The Interactive Fly

Genes involved in tissue and organ development

Fat body and the regulation of metabolism

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
A nutrient sensor mechanism controls Drosophila growth
Growth-blocking peptides as nutrition-sensitive signals for insulin secretion and body size regulation
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
Remote control of insulin secretion by fat cells in Drosophila
An obligatory role for neurotensin in high-fat-diet-induced obesity
High amylose starch consumption induces obesity in Drosophila melanogaster and metformin partially prevents accumulation of storage lipids and shortens lifespan of the insects
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
A somatic piRNA pathway in the Drosophila fat body ensures metabolic homeostasis and normal lifespan
Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes
Drosophila cytokine Unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion
Regulation of energy stores and feeding by neuronal and peripheral CREB activity in Drosophila
MEF2 is an in vivo immune-metabolic switch
Mio/dChREBP coordinately increases fat mass by regulating lipid synthesis and feeding behavior in Drosophila
The control of lipid metabolism by mRNA splicing in Drosophila
Lipoproteins in Drosophila melanogaster--assembly, function, and influence on tissue lipid composition
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
CoA protects against the deleterious effects of caloric overload in Drosophila
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 melanogaster
The Drosophila HNF4 nuclear receptor promotes glucose-stimulated insulin secretion and mitochondrial function in adults
Steroid hormone signaling is essential for pheromone production and oenocyte survival
Seipin is required for converting nascent to mature lipid droplets
Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor
Thermal stress depletes energy reserves in Drosophila
High fat diet-induced TGF-beta/Gbb signaling provokes insulin resistance through the tribbles expression
Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor
Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells
Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition
Lipid droplet subset targeting of the Drosophila protein CG2254/dmLdsdh1
THADA regulates the organismal balance between energy storage and heat production

Teesalu, M., Rovenko, B. M. and Hietakangas, V. (2017). Salt-Inducible kinase 3 provides sugar tolerance by regulating NADPH/NADP+ redox balance. Curr Biol 27(3): 458-464. PubMed ID: 28132818

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).

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).

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).

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).

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).

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).

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).

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)

Palm, W., Sampaio, J. L., Brankatschk, M., Carvalho, M., Mahmoud, A., Shevchenko, A. and Eaton, S. (2012). Lipoproteins in Drosophila melanogaster--assembly, function, and influence on tissue lipid composition. PLoS Genet 8: e1002828. PubMed ID: 22844248

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).

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).

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).

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).

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).

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).


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See also: Genes expressed in mesoderm

Genes involved in tissue development

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