting-induced Bmm transcription correlates with attenuation of H3K9ac at Bmm regulatory regions. It was thus hypothesized that Relish binding to the Bmm locus leads to fasting-dependent chromatin changes, which subsequently limit transcription activation function of other factors that are induced during metabolic adaptation. Various metabolic transcription factors were assessed and Foxo, a critical regulator of lipolysis and catabolism in general, was shown to be required for Relish-dependent changes in ATGL/Bmm expression during metabolic adaptation. Firstly, Foxo (of which there is a single ortholog in Drosophila) is activated during metabolic adaptation and required for fasting-induced ATGL/Bmm expression across taxa, including in the fly fat body. Full Relish/Foxo double mutant animals (using the foxo24 allele) are synthetic lethal during metamorphosis (relE20, foxo24/relE20). However, simply reducing Foxo gene dose in NF-κB/Relish mutant flies (relE20, foxo24/relE20) completely rescues fasting-dependent increases in Bmm expression, starvation survival rates, and increases in lipolysis (accelerated loss of lipid storage) in Relish-deficient flies during metabolic adaptation. Molecular analysis of Foxo transcription activation function also showed that Foxo binding to the Bmm promoter is slightly, but significantly, elevated in Relish-deficient flies only during fasting. Furthermore, attenuating Foxo specifically in the fat body (CGGal4>UAS-FoxoRNAi) rescues the enhanced depletion of lipid storage and starvation sensitivity associated with relE20/relE20 mutant flies during fasting. Foxo transcription activation function is thus required for Relish-dependent changes in lipid metabolism, highlighting that Relish/Foxo integration and antagonism is critical to maintain TAG metabolism throughout the course of metabolic adaptation (Molaei, 2019).

In summary, this study has uncovered a role for the innate immune transcription factor Relish in governing lipid metabolism during metabolic adaptation to fasting utilizing Drosophila. Relish is required to restrain fasting-induced lipolysis and thus conserve cellular TAG levels and promote survival during metabolic adaptation, through specific repression of Bmm lipase gene induction in the fat body/adipose. Fasting-induced changes in Bmm expression and TAG metabolism are adjusted by Relish-dependent attenuation of Foxo transcriptional activation function, likely through regulation of histone acetylation (Molaei, 2019).

These findings thus suggest that association of Relish with histone modifiers (and subsequent changes in chromatin accessibility at gene regulatory regions) functions to control or limit the induced level of certain metabolic genes. Indeed, a few previous studies have highlighted that mammalian p65/RelA or Drosophila Relish can negatively regulate gene expression, including innate immune targets, through changes in chromatin. This modification of chromatin and repression of gene expression likely occurs through recruitment of HDACs (HDAC 1/2) to promoter or enhancer regions of target genes, thus influencing histone acetylation, chromatin remodeling, and function of NF-κB itself or other transcriptional activators. This study shows that Relish can bind a metabolic target gene during both feeding and fasting but that uniquely during fasting, it can influence H3K9 acetylation levels at this target gene and subsequently limit transcriptional activation function of positive regulators (such as Foxo). This allows NF-κB transcription factors precise control of specific metabolic gene expression, potentially through metabolic changes in HDAC nuclear localization, in a context-dependent manner. To this end, this study has uncovered other metabolic target genes of Relish (in unique tissues with unique metabolic functions) regulated through similar mechanisms (Molaei, 2019).

It remains unclear whether Relish binding to metabolic target genes in fed states, independent of metabolic stress, can influence gene expression and physiology. However, this may represent a constitutive or steady-state function of NF-κB (described in mammals) and thus could act as a priming mechanism that promotes the maintenance of metabolic homeostasis during acute stress (such as fasting) (Molaei, 2019).

While these data show that NF-κB can direct lipid metabolism independent of infection, Foxo (as well as HDACs) is integrated with innate immune responses through a variety of mechanisms. Additional experimental evidence suggests that Relish and, perhaps, Relish-Foxo antagonism and Bmm gene regulation manipulate TAG metabolism after a chronic systemic bacterial infection. It is thus possible that NF-κB-dependent regulation of TAG catabolism, by limiting Foxo-mediated lipolysis, also plays a role in governing innate immune homeostasis in response to pathogenic infections (Molaei, 2019).

Dietary cysteine drives body fat loss via FMRFamide signaling in Drosophila and mouse

Obesity imposes a global health threat and calls for safe and effective therapeutic options. This study found that protein-rich diet significantly reduced body fat storage in fruit flies, which was largely attributed to dietary cysteine intake. Mechanistically, dietary cysteine increased the production of a neuropeptide FMRFamide (FMRFa). Enhanced FMRFa activity simultaneously promoted energy expenditure and suppressed food intake through its cognate receptor (FMRFaR), both contributing to the fat loss effect. In the fat body, FMRFa signaling promoted lipolysis by increasing PKA and lipase activity. In sweet-sensing gustatory neurons, FMRFa signaling suppressed appetitive perception and hence food intake. This study also demonstrated that dietary cysteine worked in a similar way in mice via neuropeptide FF (NPFF) signaling, a mammalian RFamide peptide. In addition, dietary cysteine or FMRFa/NPFF administration provided protective effect against metabolic stress in flies and mice without xal abnormalities. Therefore, this study reveals a novel target for the development of safe and effective therapies against obesity and related metabolic diseases (Song, 2023).

Galphaq, Ggamma1 and Plc21C control Drosophila body fat storage

Adaptive mobilization of body fat is essential for energy homeostasis in animals. In insects, the adipokinetic hormone (Akh) systemically controls body fat mobilization. Biochemical evidence supports that Akh signals via a G protein-coupled receptor (GPCR) called Akh receptor (AkhR) using cyclic-AMP (cAMP) and Ca(2+) second messengers to induce storage lipid release from fat body cells. Recently, genetic evidence has been provided that the intracellular calcium [iCa(2+)] level in fat storage cells controls adiposity in Drosophila. However, little is known about the genes which mediate Akh signalling downstream of the AkhR to regulate changes in iCa(2+). This study used thermogenetics to provide in vivo evidence that the GPCR signal transducers G protein alpha q subunit (Galphaq), G protein gamma1 (Ggamma1) and Phospholipase C at 21C (Plc21C) control cellular and organismal fat storage in Drosophila. Transgenic modulation of Galphaq, Ggamma1 and Plc21C affected the iCa(2+) of fat body cells and the expression profile of the lipid metabolism effector genes midway and brummer resulting in severely obese or lean flies. Moreover, functional impairment of Galphaq, Ggamma1 and Plc21C antagonised Akh-induced fat depletion. This study characterizes Galphaq, Ggamma1 and Plc21C as anti-obesity genes and supports the model that Akh employs the Galphaq/Ggamma1/Plc21C module of iCa(2+) control to regulate lipid mobilization in adult Drosophila (Baumbach, 2014).

The regulation of triglyceride storage by ornithine decarboxylase (Odc1) in Drosophila

Polyamines are low molecular weight, organic cations that play a critical role in many major cellular processes including cell cycle regulation and apoptosis, cellular division, tissue proliferation, and cellular differentiation; however, the functions of polyamines in regulating the storage of metabolic fuels such as triglycerides and glycogen is poorly understood. To address this question, focus was placed on the Drosophila homolog of ornithine decarboxylase (Odc1), the first rate-limiting enzyme in the synthesis of polyamines. Mutants in Odc1 are lethal, but heterozygotes were viable to adulthood. Odc1 heterozygotes appeared larger than their genetic background control flies and consistent with this observation, weighed more than the controls. However, the increased weight was not due to increased food consumption as heterozygotes ate less than the controls. Interestingly, Odc1 heterozygous flies had augmented triglyceride storage, and this lipid phenotype was due to increased triglyceride storage per cell and an increase in the number of fat cells produced. Odc1 heterozygous flies also displayed increased expression of the lipid synthesis genes fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), suggesting increased lipid synthesis was the cause of the augmented triglyceride phenotype. These results provide a link between the expression of Odc1 and triglyceride storage suggesting that the polyamine pathway plays a role in regulating lipid metabolism (Leon, 2019).

The role of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb27C in regulating lipid storage in the Drosophila fat body

The storage of excess nutrients as triglycerides is essential for all organisms to survive when food is scarce; however, the mechanisms by which triglycerides are stored are not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing that are important in the regulation of triglyceride storage. This lab has identified a number of splicing factors important for regulating lipid metabolism; however, the full complement of splicing proteins involved in achieving metabolic homeostasis is unknown. Heterogeneous nuclear ribonucleoproteins (hnRNPs), RNA binding proteins that inhibit the splicing of introns by preventing the assembly of splicing complexes, have no established metabolic functions. To assess any metabolic functions of hnRNPs, the GAL4/UAS system was used to induce RNAi to six hnRNP's: hnRNP-K, rumpelstiltskin (rump), smooth (sm), Hrb27C (also referred to as Hrp48), Hrb98DE, and Hrb87F in the Drosophila fat body. Decreasing the levels of hnRNP-K and rump resulted in a decrease in triglyceride storage, whereas decreasing the levels of sm, Hrb27C, and Hrb98DE resulted in an increase in triglyceride storage. The excess triglyceride phenotype in Hrb27C-RNAi flies resulted from both an increase in the number of fat body cells and the amount of fat stored per cell. In addition, both the splicing of the beta-oxidation gene, CPT1, and the expression of the lipase brummer (bmm) was altered in flies with decreased Hrb27C, providing insight into the lipid storage phenotype in these flies. Together, these results suggest that the hnRNP family of splicing factors have varying metabolic functions and may act on specific metabolic genes to control their expression and processing (Bhogal, 2020)

Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements

Collagens are large secreted trimeric proteins making up most of the animal extracellular matrix. Secretion of collagen has been a focus of interest for cell biologists in recent years because collagen trimers are too large and rigid to fit into the COPII vesicles mediating transport from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific mechanisms to create enlarged ER-to-Golgi transport carriers have been postulated, including cargo loading by conserved ER exit site (ERES) protein Tango1. This study reports an RNAi screening for genes involved in collagen secretion in Drosophila. In this screening, distribution of GFP-tagged Collagen IV was examined in live animals, and 88 gene hits were found for which the knockdown produced intracellular accumulation of Collagen IV in the fat body, the main source of matrix proteins in the larva. Among these hits, only two affected collagen secretion specifically: PH4alphaEFB and Plod, encoding enzymes known to mediate posttranslational modification of collagen in the ER. Every other intracellular accumulation hit affected general secretion, consistent with the notion that secretion of collagen does not use a specific mode of vesicular transport, but the general secretory pathway. Included in the hits are many known players in the eukaryotic secretory machinery, like COPII and COPI components, SNAREs and Rab-GTPase regulators. Further analysis of the involvement of Rab-GTPases in secretion shows that Rab1, Rab2 and RabX3, are all required at ERES, each of them differentially affecting ERES morphology. Abolishing activity of all three by Rep knockdown, in contrast, led to uncoupling of ERES and Golgi. Additionally a characterization of a screening hit, trabuco (tbc), is presented, encoding an ERES-localized TBC domain-containing Rab-GAP. Finally, the success is discussed of this screening in identifying secretory pathway genes in comparison to two previous secretion screenings in Drosophila S2 cells (Ke, 2018).

Meep, a Novel Regulator of Insulin Signaling, Supports Development and Insulin Sensitivity via Maintenance of Protein Homeostasis in Drosophila melanogaster

Insulin signaling is critical for developmental growth and adult homeostasis, yet the downstream regulators of this signaling pathway are not completely understood. Using the model organism Drosophila melanogaster, a genomic approach was undertaken to identify novel mediators of insulin signaling. These studies led to the identification of Meep, encoded by the gene CG32335. Expression of this gene is both insulin receptor- and diet-dependent. Meep was specifically required in the developing fat body to tolerate a high-sugar diet (HSD). Meep is not essential on a control diet, but when reared on an HSD, knockdown of meep causes hyperglycemia, reduced growth, developmental delay, pupal lethality, and reduced longevity. These phenotypes stem in part from Meep's role in promoting insulin sensitivity and protein stability. This work suggests a critical role for protein homeostasis in development during overnutrition. Because Meep is conserved and obesity-associated in mammals, future studies on Meep may help to understand the role of proteostasis in insulin-resistant type 2 diabetes (Pereira, 2020).

Tumor induction in Drosophila imaginal epithelia triggers modulation of fat body lipid droplets

Understanding of cancer-specific metabolic changes is currently unclear. In recent years, the fruit fly Drosophila melanogaster with its powerful genetic tools has become an attractive model for studying both tumor autonomous and the systemic processes resulting from the tumor growth. This study investigated the effect of tumorigenesis on the modulation of lipid droplets (LDs) in the larval fat bodies (mammalian equivalent of adipose tissue). Notch signaling was overexpressed alone or in combination with the developmental regulator Myocyte enhancer factor 2 (Mef2) using wing-specific and eye-specific drivers, the size of LDs in the fat body of the different tumor bearing larvae was quantified, and the expression of genes associated with lipolysis and lipogenesis was estimated. Hyperplastic and neoplastic tumor induced by overexpression of Notch and co-expression of Notch and Mef2 respectively were found to trigger impaired lipid metabolism marked by increased size of fat body LDs. The impaired lipid metabolism in tumor carrying larvae is linked to the altered expression of genes that participate in lipolysis and lipogenesis. These findings reveal modulation of LDs as one of the host's specific response upon tumor initiation. This information could potentially uncover mechanisms for designing innovative approaches to modulate cancer growth (Harsh, 2020).

The heterogeneous nuclear ribonucleoprotein (hnRNP) Glorund functions in the Drosophila fat body to regulate lipid storage and transport

The availability of excess nutrients in Western diets has led to the overaccumulation of these nutrients as triglycerides, a condition known as obesity. The full complement of genes important for regulating triglyceride storage is not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing as important lipid storage regulators. Previous work showed that a group of splicing factors called heterogeneous nuclear ribonucleoproteins (hnRNPs) regulate lipid metabolism in the fly fat body; however, the identities of all the hnRNPs that function to control triglyceride storage are not known. This study used the GAL4/UAS system to induce RNAi to the hnRNP glorund (glo) in the Drosophila fat body to assess whether this hnRNP has any metabolic functions. Decreasing glo levels resulted in less triglycerides being stored throughout the fly. Interestingly, decreasing fat body glo expression resulted in increased triglyceride storage in the fat body, but blunted triglyceride storage in non-fat body tissues, suggesting a defect in lipid transport. Consistent with this hypothesis, the expression of apolipophorin (apolpp), microsomal triglyceride transfer protein (mtp), and apolipoprotein lipid transfer particle (apoltp), apolipoprotein genes important for lipid transport through the fly hemolymph, was decreased in glo-RNAi flies, suggesting that glo regulates the transport of lipids from the fly fat body to surrounding tissues. Together, these results indicate that glorund plays a role in controlling lipid transport and storage and provide additional evidence of the link between gene expression and the regulation of lipid metabolism (Kolasa, 2021).

E2F/Dp inactivation in fat body cells triggers systemic metabolic changes

The E2F transcription factors play a critical role in controlling cell fate. In Drosophila, the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. This study shows that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently rescued animal lethality. Collectively, these data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development (Zappia, 2021).

Fat body Ire1 regulates lipid homeostasis through the Xbp1s-FoxO axis in Drosophila

The endoplasmic reticulum (ER)-resident transmembrane protein kinase/RNase Ire1 is a conserved sensor of the cellular unfolded protein response and has been implicated in lipid homeostasis, including lipid synthesis and transport, across species. This study reports a novel catabolic role of Ire1 in regulating lipid mobilization in Drosophila. Ire1 is activated by nutrient deprivation, and, importantly, fat body-specific Ire1 deficiency leads to increased lipid mobilization and sensitizes flies to starvation, whereas fat body Ire1 overexpression results in the opposite phenotypes. Genetic interaction and biochemical analyses revealed that Ire1 regulates lipid mobilization by promoting Xbp1s-associated FoxO degradation and suppressing FoxO-dependent lipolytic programs. These results demonstrate that Ire1 is a catabolic sensor and acts through the Xbp1s-FoxO axis to hamper the lipolytic response during chronic food deprivation. These findings offer new insights into the conserved Ire1 regulation of lipid homeostasis (Zhao, 2021).

Drosophila STING protein has a role in lipid metabolism

Stimulator of interferon genes (STING) plays an important role in innate immunity by controlling type I interferon response against invaded pathogens. This work describes a previously unknown role of STING in lipid metabolism in Drosophila. Flies with STING deletion are sensitive to starvation and oxidative stress, have reduced lipid storage and downregulated expression of lipid metabolism genes. Drosophila STING was found to interact with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). ACC and FASN also interact with each other, indicating that all three proteins may be components of a large multi-enzyme complex. The deletion of Drosophila STING leads to disturbed ACC localization and decreased FASN enzyme activity. Together, these results demonstrate a previously undescribed role of STING in lipid metabolism in Drosophila (Akhmetova, 2021).

STimulator of INterferon Genes (STING) is an endoplasmic reticulum (ER)-associated transmembrane protein that plays an important role in innate immune response by controlling the transcription of many host defense genes. The presence of foreign DNA in the cytoplasm signals a danger for the cell. This DNA is recognized by specialized enzyme, the cyclic GMP-AMP synthase (cGAS), which generates cyclic dinucleotide (CDN) signaling molecules. CDNs bind to STING activating it, and the following signaling cascade results in NF-κB- and IRF3-dependent expression of immune response molecules such as type I interferons (IFNs) and pro-inflammatory cytokines. Bacteria that invade the cell are also known to produce CDNs that directly activate STING pathway. Additionally, DNA that has leaked from the damaged nuclei or mitochondria can also activate STING signaling and inflammatory response, which, if excessive or unchecked, might lead to the development of autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis (Akhmetova, 2021).

STING homologs are present in almost all animal phyla. This protein has been extensively studied in mammalian immune response; however, the role of STING in the innate immunity of insects has been just recently identified. Fruit fly D. melanogaster STING homolog is encoded by the CG1667 gene, which is refered to as dSTING. dSTING displays anti-viral and anti-bacterial effects that however are not all encompassing. Particularly, it has been shown that dSTING-deficient flies are more susceptible to Listeria infection due to the decreased expression of antimicrobial peptides (AMPs) – small positively charged proteins that possess antimicrobial properties against a variety of microorganisms. dSTING has been shown to attenuate Zika virus infection in fly brains and participate in the control of infection by two picorna-like viruses (DCV and CrPV) but not two other RNA viruses FHV and SINV or dsDNA virus IIV6. All these effects are linked to the activation of NF-κB transcription factor Relish (Akhmetova, 2021).

The immune system is tightly linked with metabolic regulation in all animals, and proper re-distribution of the energy is crucial during immune challenges. In both flies and humans, excessive immune response can lead to a dysregulation of metabolic stores. Conversely, the loss of metabolic homeostasis can result in weakening of the immune system. The mechanistic links between these two important systems are integrated in Drosophila fat body. Similarly to mammalian liver and adipose tissue, insect fat body stores excess nutrients and mobilizes them during metabolic shifts. The fat body also serves as a major immune organ by producing AMPs during infection. There is an evidence that the fat body is able to switch its transcriptional status from 'anabolic' to 'immune' program. The main fat body components are lipids, with triacylglycerols (TAGs) constituting approximately 90% of the stored lipids. Even though most of the TAGs stored in fat body comes from the dietary fat originating from the gut during feeding, de novo lipid synthesis in the fat body also significantly contributes to the pool of storage lipids (Akhmetova, 2021).

Maintaining lipid homeostasis is crucial for all organisms. Dysregulation of lipid metabolism leads to a variety of metabolic disorders such as obesity, insulin resistance and diabetes. Despite the difference in physiology, most of the enzymes involved in metabolism, including lipid metabolism, are evolutionarily and functionally conserved between Drosophila and mammals. Major signaling pathways involved in metabolic control, such as insulin system, TOR, steroid hormones, FOXO, and many others, are present in fruit flies. Therefore, it is not surprising that Drosophila has become a popular model system for studying metabolism and metabolic diseases. With the availability of powerful genetic tools, Drosophila has all the advantages to identify new players and fill in the gaps in understanding of the intricacies of metabolic networks (Akhmetova, 2021).

This work describes a novel function of dSTING in lipid metabolism. Flies with a deletion of dSTING were found to be sensitive to the starvation and oxidative stress. Detailed analysis reveals that dSTING deletion results in a significant decrease in the main storage metabolites, such as TAG, trehalose, and glycogen. Two fatty-acid biosynthesis enzymes were identified – acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) – as the interacting partners for dSTING. Moreover, this study also found that FASN and ACC interacted with each other, indicating that all three proteins might be components of a large complex. Importantly, dSTING deletion leads to the decreased FASN activity and defects in ACC cellular localization suggesting a direct role of dSTING in lipid metabolism of fruit flies (Akhmetova, 2021).

STING plays an important role in innate immunity of mammals, where activation of STING induces type I interferons (IFNs) production following the infection with intracellular pathogens. However, recent studies showed that the core components of STING pathway evolved more than 600 million years ago, before the evolution of type I IFNs. This raises the question regarding the ancestral functions of STING. In this study it was found that STING protein is involved in lipid metabolism in Drosophila. The deletion of Drosophila STING (dSTING) gene rendered flies sensitive to the starvation and oxidative stress. These flies have reduced lipid storage and downregulated expression of lipid metabolism genes. It was further shown that dSTING interacted with the lipid synthesizing enzymes ACC and FASN suggesting a possible regulatory role in the lipid biosynthesis. In the fat body, main lipogenic organ in Drosophila, dSTING co-localized with both ACC and FASN in a cortical region of the ER. dSTING deletion resulted in the disturbed ACC localization in fat body cells and greatly reduced the activity of FASN in the in vitro assay (Akhmetova, 2021).

Importantly, it was also observed that ACC and FASN interacted with each other. Malonyl-CoA, the product of ACC, serves as a substrate for the FASN reaction of fatty acid synthesis. Enzymes that are involved in sequential reactions often physically interact with each other and form larger multi-enzyme complexes, which facilitates the substrate channeling and efficient regulation of the pathway flux. There are several evidences of the existence of the multi-enzyme complex involved in fatty acid biosynthesis. ACC, ACL (ATP citrate lyase), and FASN physically associated in the microsomal fraction of rat liver. Moreover, in the recent work, a lipogenic protein complex including ACC, FASN, and four more enzymes was isolated from the oleaginous fungus Cunninghamella bainieri. It is possible that a similar multi-enzyme complex exists in Drosophila and other metazoan species, and it would be of great interest to identify its other potential members (Akhmetova, 2021).

How does STING exerts its effect on lipid synthesis? Recently, the evidence has emerged for the control of the de novo fatty acid synthesis by two small effector proteins – MIG12 and Spot14. MIG12 overexpression in livers of mice increased total fatty acid synthesis and hepatic triglyceride content. It has been shown that MIG12 protein binds to ACC and facilitates its polymerization thus enhancing the activity of ACC. For Spot14, both the activation and inhibition of de novo lipogenesis have been reported, depending upon the tissue type and the cellular context. Importantly, there is an evidence that all four proteins – ACC, FASN, MIG12, and Spot14 – exist as a part of a multimeric complex. It is plausible to suggest that Drosophila STING plays a role similar to MIG12 and/or Spot14 in regulating fatty acid synthesis. It is proposed that dSTING might ‘anchor’ ACC and FASN possibly together with other enzymes at the ER membrane. The resulting complex facilitates fatty acid synthesis by allowing for a quicker transfer of malonyl-CoA product of ACC to the active site of FASN. In dSTINGΔ mutants, ACC loses its association with some regions of the ER resulting in the weakened interaction between ACC and FASN. Less FASN immunoprecipitated with ACC in dSTINGΔ mutants compared to control flies, and the opposite effect was found in flies expressing GFP-tagged dSTING (Akhmetova, 2021).

It has been shown that de novo synthesis of fatty acids continuously contributes to the total fat body TAG storage in Drosophila. It is hypothesized that the reduced fatty acid synthesis due to the lowered FASN enzyme activity in dSTINGΔ deletion mutants might be responsible for the decreased TAG lipid storage and starvation sensitivity phenotypes. Sensitivity to oxidative stress might also be explained by the reduced TAG level. Evidences exist that the lipid droplets (consisting mainly of TAGs) provide protection against reactive oxygen species. Furthermore, flies with ACC RNAi are found to be sensitive to the oxidative stress (Akhmetova, 2021).

In addition to its direct role in ACC/FASN complex activity, STING might also affect a phosphorylation status of ACC and/or FASN. Both proteins are known to be regulated by phosphorylation/dephosphorylation. In mammals, STING is an adaptor protein that transmits an upstream signal by interacting with kinase TBK1 (TANK-binding kinase 1). When in a complex with STING, TBK1 activates and phosphorylates IRF3 allowing its nuclear translocation and transcriptional response. It is possible that in Drosophila, STING recruits a yet unidentified kinase that phosphorylates ACC and/or FASN thereby changing their enzymatic activity (Akhmetova, 2021).

Drosophila STING itself could also be regulated by the lipid- synthesizing complex. STING palmitoylation was recently identified as a posttranslational modification necessary for STING signaling in mice. In this way, palmitic acid synthesized by FASN might participate in the regulation of dSTING possibly providing a feedback loop (Akhmetova, 2021).

The product of ACC – malonyl-CoA – is a key regulator of the energy metabolism. During lipogenic conditions, ACC is active and produces malonyl-CoA, which provides the carbon source for the synthesis of fatty acids by FASN. In dSTING knockout, FASN activity is decreased and malonyl-CoA is not utilized and builds up in the cells. Malonyl-CoA is also a potent inhibitor of carnitine palmitoyltransferase CPT1, the enzyme that controls the rate of fatty acid entry into the mitochondria, and hence is a key determinant of the rate of fatty acid oxidation. Thus, a high level of malonyl-CoA results in a decreased fatty acid utilization for the energy. This might explain the down-regulation of lipid catabolism genes that was observed in dSTINGΔ mutants. A reduced fatty acid oxidation in turn shifts cells to the increased reliance on glucose as a source of energy. Consistent with this notion, an increased glucose level was observed in fed dSTINGΔ mutant flies, as well as increased levels of phosphoenolpyruvate (PEP). PEP is produced during glycolysis, and its level was shown to correlate with the level of glucose. A reliance on glucose for the energy also has a consequence of reduced incorporation of glucose into trehalose and glycogen for storage, and therefore, lower levels of these storage metabolites, which was observed. To summarize, based on the current findings, a model is presented in (see Model of dSTING deletion effect on Drosophila metabolism), which suggests a direct involvement of dSTING in the regulation of lipid metabolism (Akhmetova, 2021).

Based on the data, dSTING interacts with lipid synthesizing enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In the absence of dSTING, the activity of FASN is reduced which results in decreased de novo fatty acid synthesis and triglyceride (TAG) synthesis. Low TAG level in turn lead to sensitivity to starvation and oxidative stress. Reduced FASN activity in dSTING mutants also results in ACC product malonyl-CoA build-up in the cells leading to the inhibition of the fatty acid oxidation in mitochondria. Reduced fatty acid oxidation shifts cells to the increased reliance on glucose as a source of energy resulting in reduced glycogen and trehalose levels in dSTING mutants. Palmitic acid synthesized by FASN might participate in the regulation of dSTING via palmitoylation possibly providing a feedback loop (Akhmetova, 2021).

Recent studies show that in mammals, the STING pathway is involved in metabolic regulation under the obesity conditions. The expression level and activity of STING were upregulated in livers of mice with high-fat diet-induced obesity. STING expression was increased in livers from nonalcoholic fatty liver disease (NAFLD) patients compared to control group. In nonalcoholic steatohepatitis mouse livers, STING mRNA level was also elevated. Importantly, STING deficiency ameliorated metabolic phenotypes and decreased lipid accumulation, inflammation, and apoptosis in fatty liver hepatocytes (Akhmetova, 2021).

Despite the accumulating evidences, the exact mechanism of STING functions in metabolism is not completely understood. The prevailing hypothesis is that the obesity leads to a mitochondrial stress and a subsequent mtDNA release into the cytoplasm, which activates cGAS-STING pathway. The resulting chronic sterile inflammation is responsible for the development of NAFLD, insulin resistance, and type 2 diabetes. In this case, the effect of STING on metabolism is indirect and mediated by inflammation effectors. The data presented in the current study strongly suggest that in Drosophila, STING protein is directly involved in lipid metabolism by interacting with the enzymes involved in a lipid biosynthesis. This raises the question if the observed interaction is unique for Drosophila or it is also the case for mammals. Future work is needed to elucidate the evolutionary aspect of STING role in metabolism. Understanding the relationships between STING and lipid metabolism may provide insights into the mechanisms of the obesity-induced metabolism dysregulation and thereby suggest novel therapeutic strategies for metabolic diseases (Akhmetova, 2021).

STING controls energy stress-induced autophagy and energy metabolism via STX17

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. This study reports that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 (Syntaxin 17) interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, this study demonstrates a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17 (Rong, 2022).

This study demonstrates the crucial role of STING in glucose metabolism through its negative regulation in energy stress-induced autophagy. The connection between autophagy and the cGAS-STING pathway has been intensively investigated with a focus on pathogens, DNA, or cyclic dinucleotides-induced autophagy through a non-canonical pathway. This study found that STING also plays a crucial role in energy stress-induced autophagy, especially in autophagosome-lysosome fusion through its interaction with the autophagic SNARE STX17. In the unstressed conditions, STING physically interacts with STX17 and sequesters it at ER. This interaction is disrupted by STING activation (DNA treatment etc) or autophagy stimuli (energy stress, etc.), which leads to STX17 translocation to autophagosomes, assembly of autophagic SNARE complex, and promotion of autophagosomal fusion with lysosomes. STING-regulated energy stress-induced autophagy has at least two effects, to facilitate elimination of DNA and microbes in immune cells and to boost energy metabolism in non-immune cells (Rong, 2022).

STX17 is also important for autophagosome biogenesis. How the functions of STX17 in autophagy initiation and autophagosome-lysosome fusion is differentiated is an interesting question. It is proposed that STX17 phosphorylation by TBK1, as described by Kumar (2019), likely separates these two functions. TBK1-phosphorylated STX17 translocates from Golgi to mPAS, which is not controlled by STING, while the portion of STX17 that is not phosphorylated by TBK1 interacts with STING at ER/ERGIC (endoplasmic-reticulum-Golgi intermediate compartment), and this interaction is disrupted by autophagic stress likely through TBK1-independent regulatory events, which leads to translocation of this portion of STX17 from ER/ERGIC to complete autophagosomes. These observations nicely reconcile the different functions of STX17 in autophagy initiation and maturation (Rong, 2022).

STING-regulated energy stress-induced autophagy is different from previously reported STING-mediated non-canonical autophagy in several aspects: (1) different membrane trafficking pathways are utilized. Triggered by PAMPs (pathogen-associated molecular patterns), STING translocates to single bilayer membrane vesicles positive for LC3, but these STING-LC3 positive vesicles are negative for STX17; (2) PAMPs-triggered STING-mediated autophagy is independent of BECN1, ULK1, and Atg9a. STX17 neither localizes to STING-positive vesicles nor is it required for STING trafficking and degradation; (3) PAMPs-induced non-canonical autophagy is compromised when STING is absent; while canonical autophagy is further activated in the absence of STING given that more STX17 is released from ER; (4) PAMPs-induced STING-dependent autophagy activation is limited to immune cells, but STING-regulated canonical autophagy functions broadly in both immune and non-immune cells (Rong, 2022).

STING is a crucial regulator in the cancer-immunity cycle, and activation of STING represents a promising strategy for cancer therapy. This study suggests, in addition to immunity regulation, activation of STING also promotes energy stress-induced autophagy by releasing STX17 from ER. How autophagy activation contributes to STING mediates signaling remained to be investigated. At least, this study indicates that STING might play an unexpected broader role in energy metabolism due to its regulation of energy stress-induced autophagy. Autophagy has been implicated in a broad spectrum of human diseases, and STING also expresses and functions in non-immune tissues, suggesting that the regulatory effect of STING on autophagy might contribute to the pathogenesis of autophagy-related diseases and immune functions (Rong, 2022).

Histone acetyltransferase NAA40 modulates acetyl-CoA levels and lipid synthesis

Epigenetic regulation relies on the activity of enzymes that use sentinel metabolites as cofactors to modify DNA or histone proteins. Thus, fluctuations in cellular metabolite levels have been reported to affect chromatin modifications. However, whether epigenetic modifiers also affect the levels of these metabolites and thereby impinge on downstream metabolic pathways remains largely unknown. This study tested this notion by investigating the function of N-alpha-acetyltransferase 40 (NAA40), the enzyme responsible for N-terminal acetylation of histones H2A and H4, which has been previously implicated with metabolic-associated conditions such as age-dependent hepatic steatosis and calorie-restriction-mediated longevity. Using metabolomic and lipidomic approaches, this study found that depletion of NAA40 in murine hepatocytes leads to significant increase in intracellular acetyl-CoA levels, which associates with enhanced lipid synthesis demonstrated by upregulation in de novo lipogenesis genes as well as increased levels of diglycerides and triglycerides. Consistently, the increase in these lipid species coincide with the accumulation of cytoplasmic lipid droplets and impaired insulin signalling indicated by decreased glucose uptake. However, the effect of NAA40 on lipid droplet formation is independent of insulin. In addition, the induction in lipid synthesis is replicated in vivo in the Drosophila melanogaster larval fat body. Finally, supporting these results, this study found a strong association of NAA40 expression with insulin sensitivity in obese patients. Overall, these findings demonstrate that NAA40 affects the levels of cellular acetyl-CoA, thereby impacting lipid synthesis and insulin signalling. This study reveals a novel path through which histone-modifying enzymes influence cellular metabolism with potential implications in metabolic disorders (Charidemou, 2022).

Sex determination gene transformer regulates the male-female difference in Drosophila fat storage via the adipokinetic hormone pathway

Sex differences in whole-body fat storage exist in many species. For example, Drosophila females store more fat than males. Yet, the mechanisms underlying this sex difference in fat storage remain incompletely understood. This study identified a key role for sex determination gene transformer (tra) in regulating the male-female difference in fat storage. Normally, a functional Tra protein is present only in females, where it promotes female sexual development. This study shows that loss of Tra in females reduced whole-body fat storage, whereas gain of Tra in males augmented fat storage. Tra's role in promoting fat storage was largely due to its function in neurons, specifically the Adipokinetic hormone (Akh)-producing cells (APCs). Analysis of Akh pathway regulation revealed a male bias in APC activity and Akh pathway function, where this sex-biased regulation influenced the sex difference in fat storage by limiting triglyceride accumulation in males. Importantly, Tra loss in females increased Akh pathway activity, and genetically manipulating the Akh pathway rescued Tra-dependent effects on fat storage. This identifies sex-specific regulation of Akh as one mechanism underlying the male-female difference in whole-body triglyceride levels, and provides important insight into the conserved mechanisms underlying sexual dimorphism in whole-body fat storage (Wat, 2021).

This study used the fruit fly Drosophila melanogaster to improve the knowledge of the mechanisms underlying the male-female difference in whole-body triglyceride levels. The presence of a functional tra protein in females, which directs many aspects of female sexual development, promotes whole-body fat storage. Tra's ability to promote fat storage arises largely due to its function in neurons, where the APCs were identified as one neuronal population in which tra function influences whole-body triglyceride levels. Examination of Akh/AkhR mRNA levels and APC activity revealed several differences between the sexes, where these differences lead to higher Akh pathway activity in males than in females. Genetic manipulation of APCs and Akh pathway activity suggest a model in which the sex bias in Akh pathway activity contributes to the male-female difference in fat storage by limiting whole-body triglyceride storage in males. Importantly, this study showed that tra function influences Akh pathway activity, and that Akh acts genetically downstream of tra in regulating whole-body triglyceride levels. This reveals a previously unrecognized genetic and physiological mechanism that contributes to the sex difference in fat storage (Wat, 2021).

One key finding from this study was the identification of sex determination gene tra as an upstream regulator of the male-female difference in fat storage. In females, a functional Tra protein promotes fat storage, whereas lack of tra in males leads to reduced fat storage. While an extensive body of literature has demonstrated important roles for tra in regulating neural circuits, behavior, abdominal pigmentation, and gonad development, uncovering a role for tra in regulating fat storage significantly extends understanding of how sex differences in metabolism arise. Given that sex differences exist in other aspects of metabolism (e.g., oxygen consumption), this new insight suggests that more work will be needed to determine whether tra contributes to sexual dimorphism in additional metabolic traits. Indeed, one study showed that tra influences the sex difference in adaptation to hydrogen peroxide stress. Beyond metabolism, tra also regulates multiple aspects of development and physiology such as intestinal stem cell proliferation, carbohydrate metabolism, body size, phenotypic plasticity, and lifespan responses to dietary restriction. Because some, but not all, of these studies identify a cell type in which tra function influences these diverse phenotypes, future studies will need to determine which cell types and tissues require tra expression to establish a female metabolic and physiological state. Indeed, recent single-cell analyses reveal widespread gene expression differences in shared cell types between the sexes (Wat, 2021).

Identifying neurons as the anatomical focus of Tra's effects on fat storage was another key finding from this study. While many sexually dimorphic neural circuits related to behavior and reproduction have been identified, less is known about sex differences in neurons that regulate physiology and metabolism. Indeed, while many studies have identified neurons that regulate fat metabolism, these studies were conducted in single- or mixed-sex populations. Because male-female differences in neuron number, morphology, and connectivity have all been described across the brain and ventral nerve cord, a detailed analysis of neuronal populations that influence metabolism will be needed in both sexes to understand how neurons contribute to the sex-specific regulation of metabolism and physiology. Indeed, while identification of a role for APC sexual identity in regulating the male-female difference in fat storage represents a significant step forward in understanding how sex differences in neurons influence metabolic traits, more knowledge is needed of how tra regulates sexual dimorphism in this critical neuronal subset. For example, while this study showed that females normally have lower Akh mRNA levels and APC activity, it remains unclear how the presence of tra regulates these distinct traits. tra may regulate Akh mRNA levels via known target genes fruitless (fru) and doublesex (dsx) , or alternatively through a fru- and dsx-independent pathway. To influence the sex difference in APC activity and Akh release, tra may regulate factors such as ATP-sensitive potassium (KATP) channels and 5' adenosine monophosphate-activated protein kinase (AMPK)-dependent signaling, both of which are known to modulate APC activity. Future studies will therefore need to investigate Tra-dependent changes to KATP channel expression and function in APCs, and characterize Tra's effects on ATP levels and AMPK signaling within APCs (Wat, 2021).

Additional ways to learn more about the sex-specific regulation of fat storage by the APCs will include examining how sexual identity influences physical connections between the APCs and other neurons, and monitoring APC responses to circulating hormones. For example, there are physical connections between Corazonin- and Neuropeptide F (NPF)-positive (CN) neurons and APCs in adult male flies, and between the APCs and a bursicon-α-responsive subset of DLgr2 neurons in females. These connections inhibit APC activity: CN neurons inhibit APC activity in response to high hemolymph sugar levels, whereas binding of bursicon-α to DLgr2 neurons inhibits APC activity in nutrient-rich conditions. Future studies will therefore need to determine whether these physical connections exist in both sexes. Further, it will be important to identify male-female differences in circulating factors that regulate the APCs. While gut-derived Allatostatin C (AstC) was recently shown to bind its receptor on the APCs to trigger Akh release, loss of AstC affects fat metabolism and starvation resistance only in females. This suggests sex differences in AstC-dependent regulation of fat metabolism may exist (Wat, 2021).

Given that gut-derived NPF binds to its receptor on the APCs to inhibit Akh release, that skeletal muscle-derived unpaired 2 (upd2) regulates hemolymph Akh levels, and that circulating peptides such as Allatostatin A (AstA), Drosophila insulin-like peptides (Dilps), and activin ligands influence Akh pathway activity, it is clear that a systematic survey of circulating factors that modulate Akh production, release, and Akh pathway activity in each sex will be needed to fully understand the sex-specific regulation of fat storage. Another important point to address in future studies will be confirming results from previous studies that the fat body is the main anatomical focus of Akh-dependent regulation of fat storage. Given that the sex-biased effects of triglyceride lipase bmm arise from a male-female difference in the cell type-specific requirements for bmm function, it will be important to determine which cell types mediate Akh's effects on fat storage in each sex. This line of enquiry will also clarify the underlying processes that support increased fat storage in females. At present, it remains unclear whether the higher whole-body fat storage in females is caused by lower fat breakdown, increased lipogenesis, or both. Given that Akh pathway activity plays a role in regulating both lipolysis and lipogenesis in Drosophila and other insects, it will be important to identify the cellular mechanism underlying Akh's effects on the sex difference in fat storage (Wat, 2021).

Beyond fat metabolism, it will be important to extend understanding of how sex-specific Akh regulation affects additional Akh-regulated phenotypes. Given that Akh affects fertility and fecundity, future studies will need to determine whether these phenotypes are due to Akh-dependent regulation of fat metabolism, or due to direct effects of Akh on gonads. Similarly, while Akh has been linked with the regulation of lifespan, carbohydrate metabolism, starvation resistance, locomotion, immune responses, cardiac function, and oxidative stress responses, most studies were performed in mixed- or single-sex populations. Additional work is therefore needed to determine how changes to Akh pathway function affect physiology, carbohydrate levels, development, and life history in each sex. Importantly, the lessons learned may also extend to other species. Akh signalling is highly conserved across invertebrates, and is functionally similar to the mammalian β-adrenergic and glucagon systems. Because sex-specific regulation of both glucagon and the β-adrenergic systems have been described in mammalian models and in humans, detailed studies on sex-specific Akh regulation and function in flies may provide vital clues into the mechanisms underlying male-female differences in physiology and metabolism in other animals (Wat, 2021).

A pleiotropic chemoreceptor facilitates the production and perception of mating pheromones

Optimal mating decisions depend on the robust coupling of signal production and perception because independent changes in either could carry a fitness cost. However, since the perception and production of mating signals are often mediated by different tissues and cell types, the mechanisms that drive and maintain their coupling remain unknown for most animal species.This study shows that in Drosophila, behavioral responses to, and the production of, a putative inhibitory mating pheromone are co-regulated by Gr8a, a member of the Gustatory receptor gene family. Specifically, through behavioral and pheromonal data, this study found that Gr8a independently regulates the behavioral responses of males and females to a putative inhibitory pheromone, as well as its production in the fat body and oenocytes of males. Overall, these findings provide a relatively simple molecular explanation for how pleiotropic receptors maintain robust mating signaling systems at the population and species levels (Vernier, 2023).

Sima, a Drosophila homolog of HIF-1alpha, in fat body tissue inhibits larval body growth by inducing Tribbles gene expression

Limited oxygen availability impairs normal body growth, although the underlying mechanisms are not fully understood. In Drosophila, hypoxic responses in the larval fat body (FB) disturb the secretion of insulin-like peptides from the brain, inhibiting body growth. However, the cell-autonomous effects of hypoxia on the insulin-signaling pathway in larval FB have been underexplored. This study aimed to examine the effects of overexpression of Sima, a Drosophila hypoxia-inducible factor-1 (HIF-1) α homolog and a key component of HIF-1 transcription factor essential for hypoxic adaptation, on the insulin-signaling pathway in larval FB. Forced expression of Sima in FB reduced the larval body growth with reduced Akt phosphorylation levels in FB cells and increased hemolymph sugar levels. Sima-mediated growth inhibition was reversed by overexpression of TOR or suppression of FOXO. After Sima overexpression, larvae showed higher expression levels of Tribbles, a negative regulator of Akt activity, and a simultaneous knockdown of Tribbles completely abolished the effects of Sima on larval body growth. Furthermore, a reporter analysis revealed Tribbles as a direct target gene of Sima. These results suggest that Sima in FB evokes Tribbles-mediated insulin resistance and consequently protects against aberrant insulin-dependent larval body growth under hypoxia (Noguchi, 2021).

orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control

Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. This study identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, it was demonstrated that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, this study shows that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of β-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response (Fernandez-Acosta, 2022).

Cytoophidia coupling adipose architecture and metabolism

Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. This study shows that the metabolic enzyme CTP synthase (CTPS) forms filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, causes impaired adipocyte adhesion and defective adipose tissue architecture. Moreover, CTPS influences integrin distribution and dot-like deposition of type IV collagen (Col IV). Col IV-integrin signaling reciprocally regulates the assembly of cytoophidia in adipocytes. These results demonstrate that a positive feedback signaling loop containing both cytoophidia and integrin adhesion complex couple tissue architecture and metabolism in Drosophila adipose tissue (Liu, 2022).

Microscopic and biochemical monitoring of endosomal trafficking and extracellular vesicle secretion in an endogenous in vivo model

Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. This study present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. It was furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion (Linnemannstons, 2022).

Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting

Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. This study found that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. It is proposed that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program (Parkhitko, 2022).

Maintaining cellular homeostasis upon nutrient shortage is an important challenge for all animals. Decreased activity of TORC1 is necessary to limit translation, reduce growth rates, and promote autophagy. Conversely, minimal TORC1 activity is required to promote lysosomal biogenesis, thus maintaining autophagic degradation necessary for survival. Using Drosophila as an in vivo model, this study found that TORC1 reactivation upon fasting integrates the biosynthesis of amino acids from anaplerotic inputs into the control of growth. The regulation of aspartate abundance appears to be critical during this process, possibly because it serves as a cataplerotic precursor for various macromolecules, including other amino acids and nucleotides, which in turn impinge on TORC1 activity. Cysteine recycling through the lysosome may fuel acetyl-CoA synthesis and prevent reactivation of TORC1 above a threshold that would compromise autophagy and survival during fasting. Reactivation of TORC1 during fasting was not passively controlled by the extent of amino acid remobilized from the lysosome. Instead, cysteine metabolism supported an increased incorporation of the carbons from these remobilized amino acids into the TCA cycle. It is therefore proposed that the remobilized amino acids may be transiently stored in the form of TCA cycle intermediates compartmentalized in the mitochondria, thereby restricting their accessibility. The regulation of TORC1 activity over a fasting period appears to be a combination of activating and suppressing cues that conciliate autophagy with anabolism. This process is self-regulated by autophagy, because autophagic protein degradation controls cystine availability through the lysosomal cystinosin transporter. Thus, in contrast to fed conditions, in which amino acid transporters at the plasma membrane maintain high cytosolic concentration of leucine and arginine that can directly be sensed by members of the TORC1 machinery, TORC1 reactivation in prolonged fasting is regulated indirectly by lysosome-mitochondrial cross-talk. Because cystinosin has also been shown to physically interact with several components of lysosomal TORC1 in mammalian cells, additional layers of regulation are conceivable during this process (Parkhitko, 2022).

Multiple functions of cysteine impinge on cellular metabolism, including transfer RNA thiolation, the generation of hydrogen sulfide, the regulation of hypoxia-inducible factor (HIF), and its antioxidant function through glutathione synthesis. Supplementation with cysteine or modified molecules such as N-acetyl-cysteine (NAC) can be used to efficiently buffer oxidative stress and perhaps alleviate symptoms of diseases that promote oxidative stress or glutathione deficiency, including cystinosis. Cysteine or NAC treatment extends the life span in flies, worms, and mice, and mice fed NAC show a sudden drop in body weight similar to that caused by dietary restriction. The results indicate that cysteine may not only act through its antioxidant function but also by restricting the availability of particular amino acids and limiting mTOR activity, processes known to extend life span. Moreover, this study shows that CoA is a main fate of cysteine that affects oxidative metabolism in the mitochondria, which is the main source of reactive oxygen species (ROS). Thus, the antioxidant function of cysteine also might be coupled to its effects on the mitochondria to buffer ROS production (Parkhitko, 2022).

In summary, this study demonstrate that cysteine metabolism acts in a feedback loop involving de novo CoA synthesis, the TCA cycle, and amino acid metabolism to limit TORC1 reactivation upon prolonged fasting. This pathway may be particularly important for developing organisms that must maintain autophagy and balance growth and survival during periods of food shortage (Parkhitko, 2022).

Progressive transcriptional changes in metabolic genes and altered fatbody homeostasis in Drosophila model of Huntington's disease

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder marked by progressive neuronal atrophy, particularly in striatum and cerebral cortex. Although predominant manifestations of the disease include loss in the triad of motor, cognitive and behavioral capabilities, metabolic dysfunction in patients and HD models are being increasingly recognized. Patients display progressive body weight loss, which aggravates the disease and leads to cachexia in the terminal stages. Using the Drosophila model of HD, it was earlier reported that diseased flies exhibit an atypical pattern of lipid gain and loss with progression along with exhibiting extensive mitochondrial dysfunction, impaired calcium homeostasis and heightened apoptosis in the fatbody. This study first monitored the structural changes that abdominal fatbody undergoes with disease progression. Further, the transcriptional changes of key metabolic genes in whole fly were checked as well as genes regulating mitochondrial function, apoptosis, autophagy and calcium homeostasis in the abdominal fatbody. Extensive alterations were found in whole-body and fatbody-specific transcriptional profile of the diseased flies, which was in consort with their stage-specific physiological state. Additionally, lysosome-mediated autophagy was assessed in the fatbody of diseased flies in order to ascertain the mechanisms contributing to fatbody atrophy at the terminal stage. Interestingly, elevated autophagy was found in fatbody of flies throughout disease progression. This study provides new insights into the effect on peripheral metabolism due to degeneration of neurons in the neurodegenerative disease, thereby discerns novel mechanisms leading to cachexia in diseased flies and advocates for the need of managing metabolic dysfunctions in HD (Singh, 2022).

The splicing factor 9G8 regulates the expression of NADPH-producing enzyme genes in Drosophila

Excess nutrients are stored as triglycerides, mostly as lipid droplets found in adipose tissue. Previous studies have characterized a group of splicing factors called serine/arginine rich (SR) proteins that function to identify intron/exon borders in regulating metabolic homeostasis in the Drosophila fat body. Decreasing the function of one SR protein, 9G8, causes an increase in triglyceride storage; however, the full complement of genes regulated by 9G8 to control metabolism is unknown. To address this question, RNA sequencing was performed on Drosophila fat bodies with 9G8 levels reduced by RNAi. Differential expression and differential exon usage analyses revealed several genes involved in the immune response, xenobiotic biology, protein translation, sleep, and lipid and carbohydrate metabolism whose expression or splicing is altered in 9G8-RNAi fat bodies. One gene that was both downregulated and had altered splicing in 9G8-RNAi fat bodies was Zwischenferment (Zw), the Drosophila homolog of human glucose 6-phosphate dehydrogenase (G6PD). G6PD regulates flux of glucose 6-phosphate (G6P) into the pentose phosphate pathway, which generates NADPH, a coenzyme for lipid synthesis. Interestingly, the other NADPH-producing enzyme genes in Drosophila (phosphogluconate dehydrogenase, isocitrate dehydrogenase and malic enzyme) were also decreased in 9G8-RNAi flies. Together, these findings suggest that 9G8 regulates several classes of genes and may regulate NADPH-producing enzyme genes to maintain metabolic homeostasis (Weidman, 2022).

Transportin-serine/arginine-rich (Tnpo-SR) proteins are necessary for proper lipid storage in the Drosophila fat body

After a meal, excess nutrients are stored within adipose tissue as triglycerides in structures called lipid droplets. Previous genome-wide RNAi screens have identified that mRNA splicing factor genes are required for normal lipid droplet formation in Drosophila cells. Previous work has shown that mRNA splicing factors called serine/arginine-rich (SR) proteins are important for triglyceride storage in the Drosophila fat body. SR proteins shuttle in and out of the nucleus with the help of proteins called Transportins (Tnpo-SR); however, whether this transport is important for SR protein-mediated regulation of lipid storage is unknown. The purpose of this study is to characterize the role of Tnpo-SR proteins in regulating lipid storage in the Drosophila fat body. Decreasing Tnpo-SR in the adult fat body resulted in an increase in triglyceride storage and consistent with this phenotype, Tnpo-SR-RNAi flies also have increased starvation resistance. In addition, the lipid accumulation in Tnpo-SR-RNAi flies is the result of increased triglyceride stored in each fat body cell and not due to increased food consumption. Interestingly, the splicing of CPT1, an enzyme important for the β-oxidation of fatty acids, is altered in Tnpo-SR-RNAi fat bodies. The isoform that produces the less catalytically active form of CPT1 accumulates in fat bodies where Tnpo-SR levels are decreased, suggesting a decrease in lipid breakdown, potentially causing the excess triglyceride storage observed in these flies. Together, these data suggest that the transport of splicing proteins in and out of the nucleus is important for proper triglyceride storage in the Drosophila fat body (Nagle, 2022).

Endoplasmic reticulum-associated protein degradation contributes to Toll innate immune defense in Drosophila melanogaster

In Drosophila, the endoplasmic reticulum-associated protein degradation (ERAD) is engaged in regulating pleiotropic biological processes, with regard to retinal degeneration, intestinal homeostasis, and organismal development. The extent to which it functions in controlling the fly innate immune defense, however, remains largely unknown. This study shows that blockade of the ERAD in fat bodies antagonizes the Toll but not the IMD innate immune defense in Drosophila. Genetic approaches further suggest a functional role of Me31B in the ERAD-mediated fly innate immunity. Moreover, evidence is provided that silence of Xbp1 other than PERK or Atf6 partially rescues the immune defects by the dysregulated ERAD in fat bodies. Collectively, this study uncovers an essential function of the ERAD in mediating the Toll innate immune reaction in Drosophila (Zhu, 2022).

The ESCRT-III Protein Chmp1 Regulates Lipid Storage in the Drosophila Fat Body
Defects in how excess nutrients are stored as triglycerides can result in several diseases including obesity, heart disease, and diabetes. Understanding the genes responsible for normal lipid homeostasis will help understand the pathogenesis of these diseases. RNAi screens performed in Drosophila cells identified genes involved in vesicle formation and protein sorting as important for the formation of lipid droplets; however, all of the vesicular trafficking proteins that regulate lipid storage are unknown. This study characterized the function of the Drosophila Charged multivesicular protein 1 (Chmp1) gene in regulating fat storage. Chmp1 is a member of the ESCRT-III complex that targets membrane localized signaling receptors to intralumenal vesicles in the multivesicular body of the endosome and then ultimately to the lysosome for degradation. When Chmp1 levels are decreased specifically in the fly fat body, triglyceride accumulates while fat-body-specific Chmp1 overexpression decreases triglycerides. Chmp1 controls triglyceride storage by regulating the number and size of fat body cells produced and not by altering food consumption or lipid metabolic enzyme gene expression. Together, these data uncover a novel function for Chmp1 in controlling lipid storage in Drosophila and supports the role of the endomembrane system in regulating metabolic homeostasis (Fruin, 2022).

Regulation of feeding and energy homeostasis by clock-mediated Gart in Drosophila

Feeding behavior is essential for growth and survival of animals; however, relatively little is known about its intrinsic mechanisms. This study demonstrates that Gart is expressed in the glia, fat body, and gut and positively regulates feeding behavior via cooperation and coordination. Gart in the gut is crucial for maintaining endogenous feeding rhythms and food intake, while Gart in the glia and fat body regulates energy homeostasis between synthesis and metabolism. These roles of Gart further impact Drosophila lifespan. Importantly, Gart expression is directly regulated by the CLOCK/CYCLE heterodimer via canonical E-box, in which the CLOCKs (CLKs) in the glia, fat body, and gut positively regulate Gart of peripheral tissues, while the core CLK in brain negatively controls Gart of peripheral tissues. This study provides insight into the complex and subtle regulatory mechanisms of feeding and lifespan extension in animals (He, 2023).

Feeding is a necessary behavior for animals to grow and survive, with a characteristic of taking food regularly. The quality and quantity of feeding directly impact the normal growth and development of animals. Time-restricted feeding or fasting is beneficial for preventing obesity, alleviating inflammation, and attenuating cardiac diseases and even has antitumor effects. Metabolic syndrome has become a global health problem. Shift work and meal irregularity disrupt circadian rhythms, with an increased risk of developing metabolic syndrome. The maintenance of the daily feeding rhythm is very important in metabolic homeostasis.Irregular feeding perturbs circadian metabolic rhythms and results in adverse metabolic consequences and chronic diseases (He, 2023).

Most behaviors in animals are synchronized to a ~24 h (circadian) rhythm induced by circadian clocks in both the central nervous system and peripheral tissues. Circadian rhythmic behaviors, such as feeding and locomotion, are involved in complex connections and specific output pathways under the control of the circadian clock. Although the core clock feedback loop has been well established in recent decades, the crucial genes responsible for rhythmic feeding regulation as well as for the interrelation between the core clocks and feeding are still unclear (He, 2023).

To increase the understanding of how the circadian clock regulates feeding and metabolism, this study sought to identify the output genes in the circadian feeding and metabolism control network, in which the model animal Drosophila provides special advantages to explore the mechanistic underpinnings and the complex integration of these primitive responses. Previous studies confirmed that one of juvenile hormone receptors, methoprene tolerance (Met), is important for the control of neurite development and sleep behavior in Drosophila. Many genes related to metabolic regulation have attracted attention in the transcriptome data from Met27, a Met-deficient fly line, in which this study focused on the target genes regulated by CLOCK/CYCLE (CLK/CYC). As a basic Helix-Loop-Helix-Per-ARNT-Sim (bHLH-PAS) transcription factor with a canonical binding site “CACGTG," the CLK/CYC heterodimer is a crucial core oscillator that regulates circadian rhythms (He, 2023).

The Gart trifunctional enzyme, a homologous gene of adenosine-3 in mammals, is a trifunctional polypeptide with the activities of phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, and phosphoribosylaminoimidazole synthetase. Gart in astrocytes of vertebrates plays a role in the lipopolysaccharide-induced release of proinflammatory factors (Zhang, 2014), and Gart expressed in the liver and heart is required for de novo purine synthesis. However, there is no information yet for Gart's functions in feeding rhythm. In this study, Gart was identified as a candidate that was controlled by the core clock gene CLK/CYC heterodimer and was found to be related to feeding behavior in Drosophila. Thus this study focused Gart studies on the role of feeding rhythms and further regulatory mechanisms. This study provides a critical foundation for understanding the complex feeding mechanism. (He, 2023).

In animals, hundreds of genes exhibit daily oscillation under clock regulation, and some of them are involved in metabolic functions. This study identified a CLK/CYC-binding gene, Gart, which is involved in feeding rhythms and energy metabolism independent of locomotor rhythms. Previous research reported that blocking CLK in different tissues yields different phenotypes. This study found that MET, like CYC, can combine with CLK to regulate the transcription of Gart. Knocking down Gart in different tissues exhibits different phenotypes, and Gart in different tissues can rescue the phenotype caused by CLK deletion; thus, the phenomenon caused by CLK deletion is due to the change in Gart (He, 2023).

CLK regulates the feeding rhythms of Drosophila, and its loss can cause disorders of feeding rhythms and abnormal energy storage. Tim01, Cry01, and Per01 mutants have significantly lower levels of truactkglycerides (TAGs). The maintenance of energy homeostasis is achieved by a dynamic balance of energy intake (feeding), storage, and expenditure (metabolic rate), which are crucial factors for longevity and resistance to adverse environments in Drosophila. Additionally, studies have shown that mutations of Timeless and per shorten the adult lifespan of Drosophila. This study further reveals that peripheral CLKs control the oscillation of Gart among different peripheral tissues; however, core CLKs in the brain can negatively regulate Gart expression in peripheral tissues, indicating that a complex and refined network regulatory system exists between CLK and Gart in the brain and in different peripheral tissues to coordinate feeding behavior and energy homeostasis in Drosophila and that it further affects sensitivity to starvation and longevity. These novel findings enrich the network of regulatory mechanisms by the clocks-Gart pathway on feeding, energy homeostasis, and longevity (He, 2023).

Glial cells have vital functions in neuronal development, activity, plasticity, and recovery from injury. This study reveals that flies lacking Gart in glial cells display a significant decline in the viability of Drosophila under starvation, caused by a decrease in energy storage that puts flies under a state of energy deficit. This discovery extends the functions of glial cells in feeding, energy storage, and starvation resistance controlled by Gart (He, 2023).

The fat body is the primary energy tissue for the storage of fuel molecules, such as TAG and glycogen, which play an important role in the regulation of metabolic homeostasis and provide the most energy during starvation. Indeed, functional defects of the fat body increase starvation sensitivity in Drosophila. In this study, flies lacking Gart in the fat body led to decreased energy storage, which reduces the survival rate and the survival time under starvation conditions. However, flies lacking gut Gart still maintain normal energy storage, which is not sensitive to food shortage or starvation. In addition, this study found that although high temperature can stimulate the food intake of Drosophila, which is consistent with previous reports, it does not affect the feeding rhythm (He, 2023).

This study reveals that Gart in the glia and the fat body collectively regulate the homeostasis of energy intake, storage, and expenditure, thereby influencing the viability of flies under starvation stress. Although Gart in the gut strongly influences feeding behavior, it does not play similar functions as the glia and the fat body in adversity resistance. This occurs possibly because the gut has vital roles in digestion and absorption, while the fat body has crucial functions in energy metabolism. In addition, Gart in the glia and the fat body has biased roles in the synthesis of glycogen and TAG, despite having similar functions in energy storage. The biased role of the glia and the fat body may be coordinated to provide effective energy homeostasis. These findings provide new insight into how specific circadian coordination of various tissues modulates adversity resistance and aging (He, 2023).

Such robust findings in Drosophila suggest that a decrease in lifespan and an increase in sensitivity to starvation in Drosophila is a faithful readout of disordered feeding rhythms and abnormal metabolism. Gart effects on metabolism in both glia cells and the fat body indicate the intricacy of the circadian network and energy homeostasis. It is crucial for animals to have a well-organized network to coordinate and ensure that these various tissue regions are in a normal state (He, 2023).

This study has demonstrated that CLK regulates feeding, energy homeostasis, and longevity via Gart. Even though attempts were made to explore more comprehensively how Gart coordinates and regulates the physiological functions in different tissues of D. melanogaster, there are still some limitations. For instance, it is still unclear that how Gart achieves functional differentiation in different tissues, as well as whether Gart regulates lifespan through autophagy and/or bacterial content or not, which are two critical factors related to lifespan. These future studies are of great significance for understanding the relationship between feeding and longevity regulated by Gart (He, 2023).

A Novel Drosophila Model to Investigate Adipose tissue Macrophage Infiltration (ATM) and Obesity highlights the Therapeutic Potential of Attenuating Eiger/TNFα Signaling to Ameliorate Insulin Resistance and ATM

This study presents a novel Drosophila model to investigate the mechanisms underlying adipose tissue macrophage(ATM) infiltration. This study demonstrated the therapeutic potential of attenuating Eiger/TNFα signaling to ameliorate insulin resistance and ATM. To study ATM infiltration and its consequences, a novel Drosophila model (OBL) was developed that mimics key aspects of human adipose tissue. Genetic manipulation was used to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic, and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy, and high levels of circulating glucose. Moreover, a significant infiltration of immune cells (hemocytes) in the fat bodies was observed, accompanied by insulin resistance and systemic metabolic dysregulation. Furthermore, it was found that attenuation of Eiger/TNFα signaling and using metformin and anti-oxidant bio-products like anthocyanins led to a reduction in ATM infiltration and improved insulin sensitivity. These data suggest that the key mechanisms that trigger immune cell infiltration into adipose tissue are evolutionarily conserved and may provide the opportunity to develop Drosophila models to better understand pathways critical for immune cell recruitment into adipose tissue, in relation to the development of insulin resistance in metabolic diseases such as obesity and type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). This OBL model can also be a valuable tool and provide a platform either to perform genetic screens or to test the efficacy and safety of novel therapeutic interventions for these diseases (Mirzoyan, 2023).

Fear-of-intimacy-mediated zinc transport is required for Drosophila fat body endoreplication

Endoreplication is involved in the development and function of many organs, the pathologic process of several diseases. However, the metabolic underpinnings and regulation of endoreplication have yet to be well clarified. This study showed that a zinc transporter fear-of-intimacy (foi) is necessary for Drosophila fat body endoreplication. foi knockdown in the fat body led to fat body cell nuclei failure to attain standard size, decreased fat body size and pupal lethality. These phenotypes could be modulated by either altered expression of genes involved in zinc metabolism or intervention of dietary zinc levels. Further studies indicated that the intracellular depletion of zinc caused by foi knockdown results in oxidative stress, which activates the ROS-JNK signaling pathway, and then inhibits the expression of Myc, which is required for tissue endoreplication and larval growth in Drosophila. These results indicated that FOI is critical in coordinating fat body endoreplication and larval growth in Drosophila. This study provides a novel insight into the relationship between zinc and endoreplication in insects and may provide a reference for relevant mammalian studies (Ji, 2023).

FGF signaling promotes spreading of fat body precursors necessary for adult adipogenesis in Drosophila

Knowledge of adipogenetic mechanisms is essential to understand and treat conditions affecting organismal metabolism and adipose tissue health. In Drosophila, mature adipose tissue (fat body) exists in larvae and adults. In contrast to the well-known development of the larval fat body from the embryonic mesoderm, adult adipogenesis has remained mysterious. Furthermore, conclusive proof of its physiological significance is lacking. This study shows that the adult fat body originates from a pool of undifferentiated mesodermal precursors that migrate from the thorax into the abdomen during metamorphosis. Through in vivo imaging, it was found that these precursors spread from the ventral midline and cover the inner surface of the abdomen in a process strikingly reminiscent of embryonic mesoderm migration, requiring fibroblast growth factor (FGF) signaling as well. FGF signaling guides migration dorsally and regulates adhesion to the substrate. After spreading is complete, precursor differentiation involves fat accumulation and cell fusion that produces mature binucleate and tetranucleate adipocytes. Finally, this study shows that flies where adult adipogenesis is impaired by knock down of FGF receptor Heartless or transcription factor Serpent display ectopic fat accumulation in oenocytes and decreased resistance to starvation. These results reveal that adult adipogenesis occurs de novo during metamorphosis and demonstrate its crucial physiological role (Lei, 2023).

The role of SR protein kinases in regulating lipid storage in the Drosophila fat body

The survival of animals during periods of limited nutrients is dependent on the organism's ability to store lipids during times of nutrient abundance. However, the increased availability of food in modern western society has led to an excess storage of lipids resulting in metabolic diseases. To better understand the genes involved in regulating lipid storage, genome-wide RNAi screens were performed in cultured Drosophila cells and one group of genes identified includes mRNA splicing factor genes. A group of splicing factors important for intron/exon border recognition known as SR proteins are involved in controlling lipid storage in Drosophila; however, how these SR proteins are regulated to control lipid storage is not fully understood. This study focussed on two SR protein kinases (SRPKs) in Drosophila: SRPK and SRPK79D. Decreasing the expression of these genes specifically in the adult fat body using RNAi resulted in lower levels of triglycerides and this is due to a decrease in the amount of fat stored per cell, despite having more fat cells, when compared to control flies. Decreasing SRPK and SRPK79D levels in the fat body leads to altered splicing of the β-oxidation gene, carnitine palmitoyltransferase 1 (CPT1), resulting in increased production of a more active enzyme, which would increase lipid breakdown and be consistent with the lean phenotype observed in these flies. In addition, flies with decreased SRPK and SRPK79D levels in their fat bodies eat less, which may also contribute to the decreased triglyceride phenotype. Together, these findings provide evidence to support that lipid storage is controlled by the phosphorylation of factors involved in mRNA splicing (Mercier, 2023).

The exchangeable apolipoprotein Nplp2 sustains lipid flow and heat acclimation in Drosophila

In ectotherms, increased ambient temperature requires the organism to consume substantial amounts of energy to sustain a higher metabolic rate, prevent cellular damage, and respond to heat stress. This study identifies a heat-inducible apolipoprotein required for thermal acclimation in Drosophila. Neuropeptide-like precursor 2 (Nplp2) is an abundant hemolymphatic protein thought to be a neuropeptide. In contrast, Nplp2 contributes to lipid transport, functioning as an exchangeable apolipoprotein. More precisely, Nplp2-deficient flies accumulate lipids in their gut, have reduced fat stores, and display a dyslipoproteinemia, showing that Nplp2 is required for dietary lipid assimilation. Importantly, Nplp2 is induced upon thermal stress and contributes to survival upon heat stress. It is proposed that Nplp2 associates with lipoprotein particles under homeostatic and high energy-demand conditions to optimize fat transport and storage. This study also shows that modulation of the lipid uptake and transport machinery is part of an integrated cytoprotective response (Rommelaere, 2019).

The SR proteins SF2 and RBP1 regulate triglyceride storage in the fat body of Drosophila

In Western societies where food is abundant, these excess nutrients are stored as fats mainly in adipose tissue. Fats are stored in structures known as lipid droplets, and a genome-wide screen performed in Drosophila cells has identified several genes that are important for the formation of these droplets. One group of genes found during this screen included those that regulate mRNA splicing. Previous work has identified some splicing factors that play a role in regulating fat storage; however, the full complement of splicing proteins that regulate lipid metabolism is still unknown. In this study, the levels of a number of serine-arginine (SR) domain containing splicing factors (RSF1, RBP1, RBP1-like, SF2 and Srp-54) were decreased using RNAi in the adult fat body to assess their role in the control of Drosophila metabolism. Decreasing SF2 and RBP1 showed increased triglycerides, while inducing RNAi towards RSF1, RBP1-Like and Srp-54 had no effect on triglycerides. Interestingly, the increased triglyceride phenotype in the SF2-RNAi flies was due to an increase in the amount of fat stored per cell while the RBP1-RNAi flies have more fat cells. In addition, the splicing of the beta-oxidation enzyme, CPT1, was altered in the SF2-RNAi flies potentially promoting the increased triglycerides in these animals. Together, this study identifies novel splicing factors responsible for the regulation of lipid storage in the Drosophila fat body and contributes to understanding of the mechanisms, which influence the regulation of fat storage in adipose-like cells (Bennick, 2019).

Lactate production is a prioritised feature of adipocyte metabolism

Adipose tissue is essential for whole-body glucose homeostasis, with a primary role in lipid storage. It has been previously observed that lactate production is also an important metabolic feature of adipocytes, but its relationship to adipose and whole-body glucose disposal remains unclear. Therefore, using a combination of metabolic labeling techniques, this study closely examined lactate production of cultured and primary mammalian adipocytes. Insulin treatment increased glucose uptake and conversion to lactate, with the latter responding more to insulin than did other metabolic fates of glucose. However, lactate production did not just serve as a mechanism to dispose of excess glucose, since it was also observed that lactate production in adipocytes did not solely depend on glucose availability and even occurred independently of glucose metabolism. This suggests that lactate production is prioritized in adipocytes. Furthermore, knocking down lactate dehydrogenase specifically in the fat body of Drosophila flies lowered circulating lactate and improved whole-body glucose disposal. These results emphasize lactate production is an additional metabolic role of adipose tissue beyond lipid storage and release (Krycer, 2019).

Drosophila Snazarus regulates a lipid droplet population at plasma membrane-droplet contacts in adipocytes

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Using Drosophila fat body (FB) adipocytes, this study characterized spatially distinct LD populations that are maintained by different lipid pools. Peripheral LDs (pLDs) were identified that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. Sorting nexin CG or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of Snz perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a Desaturase 1-dependent pathway. It is proposed that Drosophila adipocytes maintain spatially distinct LD populations, and Snz is identified as a regulator of LD organization and inter-organelle crosstalk (Ugrankar, 2019).

Life presents energetic and metabolic challenges, and metazoans have developed specialized nutrient-storing organs to maintain energy homeostasis and buffer against the ever-changing availability of dietary nutrients. Drosophila melanogaster is a key model organism to study energy homeostasis as many aspects of mammalian metabolism are conserved in the fly. The major energy-storage organ of insects is the fat body (FB), a central metabolic tissue that exhibits physiological functions analogous to the mammalian adipose tissue and liver including nutrient storage, endocrine secretion, and immune response. Consequently, the FB makes intimate contact with both the gut where dietary nutrients re-absorbed and circulating hemolymph that transports lipids between organs. Drosophila larvae feed continuously to promote an increase in animal mass, and absorb dietary nutrients into the FB to store these as glycogen or triacylglyceride (TAG) that is incorporated into cytoplasmic lipid droplets (LDs). TAG storage ultimately requires LD biogenesis on the surface of the endoplasmic reticulum (ER), the primary site of TAG synthesis (Wilfling, 2014). During development or when nutrients are scarce, FB cells adapt their metabolism to mobilize LDs via cytoplasmic lipases. These mobilized lipids are delivered to other organs in the hemolymph via protein shuttles called lipophorin (Lpp) particles that are analogous to mammalian VLDL particles, but how LD mobilization is related to Lpp particle lipid loading remains poorly understood (Ugrankar, 2019).

The mechanisms that govern lipid flux across the FB cell plasma membrane (PM) also remain poorly characterized, but are essential for lipid export as well as lipid uptake and storage in LDs. In insects, the internalization of hemolymph lipids into both the FB and imaginal discs is unaffected when endocytosis is blocked, suggesting a non-vesicular uptake mechanism. In line with this, Lpp proteins are not degraded via endolysosomal trafficking within the FB, consistent with a model where Lpp particles can donate and receive lipids directly at the FB cell surface. Furthermore, Lpp particles primarily transport diacylglyceride (DAG), suggesting Lpp-derived lipids are processed during their uptake and delivery to the ER by ER-resident acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes, which convert DAG to TAG. In addition to storing extracellular Lpp-derived lipids, FB cells also generate their own lipids via fatty acid de novo lipogenesis. FB cells deficient in fatty acid synthesis (FAS) enzymes exhibit severe lipodystrophy, indicating FB cells somehow balance the storage of Lpp-derived and de novo synthesized lipids to maintain fat homeostasis (Ugrankar, 2019).

Due to their specialized function in lipid uptake and storage, many fat-storing cells exhibit a unique surface architecture: their PM is densely pitted with invaginations that increase the surface area exposed to the extracellular space. In mammals, up to half the surface of white adipocytes is decorated with caveolae, invaginations that organize surface receptors as well as promote lipid and nutrient absorption. Surprisingly, Drosophila do not encode caveolin genes that are required to form caveolae. Nevertheless, Drosophila FB adipocytes exhibit their own intricate networks of surface invaginations that are stabilized by the cortical actin network. Perturbing this cortical actin network disrupts FB lipid homeostasis, suggesting a functional connection between FB surface architecture and lipid storage (Ugrankar, 2019).

Although LDs serve as organelle-scale lipid reservoirs, how cells organize their LD stores to balance storage with efficient mobilization is largely unresolved. An intuitive mechanism to organize LDs is to attach them to other organelles, as this allows them to exchange lipids with these organelles as well as potentially compartmentalize them in distinct regions of the cell interior. Recent work using Saccharomyces cerevisiae reveal that even simple yeast contain functionally distinct LD sub-populations that are spatially compartmentalized. This compartmentalization is achieved by LD-organizing proteins that bind to LDs and cluster them adjacent to the yeast vacuole/lysosome. One such organizing protein is Mdm1, an ER-anchored protein that binds to LDs and attaches them to the vacuole/lysosome surface. Mdm is highly conserved in Drosophila as CG1514/Snazarus (Snz), originally characterized as a longevity-associated gene of unknown function that is highly expressed in the Drosophila FB (Suh, 2008). Both yeast and human Snz homologs bind to LDs and regulate LD homeostasis, but the function of Snz in Drosophila remains unclear (Hariri, 2019; Datta, 2019). This study investigated how FB cells functionally and spatially organize their LD stores. FB cells contain functionally distinct LD populations that are spatially segregated into regions of the cell interior. These LD populations require distinct lipid pools for their maintenance, with LDs in the cell periphery (peripheral LDs, pLDs) requiring Lpp-dependent trafficking, whereas LDs further in the cell interior (medial LDs, mLDs) are maintained by FASN1-dependent de novo lipogenesis within the FB. Snz was also characterized as a novel regulator of pLD homeostasis that localizes to ER-PM contacts and promotes LD growth and TAG storage (Ugrankar, 2019).

Professional fat-storing cells must organize their fat reserves to balance long- term storage with the ability to efficiently mobilize lipids during energetic crises like starvation or metamorphosis. How this organization is achieved is unknown but presents significant spatial and metabolic challenges for the cell. This study reports that Drosophila FB adipocytes contain functionally distinct LD populations that are spatially segregated in the cell cytoplasm. A pLD population is maintained adjacent to the cell surface and makes intimate contact with the PM. pLD size and abundance are altered in response to fasting, suggesting pLDs are mobilized to provide circulating lipids for other larval tissues. Consistent with this, loss of lipophorin (Lpp) particles by ApoLppRNAi impacts pLD abundance and morphology, suggesting pLD maintenance requires some aspect of Lpp lipid trafficking. FB cells also contain a larger mLD population in the cell mid-plane region that is unaffected by loss of Lpp, but is drastically affected by loss of FASN1-mediated de novo lipogenesis in the FB. Remarkably, pLDs were still observed docked on the inner surface of the PM in FASN1RNAi FB tissue, further suggesting that pLDs contain lipids derived from extracellular sources that may be delivered into the FB via Lpp-dependent trafficking. This study also found that pLDs and mLDs are differentially dependent on perilipins, with mLDs relying on LSD for their morphology whereas pLDs require LSD2. Finally, this study has identified Snz as a LD-associated protein that is required for proper LD homeostasis in the FB. Snz localizes to ER-PM contacts in the FB cell periphery, and its over-expression increases TAG storage, consistent with a model whether Snz regulates ER-PM inter-organelle crosstalk that promotes lipid storage in LDs. In line with this, Snz functionally interacts with the ER- resident FA desaturase DESAT1, which is required for Snz-driven TAG accumulation (Ugrankar, 2019).

LDs have long been observed to be tethered to other organelles such as mitochondria and peroxisomes, and this impacts their sub-cellular distribution as well as their ability to exchange lipids with these organelles. Recent studies have identified specific proteins that bind to the surfaces of LDs, and mediate their attachment to other cellular organelles. Among these, yeast Mdm directly binds to nascent LDs, and promotes their attachment to the yeast vacuole. This Mdm1- vacuole interaction is critical for defining this LD positioning, as replacement of Mdm1's vacuole-binding PX domain with a PM-binding domain re-localizes Mdm to ER-PM contact sites and causes LDs to bud instead from the cortical ER adjacent to the PM (Hanaa Hariri, 2019). In addition to its role as an organelle tether, Mdm also positively regulates LD biogenesis by recruiting the fatty acyl-CoA ligase Faa to the ER surface, where it induces the incorporation of FAs into TAG in the LD (Hanaa Hariri, 2019). This study finds that Snz may function similarly to Mdm in both the spatial positioning of LDs, as well as promoting LD biogenesis. Snz localizes to the FB cell periphery and co-localizes with the ER-PM contact site biomarker dMAPPER, suggesting it enriches at regions of close contact between the ER and PM and potentially functions as an inter-organelle tether. Consistent with this, the Snz PX domain binds to liposomes containing phospholipids normally enriched on the PM, and exhibits a non-canonical lipid binding surface that mediates electrostatic interactions with phospholipids that would be present on the PM. This suggests a model where Snz functions in some aspect of functional coupling between the ER and PM, and potentially assists in lipid uptake from the hemolymph. Snz may also help to localize ER-resident lipid processing enzymes such as DESAT to the cell periphery, creating a localized pool of FA processing enzymes in the peripheral ER network which can efficiently process lipids prior to their incorporation into TAG. Consistent with this, Snz over-expression in the FB promotes TAG storage, and Snz can directly associate with LDs (Ugrankar, 2019).

Once thought to be pathogenic, adipocyte fat stores have more recently been proposed to act as metabolic buffers that protect against caloric overload and serve as sinks to reduce circulating lipids and sugars. As such, factors that enhance adipocyte fat storage may be protective against insulin insensitivity, organismal lipotoxicity, and other T2D-like pathologies. The data are consistent with this model, and indicate that Snz up-regulation promotes TAG storage in the FB through a DESAT1-dependent pathway. These elevated TAG stores not only prolong organismal survival during sustained fasting, but also promote organismal homeostasis that extends Drosophila lifespan, as well as buffers the pathological effects of chronic HSD (Ugrankar, 2019).

Collectively, these data support a model where Snz-associated pLDs provide a spatially-compartmentalized sink for lipids derived from the extracellular hemolymph. This pLD population may serve multiple functions. It could allow FB cells to quickly and efficiently process and store incoming Lpp-derived lipids from the hemolymph, thus avoiding their potentially lipotoxic accumulation in the cytoplasm. This would promote general cellular homeostasis and could minimize FA lipotoxicity during elevated lipid uptake. In addition, the high surface-to-volume size ratio of small pLDs may promote their efficient mobilization by cytoplasmic lipases during fasting. Since they are near the surface, pLD mobilization could also allow liberated FAs to be efficiently transferred to Lpp particles docked on the FB surface, where they can be subsequently trafficked to other organs. Snz has clear mammalian homologs including SNX which also bind PM-associated phospholipids. Whether SNX or other Snz homologs are also able to interact with LDs in distinct regions of the mammalian cell interior is unclear, but will be the focus of future studies (Ugrankar, 2019).

Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression

The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, this study shows that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status (Ingaramo, 2020).

The ability of an organism to sense nutrient stress and coordinate metabolic and physiological responses is critical for its survival. Over the last years, the p53 tumor suppressor has emerged as an important regulator of cellular metabolism, and its activation has been regularly observed in response to diverse metabolic inputs, such as changes in oxygen levels or nutrient availability. It has been shown that p53 interacts with main players in key nutrient-sensing pathways, such as mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), leading to modulation of autophagy and lipid and carbohydrate metabolism. p53 restricts tumor development partially by inhibiting glycolysis, limiting the pentose phosphate pathway, and promoting mitochondrial respiration. Conversely, p53 activation can benefit tumor growth by stimulating adaptive cellular responses in nutrient-deficient conditions. p53 activation is known to induce cell-cycle arrest and promote cell survival in response to transient glucose deprivation, regulate autophagy and increase cell fitness upon fasting, and promote cancer cell survival and proliferation after serine or glutamine depletion. Therefore, p53 plays a pivotal role in the ability of cells to sense and respond to nutrient stress, functions that are important not only to control cancer development but also to regulate crucial aspects of animal physiology. Further studies concerning p53 regulation and function in response to nutrient and metabolic challenges at an organismal level would expand understanding on the role of p53 in normal animal physiology, aging, and disease (Ingaramo, 2020).

The single Drosophila ortholog of mammalian p53 (Dmp53) has also been shown to regulate tissue and metabolic homeostasis. Dmp53 regulates energy metabolism through induction of cell-cycle arrest and cell growth inhibition in response to mitochondrial dysfunction by regulating glycolysis and oxidative phosphorylation to promote cell fitness in dMyc-overexpressing cells and by modulating autophagy protecting the organism from oxidative stress. Studies in Drosophila have also identified tissue-specific roles of Dmp53 in regulating lifespan and adaptive metabolic responses impacting on animal aging and stress survival, evidencing conserved functions of p53, and positioning Drosophila p53 studies as a valuable alternative providing relevant insights on mammalian health and disease (Ingaramo, 2020).

The insulin pathway is highly conserved from mammals to Drosophila and regulates carbohydrate and lipid metabolism, tissue growth, and longevity in similar ways. Drosophila insulin-like peptides (Dilps) promote growth and maintain metabolic homeostasis through activation of a unique insulin receptor (dInR) and of a conserved intracellular insulin and insulin-like growth factor (IGF) signaling pathway (IIS). Dietary conditions tightly regulate Dilp2 production and/or secretion from the insulin-producing cells (IPCs), neuroendocrine cells analogous to pancreatic β-cells located in the fly brain. Interestingly, a nutrient-sensing mechanism in the fat body (FB), a functional analog of vertebrate adipose and hepatic tissues, non-autonomously regulates Dilp2 secretion and couples systemic growth and metabolism with nutrient availability. According to the nutritional status, the FB produces signaling molecules capable of promoting or inhibiting insulin secretion from the IPCs. Thus, a simple integrated system composed of various organs and conserved signaling pathways regulates metabolic homeostasis and organismal growth in response to nutrient availability (Ingaramo, 2020).

The FB also functions as the organism's main energy reserve and is responsible for coupling energy expenditure to nutrient status. In well-fed animals, circulating insulin activates insulin receptors in the FB and promotes energy storage in the form of glycogen and triacylglycerol (TAG). Upon limited nutrient availability, stored lipids and glycogen are broken down to supply energy for the rest of the body. Previous work showed that FB-specific inhibition of Dmp53 activity accelerated the consumption of main energy stores, reduced sugar levels, and compromised organismal survival during nutrient deprivation. The mechanism by which Dmp53 regulates metabolic homeostasis and organismal survival under nutrient stress is not entirely understood and might involve regulation of specific signaling and metabolic pathways (Ingaramo, 2020).

This study provides evidence that AMPK-dependent Dmp53 activation in the FB non-cell-autonomously regulates TOR signaling and autophagy induction upon acute starvation, which is essential for organismal survival. Dmp53 activation in response to nutritional stress is required for proper communication between the FB and IPCs by modulating the expression of the Drosophila Leptin analog, Unpaired-2 (Upd2). Elevated Upd2 levels in adipose cells of starved Dmp53-depleted animals result in sustained Dilp2 circulating levels, activation of insulin/TOR signaling, and impaired autophagy induction in the whole animal, therefore reducing survival rates upon nutrient deprivation. These results indicate that Dmp53 plays an essential role in Drosophila, integrating nutrient status with metabolic homeostasis by modulating Dilp2 circulating levels, systemic insulin signaling, and autophagy (Ingaramo, 2020).

Even though progress has been made in understanding p53 metabolic functions at the cellular level, its role in the context of a whole animal response to metabolic stress is poorly understood. This study provides evidence that Drosophila p53 is critically involved in nutrient sensing and in the orchestration of an organismal response to nutrient stress. AMPK-dependent Dmp53 activation in the FB in response to nutritional stress is required for proper communication between the FB and the IPCs by modulating the expression of Drosophila Leptin analog, Upd2. Elevated Upd2 levels and activation of JAK/STAT signaling in the brain of starved Dmp53-depleted animals result in sustained Dilp2 circulating levels, activation of insulin signaling, and impaired autophagy induction in various tissues, therefore reducing survival rates upon nutrient deprivation. These results position the AMPK-p53 axis as a key player in nutrient sensing and in regulating adaptive physiological responses to low nutrient availability by remotely controlling insulin secretion and autophagy (Ingaramo, 2020).

Studies in mice have also shown that p53 is activated under several nutrient stress conditions, such as nutrient deprivation, high-caloric diet, and high-fat diet (HFD). p53 becomes activated under nutrient deprivation and regulates expression of genes involved in mitochondrial fatty acid uptake and oxidative phosphorylation. In turn, pharmacological or genetic inhibition of p53 prevented excessive fat accumulation commonly observed under HFD and resulted in decreased expression of proinflammatory cytokines and improved insulin resistance in mice with type 2 diabetes (T2D)-like disease. Conversely, upregulation of p53 in adipose tissue caused an inflammatory response that led to insulin resistance. These results show that both mice and Drosophila p53 activation in individuals exposed to challenging nutrient conditions regulate global metabolism and directly contribute to diet-associated phenotypes (Ingaramo, 2020).

Leptin is mainly produced by adipose tissue in mice and humans, and regulates food intake, energy expenditure, and metabolism acting mostly on neuronal targets in the brain. This study has shown that Dmp53 activation in the FB under nutrient stress impacts systemic insulin signaling and autophagy induction via regulation of Upd2/Leptin expression. Notably, reduced survival of Dmp53-depleted animals to nutrient deprivation was highly reverted when inhibiting either Upd2 expression in the FB or JAK/STAT signaling in GABAergic neurons in the fly brain. Similar to Upd2, Leptin circulating levels decline during fasting conditions and are increased in animals fed with a HFD. Low Leptin levels during starvation trigger adaptive metabolic and hormonal responses, such as increased appetite and decreased energy expenditure. In HFD-fed mice, p53 activation is necessary for fat accumulation in the liver and adipose tissue, indicating that p53 is essential for coordinating energy expenditure and storage in response to nutrient availability (Liu, 2017). Reduced expression of p53 target genes, such as GLUT4 and SIRT1, has been proposed to reduce NAD+ levels and energy expenditure, leading to obesity (Liu, 2017). Alternatively, p53 activation in adipose cells could regulate Leptin expression, which is known to act on the CNS to reduce food intake and enhance energy expenditure, thus limiting obesity in times of nutrient abundance. Further investigations into the role of adipose tissue p53 activity in modulating physiological and metabolic responses to stress will be necessary to have a better picture of the role p53 plays in the development of metabolic disorders, such as obesity and T2D. Of importance, based on conserved adipose tissue-specific functions of p53 and signaling pathways involved, studies in Drosophila are likely to provide insights relevant to mammalian health and disease (Ingaramo, 2020).

In the past decade, significant interest has been raised in understanding non-canonical functions of p53 that might have potential roles in tumor suppression. The fact that p53 is activated in the adipose tissue of obese animals, along with the results presented concerning a putative direct role of p53 in controlling Upd2/Leptin expression, demonstrates the importance of p53 in regulating metabolism. This is particularly interesting given that epidemiological studies over the last few decades have shown a strong influence of obesity on cancer risk and that increased Leptin can have hormone-like functions affecting tumor development. In this context, the results give insights toward the molecular understanding of p53 activation under metabolic stress and its possible role in tumor suppression acting at either local or organismal level (Ingaramo, 2020).

TOR and AMPK play essential roles in nutrient sensing, are important regulators of energy balance at both cellular and whole-body levels, and have been shown to interact with p53. Previously showed that TOR inhibition following long starvation treatments (24-48 h) contributes to Dmp53 activation, mainly by alleviating miRNA-mediated targeting of Dmp53 in the FB (Barrio, 2014). This work, demonstrated that rapid activation of Dmp53 is dependent on AMPK and absolutely required for metabolic and physiological changes that promote organismal resistance to nutrient deprivation. This short-term activation of Dmp53 by AMPK could be part of a dual mechanism along with previously demonstrated long-term activation by lack of TOR, and both of these regulating mechanisms may be important for establishing a rapid response to transient acute nutrient stress also guaranteeing a sustained response when facing a much longer nutrient-deprived period. Given that activated Dmp53 reduces Upd2 expression, systemic insulin, and TOR signaling, it would be reasonable to speculate that Dmp53-dependent TOR inhibition constitutes a positive feedback loop to reinforce Dmp53 activation upon long-term starvation conditions. Therefore, the results place p53 in a crucial position connecting nutrient sensing pathways to endocrine mechanisms, as part of a possible physiological feedback mechanism (Ingaramo, 2020).

Drosophila AMPK activation has been shown to extend lifespan and promote tissue proteostasis through non-cell-autonomous regulation of autophagy. Given that Dmp53, acting downstream of AMPK under nutrient stress, non-cell-autonomously regulates Dilp2 levels and autophagy, it will be interesting to determine whether p53, and perhaps its direct phosphorylation by AMPK, is also required for extending organismal lifespan upon tissue-specific AMPK activation (Ingaramo, 2020).

FOXO-mediated repression of Dicer1 regulates metabolism, stress resistance, and longevity in Drosophila

The adipose tissue plays a crucial role in metabolism and physiology, affecting animal lifespan and susceptibility to disease. This study presents evidence that adipose Dicer1 (Dcr-1), a conserved type III endoribonuclease involved in miRNA processing, plays a crucial role in the regulation of metabolism, stress resistance, and longevity. The results indicate that the expression of Dcr-1 in murine 3T3L1 adipocytes is responsive to changes in nutrient levels and is subject to tight regulation in the Drosophila fat body, analogous to human adipose and hepatic tissues, under various stress and physiological conditions such as starvation, oxidative stress, and aging. The specific depletion of Dcr-1 in the Drosophila fat body leads to changes in lipid metabolism, enhanced resistance to oxidative and nutritional stress, and is associated with a significant increase in lifespan. Moreover, mechanistic evidence is provided showing that the JNK-activated transcription factor FOXO binds to conserved DNA-binding sites in the dcr-1 promoter, directly repressing its expression in response to nutrient deprivation. These findings emphasize the importance of FOXO in controlling nutrient responses in the fat body by suppressing Dcr-1 expression. This mechanism coupling nutrient status with miRNA biogenesis represents a novel and previously unappreciated function of the JNK-FOXO axis in physiological responses at the organismal level (Sanchez, 2023).

Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells

Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. This study found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology (Barber, 2016).

Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition

The general transcription factor TBP (TATA-box binding protein) and its associated factors (TAFs) together form the TFIID complex, which directs transcription initiation. Through RNAi and mutant analysis, this study identified a specific TBP family protein, TRF2, and a set of TAFs that regulate lipid droplet (LD) size in the Drosophila larval fat body. Among the three Drosophila TBP genes, trf2, tbp and trf1, only loss of function of trf2 results in increased LD size. Moreover, TRF2 and TAF9 regulate fatty acid composition of several classes of phospholipids. Through RNA profiling, TRF2 and TAF9 were found to affect the transcription of a common set of genes, including peroxisomal fatty acid beta-oxidation-related genes that affect phospholipid fatty acid composition. Knockdown of several TRF2 and TAF9 target genes results in large LDs, a phenotype which is similar to that of trf2 mutants. Together, these findings provide new insights into the specific role of the general transcription machinery in lipid homeostasis (Fan, 2017).

This study reveals a rather specific role of TRF2 and TAFs, which are general transcription factors, in regulating LD size. In addition, TRF2 and TAF9 affect phospholipid fatty acid composition, most likely through ACOX genes which mediate peroxisomal fatty acid β-oxidation (Fan, 2017).

By binding to their responsive elements in target genes, specific transcription factors like SREBP (see Drosophila Srebp), PPARs and NHR49, play important roles in lipid metabolism. It is interesting to find that the general transcription machineries, in this case TRF2 and core TAFs, also exhibit specificity in regulating lipid metabolism. In the Drosophila late 3rd instar larval fat body, defects in trf2 cause increased LD size, whereas mutation of the other two homologous genes, tbp and trf1, have no obvious effects on lipid storage. Inactivation of taf genes causes a similar phenotype to trf2 mutation, suggesting that TRF2 may associate with these TAF proteins to direct transcription of specific target genes. Moreover, trf2 mutants have large LDs at both 2nd and early 3rd instar larval stages, suggesting that general transcription factors are also required at early developmental stages for LD size regulation. Interestingly, taf9 mutants have no obvious phenotype at these stages. It is possible that TAF9 may act as an accessory factor compared to promoter-binding TRF2. This is consistent with the fact that less genes are affected in taf9 mutants than trf2 mutants in RNA-seq analysis. It was also found that knockdown of trf2 in larval and adult fat body leads to different LD phenotype. This may be due to different lipid storage status or different LD size regulatory mechanisms between larval and adult stages (Fan, 2017).

The finding of this study adds to the growing evidence supporting a specific role of general transcription factors in lipid homeostasis. For example, knockdown of RNA Pol II subunits such as RpII140 and RpII33 leads to small and dispersed LDs in Drosophila S2 cells. Mutation in DNA polymerase δ (POLD1) leads to lipodystrophy with a progressive loss of subcutaneous fat. Furthermore, TAF8 and TAF7L were reported to be involved in adipocyte differentiation. Moreover, previous studies showed that several subunits of the Mediator complex interact with specific transcription factors and play important roles in lipid metabolism. Added together, these lines of evidence strongly support essential and specific roles of the core/basal transcriptional machinery components in lipid metabolism (Fan, 2017).

Using RNA-seq analysis, rescue experiments and ChIP-qPCR, identified several target genes regulated by TRF2 and TAF9. It is possible that other genes may regulate LD size but were missed in the RNA-seq analysis and RNAi screening assay because of either insufficient alterations in genes expression (lower than the twofold threshold) or low efficiency of RNAi. Among all the verified target genes of TRF2 and TAF9,CG10315, which strongly rescues the trf2G0071 mutant phenotype when overexpressed and encodes the eukaryotic translation initiation factor eIF2B-δ, may be a good candidate for further study. Although they are best known for their molecular functions in mRNA translation regulation, eIFs have been implicated in several other processes, including cancer and metabolism. For example, in yeast, eIF2B physically interacts with the VLCFA synthesis enzyme YBR159W. In adipocytes, eIF2α activity is correlated with the anti-lipolytic and adipogenesis inhibitory effects of the AMPK activator AICAR. In addition, given the evidence that some eIFs, such as eIF4G and eIF-4a, localize on LDsand knockdown of some eIFs, including eIF-1A, eIF-2β, eIF3ga, eIF3-S8 and eIF3-S9, results in large LDs in Drosophila S2 cells, it is important to further explore the specific mechanisms of these eIFs in LD size regulation (Fan, 2017).

Although TRF2 exists widely in metazoans and shares sequence homology in its core domain with TBP, it recognizes sequence elements distinct from the TATA-box. A previous study has investigated TRF2- and TBP-bound promoters throughout the Drosophila genome in S2 cells and revealed that some sequence elements, such as DRE, are strongly associated with TRF2 occupancy while the TATA-box is strongly associated with TBP occupancy (Isogai, 2007). This study also identified that DRE is significantly enriched in extended promoters of the 181 target genes. The distribution of TATA-boxes in the core promoters of the 181 target genes compared with all genes was further explored, and it was found that the TATA-box is not enriched in the core promoters of TRF2 target genes. The proportion of TATA-box is 0.155 (75 of 484 isoforms) for the 181 target genes while the proportion is 0.217 (7849 of 36099 isoforms) for all genes as the background. These results suggest that TRF2 and TAF9 may regulate the expression of a subset of genes by recognizing specific sequence elements such as DRE but not the TATA-box (Fan, 2017).

This study shows that expression of peroxisomal fatty acid β-oxidation pathway genes, including two acyl-CoA oxidase (ACOX) genes, CG4586 and CG9527, the β-ketoacyl-CoA thiolase gene CG9149, and the enoyl-CoA hydratase gene CG9577, is regulated by TRF2 and TAF9. Lipidomic analysis indicates that in the fat body of trf2 and taf9 RNAi, many phospholipids, such as PA, PC, PG and PI, contain more long chain fatty acids. Furthermore, knockdown of CG4586 and CG9527 in the fat body also causes similar changes.

These results coincide with the function of ACOX, which is implicated in the peroxisomal fatty acid β-oxidation pathway for catabolizing very long chain fatty acids and some long chain fatty acids. Similar to these findings, a previous study found that defective peroxisomal fatty acid β-oxidation resulted in enlarged LDs in C. elegans and blocked catabolism of LCFAs, such as vaccenic acid, which probably contributed to LD expansion in mutant worms. Since overexpressing CG4586 or CG9527 only marginally rescues the enlarged LD phenotype of trf2 mutants, it remains to be determined whether the increased level of long chain fatty acid-containing phospholipids contributes to LD size. Regarding the regulation of fatty acid chain length in phospholipids, a recent study reported that there was increased acyl chain length in phospholipids of lung squamous cell carcinoma accompanied by significant changes in the expression of fatty acid elongases (ELOVLs) compared to matched normal tissues. A functional screen followed by phospholipidomic analysis revealed that ELOVL6 is mainly responsible for phospholipid acyl chain elongation in cancer cells. The current findings provide new clues about the regulation of fatty acid chain length in phospholipids. ELOVL and the peroxisomal fatty acid β-oxidation pathway may represent two opposing regulators in determining fatty acid chain length in vivo (Fan, 2017).

Previous studies have shown that TRF2 is involved in specific biological processes including embryonic development, metamorphosis, germ cell differentiation and spermiogenesis. The current results reveal a novel function of TRF2 in the regulation of specialized transcriptional programs involved in LD size control and phospholipid fatty acid composition. Since TRF2 is conserved among metazoans, its role in the regulation of lipid metabolism may be of considerable relevance to various organisms including mammals. These findings may provide new insights into both the regulation of lipid metabolism and the physiological functions of TRF2 (Fan, 2017).

Lipid droplet subset targeting of the Drosophila protein CG2254/dmLdsdh1

Lipid droplets (LDs) are the principal organelles of lipid storage. They consist of a hydrophobic core of storage lipids, surrounded by a phospholipid monolayer with proteins attached. While some of these proteins are essential to regulate cellular and organismic lipid metabolism, key questions concerning LD protein function, such as their targeting to LDs, are still unanswered. Intriguingly, some proteins are restricted to LD subsets by an as yet unknown mechanism. This finding makes LD targeting even more complex. This study characterized the Drosophila protein CG2254 which targets LD subsets in cultured cells and different larval Drosophila tissues, where the prevalence of LD subsets appears highly dynamic. An amphipathic amino acid stretch was shown to mediate CG2254 LD localization. Additionally, a juxtaposed sequence stretch was identified limiting CG2254 localization to LD subsets. This sequence is sufficient to restrict a chimeric protein - consisting of the subset targeting sequence introduced to an otherwise pan LD localized protein sequence - to LD subsets. Based on its subcellular localization and annotated function, it is suggested to rename CG2254 to Lipid droplet subset dehydrogenase 1 (Ldsdh1) (Thul, 2017).

THADA regulates the organismal balance between energy storage and heat production

Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. This study uncovered the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution (Moraru, 2017).

Obesity has reached pandemic proportions, with 13% of adults worldwide being obese. Although the modern diet triggers this phenotype, 60%-70% of an individual's susceptibility to obesity is genetic. The underlying evolutionary drivers that cause susceptibility vary from person to person and are not clear. Since obesity is most prevalent in populations that have adapted to warm climates, an emerging theory proposes that populations in warm climates evolved low metabolic rates to reduce heat production, making them prone to obesity. In contrast, populations in cold climates evolved high energy consumption for thermogenesis, making them more resistant to obesity. This theory predicts the existence of genes that have been selected in the human population by climate adaptation which regulate the balance between heat production and energy storage (Moraru, 2017).

The gene Thyroid Adenoma Associated (THADA) has played an important role in human evolution. Comparison of the Neanderthal genome with the genomes of current humans reveals that SNPs in THADA were the most strongly positively selected SNPs genome-wide in the evolution of modern humans. Furthermore, as hominins left Africa circa 70,000 years ago, they adapted to colder climates. Genome-wide association studies (GWAS) identified THADA as one of the top genes that was evolutionarily selected in response to cold adaptation, suggesting a link between THADA and energy metabolism. THADA was also identified as one of the top risk loci for type 2 diabetes by GWAS Although follow-up studies could not confirm an association between THADA SNPs and various aspects of insulin release or insulin sensitivity, some studies did find an association between THADA and pancreatic β-cell response or marginal evidence for an association with body mass index. In sum, THADA has been connected to both metabolism and adaptation to climate. Nonetheless, nothing is known about the function of THADA in animal biology, at the physiological or the molecular level. Animals lacking THADA function have not yet been described. An analysis of the amino acid sequence of THADA provides little or no hints regarding its molecular function (Moraru, 2017).

To study the function of THADA, THADA knockout flies were generated. THADA knockout animals are obese and produce less heat than controls, making them sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and regulates organismal metabolism via calcium signaling. In addition to unveiling the physiological role and molecular function of this medically relevant gene, the results also show that one gene that has been strongly selected during human evolution in response to environmental temperature plays a functional role in regulating the balance between heat production and energy storage, affecting the propensity to become obese (Moraru, 2017).

This study reports the physiological and molecular function of THADA in animals. THADA mutants were found to be obese, sensitive to the cold, and have reduced heat production compared with controls. THADA interacts physically with SERCA and modulates its activity. The combination of improved calcium pumping and cold sensitivity of THADA mutants indicates that THADA acts as an SERCA uncoupler, similar to sarcolipin. This interaction between THADA and SERCA appears to be an important part of THADA function, since the obesity phenotype of THADA mutants can be rescued by mild SERCA knockdown (Moraru, 2017).

Calcium signaling is increasingly coming into the spotlight as an important regulator of organismal metabolism. In a genome-wide in vivo RNAi screen in Drosophila to search for genes regulating energy homeostasis, calcium signaling was the most enriched gene ontology category among obesity-regulating genes (Baumbach, 2014). Cytosolic calcium levels can alter organismal adiposity by more than 10-fold (from 15% to 250% of control levels) (Baumbach, 2014), indicating that it is an important regulator of organismal metabolism. In line with these numbers, THADAKO flies have 250% the triglyceride levels of control flies. The phenotypes observed for other regulators of calcium signaling all point in the same general direction that high ER calcium leads to hyperphagia and obesity. Likewise, mice heterozygous for a mutation in IP3R are susceptible to developing glucose intolerance on a high-fat diet (Moraru, 2017).

The molecular mechanisms by which ER calcium regulates organismal metabolism are not yet fully understood, but this important question will surely be the subject of intense research in the near future. Calcium levels are known to regulate activity of tricarboxylic acid cycle enzymes such as α-ketoglutarate dehydrogenase, isocitrate dehydrogenase, and pyruvate dehydrogenase, which could explain part of the effect of calcium on metabolism (Moraru, 2017).

THADA mutation leads to obesity due to roles of THADA both in the fat body and in neurons. This has also been observed for IP3R mutants. Calcium signaling regulates lipid homeostasis directly and cell-autonomously in the fat body, as observed in seipin mutants (Bi, 2014) or when Stim expression was modulated specifically in fat tissue. In addition, it regulates feeding via the CNS. Interestingly, while THADA mutant females have elevated glycogen levels, THADA mutant males do not. It is not known why this is the case: it could be due to the higher energetic demand in females compared with males, leading to stronger metabolic phenotypes in females, or THADA might regulate glycogen metabolism differently in the two sexes (Moraru, 2017).

GWAS identified THADA as one of the top risk loci for type 2 diabetes. The data presented in this study indicates that THADA regulates lipid metabolism and feeding, suggesting that the association between THADA and diabetes may be causal in nature. THADA mutant flies develop obesity, but have normal circulating sugar levels under standard laboratory food conditions. Interestingly, mouse mutants for IP3R likewise do not become insulin resistant under a regular diet, but do become insulin resistant on a high-fat diet. Combined, these data suggest that the primary effect of altered THADA activity and calcium signaling is on lipid metabolism, and that a combination with high-fat feeding may be required to lead to type 2 diabetes over time. This could potentially explain why follow-up association studies did not find links between THADA and insulin sensitivity but did find links between THADA and adiposity (Moraru, 2017 and references therein).

Insects are ectotherms, meaning that their internal physiological sources of heat are not sufficient to control their body temperature. Nonetheless they do produce heat, and the main sources of heat are either of muscular origin due to movement or shivering, or of biochemical origin from futile cycles that consume ATP with no net work. For instance, bumblebees preheat their flight muscles by simultaneously activating phosphofructokinase and fructose 1,6-bisphosphatase, which catalyze opposing enzymatic reactions, leading to the futile hydrolysis of ATP and release of heat. Drosophila also have mitochondrial uncoupling proteins, which potentially generate a futile metabolic cycle by dissipating the mitochondrial membrane potential. It is proposed in this stduy that uncoupled hydrolysis of ATP by SERCA could constitute one additional example of such a futile cycle that produces heat. It cannot be excluded, however, that THADA knockout flies might also have changes in their evaporative heat loss contributing to their reduced thermogenesis. The thermogenic phenotypes in THADA knockout flies are relatively mild, perhaps reflecting the ectothermic nature of flies. Hence it will be of interest to study in the future the metabolic parameters of THADA knockout mice (Moraru, 2017).

The combination of cold sensitivity and obesity in THADA mutant animals is interesting in terms of the evolutionary origins of the current obesity pandemic. The prevalence of obesity is highest in populations that have adapted to warmer climates, suggesting that people in warm climates evolved reduced metabolic rates to prevent overheating, and in combination with a modern diet this reduced metabolic rate leads to obesity. Interestingly, THADA is a gene that provides support for this theory. SNPs in THADA are among the SNPs genome-wide that have been most strongly selected as humans adapted to climates of different temperatures). Indeed, comparison of the Neanderthal genome with the genomes of current humans reveals that SNPs in THADA were the most strongly positively selected SNPs genome-wide in the evolution of modern humans. The data presented in this study show that THADA simultaneously affects sensitivity to cold and obesity. Uncoupled SERCA ATPase activity is a major contributor to non-shivering thermogenesis. Similar to animals mutant for another SERCA uncoupling protein, sarcolipin, this study found that THADA mutants are sensitive to the cold. This provides a possible explanation for why evolution selected for SNPs in THADA. In addition, THADA, via SERCA, also regulates lipid homeostasis. THADA thereby provides a genetic and molecular link between climate adaptation and obesity (Moraru, 2017).

Salt-Inducible kinase 3 provides sugar tolerance by regulating NADPH/NADP+ redox balance

Nutrient-sensing pathways respond to changes in the levels of macronutrients, such as sugars, lipids, or amino acids, and regulate metabolic pathways to maintain organismal homeostasis. Consequently, nutrient sensing provides animals with the metabolic flexibility necessary for enduring temporal fluctuations in nutrient intake. Recent studies have shown that an animal's ability to survive on a high-sugar diet is determined by sugar-responsive gene regulation. It remains to be elucidated whether other levels of metabolic control, such as post-translational regulation of metabolic enzymes, also contribute to organismal sugar tolerance. Furthermore, the sugar-regulated metabolic pathways contributing to sugar tolerance remain insufficiently characterized. This study identified Salt-inducible kinase 3 (SIK3), a member of the AMP-activated protein kinase (AMPK)-related kinase family, as a key determinant of Drosophila sugar tolerance. SIK3 allows sugar-feeding animals to increase the reductive capacity of nicotinamide adenine dinucleotide phosphate (NADPH/NADP+). NADPH mediates the reduction of the intracellular antioxidant glutathione, which is essential for survival on a high-sugar diet. SIK3 controls NADP+ reduction by phosphorylating and activating Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway. SIK3 gene expression is regulated by the sugar-regulated transcription factor complex Mondo-Mlx, which was previously identified as a key determinant of sugar tolerance. SIK3 converges with Mondo-Mlx in sugar-induced activation of G6PD, and simultaneous inhibition of SIK3 and Mondo-Mlx leads to strong synergistic lethality on a sugar-containing diet. In conclusion, SIK3 cooperates with Mondo-Mlx to maintain organismal sugar tolerance through the regulation of NADPH/NADP+ redox balance (Teesalu, 2017).

A search for new genes essential for sugar tolerance resulted in the identification of Salt-inducible kinase 3 (SIK3; CG42856). The salt-inducible kinases (SIKs) belong to the family of the AMP-activated protein kinase (AMPK)-related kinases, and they are emerging as key regulators of energy metabolism and. Although SIKΔ null mutants were previously denoted to display an early larval lethal phenotype, nearly 50% of them developed to pupal stage on a low-sugar diet (LSD). In contrast, on a high-sugar diet (HSD), the development of SIKΔ larvae was strikingly impaired, leading to almost complete larval lethality. Similarly to the mutants, animals with ubiquitous knockdown of SIK3 by RNAi were highly sugar intolerant. Furthermore, SIK3 knockdown larvae survived poorly on a sugar-only diet. HSD reduced food intake in general, but there was no significant difference between control and SIKΔ mutant animals on an HSD. Earlier findings of reduced lipid levels in SIK3-deficient animals were confirmed but several additional metabolic phenotypes were also discovered. While circulating glucose remained unchanged, the SIKΔ mutants displayed elevated levels of circulating trehalose. High levels of lactate and sorbitol, two glucose-derived metabolites, also implied that glucose metabolism was disturbed in SIK3-deficient animals. Moreover, SIKΔ mutants displayed hemolymph acidification, a phenotype observed earlier in mutants of Activin encoding dawdle with impaired glucose metabolism. In conclusion, the data suggest that SIK3 is a key determinant of sugar tolerance and that its role in metabolic regulation in vivo is significantly broader than previously anticipated (Teesalu, 2017).

Similarly to SIKΔ mutants, mlx mutants display sugar intolerance and high circulating trehalose levels, as well as reduced triacylglycerol (TAG) levels. Moreover, mlx mutants also displayed high circulating sorbitol levels and low hemolymph pH. These phenotypic similarities led to an exploration of the possible functional relationship between SIK3 and Mondo-Mlx. Interestingly, the expression of SIK3 was downregulated in mlx mutants during all larval stages. The Mondo-Mlx complex is most highly expressed in the fat body and in the gut and renal (Malpighian) tubules. Consistently, the mRNA expression of SIK3 was found to be Mlx dependent in all of these tissues. To test the possible sugar-dependent regulation of SIK3, first-instar Drosophila larvae were fed with an LSD versus an HSD for 16 hr and SIK3 expression was modestly, but significantly, elevated on an HSD. mlx mutants displayed no elevation of SIK3 expression in response to dietary sugar. To explore whether SIK3 is a direct target of Mondo-Mlx, the SIK3 promoter region was examined for putative Mondo-Mlx binding sites, i.e., carbohydrate response elements (ChoREs; consensus CACGTGnnnnnCACGTG). A putative ChoRE, was found which was conserved among Drosophilae. Chromatin immunoprecipitation (ChIP) in S2 cells revealed a moderate, but significant, enrichment of Mlx on the SIK3 promoter region, and the Mlx binding was increased on high glucose. In conclusion, these results show that SIK3 gene expression is regulated by Mondo-Mlx, and the phenotypic similarities further suggest functional interplay between SIK3 and Mondo-Mlx on metabolic regulation (Teesalu, 2017).

It was observed earlier that the pentose phosphate pathway (PPP) is transcriptionally regulated by Mondo-Mlx and that PPP activity is essential for sugar tolerance and maintaining TAG levels. The phenotypic similarities of SIK3 and mlx mutants led to a hypothesis that SIK3 might also regulate PPP activity. Indeed, co-immunoprecipitation uncovered a physical interaction between SIK3 and glucose-6-phosphate dehydrogenase (G6PD; encoded by Zwischenferment; Zw), the rate-limiting enzyme of the PPP. To analyze G6PD phosphorylation, phosphate-binding tag (Phos-tag) SDS-PAGE was used. Co-expression of SIK3 induced several slow-migrating bands of G6PD, which were confirmed to be phosphorylated forms by alkaline phosphatase treatment. An in vitro kinase assay to detect the activity of SIK3 co-purified with G6PD provided further evidence of SIK3-mediated phosphorylation of G6PD (Teesalu, 2017).

To identity the phosphorylation sites of SIK3, mass spectrometric analysis of G6PD, which was affinity purified from S2 cells, was used. In total, eight high-confidence phosphorylation sites were detected, and six of them were only present upon SIK3 co-expression. These six sites may be both directly and indirectly regulated by SIK3. Since SIK3 is a serine/threonine kinase, phosphorylation of Y384 is most likely mediated by another kinase, possibly following the priming phosphorylation by SIK3. Transgenic flies of wild-type (WT) G6PD and the mutant form were generated with the six SIK3-dependent phosphorylation sites mutated into corresponding non-phosphorylatable amino acids (6xP-mut). An in vitro assay to measure G6PD enzyme activity from larval lysates revealed that WT G6PD activity was increased upon sugar feeding, while the activity of the phospho-deficient mutant was not. This was consistent with the idea that SIK3-mediated phosphorylation activates G6PD upon sugar feeding. Endogenous G6PD activity in control larvae was also elevated in response to an HSD, but this increase was not observed in SIK3 mutants or in SIK3 RNAi animals. Knockdown of G6PD served as a positive control. In accordance with Zw and SIK3 being transcriptional targets of Mondo-Mlx, an impaired sugar-induced activation of G6PD was observed in mlx mutants. However, unlike mlx mutants, SIK3 mutants did not display reduced Zw mRNA expression, which supports the idea that SIK3 regulates G6PD activity post-translationally. Furthermore, knockdown of G6PD led to elevated circulating trehalose levels, in addition to sugar intolerance and low TAG levels reported earlier (Teesalu, 2017).

The data implied that SIK3 synergizes with Mondo-Mlx to control G6PD activity. Thus, it was plausible that mondo-mlx and SIK3 interact genetically. To test this, SIK3 and mondo (encoding the essential interaction partner of Mlx) by were depleted RNAi and the development of the animals was monitored. Strikingly, ubiquitous double knockdown of Mondo and SIK3 caused a strong synthetic phenotype, leading to larval growth impairment and lethality on moderate levels (5%) of dietary sucrose. Furthermore, the SIK3, mlx double mutants displayed synergistic lethality on a sugar-only diet (Teesalu, 2017).

Since the oxidative branch of the pentose phosphate pathway is crucial in generating reductive power in the form of NADPH, it was predicted that the regulation of NADPH/NADP+ balance might be deregulated in the SIK3 mutant animals. This was the case, since the NADPH/NADP+ ratio was significantly elevated in HSD-fed control animals, but such an increase was not observed in SIK3 mutants. Similar results were obtained with mlx mutants. The reducing equivalents of NADPH are necessary for counteracting oxidative stress through the glutathione (GSH) redox couple (GSH/GSH disulfide, GSH/GSSG). In agreement with a low NADPH/NADP+ ratio, the GSH/GSSG ratio was reduced in SIK3 mutants on an HSD, as well as upon G6PD knockdown. Moreover, feeding larvae with reduced glutathione partially rescued the pupariation of SIKΔ mutants on a sugar-containing diet (Teesalu, 2017).

Drosophila genome lacks glutathione reductase, and the glutathione reduction is mediated through reduced thioredoxin. Loss-of-function of thioredoxin reductase-1, an enzyme that uses NADPH to reduce thioredoxin (and, consequently, GSH), led to significantly impaired sugar tolerance. Glutathione prevents oxidative damage of cellular biomolecules, including peroxidation of lipids. Consistent with the low GSH/GSSG ratio, the levels of lipid peroxides were significantly elevated in sugar-feeding SIK3 mutants. Furthermore, depletion of glutathione peroxidase PHGPx, a GSH-dependent enzyme involved in counteracting lipid peroxidation, led to sugar intolerance. This further corroborated the role of oxidative stress prevention in sugar tolerance (Teesalu, 2017).

This study has shown that SIK3-deficient Drosophila larvae display lethality on an HSD and thus that SIK3 is a critical mediator of sugar tolerance. While SIK3 was earlier shown to control Drosophila lipid catabolism and tissue growth, this study provides evidence for SIK3-mediated control of glucose metabolism and NADPH redox balance, thereby significantly broadening the known in vivo role of SIK3. Earlier studies have shown that Drosophila SIK3 regulates metabolism via phosphorylation of the transcriptional cofactor HDAC4 and tissue growth by phosphorylating Salvador, a component of the Hippo signaling pathway. This study observed that SIK3 forms a complex with G6PD and controls its activity by phosphorylation. Loss of SIK3-dependent phosphorylation sites prevented post-translational activation of G6PD upon sugar feeding, demonstrating the functional relevance of SIK3-mediated G6PD phosphorylation in vivo (Teesalu, 2017).

Earlier studies in mammalian cells and rats have shown G6PD to be phosphorylated by protein kinase A, which inhibits G6PD activity. It is perhaps not surprising that SIK3 and protein kinase A (PKA) might be counteracting each other on G6PD regulation since, in cAMP-response-element-binding protein (CREB)-mediated transcription, SIK family members and PKA also mediate opposing activities. PKA-mediated phosphorylation activates CREB, while SIK family members inhibit the cofactor of CREB, CRTC (CREB-regulated transcription coactivator). Furthermore, PKA phosphorylates and inhibits Drosophila SIK3, while SIK3 is activated by insulin-mediated phosphorylation. This study revealed an additional layer of SIK3 regulation by observing that SIK3 gene expression is reduced in mlx mutants. A binding site for Mlx was identified in the SIK3 promoter, suggesting that SIK3 is a direct Mondo-Mlx target, although indirect mechanisms cannot be ruled out. Given the relatively modest increase of SIK3 expression on an HSD, it is also likely that post-translational mechanisms are involved in the sugar-induced activation of SIK3. It was recently shown that Mondo-Mlx transcriptionally activates the pentose phosphate pathway, including the G6PD-encoding gene Zw. Thus, Mondo-Mlx and SIK3 appear to form a regulatory circuit, which converges on the control of G6PD. Such dual regulation through gene expression and phosphorylation is likely to increase the dynamic range of G6PD activation upon sugar feeding and thereby extend the range of tolerated dietary sugar. Indeed, simultaneous RNAi-mediated inhibition of SIK3 and Mondo-Mlx had devastating consequences, leading to early larval lethality on moderate (5%) sugar levels. It will be interesting to learn whether the convergent control via gene expression and phosphorylation will also involve other sugar-regulated genes (Teesalu, 2017).

One of the key findings of this study is the dynamic control of NADPH-GSH reductive capacity in response to sugar feeding and its importance on sugar tolerance. Larvae lacking SIK3 were unable to elevate their NADPH/NADP+ ratio and displayed signs of oxidative stress on an HSD. Inhibition of glutathione reduction by RNAi against thioredoxin reductase-1 conferred animals intolerant to an HSD, while having no impact on animals on an LSD, and the feeding of glutathione increased the survival of SIK3 mutants specifically on a sugar-containing diet. This study, together with earlier findings, supports a model where sugar-sensing pathways synchronously coordinate the activities of several pathways that mediate safe elimination and storage of the excess carbon skeletons provided by dietary sugars. This includes activation of glycolytic and lipogenic gene expression programs, as well as an increase of NADPH reductive capacity through G6PD activation. The need for elevated GSH reductive capacity on HSD might stem from the challenge posed by reactive metabolic intermediates, such as methylglyoxal, formed during high glycolytic activity. On the other hand, de novo lipogenesis requires a high degree of NADPH, which would impair the proper function of the GSH-mediated prevention of oxidative stress, unless the generation of reductive capacity is simultaneously increased. Future studies will elucidate whether other pathways regulating NADPH/NADP+ balance contribute to sugar tolerance (Teesalu, 2017).

Juvenile hormone and 20-hydroxyecdysone coordinately control the developmental timing of matrix metalloproteinase-induced fat body cell dissociation

Tissue remodeling is a crucial process in animal development and disease progression. Coordinately controlled by the two main insect hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), tissues are remodeled context-specifically during insect metamorphosis. Previous work has discovered that two matrix metalloproteinases (Mmps) cooperatively induce fat body cell dissociation in Drosophila. However, the molecular events involved in this Mmps-mediated dissociation are unclear. This study reports that JH and 20E coordinately and precisely control the developmental timing of Mmps-induced fat body cell dissociation. During the larval-prepupal transition, the anti-metamorphic factor Kr-h1 was found to transduce JH signaling, which directly inhibited Mmps expression and activated expression of tissue inhibitor of metalloproteinases (timp), and thereby suppressed Mmps-induced fat body cell dissociation. It is also noted that upon a decline in the JH titer, a prepupal peak of 20E suppresses Mmps-induced fat body cell dissociation through the 20E primary-response genes, E75 and Blimp-1, which inhibited expression of the nuclear receptor and competence factor betaftz-F1. Moreover, upon a decline in the 20E titer, betaftz-F1 expression was induced by the 20E early-late response gene DHR3, and then betaftz-F1 directly activated Mmps expression and inhibited timp expression, causing Mmps-induced fat body cell dissociation during 6-12 hrs after puparium formation. In conclusion, coordinated signaling via JH and 20E finely tunes the developmental timing of Mmps-induced fat body cell dissociation. These findings shed critical light on hormonal regulation of insect metamorphosis (Jia, 2017).

MMPs and tissue inhibitor of metalloproteinases (TIMPs) play crucial roles in regulating tissue remodeling in both vertebrates and Drosophila. Previous work has demonstrated the collaborative functions of Mmp1 and Mmp2 in inducing fat body cell dissociation in Drosophila. timp mutant adults show autolyzed tissue in the abdominal cavity and inflated wings, a phenotype consistent with the role of timp in BM integrity and remodeling. The current study clarified the role of timp in inhibiting the enzymatic activity of Mmps and thus, Mmp-induced fat body cell dissociation. In mammals, Mmps activity in vivo is controlled at different levels, including the regulation by gene expression, the zymogens activation, and the inhibition of active enzymes by TIMPs. These studies unify the important inhibitory roles of timp/TIMP in regulating tissue remodeling in both Drosophila and mammals. In addition to regulating Mmp expression, JH and 20E signals differentially regulate timp expression, with the stimulatory role of Kr-h1 and the inhibitory role of βftz-F1. Because timp inhibits the enzymatic activity of Mmps in the Drosophila fat body, it is concluded that JH and 20E coordinately control Mmps activity at both the mRNA and enzymatic levels (Jia, 2017).

Previously work has show the requirement of both JH and its receptors to inhibit fat body cell dissociation in Drosophila. This study demonstrated the ability of Kr-h1 to transduce JH signaling to decrease Mmp expression and to induce timp expression during larval-prepupal transition. Moreover, a Kr-h1-binding sites (KBS) was identified in the Mmp1 promoter, indicating that Kr-h1 directly represses Mmp1 expression. Interestingly, Kr-h1 expression gradually increases from initiation of wandering (IW) to 3 h APF when induced by JH and 20E in an overlapping manner, thus inhibiting the enzymatic activity of Mmps and Mmp-induced fat body cell dissociation during the larval-prepupal transition. Moreover, Kr-h1 acts as an anti-metamorphic factor by inhibiting 20E signaling. It is proposed, in addition to directly affecting the expression of Mmps and timp, that Kr-h1 might also indirectly regulate their expression by inhibiting 20E signaling (Jia, 2017).

Two consecutive 20E pulses control timely metamorphosis in Drosophila. Together with previous findings, the current results show that the conserved 20E transcriptional cascade precisely controls the timing of Mmp-induced fat body cell dissociation. In general, the first 20E signal pulse plays an inhibitory role during the larval-prepupal transition; however, it is a prerequisite for the expression of βftz-F1, which induces the second 20E signal pulse during the prepupal-pupal transition and the expression of Mmps. Because of the requirement for the first 20E signal pulse, blockade of the 20E receptor prevents fat body cell dissociation. When JH titer declines, the prepupal peak of 20E activates expression of two 20E primary-response genes, E75 and Blimp-1, to inhibit fat body cell dissociation: E75 represses DHR3 transactivation of βftz-F1 expression, and Blimp-1 directly represses βftz-F1 expression. During the prepupal-pupal transition, DHR3 directly induces βftz-F1 expression from 6 h APF to 12 APF. Before pupation, βftz-F1 induces Mmp expression and represses timp expression. Moreover, an FBS was identified in the Mmp2 promoter, demonstrating that βftz-F1 directly induces Mmp2 expression. Finally, within 6 h before pupation, Mmp1 and Mmp2 cooperatively induce fat body cell dissociation, with each assuming a distinct role (Jia, 2017).

Insect metamorphosis is coordinately controlled by JH and 20E, whereas the hormonal control of tissue remodeling is strictly context-specific. Different larval tissues and adult organs might have distinct, yet precise, developmental fates and timing. Knowledge regarding this question is poor. Based on previous preliminary information, this study clarified the detailed molecular mechanisms by which JH and 20E precisely control the developmental timing of Mmp-induced fat body cell dissociation at both mRNA and enzymatic levels in Drosophila, and a working model is provided of hormonal control of tissue remodeling in animals (see Model showing developmental timing of Mmp-induced fat body cell dissociation is coordinately and precisely controlled by JH and 20E in Drosophila). In summary, at first, Kr-h1 transduces JH signaling to inhibit Mmp-induced fat body cell dissociation during larval-prepupal transition. Then when JH titer declines, the prepupal peak of 20E suppresses Mmp-induced fat body cell dissociation through E75 and Blimp-1, which inhibit βftz-F1 expression. Finally, until 20E titer declines, DHR3 induces βftz-F1 expression, and βftz-F1 covers the 20E-triggered transcriptional cascade to activate Mmp-induced fat body cell dissociation within 6 h before pupation. This study provides an excellent sample for better understanding the hormonal regulation of insect metamorphosis (Jia, 2017).

Fat body cells are motile and actively migrate to wounds to drive repair and prevent infection

Adipocytes have many functions in various tissues beyond energy storage, including regulating metabolism, growth, and immunity. However, little is known about their role in wound healing. This study used live imaging of fat body cells, the equivalent of vertebrate adipocytes in Drosophila, to investigate their potential behaviors and functions following skin wounding. Pupal fat body cells are not immotile, as previously presumed, but actively migrate to wounds using an unusual adhesion-independent, actomyosin-driven, peristaltic mode of motility. Once at the wound, fat body cells collaborate with hemocytes, Drosophila macrophages, to clear the wound of cell debris; they also tightly seal the epithelial wound gap and locally release antimicrobial peptides to fight wound infection. Thus, fat body cells are motile cells, enabling them to migrate to wounds to undertake several local functions needed to drive wound repair and prevent infections (Franz, 2018).

The data show that FBCs, Drosophila adipocytes, are recruited to wounds in pupae where they have multiple local roles in wound healing. The observation that FBCs are motile cells that actively migrate to wounds is unexpected and has not previously been made for adipocytes in any other organism. However, these findings raise the interesting question as to whether vertebrate adipocytes might also have the capacity to migrate. In that regard, a recent mammalian wound study found that adipocytes repopulate murine wounds, and suggested that some may have migrated from distant sites. It will be fascinating to discover whether some sub-populations of vertebrate adipocytes are indeed motile and whether they utilize similar migratory strategies to those highlighted in Drosophila FBCs (Franz, 2018).

The mode of motility that was observed for FBCs moving through the hemolymph to wounds is unusual, since it does not appear to involve the use of standard lamellipodia or blebs, utilized by most known migrating cells as they crawl in an adhesion-dependent fashion over substrates and through a milieu of extracellular matrix. Adhesion-independent migration has recently emerged as an alternative migration mode that has now been described for several other types of cells, including ameba, lymphocytes, and some cancer cells. Four models have been proposed for adhesion-independent migration: force transmission driven by 'chimneying' between two opposing substrate faces, the intercalation of lateral cell protrusions with gaps in the substrate, non-specific friction between cell and substrate, and swimming by noncyclic cell shape deformations. Only the last of these is entirely independent of any interactions with (or close proximity to) a solid substrate and hence best describes the observation of the migration of FBCs through hemolymph to wounds, since no significant interactions of these cells are seen with any substrate or other cells as they migrate. Similar to FBCs, several other cell types have been reported to migrate by swimming, when they are required to move through viscous fluid: amebae and neutrophils have been shown to swim when in viscous solution and lymphocytes are known to migrate using contraction waves when in suspension. However, the exact mechanism by which these swimming cells generate internal forces and how these forces are transduced to the extracellular environment to generate forward movement is still unknown. A recent study has shed some light on how internal forces are generated during another type of adhesion-independent migration; it showed that the migration of Walker carcinoma cells in confinement is driven by cyclical rearward flow of cortical actin that is coupled to the substrate through friction. This migration depends on the contractility of cortical actin at the rear of the cells. Moreover, rearward flow of cortical actin has also been described for the oscillatory behavior of detached cells and cell fragments, as well as for the stable-bleb cell migration of zebrafish germ layer progenitor cells. This is strikingly similar to the rearward peristaltic actin waves observed in FBCs migrating to wounds, suggesting that this could be the mechanism of force generation in FBCs also (Franz, 2018).

However, it still remains unclear how such an intracellular force might be transduced to the extracellular environment to drive forward movement of FBCs. It has previously been presumed that, while swimming works for large multicellular organisms, it cannot operate at the microscopic cell level, where viscous forces are many orders of magnitude higher than inertial forces and hence geometrically reciprocal cell shape changes may not generate propulsive forces. However, this view has been challenged and may only be true for simple Newtonian fluids, like water, which the hemolymph that FBCs swim through is clearly not. Moreover, swimming in a non-Newtonian fluid is thought to be possible if the cell shape changes of migrating cells are nonreciprocal, which might be true for FBCs migrating to wounds. It is also possible that FBCs, in addition to swimming, make use of other mechanisms to migrate. The hemolymph is relatively densely packed with cells including hemocytes and other FBCs, and FBCs are adjacent to the epithelium and muscle, depending on the location in the body. Although no contacts were observed, it is possible that the close proximity of FBCs with other cells and tissues en route to a wound might enable them to occasionally generate additional frictional forces like the ones reported for non-adherent Walker cells migrating in a confined microfluidics channel, which may also contribute to their swimming motility (Franz, 2018).

This study shows that FBCs play multiple local roles in driving wound repair and preventing wound infection. Some of these local functions might also partially extrapolate to the vertebrate wound scenario. Drosophila FBCs have long been known to systemically produce a variety of AMPs following infection and this study reveals that, during wound infection, FBCs migrate to wounds to release AMPs locally. A recent study has shown that mouse adipocytes are able to produce AMPs following bacterial skin infections. Hence, it would be interesting to examine whether mammalian adipocytes, like Drosophila FBCs, play a local role during wound healing in delivering AMPs to fight wound infection (Franz, 2018).

Given the finding that hemocytes and FBCs collaborate during the wound repair process to clear cell debris and fight infection, it is tempting to speculate that these two cell types communicate with each other during vertebrate wound healing also. Interestingly, in recent years several mammalian studies have uncovered complex interactions between adipocytes and macrophages in white adipose tissue (WAT), with important implications for tissue regeneration and disease. One example is obesity-induced inflammation and insulin resistance, where, upon overnutrition, the adipocytes in visceral WAT are thought to release chemokines to stimulate macrophage recruitment into fat tissue, leading to smoldering inflammation and subsequently insulin resistance. This is believed to be due to proinflammatory macrophages releasing cytokines that attenuate insulin signaling in various cell types, including adipocytes. In support of these mammalian reports, a recent study in the fly showed that animals fed a lipid-rich diet display reduced insulin sensitivity and lifespan, and both of these effects are mediated by hemocytes (Franz, 2018).

Thus interactions between adipocytes and immune cells appear to be key in many diseases, including type 2 diabetes, and it is believed that important insights into these links may be provided by future studies of the functional relationship and communication between FBCs and hemocytes during pupal wound repair in flies (Franz, 2018).

These studies in Drosophila pupae point to novel behaviors and functions for FBCs in Drosophila and open up genetic opportunities to further understanding of the important roles played by adipocytes in repair and regeneration (Franz, 2018).

Inherent constraints on a polyfunctional tissue lead to a reproduction-immunity tradeoff

Single tissues can have multiple functions, which can result in constraints, impaired function, and tradeoffs. The insect fat body performs remarkably diverse functions including metabolic control, reproductive provisioning, and systemic immune responses. How polyfunctional tissues simultaneously execute multiple distinct physiological functions is generally unknown. Immunity and reproduction are observed to trade off in many organisms but the mechanistic basis for this tradeoff is also typically not known. This study investigated constraints and trade-offs in the polyfunctional insect fat body. Using single-nucleus sequencing, it was determined that the Drosophila melanogaster fat body executes diverse basal functions with heterogenous cellular subpopulations. The size and identity of these subpopulations are remarkably stable between virgin and mated flies, as well as before and after infection. However, as an emergency function, the immune response engages the entire tissue and all cellular subpopulations produce induce expression of defense genes. Reproductively active females who were given bacterial infection exhibited signatures of ER stress and impaired capacity to synthesize new protein in response to infection, including decreased capacity to produce antimicrobial peptides. Transient provision of a reversible translation inhibitor to mated females prior to infection rescued general protein synthesis, specific production of antimicrobial peptides, and survival of infection. The commonly observed tradeoff between reproduction and immunity appears to be driven, in D. melanogaster, by a failure of the fat body to be able to handle simultaneous protein translation demands of reproductive provisioning and immune defense. It is suggested that inherent cellular limitations in tissues that perform multiple functions may provide a general explanation for the wide prevalence of physiological and evolutionary tradeoffs (Gupta, 2022).

Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. This study characterizes the Drosophila tuSz mutant strain, which mounts an aberrant immune response against its own fat body. This study demonstrates that this autoimmunity is the result of two mutations: 1) a mutation in the Glucosidase 1/GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals (Mortimer, 2021).

This work has investigated the Drosophila tuSz1 mutant strain. tuSz1 is a temperature-sensitive mutant, and at the restrictive temperature, posterior fat body tissue is melanotically encapsulated by hemocytes in a reaction similar to the antiparasitoid immune response. The tuSz1 phenotype is caused by two tightly linked mutations: a nonconditional, dominant gain-of-function mutation in hop that leads to ectopic immune activation and a temperature-sensitive, recessive mutation in GCS1 that leads to loss of protein N-glycosylation of the ECM overlaying the posterior fat body. These data lead to a a proposal of a two-step model in which immune activation and the loss of SAMP presentation/recognition are both necessary for the breakdown of self-tolerance. In a naïve wild-type larva, neither condition is met and self-tolerance is maintained. In the case of the tuSz1 mutant, the posterior fat body lacks appropriate ECM protein N-glycosylation and is targeted by constitutively activated hemocytes for encapsulation. This two-step model is also reflected in the hopTum mutant background, in which the simultaneous disruption of N-glycosylation in this immune-activated background results in tissue self-encapsulation similar to the tuSz1 mutant (Mortimer, 2021).

Models describing the necessity for two independent signals in fly encapsulation responses. (A) Homeostasis is maintained in naive wild-type larvae. (B) In tuSz1 mutant larvae, immune cells are inappropriately activated by JAK-STAT pathway activation due to the hopSz gain-of-function mutation. The loss of protein N-glycosylation in posterior fat body tissue due to the GCS1Sz mutation leads to loss of self-tolerance and tissue encapsulation. (C) In the model of self-tolerance described in a previous study, the coupled phenotypes of loss of cell integrity and loss of ECM integrity are sufficient to disrupt self-tolerance. (D) Immune cells are activated following parasitoid wasp infection, presumably due to the wound-mediated activation of JAK-STAT signaling. SAMP-presenting host tissues are protected from encapsulation, and wasp eggs may be targeted for encapsulation because they are missing the ECM N-glycosylation SAMP (Mortimer, 2021).

Interestingly, previous work also documented the necessity of at least two signals for self-encapsulation in Drosophila. In that case, both the loss of the ECM (with its glycosylated proteins) and the disrupted positional integrity of the underlying fat body cells (potentially mimicking a wound) were required for immune cells to become activated and encapsulate the self tissue. A similar loss of ECM and underlying cell integrity was also found in the classical melanotic tumor mutant tu(2)W. This model, in which at least two factors are required for self-encapsulation, may explain why the several classically described self-encapsulation mutants, unlike virtually all other types of visible Drosophila mutants, were never successfully mapped (Mortimer, 2021).

The disruption of either factor in the two-step model in isolation is not sufficient to cause self-encapsulation. This can be seen in parasitoid wasp infected larvae; the wounding associated with parasitoid infection leads to immune activation, but in the absence of SAMP disruption, the fly is able to specifically encapsulate the parasitoid egg while protecting against self-encapsulation. Conversely, while internal tissue damage in a naive larva does attract hemocyte interactions, in the absence of an immune stimulus, this does not lead to self-encapsulation, but rather the hemocytes attempt to repair the damaged tissue. That blood cells err on the side of fixing disrupted self tissue rather than treating it as pathogenic and encapsulating it unless another stress signal is also present suggests that flies may have evolved a multi-input system to safeguard against spurious encapsulation (Mortimer, 2021).

D. melanogaster immune responses have proven to be an excellent model for understanding the mechanisms underlying conserved innate immune responses, including those of humans. Findings on Drosophila self-tolerance may also be relevant to human innate self-tolerance. Indeed, data from a range of studies are consistent with the idea that protein glycosylation is a mediator of vertebrate immune responses, and cell-surface glycans have been proposed as candidate SAMPs for the innate immune response to distinguish healthy self tissues from aberrant or foreign tissues even if the mechanisms are not entirely understood. Protein-linked sugar groups should presumably fit this role well, as they can take on diverse combinations of sugar residues and branching patterns (Mortimer, 2021).

Protein N-glycosylation is a complex multistep process that begins with the addition of a presynthesized glycosyl precursor to the protein at an asparagine residue. This nascent glycan is then trimmed back to a core glycan structure, a process that is initiated by the activity of GCS1. The core glycan is then elaborated with the addition of multiple carbohydrate groups to give rise to a variety of final structures, with hybrid and complex type N-glycans among the most prevalent. Glycan elaboration begins with the activity of Mgat1, which leads to the production of hybrid type N-glycans. These hybrid N-glycans can be further processed by the α-mannosidase-I and -II family of enzymes to produce paucimannose N-glycans, which can serve as complex N-glycan precursors and are further elaborated by downstream enzymes to give rise to the final complex N-glycan structure. The current data suggest that disruption of any of the genes encoding key N-glycan-processing enzymes will be associated with the loss of self-tolerance in Drosophila. Similarly, the loss of the α-mannosidase-II (αM-II) gene in mice is linked with the development of an autoimmune phenotype that is likened to systemic lupus erythematosus. Like the tuSz1 mutant, the αM-II mouse phenotype arises due to alterations in protein N-glycosylation in nonimmune tissues and is mediated by innate immune cells. Altered patterns of protein N-glycosylation are also observed in additional mouse models of autoimmune disease and have been linked to autoimmune disease in human patients (Mortimer, 2021).

The ECM is a conserved structure made up of numerous proteins, many of which are N-glycosylated, including laminin and collagen. A role for the ECM in mediating self-tolerance has been previously proposed: The encapsulation of self tissues in D. melanogaster is also observed following RNA interference (RNAi) knockdown of genes encoding the ECM proteins laminin and collagen, supporting the idea that SAMPs reside in the ECM. The role of the ECM in self-tolerance is further emphasized by tissue transplantation studies in Drosophila. Drosophila larvae are largely tolerant of conspecific tissue transplants, but this tolerance is abolished when tissues are first treated with collagenase to disrupt the ECM, leading to the specific encapsulation of treated tissues. Reactivity to ECM proteins is also associated with various forms of human autoimmune disease. Based on these data, it is proposed that the N-glycosylation of ECM proteins may serve as a conserved self-tolerance signal for innate immune mechanisms and that loss of ECM protein N-glycosylation may lead to loss of self-tolerance and, consequently, autoimmune disease in a diverse range of species (Mortimer, 2021).

An alternative means by which hosts can recognize pathogens is missing-self recognition. Instead of tracking pathogen diversity with numerous recognition receptors, as in nonself recognition, missing-self recognition does not rely on tracking pathogens at all, but only on specifically recognizing self and attacking tissues that lack the self signal. Further, unlike germ line-encoded forms of nonself recognition, missing-self recognition systems allow host species to respond to novel pathogen types that they have never encountered in their evolutionary history. Protein glycosylation plays an important role in the handful of identified missing-self recognition systems of vertebrates. The most well-known case of missing-self recognition involves the interaction between vertebrate NK cells and host MHC class I (MHCI) proteins. All vertebrate cells produce MHCI to display any possible antigens present in their cytoplasm to T cells, but intracellular pathogens often suppress host cell MHCI expression to prevent their molecules from being displayed. NK cells are lymphoid-type cells that induce cytolysis in infected host cells. In an uninfected state, recognition of properly glycosylated MHCI inhibits NK cell cytolysis of host cells, but in an infected state in which host cells are missing the MHCI self signal, the NK cell inhibitory receptors fail to recognize 'self,' and the infected host cells are lysed, an effective means of killing intracellular pathogens that have suppressed host MHC signaling (Mortimer, 2021).

As of yet, there are no examples of missing-self immune recognition systems in invertebrates, and it has been hypothesized that invertebrate immune systems rely largely on PRRs for nonself recognition of pathogens. Still, invertebrates do mount immune responses against a variety of inanimate objects like oil droplets, sterile nylon, and charged chromatography beads as well as tissue transplants from other insect species. All of these foreign bodies presumably lack distinct PAMPs, suggesting that invertebrates have some sort of missing-self recognition system. Additionally, while multiple antimicrobial PRRs have been identified in the Drosophila genome, PRRs targeting macroparasites like parasitoid wasps have not yet been discovered. The current model of self-recognition suggests that following parasitoid infection, activated immune cells assess all exposed tissue surfaces for the self-tolerance glycan signal and that the absence of this Drosophila SAMP on parasitoid wasp eggs might be the cue that targets them for melanotic encapsulation (Mortimer, 2021).

Down-regulation of a cytokine secreted from peripheral fat bodies improves visual attention while reducing sleep in Drosophila

Sleep is vital for survival. Yet under environmentally challenging conditions, such as starvation, animals suppress their need for sleep. Interestingly, starvation-induced sleep loss does not evoke a subsequent sleep rebound. Little is known about how starvation-induced sleep deprivation differs from other types of sleep loss, or why some sleep functions become dispensable during starvation. This study demonstrate that down-regulation of the secreted cytokine unpaired 2 (upd2) in Drosophila flies may mimic a starved-like state. A genetic knockdown strategy to investigate the consequences of upd2 on visual attention and sleep in otherwise well-fed flies, thereby sidestepping the negative side effects of undernourishment. Knockdown of upd2 in the fat body (FB) is sufficient to suppress sleep and promote feeding-related behaviors while also improving selective visual attention. Furthermore, this study shows that this peripheral signal is integrated in the fly brain via insulin-expressing cells. Together, these findings identify a role for peripheral tissue-to-brain interactions in the simultaneous regulation of sleep quality and attention, to potentially promote adaptive behaviors necessary for survival in hungry animals (Ertekin, 2020).

The Nuclear Receptor Seven Up Regulates Genes Involved in Immunity and Xenobiotic Response in the Adult Drosophila Female Fat Body

There is increasing evidence that nuclear receptor signaling in peripheral tissues can influence oogenesis. It was previously shown that the Drosophila nuclear receptor Seven up (Svp) is required in the adult fat body to regulate distinct steps of oogenesis; however, the relevant downstream targets of Svp remain unknown. This study took an RNA sequencing approach to identify candidate Svp targets specifically in the adult female fat body that might mediate this response. svp knockdown in the adult female fat body significantly downregulated immune genes involved in the first line of pathogen defense, suggesting a role for Svp in stimulating early immunity. In addition, it was found that Svp transcriptionally regulates genes involved in each step of the xenobiotic detoxification response. Based on these findings, a testable model is proposed in which Svp functions in the adult female fat body to stimulate early defense against pathogens and facilitate detoxification as part of its mechanisms to promote oogenesis (Weaver, 2020).

Kynurenine Metabolism in the Fat Body Non-autonomously Regulates Imaginal Disc Repair in Drosophila

issue interactions are critical for maintaining homeostasis; however, little is known about how remote tissue regulates regeneration. Previous work established a genetic dual system that induces cell ablation in Drosophila larval imaginal discs and simultaneously manipulates genes in non-damaged tissues. Using humoral metabolome analysis and a genetic damage system, this study found that the Tryptophan (Trp)-Kynurenine (Kyn) pathway in the fat body is required for disc repair. Genetic manipulation of Trp-Kyn metabolism in the fat body impaired disc regeneration without affecting wing development. In particular, the fat body-derived humoral kynurenic acid (KynA) was required for disc repair. The impairment of S-adenosylmethionine (SAM) synthesis from methionine (Met) in the fat body hampers the maintenance of KynA levels in hemolymph at the early stage of disc repair, suggesting a connection between Met-SAM and Trp-Kyn metabolisms. These data indicate KynA from the fat body acts as a permissive metabolite for tissue repair and regeneration (Kashio, 2020).

Fat-body brummer lipase determines survival and cardiac function during starvation in Drosophila melanogaster

The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. Evidence is provided that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, it is suggested that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, this study identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress (Blumrich, 2021).

Tryptophan regulates Drosophila zinc stores

Zinc deficiency is commonly attributed to inadequate absorption of the metal. Instead, this study shows that body zinc stores in Drosophila melanogaster depend on tryptophan consumption. Hence, a dietary amino acid regulates zinc status of the whole insect—a finding consistent with the widespread requirement of zinc as a protein cofactor. Specifically, the tryptophan metabolite kynurenine is released from insect fat bodies and induces the formation of zinc storage granules in Malpighian tubules, where 3-hydroxykynurenine and xanthurenic acid act as endogenous zinc chelators. Kynurenine functions as a peripheral zinc-regulating hormone and is converted into a 3-hydroxykynurenine–zinc–chloride complex, precipitating within the storage granules. Thus, zinc and the kynurenine pathway—well-known modulators of immunity, blood pressure, aging, and neurodegeneration—are physiologically connected (Garay, 2022).

Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila

Adapting to changes in food availability is a central challenge for survival. Glucose is an important resource for energy production, and therefore many organisms synthesize and retain sugar storage molecules. In insects, glucose is stored in two different forms: the disaccharide trehalose and the branched polymer glycogen. Glycogen is synthesized and stored in several tissues, including in muscle and the fat body. Despite the major role of the fat body as a center for energy metabolism, the importance of its glycogen content remains unclear. This study showed that glycogen metabolism is regulated in a tissue-specific manner under starvation conditions in the fruit fly Drosophila. The mobilization of fat body glycogen in larvae is independent of Adipokinetic hormone (Akh, the glucagon homolog) but is regulated by sugar availability in a tissue-autonomous manner. Fat body glycogen plays a crucial role in the maintenance of circulating sugars, including trehalose, under fasting conditions. These results demonstrate the importance of fat body glycogen as a metabolic safeguard in Drosophila (Yamada, 2018).

An autonomous metabolic role for Split ends

Preventing obesity requires a precise balance between deposition into and mobilization from fat stores, but regulatory mechanisms are incompletely understood. Drosophila Split ends (Spen) is the founding member of a conserved family of RNA-binding proteins involved in transcriptional regulation and frequently mutated in human cancers. This study found that manipulating Spen expression alters larval fat levels in a cell-autonomous manner. Spen-depleted larvae had defects in energy liberation from stores, including starvation sensitivity and major changes in the levels of metabolic enzymes and metabolites, particularly those involved in beta-oxidation. Spenito, a small Spen family member, counteracted Spen function in fat regulation. Finally, mouse Spen and Spenito transcript levels scaled directly with body fat in vivo, suggesting a conserved role in fat liberation and catabolism. This study demonstrates that Spen is a key regulator of energy balance and provides a molecular context to understand the metabolic defects that arise from Spen dysfunction (Hazegh, 2017).

This work provides the first detailed investigation of a fat regulatory role for Spen in any organism, and the first evidence that Nito also functions in this process. Spen depletion in the fat body drastically increased stored fat. Spen has been implicated in multiple pathways involved in endocrine signaling, including Notch, Wingless, and nuclear receptor signaling. This study found it unlikely that nuclear receptor pathways are relevant to the fat regulatory role this study defines, because upon Spen depletion or overexpression consistent changes in the expression of genes that are targets of those pathways were not observed. Furthermore, the lack of phenotypes involving fat storage per se upon overexpression of C-terminal Spen-SPOConly domain argues against a role for Wg signaling, in which the same construct has potent dominant negative effects. Conversely, whereas a C-terminally truncated version of mSpen has little effect on Notch signaling, the strong fat phenotypes resulting from Spen-ΔSPOC overexpression suggest that Spen does not regulate fat via the Notch pathway (Hazegh, 2017).

Notably, Spen KD larvae also exhibited behavioral changes (increased food intake, decreased locomotion) that may have contributed to the fat increase. Thus, in addition to direct roles in fat accumulation within fat storage cells, Spen may be involved in a cross-talk pathway between the FB and the brain. However, a model is strongly support wherein increased food intake is instead an attempt to compensate for a condition of 'perceived starvation' resulting from an inability to access energy stores. Similarly, a lack of available energy may restrict locomotion. This hypothesis is further strengthened by the observation that Spen overexpression was sufficient to deplete stored fat but did not cause opposing behavioral phenotypes (Hazegh, 2017).

Mosaic analysis confirmed an autonomous role for Spen in FB cells. Spen KD in clones throughout the FB showed a striking increase in LD size. Larger LDs normally have lower surface tension, and the stored fat is easier to access. LD remodeling in WT animals is a highly regulated process involving specific factors, some of which were identified in a genome-wide RNAi screen in cultured Drosophila S2 cells. Notably, RNAseq data revealed that the products of several LD-regulating genes were significantly altered by Spen depletion, including l(2)01289 (~7-fold decreased), CG3887 (1.3-fold decreased), and eIF3-S9 (1.5-fold increased). While it is unclear if these changes are direct effects of Spen depletion, they may explain why LDs in Spen KD larvae are large yet apparently inaccessible, resulting in starvation sensitivity (Hazegh, 2017).

Consistent with the observed changes in FB cell and LD morphology and starvation sensitivity, changes in metabolites and gene expression in Spen KD larvae pointed to a drastic defect in lipid catabolism. Defects in β-oxidation were the most obvious, in part because the opposite effects were observed upon FB-restricted Spen overexpression. Spen depletion led to a decrease in the levels of free and acyl-conjugated carnitine, as well as of transcripts of three of the four enzymes necessary to break down acyl-carnitines into free fatty acids. Three lipases were also downregulated, which likely further contributes to an inability to convert energy stored as TAGs into usable forms. While an apparent upregulation of gluconeogenesis is evident, as supported by alterations in aspartate and PEPCK expression, these processes may be unable to completely compensate for decreased trehalose utilization, and these defects may contribute to the lethargy phenotype resulting from Spen KD. Consequently, surviving the loss of Spen may require breakdown of protein into free amino acids in order to anaplerotically replenish the TCA cycle, consistent with changes in expression of proteases, the observed decrease in many free amino acids, as well as increases in protein catabolism and collagen turnover markers (N-acetylmethionine and hydroxyproline). Of note, sustained proteolysis is a marker of aging and inflammation, a phenotype that has been previously associated with decreased locomotion in human and mouse models of physical activity, suggesting potential future ramifications of Spen’s role in metabolism with respect to aging/inflammation research. Finally, the observed decrease in glycogen levels upon Spen KD supports a model wherein glycogen is used as a carbohydrate source (in lieu of decreased levels of trehalose) to fuel glycolysis. The overall metabolic defects described in this study are distinctly different from what has been observed upon manipulation of other fat regulators (e.g., Sir2), suggesting that Spen operates in a previously undescribed pathway (Hazegh, 2017).

The results with Spen and Nito truncations provide additional mechanistic insight into how these proteins function in fat regulation. Overexpressing Spen-ΔSPOC reversed the phenotype of full-length Spen overexpression, and instead resulted in similar phenotypes to Spen depletion. Nito-ΔC overexpression had the same effects: larvae arrested development and FB clones mimicked starvation even when dietary nutrients were abundant. Overexpression of the Spen-SPOConly construct had no effect on FB cells, as was the case for Nito-ΔN. Thus only Spen harboring the RRMs and the SPOC domain was able to promote fat depletion when overexpressed. Conversely, only truncated forms of Spen or Nito that retain the RRMs dominantly perturbed both FB cell viability and organismal resistance to starvation (Hazegh, 2017).

Recent studies of X chromosome inactivation found that mSpen RRMs mediate binding to the lncRNA Xist. Rbm15 (mNito) also binds Xist, and is required for N6-methyladenosine (m6A) modification of that lncRNA, which is in turn required for its ability to repress X chromosome transcription. Nito is a subunit of the Drosophila m6A methyltransferase complex and is required for RNA binding by that complex; Nito knockdown severely decreases global m6A modification of mRNA (Lence, 2016). Interestingly, the m6A demethylase FTO/ALKBH9 was the first human obesity susceptibility gene identified by genome-wide association studies, but the relevant nucleic acid target(s) remain unknown. This work provides the first hint that an RNA bound by Spen and/or Nito may be a key FTO substrate (Hazegh, 2017).

These findings lead to a model for Spen and Nito function in the regulation of fat storage. Spen binds via its RRMs to one or more RNAs and, via recruitment of other factors, promotes the expression of enzymes key for mobilization of energy stored as fat (e.g. lipases). The mechanism of activation may be direct or indirect, and via alternative splicing, activation/repression of transcription, or effects on RNA stability and/or translation. Moreover, RNA binding partners may be mRNA or non-coding RNA. Future work will be required to make these distinctions. It is proposed that the Spen SPOC domain is critical for this function, but undefined domains in between the N-terminal RRMs and C-terminal SPOC domain are also important, and these are not shared with Nito. It is proposed that Nito binds via its RRMs the same or a largely overlapping set of RNAs, and also recruits additional factors via its SPOC domains, but-either because it fails to recruit specific factors recruited by Spen, or because it recruits other factors not recruited by Spen—Nito ultimately inhibits/represses the same energy-storage-mobilizing enzymes that are activated by Spen. Overexpressed Spen or Nito fragments containing RRMs sequester target RNAs away from endogenous full-length Spen and the other effectors of fat storage control. Finally, the findings in mouse adipose tissue that mSpen and mNito both increase in expression when a HFD drives fat accumulation lead to the belief that in WT animals Nito acts as a counterbalance to Spen in order to fine-tune fat storage. Future studies delving into more mechanistic details may lead to treatments for obesity and related metabolic disorders that result from perturbation of the pathway that was elucidated here (Hazegh, 2017).


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See also: Genes expressed in mesoderm

Genes involved in tissue development

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