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

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

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

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

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

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. 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. 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. 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. Adult IPCs, however, are responsive to glucose for DILP2 release. 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).

A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. This study has identified a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (Dilp2) and the other extends to Adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and Dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. It is proposed that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of Dilp2 and suppressing the release of AKH when haemolymph glucose levels are high (Oh, 2019).

Glucose-sensing neurons respond to glucose or its metabolites, which act as signalling cues to regulate their neuronal activity. According to the glucostatic hypothesis proposed in 1953, feeding and related behaviours are regulated by neurons in the brain that sense changes in glucose levels in the blood. Despite the discovery of glucose-sensing neurons in the hypothalamus through electrophysiological methods more than ten years later, the physiological role of these neurons remained unclear until recently, when a population of glucose-excited neurons in the Drosophila brain were determined to function as an internal nutrient sensor to mediate the animal's consumption of sugar (Dus, 2015). A large number of glucose-sensing neurons appear to be present in animals; it is speculated that these neurons mediate physiological functions that are critical for the wellbeing of the animal, including glucose homeostasis. This study reports the identification of a pair of glucose-excited neurons in the Drosophila brain that maintain glucose homeostasis by coordinating the activity of the two key hormones involved in the process: insulin and glucagon (Oh, 2019).

To identify neurons that respond to sugar on the basis of its nutritional value, a two-choice assay was used to screen Vienna tiles (VT)-Gal4 Drosophila lines that had been crossed to UAS-Kir2.1, tub-Gal80ts flies (inward-rectifier potassium ion channel allele Kir2.1 with tubulin-temperature-sensitive Gal80) for defects in their ability to select nutritive D-glucose over non-nutritive L-glucose. Two independent Gal4 lines, VT58471 and VT43147-Gal4, were isolated that failed to select D-glucose after periods of starvation and appeared to contain dorsolateral cells that resemble those that are labelled by the corazonin (Crz)-Gal4 line. Flies in which Crz-Gal4-expressing neurons had been inactivated failed to select D-glucose even when starved. These results suggest that the dorsolateral neurons labelled by Crz-Gal4 and two candidate Gal4 lines mediate the behavioural response to sugar (Oh, 2019).

A Crz antibody was used to confirm the identity of the dorsolateral neurons. A previous study demonstrated that a subset of Crz-expressing neurons also express short neuropeptide F (sNPF). Immunolabelling revealed that the dorsolateral neurons expressing Crz indeed express sNPF. On the basis of these findings, these Crz+sNPF+ neurons were named CN neurons. To restrict Gal4 expression to a few cells that include the dorsolateral neurons, T58471-Gal4 was crossed to choline acetyltransferase (ChAT)-Gal80, generating CN-Gal4, which unambiguously labelled a pair of CN neurons when crossed to UAS-mCD8::GFP. Flies in which these dorsolateral neurons were inactivated using CN-Gal4 failed to select D-glucose when starved. Each CN cell body projects an axon that bifurcates to form two major branches. One branch (axon 1) projects to the pars intercerebralis (PI) region of the brain and the other branch (axon 2) projects ventrolaterally towards the corpora cardiaca (CC). An intersectional approach was used to define these projections further, thereby validating that axon 1 innervates the PI and axon 2 projects to the CC. This approach was also used to induce the expression of tetanus toxin (TNT) to silence a pair of CN neurons. These flies failed to choose D-glucose even after starvation when CN neurons were inactivated. This provided further evidence of the contribution of the pair of the dorsolateral CN neurons to glucose-evoked behaviour (Oh, 2019).

Attempts were made to determine whether CN neurons respond to glucose and other sugars. Calcium-imaging studies using ex vivo brain preparations of flies carrying the calcium indicator UAS-GCaMP6s14 and CN-Gal4 revealed that CN neurons were robustly activated by D-glucose with substantial calcium oscillations. CN neurons also responded to D-trehalose and D-fructose, which are found in the haemolymph, but failed to respond to (1) the non-nutritive sugar L-glucose; (2) the non-haemolymph sugar sucrose; and (3) the non-sugar nutrients amino acids. D-Glucose and D-trehalose are key sugars in the haemolymph, although D-trehalose stimulates the activity of CN neurons only after a substantial delay (about 12 min), possibly because it requires additional metabolic steps to be converted to glucose. D-Fructose applied at 20 mM activated CN neurons, although the concentration of D-fructose in the haemolymph is much lower (<2 mM). These findings suggest that the pair of CN neurons responds only to D-glucose under normal physiological conditions (Oh, 2019).

It was next determined whether activation of CN neurons by D-glucose requires glucose metabolism inside the cell. Exposing the brain to D-glucose mixed with 2DG, phlorizin or nimodipine, which inhibits glycolysis, glucose transport or voltage-gated calcium channels, respectively, blunted the glucose-induced stimulation of CN neurons. In the presence of pyruvate (an end product of glycolysis), the CN neurons demonstrated activity similar to that seen in the presence of other haemolymph sugars. Application of the ATP-sensitive potassium channel (KATP)16 blocker glibenclamide resulted in activation of CN neurons (Fig. 2b, c). Furthermore, glucose-induced calcium transients of these neurons were not abrogated by the application of the sodium-channel blocker tetrodotoxin (TTX). Using RNA-mediated interference (RNAi) lines, it was also determined that glucose transporter 1 (Glut1), hexokinase C (Hex-C), a subunit of the KATP channel (SUR1) and the voltage-gated calcium channel are required in CN neurons for the two-choice behaviour. Consistent with the behavioural results, the glucose-induced calcium response of CN neurons requires Glut1, SUR1 and a voltage-gated calcium channel, further supporting the role of the intracellular glucose metabolic pathway in stimulating CN neuronal activity (Oh, 2019).

The calcium-dependent nuclear import of LexA (CaLexA) system was used to measure cellular activity in CN neurons in intact flies; GFP signal driven by the CaLexA system in starved flies was significantly reduced compared to the signal in fed flies, and the signal was restored when starved flies were refed D-glucose. These results suggest that the activity of CN neurons is stimulated by the increase in glucose levels observed under fed conditions. In addition to the altered CaLexA signals, the effect of glucose on the number and intensity of synaptotagmin (Syt)-GPF+ puncta in fed, starved and refed animals. The Syt-GPF+ signals decreased significantly in axon 1 in starved animals and returned to normal levels after the flies were fed with D-glucose. However, this nutrient-dependent plasticity was not observed in Crz-Gal4-labelled axonal processes that did not originate from the dorsolateral CN neurons (Oh, 2019).

Next attempts were made to determine whether CN neurons are coupled with IPCs20 at the synaptic level. A modified GFP reconstitution across synaptic partners (GRASP) method was used, and the GRASP signals were found to be visible around the synapse between CN neurons and insulin-producing cells (IPCs), indicating physical coupling between CN neurons and IPCs (Oh, 2019).

To determine whether the coupling between CN neurons and IPCs is functional, ATP-gated P2X2 purine receptors were used in CN neurons and the calcium indicator GCaMP6s14 in IPCs, and then CN neurons were stimulated using ATP while recording from the IPCs. ATP-induced CN-neuron activity was accompanied by a significant increase in the amplitude of GCaMP signals in the IPCs in fed flies; this effect was reduced in starved flies. This finding supports the hypothesis that the nutrient-dependent synaptic changes observed between CN neurons and IPCs have functional consequences. The CN neurons did not appear to be functionally coupled to glucose-excited diuretic hormone 44 (Dh44) neurons. Furthermore, whether CN neuronal activity is required for Dilp2 secretion from IPCs was tested. A significant reduction in the intensity of Dilp2 immunoreactivity was observed in the IPCs of fed control flies, but not in fed flies in which CN neurons had been inactivated. These results suggest that an excitatory signal from the CN neurons contributes to the secretion of Dilp2 from IPCs in response to increased glucose levels. Using mass spectrometry and dot blot assay, it was further validated that the flies carrying CN-Gal4 and UAS-Kir2.1 had lower Dilp2 levels circulating in the haemolymph than wild-type flies, in contrast to the higher dilp2 levels found in IPCs (Oh, 2019).

To further clarify the role of CN neurons in mediating glucose-evoked activity in IPCs, CN neurons were inactivated by expressing TNT13, and then the responsiveness of IPCs to glucose was evaluated. The amplitude of calcium signals in IPCs that had been exposed to D-glucose was significantly reduced when the CN neurons were inactivated. Furthermore, it was found that IPCs harbour at least three subpopulations of neurons with distinct responses to glucose or KATP channel blocker. These findings suggest that CN neuronal activity is required for the majority of IPCs to respond to glucose (Oh, 2019).

To determine whether nutrient-dependent plasticity also occurs in axon 2 of the CN neurons, the number and intensity of Syt-GFP+ puncta was monitored before and after feeding flies with D-glucose. A significant reduction was observed in these parameters in starved flies, and a restoration to normal levels was found after refeeding starved flies with D-glucose. This raised the possibility of coupling between CN neurons and AKH-producing cells. Using a modified GRASP method, GRASP fluorescent signals were observed around AKH-producing cells. To determine whether there is any functional connectivity between these cells, the CN neurons were activated while monitoring the activity of AKH-producing cells, and calcium transients found in the AKH-producing cells appeared to decrease during activation of CN neurons (Oh, 2019).

To probe this observation further, the Arclight receptor, which increases fluorescent signals when cells become hyperpolarized, was expressed in AKH-producing cells, and P2X2 receptors were expressed in CN neurons. When the CN neurons were activated using ATP, the Arclight fluorescence intensity in fed flies increased significantly compared with that in starved flies, validating the occurrence of nutrient-dependent changes in the synapses between CN neurons and AKH-producing cells. Notably, when CN neurons were inactivated, the intracellular AKH levels decreased significantly compared with controls. Using mass spectrometry and dot blot assay, significantly higher levels of AKH were expressed in haemolymph of flies carrying CN-Gal4 and UAS-Kir2.1 compared with those in control flies. These findings suggest that CN neuronal activity inhibits the release of AKH from the CC and the increase of AKH levels in haemolymph (Oh, 2019).

Next the identities were investigated of the key neurotransmitters in axon 1 and axon 2 for regulating the functionally opposing synaptic activities. The role of Crz and sNPF was tested in the two-choice behaviour using RNAi lines, and sNPF in CN neurons and sNPF receptor in the postsynaptic IPCs, but not Crz or its receptor, were found to be important. sNPF but not Crz levels in CN neurons were significantly reduced when CN neurons were exposed to D-glucose. Approximately a half of the IPCs that had responded to glucose failed to respond glucose when the dominant-negative allele of sNPF receptor was expressed in IPCs. Furthermore, it was observed that intracellular AKH levels remained high in AKH-producing cells in fed control flies, but declined significantly in fed flies in which the function of sNPF receptor was inhibited in AKH-producing cells (Oh, 2019).

Finally, whether sNPF alters activity of IPCs and/or the CC was determined. The activity of IPCs was significantly stimulated by the application of sNPF26, whereas CC activity was significantly inhibited by sNPF. These functionally opposing effects of sNPF are probably mediated by Gq in IPCs and by Gi/o in AKH-producing cells via the sNPF receptor, which is a G-protein-coupled receptor. Exposing the brain to U73122, a PLC inhibitor that inhibits the Gq pathway, eliminated the glucose-evoked activation of IPCs, but had no effect on sNPF-induced inhibition of AKH-expressing cells. Conversely, exposing the brain to pertussis toxin, a Gi inhibitor, blunted the sNPF-induced inhibition of AKH-producing cells, but had no effect on the glucose-evoked activation of IPCs. These results indicate that axon 1 and axon 2 can have opposing synaptic activities through a mechanism involving the same neurotransmitter and receptor but with distinct downstream factors coupled with opposing outputs (Oh, 2019).

To determine whether CN neuronal activity can alter circulating sugar levels in flies, circulating concentrations of glucose and trehalose in haemolymph were monitored; they were significantly increased in flies in which CN neurons were inactivated compared with controls. This finding illustrates that dysfunctional CN neuronal input to IPCs and AKH-producing cells results in a defect in glucose homeostasis (Oh, 2019).

This study identified and characterized a pair of glucose-sensing neurons in the Drosophila brain that have an essential role in maintaining glucose homeostasis. This was achieved by counterbalancing the activities of Drosophila equivalents of insulin- and glucagon-producing cells. When food consumption leads to a rise in haemolymph sugar levels, CN neurons excite the IPCs through sNPF and its receptor, which appear to be coupled to the Gq signalling cascade to induce the secretion of Dilp2, while suppressing the release of AKH by using the same sNPF receptor, which in this case is coupled with Gi signalling pathway. It is speculated that precise control of these opposing functions is facilitated because the nutrient-dependent plastic changes arise from a single cell (Oh, 2019).

This study demonstrates how the activity of the two key endocrine systems is coordinated in metazoans and that their coordination is under the direct control of glucose-sensing neurons. Such coordination has been proposed to occur in mammals via the sympathetic and parasympathetic nerves that connect the pancreatic islets with glucose-sensing neurons in the hypothalamus and hindbrain. The finding that a large proportion of IPCs respond to glucose through CN neurons in insects raises an intriguing possibility that both direct and indirect mechanisms control endocrine function in mammals. Finally, this work may shed light on the function of glucose-sensing neurons. Further research is needed to understand how these regulatory processes are affected by excessive nutrition and other metabolic disturbances, including obesity (Oh, 2019).

A gut-derived hormone suppresses sugar appetite and regulates food choice in Drosophila

Animals must adapt their dietary choices to meet their nutritional needs. How these needs are detected and translated into nutrient-specific appetites that drive food-choice behaviours is poorly understood. This study shows that enteroendocrine cells of the adult female Drosophila midgut sense nutrients and in response release neuropeptide F (NPF), which is an ortholog of mammalian neuropeptide Y-family gut-brain hormones. Gut-derived NPF acts on glucagon-like adipokinetic hormone (AKH) signalling to induce sugar satiety and increase consumption of protein-rich food, and on adipose tissue to promote storage of ingested nutrients. Suppression of NPF-mediated gut signalling leads to overconsumption of dietary sugar while simultaneously decreasing intake of protein-rich yeast. Furthermore, gut-derived NPF has a female-specific function in promoting consumption of protein-containing food in mated females. Together, these findings suggest that gut NPF-to-AKH signalling modulates specific appetites and regulates food choice to ensure homeostatic consumption of nutrients, providing insight into the hormonal mechanisms that underlie nutrient-specific hungers (Malita, 2022).

To maintain nutritional homeostasis, animals need to match their ingestion of specific nutrients to their needs. This is achieved by modulating appetite towards the specific nutrients needed. A number of factors, including gut hormones, that regulate food consumption have been identified in both flies and mammals, and reports have also described central brain mechanisms that induce ingestion of protein food in response to amino-acid deprivation, that sense amino acids and promote food consumption and that reject food lacking essential amino acids. However, little is known about the hormonal mechanisms that regulate nutrient-specific appetite, and gut hormones that regulate selective food intake are completely unknown. The current findings indicate that, in mated female Drosophila, gut-derived NPF is a selective driver of sugar satiety and protein consumption, providing a basis for understanding these mechanisms. Hormone-based therapies that inhibit appetite offer promising new directions for weight-loss treatment. For example, Fibroblast growth factor 21 (FGF21) is a liver-derived hormone that promotes protein consumption, and it is emerging as a promising target for metabolic disorders. Uncovering appetite-regulatory hormones such as gut-derived NPF that specifically inhibit sugar consumption while promoting the intake of protein-rich foods could provide effective new weight-management strategies by promoting healthier food choices (Malita, 2022).

The SLC2-family sugar transporter Sut2 is the closest Drosophila homologue of human SLC2A7 (GLUT7), a transporter expressed mainly in the intestine whose function is poorly defined. In flies, GLUT1 is important for Bursicon secretion from the EECs, and Sut1, another SLC2-family sugar transporter protein, was recently shown to be involved in midgut NPF release in virgin females. The current results implicate Sut2 in the release of NPF from EECs in mated females and thus link it to the mechanism by which NPF-mediated gut signalling controls feeding decisions. This indicates that both Sut1 and Sut2 sugar transporters are involved in glucose-stimulated NPF secretion from the gut. In mammals, several mechanisms also regulate glucose-stimulated GLP-1 secretion from intestinal endocrine cells, which involves sodium-glucose cotransporter 1 (SGLT1), the glucose transporter GLUT2 and sweet taste receptors. Targeting of these intestinal glucose-sensing mechanisms therefore has become a focus of weight-management therapies because of its potential in regulating appetite and incretin effects. Future studies should investigate whether GLUT7, like its Drosophila homologue Sut2, affects appetite-regulatory mechanisms in the mammalian gut (Malita, 2022).

NPF is orthologous with the mammalian NPY family of gut-brain peptides, including peptide YY (PYY), pancreatic polypeptide and NPY itself, that regulate food-seeking behaviours and metabolism. Like mammalian NPY-family hormones, Drosophila NPF is expressed in both the nervous system and the gut. While NPY is abundant in the nervous system and, like brain NPF, promotes food intake, PYY is mainly produced by endocrine cells of the gut as a satiety factor. Gut-expressed PYY is homologous to NPY, and both act through specific G-protein coupled receptors, called NPY receptors (NPYRs), that are orthologous with Drosophila NPFR. Thus, in mammals, multiple NPY-family peptides from different tissues sources exert their functions on target organs through several related NPYRs, while in Drosophila, these functions may be regulated through the single peptide-receptor pair of NPF and NPFR (Malita, 2022).

The results indicate that gut-derived Drosophila NPF fulfills the function of mammalian PYY. PYY is produced by the endocrine L-cells of the gut, which, like the EECs of Drosophila, produce a context-dependent combination of multiple hormones49. The physiological role of PYY in feeding regulation has been difficult to clarify, but it is believed to act through different NPYRs on tissues including the hypothalamus and the pancreatic islets to suppress appetite. These findings show that, in flies, NPF injection strongly reduces the intake of sugar-containing food and promotes the ingestion of protein-rich food. In humans, PYY infusion also been shown to strongly reduce food intake. Although the satiety function of human PYY has made it a prime therapeutic target for potential weight management, it is not clear whether PYY regulates nutrient-specific appetite, which would be important from a therapeutic perspective. These results indicate that Drosophila gut NPF, perhaps filling the role of mammalian gut PYY, acts to mediate sugar-specific satiety, illustrating a key hormonal mechanism that underlies selective hunger by which animals adjust their intake of specific nutrients (Malita, 2022).

Feeding decisions are based on internal state and exhibit sexual dimorphism. In Drosophila, males and females differ in their preference for and intake of dietary sugar and protein6. The current findings define a complex interorgan communication system through which mating influences food choices in females. Midgut NPF was found to be involved in mediating SP-induced postmating responses in females, inhibiting sugar appetite and promoting the ingestion of protein-rich yeast food, and it was further shown that AKH is required for mediating the effects of NPF. When mated females consume dietary carbohydrates, NPF is released from the EECs and inhibits the AKH axis by directly suppressing AKH release from the AKH-producing cells (APCs) as well as by inhibiting the release of midgut AstC, a factor that stimulates AKH secretion. Furthermore, NPF acts directly on the fat body through NPFR to inhibit energy mobilization, thereby antagonizing AKH-mediated signalling in the adipose tissue. Likewise, mammalian NPY-family peptides also regulate metabolism by direct actions on adipose tissue via NPYR. Although a number of studies have demonstrated that AKH is a regulator of metabolism, the current findings uncover a key role of AKH in governing nutrient-specific feeding decisions. It is becoming clear that the APCs integrate many signals that affect AKH release, and these signals may therefore also affect food choice. The APCs therefore seem to function as a signal-integration hub, similar to the IPCs, which receive many different inputs to control insulin production and release. AstC, Bursicon and NPF from the gut control AKH expression and secretion, indicating that multiple signals, even from the same organ, converge on the APCs. These signals presumably convey different aspects of nutritional status and may act with different dynamics to regulate AKH production and/or release, or even in a redundant manner to regulate AKH signalling. Likewise, many signals released from the fat body convey similar and seemingly redundant nutritional information to the IPCs (Malita, 2022).

Recent work has also revealed a sex-specific role of AKH, with lower activity in females underlying differences in male and female metabolism. Consistent with this notion, the current results indicate that in mated females the midgut NPF system inhibits AKH signalling, suppressing intake of sugar-rich food. Furthermore, it was recently shown that in mated females, midgut-derived AstC acts in a sex-specific manner through AKH to coordinate metabolism and food intake under nutritional stress. The current work shows that NPF also works sex-specifically to sustain physiological requirements in mated females by signalling from the gut to control AKH, suggesting that the gut-AKH axis occupies a central link in the hormonal relays underlying sex-specific regulation of physiology. A recent report showed that female germline cells modulate sugar appetite, but this effect is not induced by mating and does not affect yeast feeding as this study has found for gut NPF and AKH, suggesting that it is an independent mechanism (Malita, 2022).

How nutrient signals from the gut modulate feeding is key to understanding how nutritional needs are translated into specific feeding actions to maintain balance. This study has identified a homeostatic circuit triggered by gut-derived NPF that limits sugar consumption. Similar mechanisms for sugar-induced satiety that promote protein consumption may also enable mammals to balance their intake of different nutrients with their metabolic needs. Explaining how nutrient-responsive gut hormones such as NPF affect dietary choice is important to better understand hunger and cravings for specific nutrients that may ultimately lead to obesity (Malita, 2022).

Exposure to high-sugar diet induces transgenerational changes in sweet sensitivity and feeding behavior via H3K27me3 reprogramming

Human health is facing a host of new threats linked to unbalanced diets, including high-sugar diet (HSD), which contributes to the development of both metabolic and behavioral disorders. Studies have shown that diet-induced metabolic dysfunctions can be transmitted to multiple generations of offspring and exert long-lasting health burden. Meanwhile, whether and how diet-induced behavioral abnormalities can be transmitted to the offspring remains largely unclear. This study showed that ancestral HSD exposure suppressed sweet sensitivity and feeding behavior in the offspring in Drosophila. These behavioral deficits were transmitted through the maternal germline and companied by the enhancement of H3K27me3 modifications. PCL-PRC2 complex, a major driver of H3K27 trimethylation, was upregulated by ancestral HSD exposure, and disrupting its activity eliminated the transgenerational inheritance of sweet sensitivity and feeding behavior deficits. Elevated H3K27me3 inhibited the expression of a transcriptional factor Cad and suppressed sweet sensitivity of the sweet-sensing gustatory neurons, reshaping the sweet perception and feeding behavior of the offspring. Taken together, this study uncovered a novel molecular mechanism underlying behavioral abnormalities spanning multiple generations of offspring upon ancestral HSD exposure, which would contribute to the further understanding of long-term health risk of unbalanced diet (Yang, 2023).

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. 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).

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. PAs low cost and toxicity profile make it an especially attractive target for future clinical studies (Musselman, 2016 and references therein).

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

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

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

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

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

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

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

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

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

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

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).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Drosophila STING protein has a role in lipid metabolism

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Decapentaplegic retards lipolysis during metamorphosis in Bombyx mori and Drosophila melanogaster

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

Arc1 : a regulator of triglyceride homeostasis in male Drosophila. Micro Publication Biology

Achieving metabolic homeostasis is necessary for survival, and many genes are required to control organismal metabolism. A genetic screen in Drosophila larvae identified putative fat storage genes including Arc1. Arc1 has been shown to act in neurons to regulate larval lipid storage; however, whether Arc1 functions to regulate adult metabolism is unknown. Arc1^esm18 males store more fat than controls while both groups eat similar amounts. Arc1^esm18 flies express more brummer lipase and less of the glycolytic enzyme triose phosphate isomerase, which may contribute to excess fat observed in these mutants. These results suggest that Arc1 regulates adult Drosophila lipid homeostasis (Swope, 2023).

Drosophila insulin-like peptide dilp1 increases lifespan and glucagon-like Akh expression epistatic to dilp2

The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. This study finds that dilp1 is highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Dilp1 and dilp2 single and double mutants were used to describe interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 - dilp2 double mutants. dilp1 and dilp2 interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. Repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. The data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide (Post, 2018).

Based on mutational analyses of the insulin receptor (daf-2, InR) and its associated adaptor proteins and signaling elements, numerous studies in C. elegans and Drosophila established that decreased insulin/IGF signaling (IIS) extends lifespan. Studies on how reduced IIS in Drosophila systemically slows aging also reveal systems of feedback where repressed IIS in peripheral tissue decreases DILP2 production in brain insulin-producing cells (IPC), which may then reinforce a stable state of longevity assurance. This study finds that expression of dilp1 is required for loss of dilp2 to extend longevity. This novel observation contrasts with conventional interpretations where reduced insulin ligand is required to slow aging: Elevated dilp1 is associated with longevity in dilp2 mutants, and transgene expression of dilp1 increases longevity (Post, 2018).

dilp1 and dilp2 are encoded in tandem, likely having arisen from a duplication event. Perhaps as a result, some aspects of dilp1 and dilp2 are regulated in common: Both are expressed in IPCs, are regulated by sNPF, and have strongly correlated responses to dietary composition. Nonetheless, the paralogs are differentially expressed throughout development. While dilp2 is expressed in larvae, dilp1 expression is elevated in the pupal stage when dilp2 expression is minimal. In reproductive adults, dilp1 expression decreases substantially after eclosion and dilp2 expression increases (Post, 2018).

Furthermore, DILP1 production is associated with adult reproductive diapause. IIS regulates adult reproductive diapause in Drosophila, a somatic state that prolongs survival during inclement seasons. DILP1 may stimulate these diapause pro-longevity pathways, while expression in nondiapause adults is sufficient to extend survival even in optimal environments (Post, 2018).

The current data suggest a hypothesis whereby dilp1 extends longevity in part through induction of adipokinetic hormone (AKH), which is also increased during reproductive diapause and acts as a functional homolog of mammalian glucagon. Critically, AKH secretion has been shown to increase Drosophila lifespan and to induce triacylglycerides and free fatty acid catabolism. Here, it is noted that dilp1 mutants were more sensitive to starvation than wild-type and dilp2 mutants, as might occur if DILP1 and AKH help mobilize nutrients during fasting and diapause. Mammalian insulin and glucagon inversely regulate glucose storage and glycogen breakdown, while insulin decreases glucagon mRNA expression. It is proposed that DILP2 in Drosophila indirectly regulates AKH by repressing dilp1 expression, while DILP1 otherwise induces AKH (Post, 2018).

A further connection between dilp1 and diapause involves juvenile hormone (JH). In many insects, adult reproductive diapause and its accompanied longevity are maintained by the absence of JH. Furthermore, ablation of JH-producing cells in adult Drosophila is sufficient to extend lifespan, and JH is greatly reduced in long-lived Drosophila insulin receptor mutants. In each case, exogenous treatment of long-lived flies with a JH analog (methoprene) restores survival to the level of wild-type or nondiapause controls. JH is a terpenoid hormone that interacts with a transcriptional complex consisting of Met (methoprene tolerant), Taimen, and Kruppel homolog 1 (Kr-h1). As well, JH induces expression of kr-h1 mRNA, and this serves as a reliable proxy for functionally active JH. This study finds that dilp2 mutants have reduced kr-h1 mRNA, while the titer of this message is similar to that of wild-type in dilp1 - dilp2 double mutants. DILP1 may normally repress JH activity, as would occur in diapause when DILP1 is highly expressed. Such JH repression may contribute to longevity assurance during diapause as well as in dilp2 mutant flies maintained in laboratory conditions (Post, 2018).

Does DILP1 act as an insulin receptor agonist or inhibitor? Inhibitory DILP1 could directly interact with the insulin receptor to suppress IIS, potentially even in the presence of other insulin peptides. Such action could induce programs for longevity assurance that are associated with activated FOXO. Alternatively, DILP1 may act as a typical insulin receptor agonist that induces autophosphorylation and represses FOXO. In this case, to extend lifespan, DILP1 should stimulate cellular responses distinct from those produced by other insulin peptides such as DILP2 or DILP5. Through a third potential mechanism, DILP1 may interact with binding proteins such as IMPL2 or dALS to indirectly inhibit IIS output. These distinctions may be resolvednin a future study using synthetic DILP1 applied to cells in culture (Post, 2018).

A precedent exists from C. elegans where some insulin-like peptides are thought to function as antagonists. In genetic analyses, ins-23 and ins-18 stimulate larval diapause and longevity, while ins-1 promotes Dauer formation during development and longevity in adulthood. Moreover, C. elegans ins-6 acts through DAF-2 to suppress ins-7 expression in neuronal circuits to affect olfactory learning, where ins-7 expression inhibits DAF-2 signaling. These studies propose that additional amino acid residues of specific insulin peptides contribute to their distinct functions, and notably, the B-chain of DILP1 has an extended N-terminus relative to other DILP sequences (Post, 2018).

While dFOXO and DAF-16 are intimately associated with how reduced IIS regulates aging in Drosophila and C. elegans, in the current work, the behavior of FOXO does not correspond with how longevity is controlled epistatically by dilp1 and dilp2. Mutation of dilp2 did not impact FOXO activity, as measured by expression of target genes InR and 4eBP, and interactions with dilp1 did not modify this result. Some precedence suggests only a limited role for dfoxo as the mediator of reduced IIS in aging, as dfoxo only partially rescues longevity benefits of chico mutants, revealing that IIS extends lifespan through some FOXO-independent pathways. On the other hand, dilp1 expression from a transgene in the dilp1-2 double mutant background did induce FOXO targets. Differences among these results might arise if whole animal analysis of dFOXO targets obscures its role when IIS regulates aging through actions in specific tissues. In this vein, this study found that dilp2 controls thorax ERK signaling but not AKT, suggesting that dilp2 mutants may activate muscle-specific ERK/MAPK anti-aging programs (Post, 2018).

Dilp1 and dilp2 redundantly regulate glycogen levels and blood sugar, while these dilp loci interact synergistically to modulate dilp5 expression and starvation sensitivity. In contrast, dilp1 and dilp2 interact in a classic epistatic fashion to modulate longevity and AKH. Such distinct types of genetic interactions may reflect unique ways DILP1 and DILP2 stimulate different outcomes from their common tyrosine kinase insulin-like receptor, along with outcomes based on cell-specific responses. Understanding how and what is stimulated by DILP1 in the absence of dilp2 will likely reveal critical outputs that specify longevity assurance (Post, 2018).

High-fat diet enhances starvation-induced hyperactivity via sensitizing hunger-sensing neurons in Drosophila

The function of the central nervous system to regulate food intake can be disrupted by sustained metabolic challenges such as high-fat diet (HFD), which may contribute to various metabolic disorders. Previous work has shown that a group of octopaminergic (OA) neurons mediated starvation-induced hyperactivity, an important aspect of food-seeking behavior. This study found that HFD specifically enhances this behavior. Mechanistically, HFD increases the excitability of these OA neurons to a hunger hormone named adipokinetic hormone (AKH), via increasing the accumulation of AKH receptor (AKHR) in these neurons. Upon HFD, excess dietary lipids are transported by a lipoprotein LTP to enter these OA(+)AKHR(+) neurons via the cognate receptor LpR1, which in turn suppresses autophagy-dependent degradation of AKHR. Taken together, this study has uncovered a mechanism that links HFD, neuronal autophagy, and starvation-induced hyperactivity, providing insight in the reshaping of neural circuitry under metabolic challenges and the progression of metabolic diseases (Huang, 2020).

Obesity and obesity-associated metabolic disorders such as type 2 diabetes and cardiovascular diseases have become a global epidemic. Chronic over-nutrition, especially excessive intake of dietary lipids, is one of the leading causes of these metabolic disturbances. Accumulating evidence has shown that HFD imposes adverse effects on the physiology and metabolism of liver, skeletal muscle, the adipose tissue, and the nervous system. It is therefore of importance to understand the mechanisms underlying HFD-induced changes in different organs and cell types, which will offer critical insight into the diagnosis and treatment of obesity and other metabolic diseases (Huang, 2020).

The central nervous system plays a critical role in regulating energy intake and expenditure. In rodent models, neurons located in the arcuate nucleus of the hypothalamus, particularly neurons expressing Neuropeptide Y (NPY) and Agouti-Related Neuropeptide (AgRP) or those expressing Pro-opiomelanocortin (POMC), are important behavioral and metabolic regulators. These neurons detect various neural and hormonal cues such as circulating glucose and fatty acids, leptin, and ghrelin, and modulate energy intake and expenditure accordingly. Upon the reduction of the internal energy state, NPY/AgRP neurons are activated and exert a robust orexigenic effect. Genetic ablation of NPY/AgRP neurons in neonatal mice completely abolishes food consumption whereas acute activation of these neurons significantly enhances food consumption. NPY/AgRP neurons also antagonize the function of POMC neurons that plays a suppressive role on food consumption. Taken together, these two groups of neurons, among other neuronal populations, work in synergy to ensure a refined balance between energy intake and expenditure, and hence organismal metabolism (Huang, 2020).

In spite of their critical roles, the function of the nervous system to accurately regulate appetite and metabolism may be disrupted by sustained metabolic stress, resulting in eating disorders and various metabolic diseases such as obesity and type 2 diabetes. Several lines of evidence have begun to reveal the underlying neural mechanisms. For example, HFD increases the intrinsic excitability of orexigenic NPY/AgRP neurons, induces leptin resistance, and enhances their inhibitory innervations with anorexigenic POMC neurons, altogether resulting in hypersensitivity to starvation and increased food consumption. Interestingly, besides HFD, other metabolic challenges, including maternal HFD, alcohol consumption, as well as aging, also disrupt normal food intake via affecting the excitability and/or innervation of NPY/AgRP neurons. All these interventions may contribute to the onset and progression of metabolic disorders (Huang, 2020).

Before the actual food consumption, food-seeking behavior is a critical yet largely overlooked behavioral component for the localization and occupation of desirable food sources. Food-seeking behavior has been characterized in rodent models, primarily by the elevation of locomotor activity and increased food approach of starved animals. It has been reported that NPY/AgRP neurons also play a role in food-seeking behavior. However, to ensure adequate food intake, food seeking and food consumption are temporally and spatially separated and even reciprocally inhibited. It remains largely unclear how the neural circuitry of food seeking and food consumption segregated and independently regulated in rodent models. Furthermore, it remains unknown whether HFD also affects food seeking, and if so whether its effects on both food seeking and food consumption share common mechanisms or not. To fully understand the intervention of energy homeostasis by sustained metabolic stress, it is necessary to dissect the neural circuitry underlying food seeking and examine whether and how it is affected by HFD (Huang, 2020).

Fruit flies Drosophila melanogaster share fundamental analogy to vertebrate counterparts on the regulation of energy homeostasis and organismal metabolism despite that they diverged several hundred million years ago. Therefore, it offers a good model to characterize food-seeking behavior in depth and provides insight into the regulation of energy intake and the pathogenesis of metabolic disorders in more complex organisms such as rodents and human (Huang, 2020).

Previous work showed that fruit flies exhibited robust starvation-induced hyperactivity that was directed towards the localization and acquisition of food sources, therefore resembling an important aspect of food-seeking behavior upon starvation (Yang, 2015). A small subset of OA neurons in the fly brain were identified that specifically regulated starvation-induced hyperactivity (Yu, 2016). Analogous to mammalian systems, a number of neural and hormonal cues are involved in the systemic control of nutrient metabolism and food intake in fruit flies. Among them, Neuropeptide F (NPF), short NPF (sNPF), Leucokinin, and Allatostatin A (AstA), have been shown to regulate food consumption, all of which have known mammalian homologs that regulate food intake. In particular, starvation-induced hyperactivity is regulated by two classes of neuroendocrine cells (Yu, 2016). One is functionally analogous to pancreatic α cells and produce AKH upon starvation, whereas the other produces Drosophila insulin-like peptides (DILPs), resembling the function of pancreatic β cells. These two classes of Drosophila hormones exert antagonistic functions on starvation-induced hyperactivity via the same group of OA neurons in the fly brain (Huang, 2020).

Based on these findings, this study sought to examine whether HFD disrupted the regulation of starvation-induced hyperactivity in fruit flies and aimed to investigate the underlying mechanism. HFD-fed flies became significantly more sensitive to starvation and exhibited starvation-induced hyperactivity earlier and stronger than flies fed with normal diet (ND). Meanwhile, HFD did not alter flies' food consumption, suggesting that starvation-induced hyperactivity and food consumption are independently affected by HFD. Several days of HFD treatment did not alter the production of important hormonal cues like AKH and DILPs, but rather increased the sensitivity of the OA neurons that regulated starvation-induced hyperactivity to the hunger hormone AKH. In these OA neurons, constitutive autophagy maintained the homeostasis of AKHR protein, which determined their sensitivity to AKH and hence starvation. HFD feeding suppressed neuronal autophagy via AMPK-TOR signaling and in turn increased the level of AKHR in these OA neurons. Consistently, eliminating autophagy in these neurons mimicked the effect of HFD on starvation-induced hyperactivity whereas promoting autophagy inhibited the induction of hyperactivity by starvation. Furthermore, this study also showed that a specific lipoprotein LTP and its cognate receptor LpR1 likely mediated the effect of HFD on the neuronal autophagy of OA neurons and hence its effect on starvation-induced hyperactivity. Taken together, this study uncovered a novel mechanism that linked HFD, AMPK-TOR signaling, neuronal autophagy, and starvation-induced hyperactivity, shedding crucial light on the reshaping of neural circuitry under metabolic stress and the progression of metabolic diseases (Huang, 2020).

There is accumulating evidence that notes the effect of HFD on food consumption from insects to human, which results in obesity and obesity-associated metabolic diseases. But the effect of HFD on another critical food intake related behavior, food seeking, remains largely uncharacterized. Conceptually, food-seeking behavior in the fruit fly is composed of two behavioral components, increased sensitivity to food cues, and enhanced exploratory locomotion, which altogether facilitates the localization and acquisition of desirable food sources. Previous work has shown that starvation promotes starvation-induced hyperactivity, the exploratory component of food-seeking behavior, via a small group of OA neurons in the fly brain. These hunger-sensing OA neurons sample the metabolic status by detecting two groups of functionally antagonistic hormones, AKH and DILPs, and promote starvation-induced hyperactivity (Yu, 2016; Huang, 2020).

This study has demonstrated that this behavior is compromised by metabolic challenges. After a few days of HFD feeding, flies became behaviorally hypersensitive to starvation and as a result their starvation-induced hyperactivity was greatly enhanced, despite that their food intake and expenditure were not affected. These results suggest that HFD feeding may specifically modulate the activity of the neural circuitry underlying starvation-induced hyperactivity and offers an opportunity to further elucidate the cellular and circuitry mechanisms underlying behavioral abnormalities upon metabolic challenges (Huang, 2020).

As an insect counterpart of mammalian glucagon, AKH acts as a hunger signal to activate its cognate receptor AKHR expressed in the fat body and subsequently triggers lipid mobilization and energy allocation. In the fly brain, a small number of OA neurons also express AKHR. These neurons have been shown to be responsive to starvation and modulate various behaviors including food seeking and drinking (Jourjine, 2016; Yu, 2016). In that sense, these OA+AKHR+ neurons are functionally analogous to mammalian NPY/AgRP neurons in the hypothalamus, which also senses organismal metabolic states and regulates specific food intake behaviors. This study found that OA+AKHR+ neurons exhibited higher AKHR protein accumulation and became hypersensitive to AKH after HFD feeding. Notably, HFD feeding in mammals also increases the excitability of NPY/AgRP neurons, which contributes to the hypersensitivity to starvation and increased food consumption (Vernia, 2016). Thus, HFD may exert a conserved effect in the regulation of neuronal excitability and food intake related behaviors in both fruit flies and mammals (Huang, 2020).

Autophagy, a lysosomal degradative process that maintains cellular homeostasis, is critical for energy homeostasis. Upon cellular starvation, autophagy generates additional energy supply by breaking down macromolecules and subcellular organelles. At the organismal level, autophagy also contributes to the regulation of food intake and hence organismal energy homeostasis. For example, fasting induces autophagy in NPY/AgRP neurons via fatty acid uptake and promotes AgRP expression, which in turn enhances food intake (Kaushik, 2011). In line with these results, eliminating autophagy in NPY/AgRP neurons reduces food intake and hence body weight and fat deposits (Kaushik, 2011). Conversely, loss of autophagy in POMC neurons displays increased food intake and adiposity (Coupe, 2012). Consistently, in the current study, fruit flies neuronal autophagy was critical for the function of OA+AKHR+ neurons to sense hunger and regulate starvation-induced hyperactivity (Huang, 2020).

Accumulating evidence suggests that HFD suppresses autophagy in different peripheral tissue types such as liver, skeletal muscle, and the adipose tissue. Similarly, HFD suppresses autophagy in the hypothalamus, whereas blocking hypothalamic autophagy, particularly in POMC neurons, exacerbates HFD induced obesity. This study showed that HFD suppressed neuronal autophagy in OA+AKHR+ neurons and enhanced AKHR accumulation in these neurons. As a result, OA+AKHR+ neurons became hypersensitive to starvation and promoted starvation-induced hyperactivity. It will be of interest to examine whether HFD also reduces autophagy and increases the accumulation of specific membrane receptors in mammalian NPY/AgRP neurons (Huang, 2020).

This study also sought to examine the cellular mechanism that linked HFD feeding to the reduction of autophagy. HFD feeding activated TOR signaling. TOR, a highly conserved serine-threonine kinase, controls numerous anabolic cellular processes. Yhis study found that TOR signaling was tightly associated with the activity of AKHR+ neurons and the behavioral responses upon HFD feeding. Genetic enhancement of TOR activity in AKHR+ neurons increased AKHR protein accumulation, the sensitivity of these neurons to AKH, and hence starvation-induced hyperactivity, all of which mimicked the effect of HFD feeding. Inhibiting TOR activity exerted an opposite effect. In addition, the effect of HFD on TOR signaling was found to be mediated by AMPK signaling. These results altogether suggest that AMPK-TOR signaling in AKHR+ neurons plays an important role in maintaining the homeostasis of these neurons and determining the responsiveness to HFD feeding. Similarly, rodent studies have shown that manipulating AMPK-TOR signaling results in the dysfunction of NPY/AgRP neurons as well as POMC neurons, which leads to abnormal food consumption and adiposity. It will be of interest to examine whether HFD also modulates AMPK-TOR signaling in these specific hypothalamic neurons (Huang, 2020).

This study next sought to understand how AKHR+ neurons detected HFD, or more specifically, excess lipid ingested by the flies. As an essential nutrient and important energy reserve, dietary lipids were transported via their carrier proteins, named lipoproteins, in the circulation system and regulated multiple cellular signaling pathways. Proteomic analysis helped identify one lipoprotein LTP that was enriched in flies' hemolymph after HFD feeding. Single-cell RNAseq of AKHR+ neurons identified a number of lipoprotein receptors, especially LpR1, highly expressed in these neurons. Therefore, it is proposed that AKHR+ neurons might sense HFD feeding via LTP-LpR1 signaling. Evidently, it was found that eliminating LpR1 in AKHR+ neurons could protect flies from HFD, reducing AKHR accumulation and abolishing the effect of HFD to enhance starvation-induced hyperactivity. Conversely, eliminating LpR1 in the fat body, the major lipid reservoir of flies, created diet-independent hyperlipidemia and mimicked the effect of HFD feeding on flies' starvation-induced hyperactivity. Taken together, a working model is proposed that upon HFD feeding, excess dietary lipids are transported by LTP in the hemolymph, which interacts with its cognate receptor LpR1 in OA+AKHR+ neurons. As a result, these neurons undergo a number of cellular signaling processes and eventually become hypersensitive to starvation (Huang, 2020).

To summarize, the present study establishes a link between an unhealthy diet and abnormalities of food intake related behaviors in a model organism. The underlying mechanism was also deciphered, involving intracellular AMPK-TOR signaling, reduced neuronal autophagy, accumulation of a specific hormone receptor, and increased excitability of a small group of hunger-sensing neurons. This study will shed crucial light on the pathological changes in the central nervous system upon metabolic challenges. Given that the central control of metabolism and food intake related behaviors are highly conserved across different species, it will be of importance to further examine whether similar mechanisms also mediate the effect of HFD feeding on food intake and metabolic diseases in mammals (Huang, 2020).

Regulatory roles of Drosophila Insulin-Like Peptide 1 (DILP1) in metabolism differ in pupal and adult stages

The insulin/IGF-signaling pathway is central in control of nutrient-dependent growth during development, and in adult physiology and longevity. Eight insulin-like peptides (DILP1-8) have been identified in Drosophila, and several of these are known to regulate growth, metabolism, reproduction, stress responses, and lifespan. However, the functional role of DILP1 is far from understood. Previous work has shown that dilp1/DILP1 is transiently expressed mainly during the pupal stage and the first days of adult life. The role of dilp1 in the pupa, as well as in the first week of adult life, was studied, and some comparisons were made to dilp6 that displays a similar pupal expression profile, but is expressed in fat body rather than brain neurosecretory cells. Mutation of dilp1 diminishes organismal weight during pupal development, whereas overexpression increases it, similar to dilp6 manipulations. No growth effects of dilp1 or dilp6 manipulations were detected during larval development. It was next show that dilp1 and dilp6 increase metabolic rate in the late pupa and promote lipids as the primary source of catabolic energy. Effects of dilp1 manipulations can also be seen in the adult fly. In newly eclosed female flies, survival during starvation is strongly diminished in dilp1 mutants, but not in dilp2 and dilp1/dilp2 mutants, whereas in older flies, only the double mutants display reduced starvation resistance. Starvation resistance is not affected in male dilp1 mutant flies, suggesting a sex dimorphism in dilp1 function. Overexpression of dilp1 also decreases survival during starvation in female flies and increases egg laying and decreases egg to pupal viability. In conclusion, dilp1 and dilp6 overexpression promotes metabolism and growth of adult tissues during the pupal stage, likely by utilization of stored lipids. Some of the effects of the dilp1 manipulations may carry over from the pupa to affect physiology in young adults, but the data also suggest that dilp1 signaling is important in metabolism and stress resistance in the adult stage (Liao, 2020).

The insulin/IGF signaling (IIS) pathway plays a central role in nutrient-dependent growth control during development, as well as in adult physiology and aging. More specifically, in mammals, insulin, IGFs, and relaxins act on different types of receptors to regulate metabolism, growth, and reproduction. This class of peptide hormones has been well conserved over evolution and therefore the genetically tractable fly Drosophila is an attractive model system for investigating IIS mechanisms. Eight insulin-like peptides (DILP1-8), each encoded on a separate gene, have been identified in Drosophila. The genes encoding these DILPs display differential temporal and tissue-specific expression profiles, suggesting that they have different functions. Specifically, DILP1, 2, 3, and 5 are mainly expressed in median neurosecretory cells located in the dorsal midline of the brain, designated insulin-producing cells (IPCs). The IPC-derived DILPs can be released into the open circulation from axon terminations in the corpora cardiaca, the anterior aorta, the foregut, and the crop. Genetic ablation of the IPCs reduces growth and alters metabolism, and results in increased resistance to several forms of stress and prolongs lifespan (Liao, 2020).

The functions of the individual DILPs produced by the IPCs may vary depending on the stage of the Drosophila life cycle. Already, the temporal expression patterns hint that DILP1-3 and 5 play different roles during development. Thus, whereas DILP2 and 5 are relatively highly expressed during larval and adult stages, DILP1 and 6 are almost exclusively expressed during pupal stages under normal conditions (Liao, 2020).

DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1 is different from the other insulin-like peptides tested (Liao, 2020). DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1/DILP1 expression is also high (Liu, 2016). The temporal expression profile of dilp1/DILP1 resembles that of dilp6/DILP6 although the latter peptide is primarily produced by the fat body, not IPCs. Since DILP6 was shown to regulate growth of adult tissues during pupal development, it was asked whether also DILP1 plays a role in growth control. It is known that overexpression of several of the DILPs is sufficient to increase body growth through an increase in cell size and cell number, and especially DILP2 produces a substantial increase in body weight. In contrast, not all single dilp mutants display a decreased body mass. The dilp1, dilp2, and dilp6 single mutants display slightly decreased body weight, whereas the dilp3, dilp4, dilp5, and dilp7 single mutants display normal body weight. However, a triple mutation of dilp2, 3, and 5 causes a drastically reduced body weight, and a dilp1-4,5 mutation results in a further reduction. Note that several of the above studies do not show bona fide effects on cell or organismal growth (e.g., volume or cell numbers/sizes); they only provide body mass data (Liao, 2020).

There is a distinction between how DILPs act in growth regulation. DILPs other than DILP1 and DILP6 promote growth primarily during the larval stages (both feeding and wandering stages) when their expression is high. This nutrient-dependent growth is relatively well-understood and is critical for production of the steroid hormone ecdysone and thereby developmental timing and induction of developmental transitions such as larval molts and pupariation. The growth in the pupal stage, which primarily affects imaginal discs and therefore adult tissues, is far less studied. This study investigated the role of dilp1/DILP1 in growth regulation in Drosophila in comparison to dilp6/DILP6. For this, both bona fide size of body and/or wings were determined and wet weights were provided, and thus it was possible to distinguish between growth and increase of body mass. Mutation of dilp1 diminishes body weight (but not body size), whereas ectopic dilp1 expression promotes organismal growth by increasing both weight and size during the pupal stage, similar to dilp6. Thus, we cannot unequivocally show a role of dilp1 in organismal growth, but it does regulate body mass, suggesting that dilp1 affects metabolism and energy stores. Determination of metabolic rate (MR) and respiratory quotient (RQ) as well as triacylglyceride (TAG) levels during late pupal development provides evidence that dilp1 and dilp6 increase the MR and that the associated increased metabolic cost is fueled by increased lipid catabolism (Liao, 2020).

Since dilp1/DILP1 levels are high the first week of adult life, the role of dilp1 mutation and overexpression on early adult physiology was determined, including metabolism stress resistance and fecundity. Interestingly, the newly eclosed dilp1 mutant flies are less resistant to starvation than controls and dilp2 mutants. Thus, dilp1 acts differently from other dilps for which it has been shown that reduced signaling increases survival during starvation. Also, early egg laying and female fecundity are affected by dilp1 overexpression, and in general, dilp1 manipulations produce more prominent effects in female flies (Liao, 2020).

Taken together, these data suggest that ectopic expression of dilp1/DILP1 promotes growth of adult tissues during the pupal stage, and that this process mainly utilizes stored lipids to fuel the increased MR. The DILP1 signaling also affects the metabolism in the young adult fly, and sex dimorphic effects of altered signaling on stress responses and fecundity were seen (Liao, 2020).

This study shows that dilp1 gain of function stimulates adult tissue growth and increases metabolic rate (MR) during the pupal stage, and also affects adult physiology, especially during the first days of adult life. These stages correspond to the time when dilp1 is normally expressed. The gain of function experiments in this study suggest that the developmental role of ectopic dilp1 could be similar to that of dilp6, namely, to stimulate growth of adult tissues during pupal development. It was furthermore shown that in the adult fly, dilp1 is upregulated during starvation and genetic gain and loss of function of dilp1 signaling diminishes the flies' survival under starvation conditions in a sex-specific manner. These novel findings, combined with previous data that demonstrated high levels of dilp1 during adult reproductive diapause and the role of dilp1 as a pro-longevity factor during aging, suggest a wide-ranging importance of this signaling system. Not only does dilp1 expression correlate with stages of non-feeding (or reduced feeding), these stages are also associated with lack of reproductive activity and encompass the pupa, newly eclosed flies, and diapausing flies. Under normal conditions, the transient expression of dilp1/DILP1 during the first few days of adult life may be associated with a metabolic transition (remodeling from larval to adult fat body) and the process of sexual maturation (gonad growth and differentiation). The data also suggest that dilp1 affects physiology more prominently in young female flies than in males, which might be associated with ovary maturation (Liao, 2020).

It is also interesting to note that the diminished starvation resistance in dilp1 and dilp1/dilp2 mutants is opposite to the phenotype seen after IPC ablation, mutation of dilp1-4, or diminishing IIS by other genetic interventions. Thus, in recently eclosed flies, dilp1 appears to promote starvation resistance rather than diminishing it. Furthermore, the decreased survival during starvation in female dilp1 mutants is the opposite of that shown in dilp6 mutants, indicating that dilp1 action is different from the other insulin-like peptides tested (Liao, 2020).

In Drosophila, the final body size is determined mainly by nutrient-dependent hormonal action during the larval feeding stage. However, some regulation of adult body size can also occur after the cessation of the larval feeding stage, and this process is mediated by dilp6 acting on adult tissue growth in the pupa in an ecdysone-dependent manner. This is likely a mechanism to ensure growth of adult tissues if the larva is exposed to shortage of nutrition during its feeding stage. The current findings suggest that dilp1 can function as another regulator of growth during the pupal stage. Overexpression of dilp1 promotes organismal growth in the pupa, probably at the cost of stored nutrients derived from the larval feeding stage. This is supported by respiratory quotient (RQ) data that clearly show a shift from mixed-energy substrate metabolism in control flies toward almost pure lipid catabolism at the end of pupal development in the dilp1 overexpression flies (also seen for dilp6 gain of function in these experiments). Furthermore, triacylglycerol (TAG) (but not carbohydrate) levels in dilp1 overexpression pupae were clearly decreased, which likely reflects the shift in catabolic energy substrate also seen in the RQ using respirometry. It should be noted that insects predominantly use lipids as fuel during metamorphosis and dilp1 overexpression increases lipid catabolism. This study hence suggests that dilp1 can parallel dilp6 in balancing adult tissue growth and storage of nutrient resources during pupal development. This is interesting since dilp6 is an IGF-like peptide that is produced in the nutrient sensing fat body, whereas the source of the insulin-like dilp1 is the brain IPCs (Liao, 2020).

In contrast to the dilp1 gain of function, the experiments with dilp1 mutant flies did not show a clear effect on adult body growth, only a decrease in weight. Is this a result of compensation by other DILPs? Previous work showed that young adult dilp1 mutant flies display increased dilp6 and vice versa, suggesting feedback between these two peptide hormones in adults. During the pupal stage, this feedback appears less prominent in dilp1 mutants and no effects were detected on (dilp2, dilp3), or dilp6 levels. Furthermore, overexpression of dilp1 in fat body or IPCs has no effect on pupal levels of dilp2 and dilp6. Thus, at present, there is no evidence for compensatory changes in other dilps/DILPs in pupae with dilp1 manipulations. However, under normal conditions (in wild-type pupae), dilp6 levels are far higher than those of dilp1, which could buffer the effects of changes in dilp1 signaling (Liao, 2020).

DILPs and IIS are involved in modulating responses to starvation, desiccation, and oxidative stress in Drosophila. Flies with ablated IPCs or genetically diminished IIS display increased resistance to several forms of stress, including starvation . Conversely, overexpression of dilp2 increases mortality in Drosophila. This study found that young dilp1 mutant flies displayed diminished starvation resistance. In both recently eclosed and 3-day-old flies, mutation of dilp1 decreased survival during starvation (but not in 6- to 7-day-old flies) (Liao, 2020).

Action of dilp1 in the adult fly is also linked to reproductive diapause in females, where feeding is strongly reduced, and both peptide and transcript are upregulated. Related to this, dilp1 mRNA was found to upregulated during starvation in 12-day-old flies. Furthermore, it was shown that expression of dilp1 (dilp1 rescue) increases lifespan in dilp1/dilp2 double mutants, suggesting that loss of dilp2 induces dilp1 as a factor that promotes longevity. Thus, dilp1 activity is beneficial also during adult life, even though its expression under normal conditions is very low. This pro-longevity effect of dilp1 is in contrast to dilp2, 3, and 5 and the mechanisms behind this effect are of great interest to unveil (Liao, 2020).

A previous study showed that in wild-type (Canton S) Drosophila, DILP1 expression in young adults is sex-dimorphic with higher levels in females. In line with this, starvation resistance in young flies is diminished only in female dilp1 mutant and dilp1 overexpression flies. Thus, taken together, previous work showed that dilp1 displays a sex-specific expression and this study shows female-specific function in young adult Drosophila. It is tempting to speculate that the more prominent role of dilp1 in female flies is linked to metabolism associated with reproductive physiology and early ovary maturation, which is also reflected in the dilp1 upregulation during reproductive diapause. In fact, this study shows that egg-laying increased after dilp1 overexpression, and an earlier study demonstrated a decreased egg laying in dilp1 mutant flies. Part of the sex dimorphic effects on body weight of young adults after dilp1 manipulations might be a result of a differential role of dilp1 in water homeostasis (Liao, 2020).

This study shows that IPC-derived dilp1 displays several similarities to the fat body-produced dilp6, including temporal expression pattern, growth promotion, effects on adult stress resistance and lifespan. Additionally, dilp1 may play a role in regulation of nutrient utilization and metabolism during the first few days of adult life, especially in females. At this time, larval fat body is still present and utilized as energy fuel/nutrient store and this source also contributes to egg development. Curiously, there is a change in the action of DILP1 between the pupal and adult stages from being able to stimulate growth (agonist of dInR, like DILP6) in pupae, to acting in a manner opposite to DILP2, DILP6, and other DILPs in adults in regulation of lifespan and stress responses. Only one dInR is known so far (excluding the G protein-coupled receptors for the relaxin-like DILP7 and DILP8). Thus, the mechanisms behind this apparent switch in function of DILP1 signaling remain an open question. One possibility is that DILP1 acts via different signaling pathways downstream the dInR in pupae and adults. An obvious difference between these two stages is the presence of larval-derived fat body in the pupa and during the first few days of adults and its replacement by functional adult fat body in later stages. Perhaps dInR-mediated action differs in these types of fat body when activated by DILP1. Another possibility is stage-specific expression of insulin/IGF-binding proteins such as SDR, ALS, and Imp-L2 that could affect the activity of DILP1 in particular (Liao, 2020).

In the future, it would be interesting to investigate whether DILP1 acts differently on larval/pupal and adult fat body, or act on different downstream signaling in the two stages of the life cycle. Also, the possibility that dilp1 and dilp6 interact to regulate growth and metabolism in Drosophila is worth pursuing (Liao, 2020).

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

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

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

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

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

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

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

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

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

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

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

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

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

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).

The isothermal Boltzmann-Gibbs entropy reduction affects survival of the fruit fly Drosophila melanogaster

This study presents is the first experimental evidence of the effect of isothermal changes in entropy on a living organism. In greater detail, the effect of the reduction of the total Boltzmann-Gibbs entropy (S) of the aquatic environment on the survival rate and body mass of the fruit fly Drosophila melanogaster was investigated. The tests were carried out in standard thermodynamic states at room temperature of 296.15 K and ambient atmospheric pressure of 1 bar. Two variants of entropy reduction (ΔS) were tested for ΔS = 28.49 and 51.14 J K(-1) mol(-1) compared to the blind and control samples. The entropy level was experimentally changed, using the quantum system for isothermal entropy reduction. This system is based on quantum bound entanglement of phonons and the phenomenon of phonon resonance (interference of phonon modes) in condensed matter (Silicon dioxide (SiO(2)) and single crystals of Silicon (Si(0)), Aluminum (Al(0)) plates ("chips"), glass, and water). All studied organisms were of the same age (1 day). Mortality was observed daily until the natural death of the organisms. The investigations showed that changes in the Boltzmann-Gibbs entropy affected the survival and body mass of the fruit flies. On the one hand, the reduction in entropy under isothermal conditions in the aquatic environment for ΔS = 28.49 J K(-1) mol(-1) resulted in an extension of the lifespan and an increase in the body mass of female fruit flies. On the other hand, the almost twofold reduction in this entropy for ΔS = 51.14 J K(-1) mol(-1) shortened the lives of the males. Thus, the lifespan and body mass of flies turned out to be a specific reaction of metabolism related to changes in the entropy of the aquatic environment (Gruss, 2023).

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

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

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

Using RiPI, this study has established a Drosophila obesity model based on chronic, adipose tissue-directed knockdown of Stim, which shares remarkable similarity to characteristics of human obesity. First, the visibly enlarged abdomen of the obese flies corresponds to increased waist circumference, which gains importance as meaningful parameter to assess android adiposity. Similarly, body fat accumulation causes significant weight gain, another readout to quantify obesity in rodents and humans. Second, the excessive fat accumulation correlates with climbing deficits of the obese flies, with physical fitness reduction being another hallmark of human adiposity. Moreover, obese Stim-TRiPI flies have reduced life span, which is reminiscent of the higher mortality rates in human obesity patients. Third, this study demonstrates that early-onset hyperphagia drives the positive energy balance in Stim-TRiPI flies. Consistently, increased food intake is the major driver of human obesity. Hyperphagia is linked to increased dietary glucose conversion into storage fat in obese Stim-TRiPI flies. Notably, increased food intake and elevated glucose conversion into storage lipids has also been reported after silencing obesity blocking neurons in the fly central brain (Al-Anzi, 2009). With hyperphagia being an important contributor, obesity development in Stim-RiPI flies is not monocausal. It is noteworthy that the rise in fat storage in Stim-DRiPI substantially exceeds the food intake increase. Moreover, matching the food intake of Stim-TRiPI On and Off flies still results in body fat accumulation. Importantly, there is a significantly reduced metabolic rate of Stim-DRiPI flies. Finally, the observed hyperglycemia at day 10, physical fitness reduction at day 24 and shortened life span of Stim-TRiPI On flies are associated with obesity development, similar to type 2 diabetes (T2D), exercise intolerance and mortality, which are also highly correlated with human obesity. In summary, chronic knockdown of Stim in the adult fat body causes fly obesity by a number of physiological factors culminating in organismal energy imbalance similar to mammalian adiposity (Xu, 2019).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism

Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. This study sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells (Schell, 2017).

It was first observed almost 100 years ago that, unlike differentiated cells, cancer cells tend to avidly consume glucose, but not fully oxidize the pyruvate that is generated from glycolysis. This was originally proposed to be due to dysfunctional or absent mitochondria, but it has become increasingly clear that mitochondria remain functional and critical. Mitochondria are particularly important in proliferating cells because essential steps in the biosynthesis of amino acids, nucleotide and lipid occur therein. Most proliferating stem cell populations also exhibit a similar glycolytic metabolic program, which transitions to a program of mitochondrial carbohydrate oxidation during differentiation. The first distinct step in carbohydrate oxidation is import of pyruvate into the mitochondrial matrix, where it gains access to the pyruvate dehydrogenase complex (PDH) and enters the tricarboxylic acid (TCA) cycle as acetyl-CoA. The two proteins that assemble to form the mitochondrial pyruvate carrier (MPC) have been recently described. This complex is necessary and sufficient for mitochondrial pyruvate import in yeast, flies and mammals, and thereby serves as the junction between cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. Decreased expression and activity of the MPC underlies the glycolytic program in colon cancer cells in vitro, and forced re-expression of the MPC subunits increased carbohydrate oxidation and impaired the ability of these cells to form colonies in vitro and tumours in vivo. This impairment of tumorigenicity was coincident with a loss of key markers and phenotypes associated with stem cells. This has prompted an examination of whether glycolytic non-transformed stem cells might also exhibit low MPC expression and mitochondrial pyruvate oxidation, which must increase during differentiation (Schell, 2017).

The role of the MPC was studied in the ISCs of the fruit fly Drosophila, which share key aspects of their biology with mammalian ISCs. Both MPC1 and MPC2 are expressed in all four cell types of the intestine, with the lowest level of expression in the ISCs and the highest expression in the differentiated enteroendocrine cells. Confocal imaging of intestines dissected from dMPC1 mutants revealed that the epithelium exhibits multilayering unlike the normal single-cell layer seen in controls. This is a classic overgrowth phenotype that is associated with oncogene mutations in Drosophila. Accordingly, MARCM clonal analysis was used to determine whether a specific loss of the MPC in ISCs leads to an increase in their proliferation. On average, newly divided GFP-marked dMPC1 mutant clones are more than twofold larger than control clones, which were generated in parallel using a wild-type chromosome, indicating that the MPC is required in the ISC lineage to suppress proliferation. Because GFP-marked clones could include cells that differentiate into mature enterocytes or enteroendocrine cells, clonal analysis was conducted in the absence of Notch, thereby blocking ISC differentiation. Under these conditions, an approximately twofold increase was observed in the size of dMPC1 mutant ISC clones. To confirm and extend these results, MPC function was specifically disrupted in the ISCs by using the Dl-GAL4 driver in combination with UAS-GFP, which facilitates stem cell identification. Once again, approximately twofold more GFP-marked stem cells were observed relative to controls when either dMPC1 or dMPC2 expression was disrupted by RNA-mediated interference (RNAi) along with increased ISC proliferation as detected by staining for phosphorylated histone H3 (pHH3). Similar results were obtained when RNAi was targeted to the E1 or E2 subunits of PDH to specifically disrupt the next step in mitochondrial pyruvate oxidation. Importantly, an opposite phenotype was seen when Ldh was reduced by RNAi in the ISCs or progenitor cells. Ldh suppression is known to result in a significant increase in pyruvate levels, which can promote pyruvate oxidation. Taken together with the results with Pdh RNAi, these observations support the model that the MPC limits stem cell proliferation through promoting oxidative pyruvate metabolism in the mitochondria. It also appears to be sufficient as specific overexpression of MPC1 and MPC2 in ISCs or progenitors caused a reduction in stem cell proliferation, the opposite of the loss-of-function phenotype. This can be seen in either Pseudomonas-infected intestines, which undergo rapid stem cell proliferation, or under basal conditions in aged animals. Consistent with this, MPC overexpression under basal conditions had no effects on intestinal morphology, while the intestines from infected flies displayed a fully penetrant size reduction, which is probably due to the inability of ISCs to maintain tissue homeostasis. Taken together, these results demonstrate that mitochondrial pyruvate uptake and metabolism is both necessary and sufficient in a stem cell autonomous manner to regulate ISC proliferation and maintain intestinal homeostasis in Drosophila (Schell, 2017).

Studies in Drosophila, intestinal organoids and mice provide strong evidence that the MPC is necessary and sufficient in a cell autonomous manner to suppress stem cell proliferation. Consistently, this study has demonstrated that ISCs maintain low expression of the subunits that comprise the MPC, which enforces a mode of carbohydrate metabolism wherein glucose is metabolized in the cytosol to pyruvate and other biosynthetic intermediates. This glycolytic metabolic program appears to be sufficient to drive robust and continuous stem cell proliferation. High mitochondrial content was observed in ISCs, which must be geared primarily toward biosynthetic functions and/or oxidation of other substrates such as fatty acids. Increased fatty acids, the metabolism of which is enhanced in MPC-deficient and MPC-inhibited organoids, have been shown to promote ISC expansion and proliferation via enhanced beta-catenin signalling and increasing tumour-initiating capacity. MPC expression increases following differentiation, consistent with the shift in demand from macromolecule biosynthesis to ATP production in support of post-mitotic differentiated cell function. A similar switch in MPC expression can be seen following differentiation of embryonic stem cells, haematopoietic stem cells and trophoblast stem cells. Conversely, MPC expression is reduced after reprogramming fibroblasts to induced pluripotent stem cells. This suggests that the effects of altering pyruvate flux that wad observed in this study might not be restricted to ISCs, but instead be representative of similar effects on multiple stem cell populations. Interestingly, Myc is known to drive a metabolic program that is similar to that observed following MPC loss, characterized by increased glycolysis and reliance on glutamine and fatty acid oxidation with reduced glucose oxidation. This suggests that Myc may play a role in repressing the MPC in stem cells, possibly acting downstream of Wnt/beta-catenin signalling. Consistent with this, Myc and its repressive co-factors localize to the Mpc1 promoter and Myc expression is strongly anti-correlated with Mpc1 expression (Schell, 2017).

Taken together, these studies demonstrate that changes in the MPC and mitochondrial pyruvate metabolism are required to properly orchestrate the proliferation and homeostasis of intestinal stem cells. Importantly, this metabolic program, mediated at least partially by the MPC, appears to be instructive for cell fate, rather than a downstream consequence of cell fate. Future work will define the extent to which the results presented in this study relate to those showing that diet quality and quantity can modulate ISC behaviour. It is tempting to speculate that ISC metabolism is used as a signal for increased or decreased demand for intestinal epithelium. Perhaps of most importance will be to define the mechanisms whereby altered partitioning of pyruvate metabolism affects stem cell proliferation and fate. It is speculated that the robust changes that were observed in fatty acid oxidation and histone acetylation, which are probably downstream of altered metabolite utilization for acetyl-CoA production, play an important role. While the mechanisms are not as yet defined, these studies establish a paradigm wherein mitochondrial metabolism does not merely provide a permissive context for proliferation or differentiation, but rather plays a direct and instructive role in controlling stem cell fate (Schell, 2017).

Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Comprehensive genetic characterization of mitochondrial Ca(2+) uniporter components reveals their different physiological requirements in vivo

Mitochondrial Ca(2+) uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca(2+) uniporter has opened it up to genetic analysis in model organisms. This study reports a comprehensive genetic characterization of all known uniporter components conserved in Drosophila. While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca(2+) uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmentally lethal, consistent with unregulated Ca(2+) uptake. Mutants for the neuronally restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. The data reveal the interplay among components of the mitochondrial Ca(2+) uniporter and shed light on their physiological requirements in vivo (Tufi, 2019).

The uptake of Ca2+ into mitochondria has long been established as a key regulator of an array of cellular homeostatic processes as diverse as bioenergetics and cell death. A series of seminal discoveries has elucidated the identity of the components that make up the mitochondrial Ca2+ uniporter complex. The mammalian uniporter is composed of MCU (mitochondrial calcium uniporter) as the main pore-forming protein; its paralog MCUb; a small structural component, EMRE (essential MCU regulator); and the regulatory subunits MICU1-MICU3 (mitochondrial calcium uptake 1-3). Reconstitution studies in yeast, which lacks a mitochondrial Ca2+ uniporter, have demonstrated that heterologous co-expression of MCU and EMRE is necessary and sufficient to confer uniporter activity. The family of EF-hand-containing proteins (MICU1, MICU2, and MICU3) has been shown to exhibit a gatekeeper function for the uniporter, inhibiting Ca2+ uptake at low cytoplasmic concentrations. These components are generally highly conserved across eukaryotes, including most metazoans and plants, but not in many fungi and protozoans, reflecting their ancient and fundamental role (Tufi, 2019).

Although the composition and function of the uniporter have been well characterized in vitro and in cell culture models, the physiological role of the uniporter is beginning to emerge with in vivo characterization of knockout mutants. Current data present a complex picture. Initial studies of MCU knockout mice described a viable strain with a modest phenotype in a mixed genetic background, although subsequent studies using an inbred background reported MCU loss to be lethal or semi-viable and tissue-specific conditional knockout revealed an important role in cardiac homeostasis. Similarly, loss of MICU1 in mice has a complex phenotype, varying from fully penetrant perinatal lethality to incomplete lethality with a range of neuromuscular defects that unexpectedly improve over time in surviving animals (Tufi, 2019).

One explanation for the reported phenotypic variability is that perturbing mitochondrial Ca2+ uptake can be influenced by additional factors, the most obvious being genetic background. Hence, there is a need for greater investigation into the physiological consequences of genetic manipulation of the uniporter components in a genetically powerful model system. This paper reports a comprehensive genetic analysis of the uniporter complex components that are conserved in Drosophila. This includes loss-of-function mutants for MCU, EMRE, MICU1, and MICU3 (Drosophila lack MCUb and MICU2) and corresponding inducible transgenic expression lines. Despite lacking fast Ca2+ uptake, MCU and EMRE mutants present a surprising lack of organismal phenotypes, although both mutants are short lived, with a more pronounced effect when MCU is lost. In contrast, loss of MICU1 causes developmental lethality, whereas mutants for MICU3 are viable with modest phenotypes. Performing genetic interaction studies with these strains, this study has confirmed the gatekeeper function of MICU1 is conserved in flies and reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, it was found that loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that the lethality results from MCU-independent functions. The generation of these genetic tools in Drosophila will facilitate further investigation of the functional roles of the uniporter components in vivo (Tufi, 2019).

MCU mutants are viable and fertile with no gross morphological or behavioral defects, which was initially surprising given the historical importance of mitochondrial Ca2+. Still, this corroborates another report of fly MCU mutants and is consistent with studies in mice and worms in which deletion of MCU orthologs is essentially benign at the organismal level under basal conditions. However, fly MCU mutants are significantly shorter lived than controls. This situation is mirrored by EMRE mutants, albeit with a smaller impact on lifespan. The reason for the shortened lifespans is unknown but may reflect the effects of a chronic bioenergetic deficit evident from the OCR measurements. Accordingly, MCU mutants show a greater respiration defect compared to EMRE mutants, consistent with their respective impacts on lifespan. The respiratory impairment could be due to the previously reported increase in oxidative stress that occurs in MCU mutants, which has yet to be assessed in EMRE mutants. Alternatively, the short lifespan may be due to a myriad of potential metabolic imbalances, such as disruption of NADH/NAD+ levels. Chronic adaptations may also occur through transcriptional responses. Further studies analyzing the metabolic and transcriptional changes occurring in these flies will shed light on this fundamental question (Tufi, 2019).

Nevertheless, the EMRE mutants are relatively benign at the organismal level, which corroborates the surprising viability of MCU mutants. Considering this, it is striking that flies, like mice and worms, consistently show an ability to compensate for the lack of fast mitochondrial Ca2+ uptake, suggesting the induction of some adaptive mechanism. While alternative routes of mitochondrial Ca2+ entry must exist, because matrix Ca2+ is not abolished in MCU knockout (KO) mice, proposed mechanisms are speculative, and it is unclear whether they constitute a compensatory adaptation for fast Ca2+ uptake or simply allow gradual, slow accumulation. However, rapid mitochondrial Ca2+ uptake mediated by MCU is thought to constitute a specific metabolic regulatory mechanism, e.g., to increase ATP production, under certain conditions, such as strenuous exercise or pathological conditions, which is partly evident in the MCU KO mice or heart-specific conditional KO. Such important physiological roles would not necessarily be apparent under basal conditions in flies. MCU has also been proposed to promote wound healing; however, preliminary studies did not find evidence supporting this. The current study presents a summary of the requirements of uniporter components under basal conditions, and further work will be needed to evaluate the role of the uniporter in the full range of physiological conditions (Tufi, 2019).

In seeking to understand the importance of the regulatory components of the uniporter, this study also developed loss-of-function models for MICU1 and MICU3. In contrast to MCU and EMRE mutants, loss of MICU1 results in larval lethality, which is associated with alterations in mitochondrial distribution and motility, and a reduced level of total ATP. In line with its role as the principle gatekeeper of the uniporter, coupled with excess mitochondrial Ca2+ triggering cell death, it was reasoned that the lethality was due to Ca2+ accumulation in the mitochondrial matrix through unregulated MCU-EMRE channels. Supporting this, it was observed that dual overexpression of MCU and EMRE in the eye leads to substantial loss of retinal tissue; concomitant overexpression of MICU1 is sufficient to prevent this phenotype, consistent with MICU1 re-establishing appropriately regulated uniporter channels (Tufi, 2019).

However, one observation that was most surprising was the inability of MCU or EMRE mutants to rescue the MICU1 mutant lethality. This result is particularly puzzling, because it has been shown that mice lacking MICU1, which present multiple pathogenic phenotypes, are substantially rescued by genetic reduction of EMRE levels. While the reason for the lack of rescue in flies is unclear, it is postulate that this suggests the function of MICU1 is not limited to uniporter-dependent Ca2+ uptake. It is not known whether the lethality of MICU1 mutants is specifically due to excessive mitochondrial Ca2+ levels; however, it appears to be independent of fast mitochondrial Ca2+ uptake, because this is eliminated in MCU and EMRE mutants. As noted earlier, other routes of Ca2+ uptake into mitochondria exist, but the mechanisms that regulate them are uncertain. It is possible that aberrant manganese uptake, as reported to occur in cell models, may contribute to the MICU1 mutant lethality. However, this mechanism would presumably be expected to be mitigated by loss of MCU. Nevertheless, these Drosophila models are ideally suited for unbiased genetic screening to uncover such fundamental regulatory mechanisms (Tufi, 2019).

In contrast to MICU1, loss of MICU3 was well tolerated overall at the organismal level. Functional analysis of MICU3 is extremely limited, but the neuronally restricted expression led to the expectation that these mutants might have more neurological-specific phenotypes, which was at least partly borne out. Whereas longevity of these mutants was only minimally affected, they exhibited a notable locomotor deficit even in young flies. It was initially hypothesized that MICU3 may be able to act redundantly with MICU1, but attempts to transgenically rescue MICU1 mutants by ectopic MICU3 expression were unsuccessful. This result is consistent with a report showing that MICU3 binds to MICU1 but apparently enhances mitochondrial Ca2+ uptake (Tufi, 2019).

In summary, this study presents a comprehensive analysis of the conserved components of the mitochondrial Ca2+ importer and its regulators. While loss of the various components results in dramatically different organismal phenotypes, ranging from the most severe deficit exemplified by the MICU1 mutants to the mild consequences of mutating MICU3, such diverse phenotypes mirror the situation reported in humans so far. The first described patients with MICU1 mutations exhibit a severe, complex neurological condition accompanied by muscular dystrophy and congenital myopathy, clearly associated with mitochondrial dysfunction, whereas a later study reported MICU1 patients with a relatively mild fatigue syndrome. One explanation for the reported phenotypic variability is that the consequence of perturbing mitochondrial Ca2+ uptake can be influenced by additional factors, the most obvious being genetic background. The genetic tools described in this study open up the possibility for a thorough analysis of the uniporter function in a powerful genetic model organism, which will advance understanding of the role of mitochondrial Ca2+ in health and disease (Tufi, 2019).

Electron transport chain biogenesis activated by a JNK-insulin-Myc relay primes mitochondrial inheritance in Drosophila

Oogenesis features an enormous increase in mitochondrial mass and mtDNA copy number, which are required to furnish mature eggs with an adequate supply of mitochondria and to curb the transmission of deleterious mtDNA variants. Quiescent in dividing germ cells, mtDNA replication initiates upon oocyte determination in the Drosophila ovary, which necessitates active mitochondrial respiration. However, the underlying mechanism for this dynamic regulation remains unclear. This study shows that an feedforward insulin-Myc loop promotes mitochondrial respiration and biogenesis by boosting the expression of electron transport chain subunits and of factors essential for mtDNA replication and expression, and for the import of mitochondrial proteins. Transient activation of JNK enhances the expression of the insulin receptor and initiates the insulin-Myc signaling loop. This signaling relay promotes mitochondrial biogenesis in the ovary, and thereby plays a role in limiting the transmission of deleterious mtDNA mutations. This study demonstrates cellular mechanisms that couple mitochondrial biogenesis and inheritance with oocyte development (Wang, 2019).

Mitochondria host a number of biosynthetic pathways and produce most of the cell's ATP through oxidative phosphorylation, which is carried out by the electron transport chain (ETC) complexes located on the mitochondrial inner membrane. While the majority of mitochondrial proteins are encoded on the nuclear genome, synthesized in the cytoplasm, and imported into the mitochondria, a subset of core ETC components are encoded on the mitochondrial genome (mtDNA) and synthesized inside the mitochondrial matrix. Thus, mitochondria biogenesis and ETC activity in particular, rely on the coordinated expression of both nuclear- and mtDNA-encoded mitochondrial genes. Mitochondria vary in number and activity to meet the different energy and metabolic demands of different tissues and developmental processes. Mitochondria are transmitted exclusively through the maternal lineage in most metazoans, which demands a complex regulation of mitochondrial biogenesis and ETC activity during oogenesis. Animal oocytes are hundreds of times larger than their progenitors. During this tremendous oocyte growth, mitochondria undergo prodigious biogenesis and increase mtDNA copy number over a thousand folds. The massive amount of mitochondria in the mature oocyte is necessary to power early embryonic development, as inadequate mitochondrial contents often lead to embryonic lethality. However, the mechanism by which the germline couples mitochondrial biogenesis to oocyte development remains elusive (Wang, 2019).

While furnishing mature oocytes with sufficient number of mitochondria, oogenesis also limits the transmission of harmful mtDNA mutations. The mitochondrial genome is prone to accumulating mutations because of its close vicinity to the highly mutagenic free radicals present in the mitochondrial matrix and of a lack of effective repair mechanisms. Yet, harmful mtDNA mutations are rare in populations, underscoring the presence of efficient mechanisms to limit their transmission through the female germline. It has been reported that mtDNA replication depends on active respiration in the Drosophila ovary. Healthy mitochondria with wild-type genomes propagate more vigorously than defective ones carrying harmful mutations, thereby curbing the transmission of deleterious mtDNA mutations to the next generation. Therefore, an active ETC appears to be a stress test for the functionality of mtDNA, and is essential for mtDNA selective inheritance. Nonetheless, how the activity of the ETC is regulated during oogenesis is not well understood (Wang, 2019).

Insulin signaling (IIS), an evolutionary conserved pathway that controls cell growth and proliferation, has also been shown to regulate ETC biogenesis and ATP production in human skeletal muscles. In the Drosophila ovary, IIS promotes the growth of follicles from the early to the middle stages of oogenesis. IIS activity decreases before the nurse cells dump their content into the oocyte. This decrease relieves the inhibition of GSK3, thereby shutting down mitochondrial respiration. However, oogenesis begins with germline stem cells (GSCs) that are thought not to rely on oxidative phosphorylation to ATP production. It is predicted there had to be developmental cues to activate mitochondrial respiration in the late germarium stage when mtDNA replication commences. IIS represents a logical candidate to modulate this metabolic transition in early oogenesis. Nonetheless, it remains to be explored how IIS is dynamically regulated during oogenesis and whether it is indeed involved in the aforementioned metabolic transition. Furthermore, little is known regarding how IIS modulates ETC activity and mtDNA biogenesis in general (Wang, 2019).

This study found that mitochondrial respiration is quiescent in GSCs and dividing cysts, but markedly upregulated in the late germarium, the same spatial-temporal pattern as mtDNA replication. A feedforward loop was found between IIS and Myc protein which orchestrates the transcriptional activation of respiration and mtDNA replication. Furthermore, transient JNK activity boosts insulin receptor (InR) transcription to enhance the IIS-Myc loop. This work uncovers how developmental programs couple mitochondrial biogenesis with cell growth and mitochondrial inheritance (Wang, 2019).

mtDNA replication in the Drosophila ovary relies on active respiration, suggesting that ETC activity and mtDNA replication might be subject to the same spatio-temporal regulation. This study has addressed this question and has further elucidated the developmental mechanisms regulating ETC activity and mtDNA biogenesis in the ovary. Utilizing the COX/SDH dual activity staining, it was revealed that ETC complexes are inactive in the germline stem cells (GSCs) and dividing cysts from germarium region 1 to 2A, but sharply activated in region 2B and active through stage-10 follicles. This spatial pattern mirrors that of mtDNA replication in the Drosophila ovary, supporting an essential role of mitochondrial respiration in mtDNA inheritance, both quantitively and qualitatively. It was also demonstrated that ETC activation is accompanied with an upregulation of the expression of ETC genes of both nuclear and mitochondrial origin. Interestingly, MDI, which drives the local translation of nuclear encoded mitochondrial proteins on the mitochondrial outer membrane and TFAM , which governs mtDNA replication and transcription, exhibit the same developmental pattern as mitochondrial respiration in the germarium. Collectively, these proteins would boost the biogenesis of ETC in region 2B of the germarium and in growing egg chambers. In an ovariole, different stages of developing germ cells reside in the same microenvironment and experience the same oxygen tension. Thus, the mitochondrial respiratory activity is likely to be determined by the abundance of ETC components, which itself is controlled by transcriptional activationx (Wang, 2019).

To understand how mitochondrial respiration is regulated, an RNAi screen was conducted for genes that boost COX/SDH activity in the ovary. The myc gene emerged as one of the strongest hits, and a hypomorphic allele, myc^P0, largely abolished ETC activity and mtDNA replication in the germarium. Moreover, the spatial pattern of Myc protein mirrors mtDNA replication and ETC activity, further supporting its essential role in transcriptional activation of ETC biogenesis. RNA sequencing data demonstrate that Myc broadly stimulates gene expression in the Drosophila ovary, including many nuclear-encoded ETC genes and factors required for mtDNA replication and expression. These observations are consistent with previous studies in mammals showing that MYC can promote mitochondrial biogenesis by directly elevating the expression of nuclear-encoded mitochondrial genes. Among 198 annotated human mitochondrial genes that are up-regulated by Myc overexpression, 185 have homologs in the Drosophila genome. Of note, 44.9% (101 out of 225) of the fly homologs are down-regulated in myc^P0 mutant ovaries, suggesting an evolutionarily conserved function of Myc in regulating mitochondrial biogenesis through gene expression. The finding that Myc induces ETC biogenesis and respiration is also in line with the studies in mammals demonstrating the multi-faceted roles of Myc in the regulation of mitochondria, including boosting mitochondrial biogenesis, stimulating oxidative metabolism , and regulating mitochondrial structure and dynamics (Wang, 2019).

Myc overexpression sometimes gives rise to different transcriptional output in different cell types. This observation reflects the fact that Myc-family proteins often associate with other cofactors and exert a broad and complex transcriptional role in a cell- or tissue-specific manner. This study also found that 130 transcription regulators, including Spargel Srl (fly homolog of human PGC-1) and CG32343 (fly homolog of GABPB2), were affected by the myc^P0 mutation. PGC-1 proteins belong to an evolutionarily conserved family that integrates mitochondrial biogenesis and energy metabolism with a variety of cellular processes. In Drosophila, Srl regulates the expression of a subset of nuclear encoded mitochondrial genes. Mammalian GABPB2 is a regulatory subunit of the Nuclear Respiratory Factor complex 2 that regulates the expression of a small set of nuclear encoded mitochondrial proteins. Therefore, additional tiers of transcriptional regulations downstream of Myc are likely involved in boosting ETC biogenesis (Wang, 2019).

While myc transcription is uniform in the germarium, Myc protein is elevated at region 2B and remains high until the stage-10 egg chamber, indicating that Myc abundance is mainly regulated via post transcriptional mechanisms. IIS and JNK also emerged from the RNAi screen, and both were further confirmed to be required for triggering ETC biogenesis and mtDNA replication. IIS activity, marked by both p-AKT and p-GSK3 staining, displayed a pattern similar to that of Myc. Additionally, elevated IIS activity was required to establish a high level of Myc and to activate ETC in the late germarium stage. GSK3 directly phosphorylates Myc and promotes its ubiquitination and degradation in both mammalian and fly cultured cells. Thus, IIS likely stabilizes Myc protein by inhibiting GSK activity. This result is also in line with a previous study showing that decreased IIS activity relieves the inhibition on GSK3, which leads to mitochondrial quiescence at later stages of oogenesis. Importantly, this work uncovers Myc as the downstream effector of IIS in the regulation of respiration and mtDNA biogenesis in the ovary (Wang, 2019).

It was noticed that InR transcription was down-regulated in the myc mutant ovary, suggesting a positive feedback regulation between IIS and Myc. This regulatory loop maintains high levels of both Myc protein and IIS activity in the mid-stage follicles, where massive mitochondrial biogenesis and massive cell growth take place. However, it does not explain how this loop is activated in the first place at the late germarium stages. It was found that JNK was transiently activated in germ cells in the germarium region 2B, but decreased in budding egg chambers and sharply diminished thereafter. High level and sustained JNK activity often lead to apoptosis. However, cell death is rarely observed in the germaria of flies cultured under normal conditions. Thus, JNK activation in the late germarium must be triggered by cellular processes unrelated to apoptosis. Transiently elevated JNK activity was sufficient to increase InR mRNA level, which in-turn boosted IIS activity and stabilized Myc protein. Currently, the link between JNK and IIS is not well-understood. In the metastatic Drosophila epithelium, cell survival and proliferation entail upregulation of InR expression by JNK through wingless signaling. However, no genes in the wingless signaling pathway emerged from the RNAi screen in germ cells. The molecular mechanisms that links JNK activation to InR expression in ovary remain to be explored (Wang, 2019).

The JNK-dependent transcriptional program can be activated by various cellular stresses and cell-cell signaling events. In region 2B of the germarium, the follicle cells extend and migrate laterally across the germarium to wrap around the 16 cells cyst. Thus, JNK activation in germ cells may reflect paracrine signaling from the follicle cells, for instance via TNF-α. Alternatively, the process of follicle cells enveloping and compressing the 16-cell cyst may generate mechanical stress that subsequently activates JNK. Regardless, this work uncovers a novel function of JNK in energy metabolism and mitochondrial biogenesis besides its well-established roles in controlling cell apoptosis, growth, and proliferation (Wang, 2019).

Studies in a variety of animal models have shown that reproductive aging in females is tightly associated with decreased IIS activity. Interestingly, oocytes of aged females often have higher incidence of mtDNA lesions and lower mtDNA copy number. Thus, developmental control of mitochondrial biogenesis and mtDNA replication via IIS may be a conserved mechanism in metazoans. Previous studies demonstrated that prodigious mitochondrial biogenesis during oogenesis underlies the selective inheritance of functional mtDNA by allowing proliferation competition between healthy mitochondria and mitochondria carrying deleterious mtDNA mutations. This study has shown that the JNK/IIS/Myc signaling relay governs mitochondrial biogenesis in the ovary, and thereby influences mitochondrial inheritance both quantitively and quantitively. These studies could provide a molecular framework to further understand the control of mitochondrial biogenesis and mtDNA inheritance in animals (Wang, 2019).

Downregulation of respiratory complex I mediates major signalling changes triggered by TOR activation

Mitochondrial dysfunctions belong amongst the most common metabolic diseases but the signalling networks that lead to the manifestation of a disease phenotype are often not well understood. This study identified the subunits of respiratory complex I, III and IV as mediators of major signalling changes during Drosophila wing disc development. Their downregulation in larval wing disc leads to robust stimulation of TOR activity, which in turn orchestrates a complex downstream signalling network. Specifically, after downregulation of the complex I subunit ND-49 (mammalian NDUFS2), TOR activates JNK to induce cell death and ROS production essential for the stimulation of compensatory apoptosis-induced proliferation within the tissue. Additionally, TOR upregulates Notch and JAK/STAT signalling and it directs glycolytic switch of the target tissue. These results highlight the central role of TOR signalling in mediating the complex response to mitochondrial respiratory dysfunction and they provide a rationale why the disease symptoms associated with respiratory dysfunctions are often alleviated by mTOR inhibitors (Perez-Gomez, 2020).

Mitochondria play an essential function in cellular energetic and NADH metabolism. Five protein complexes (complex I-V) of the electron transport chain (ETC) have distinct functions in the oxidation of NADH and/or FADH2, maintenance of the inner mitochondrial membrane potential and production of ATP via oxidative phosphorylation. Moreover, they serve as signalling hubs for specific cellular events including ROS-mediated signalling, apoptosis and Ca2+ signalling. Mutations in mitochondrial enzymes are the most frequent metabolic mutations present in human (Perez-Gomez, 2020).

Complex I of the ETC is the node point in the mitochondrial NADH metabolism as it mediates electron transfer from NADH to the other respiratory complexes. Therefore, complex I inhibitors have been exploited as therapeutic targets in cancer treatment, although the mechanism of action is often unclear. On the other hand, complex I inhibition can lead to increased proliferation, depending on the cell type and complex I inhibitor used. Despite the fact that mitochondrial electron transport chain disorders are one of the most common human genetic diseases, the mechanisms behind the dichotomy in the functional outputs after complex I inhibition are not well understood (Perez-Gomez, 2020).

The mTOR pathway (TOR in Drosophila) is the key integrator of cellular metabolic inputs that connects cell growth with environmental signals, including nutrient and growth factors availability. It promotes cell growth by stimulation of cellular translation, anabolic metabolism and by inhibiting autophagy. At the same time, mTOR activation can lead to apoptosis in certain contexts. The upregulation of mTOR is observed during epithelial wound healing, during aging as well as in many types of cancers. Although strong inhibition of mitochondrial respiration can cause a metabolic catastrophe and cell death connected with mTOR inhibition through the activation of AMPK, increasing evidence suggest that many types of respiratory dysfunctions are connected with increase in mTOR activity and mTOR inhibition leads to alleviation of the phenotype (Perez-Gomez, 2020).

This study found that downregulation of mitochondrial respiratory complex I, III or IV stimulates TOR activity that directs major downstream signalling events, including Notch activation, metabolic changes and apoptosis-driven proliferation. As TOR overactivation balances between stimulation of apoptosis and proliferation, presented in this study suggests a possible mechanism for the observations when complex I inhibition promotes either cell death or proliferation in different contexts. The signalling network that was identified also suggests a possible explanation why the disease symptoms associated with respiratory dysfunctions are often alleviated by mTOR inhibitors (Perez-Gomez, 2020).

Despite the fact that mutations in mitochondrial enzymes are the most frequent metabolic mutations present in human, manifested in a whole range of clinical disorders, the actual signalling networks that are triggered by malfunctioning mitochondria to develop clinical symptoms are still not well understood. The results argue that TOR pathway is the key signalling effector triggered after downregulation of complex I, III or IV in the Drosophila wing disc. TOR activity in turn activates JNK, Notch and JAK/STAT signalling, boosts glycolysis and promotes compensatory apoptosis-induced proliferation to produce profound effects on tissue size and patterning (Perez-Gomez, 2020).

By placing TOR at the top of the signalling network triggered by complex I dysfunction, this study provides a rationale for numerous observations where TOR inhibition alleviated the disease symptoms associated with mitochondrial dysfunction. For example, the maternally inherited Leigh syndrome (MILS), caused by mutation in complex I subunit Ndufs4, is associated with enhanced mTOR activity in neurons and the disease symptoms can be alleviated using chemical mTOR inhibitors. Upregulation of mTORC1 has also been described as a key component of the mitochondrial integrated stress response during mitochondrial myopathy. Alongside this line, the low survival rate of flies with mutation in the ND2 subunit of complex I can also be rescued by chemical TOR inhibition in Drosophila. Moreover, an aggressive phenotype of breast cancer that is associated with complex I mutations can be reversed via restoration of complex I function that is associated with decreased mTOR activity. Therefore, the data showing the role of TOR at the very apex of the signalling hierarchy after complex I dysfunction makes it interesting to test if similar regulatory mechanism underpins other types of mitochondrial dysfunction (Perez-Gomez, 2020).

Respiratory inhibitors are used to surpress various types of cancers although respiratory dysfunction can also promote cancer progression. Based on the current data it can be hypothesized that the types of complex I dysfunctions that stimulate cancer progression would correlate with overstimulation of mTOR activity that initiates downstream signalling events promoting apoptosis but also apoptosis-induced proliferation. This may seem contradictory as complex I inhibition usually leads to decrease in ATP:ADP ratio that in turn activates AMPK, a known suppressor of mTOR activity. However, the evidence that complex I inhibition would actually lead to downregulation of mTOR activity is surprisingly scarce and concerns mainly complex I inhibitors like biguanides (metformin, fenformin) or fenofibrate. On the contrary, there is ample of observations where mTOR is increased during mitochondrial dysfunction, supporting the results. In fact, mTOR activity can be stimulated even in the presence of active AMPK, as in the case of the Leigh syndrome caused by mutation in complex I subunit Ndufs4. By balancing between stimulation of apoptosis and proliferation, the mTOR driven signalling network identified in this study may suggest a possible mechanism for the contradictory observations where complex I inhibition was reported to promote cell death but also support proliferation depending on context (Perez-Gomez, 2020).

One remaining question is how TOR is upregulated by mitochondria. One possibility may be an activation of TOR via mitochondrial Akt signalling and TOR complexes located in mitochondria-associated endoplasmic reticulum. However, since TOR operates at the top of the signalling hierarchy after complex I downregulation, it can also be speculated that its activity could be sensitive to the primary metabolic misbalance caused by disruption of mitochondrial metabolism. Indeed, the decrease in the NAD:NADH ratio and the associated slowdown of the TCA cycle that are associated with downregulation of respiration are likely to influence the activity of protein metabolic sensors such as sirtuin deacetylases or 2-KG dependent demethylases that may in turn regulate mTOR activity, either directly or indirectly, as suggested in some other contexts (Perez-Gomez, 2020).

Through non-apoptotic roles of caspases, dying cells can release diffusible mitogens and thus signal to their neighbours and instruct them to proliferate -- a process known as apoptosis-induced proliferation (AIP). Several modes of AIP have been described in various species and tissues characterised by the use of either initiator or effector caspase to drive the signalling mechanism that in turn promotes proliferation. In Drosophila wing or eye discs, the most studied AIP models are based on targeted expression of the pro-apoptotic genes hid or rpr where the proliferation is dependent on the initiator caspases that activate ROS production and JNK activity. In this model, proliferation is also dependent on caspases, ROS production and JNK activation, however it is unique in the way it is triggered and in the way the signalling components are interconnected: (1) it is initiated by downregulation of mitochondrial respiratory complex I and therefore it has a metabolic origin (2) it is orchestrated by consequent activation of the TOR pathway (3) it is dependent on effector caspases and (4) JNK activation is upstream of cell death, not activated by the non-apoptotic roles of caspases. Examples of AIP dependent on effector caspases have been described in Drosophila postmitotic cells in the eye disc and in mammalian cells after irradiation induced cell damage but the signalling components involved and their regulatory relationship also differ from the model used in this study (Perez-Gomez, 2020).

It is important to stress that the high levels of ROS species are not produced in every cell where ND-49-RNAi is induced. Although low levels of ROS may appear as a primary consequence of ND-49 downregulation, the strong ROS signal observed in certain areas of the wing disc occurs downstream of cell death, as blocking apoptosis alongside ND-49-RNAi also eliminates the ROS signal. This is in agreement with ROS generation in other modes of AIP. Although ROS production was described with certain complex I inhibitors it does not happen when other inhibitors are used. Assembly of complex I into supercomplexes with other ETC proteins determines if ROS will be produced or not. In the current model it is obvious that the majority of ROS observed does not originate from the dysfunctional complex I but they result from apoptosis, as blocking apoptosis prevents also ROS formation (Perez-Gomez, 2020).

Taken together, the results highlight the central role of TOR pathway activation during mitochondrial dysfunction. As TOR overactivation gives identical phenotype to complex I downregulation, future studies should investigate if the results may be relevant outside of the mitochondria field, in some of the other contexts involving TOR overactivation, such as many types of cancer, wound healing or aging, with potentially important clinical implications (Perez-Gomez, 2020).

ATF4-induced Warburg metabolism drives over-proliferation in Drosophila

The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. This study shows that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. This stress adaptation is found to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. These results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity (Sorge, 2020).

Controlling cell proliferation is one of the major challenges of multicellular life, both during phases of growth in developing organisms and phases of homeostatic cell replenishment essential in adult animals. Lack of appropriate control can lead to severe disorders, including cancer, at any stage of life. While over-proliferation of transformed, cancerous cells is usually caused by inactivation of tumor suppressors and/or activation of oncogenes, it has long been noted that tumors exhibit altered cellular characteristics such as a glycolytic metabolism. While this metabolic switch has been shown to be caused by oncogene signaling, cellular metabolism is also controlled at multiple levels under normal physiological (non-transformed) conditions, including at the transcriptional level through diverse stress-response pathways. One of these is the activating transcription factor 4 (ATF4), which is known to activate a transcriptional (integrated) stress response (ISR) under various stress conditions that trigger phosphorylation of eIF2α. The ATF4 transcriptional program consists of a diverse set of genes with cytoprotective function, but chronic activation induces apoptosis indirectly through transcription of the mammalian ATF4 target CHOP. Yet, ATF4 activation has been detected in several human tumors, especially in hypoxic or nutrient-deprived regions, where ATF4 has been attributed with pro-survival and pro-proliferative effects. Interestingly, a recent study showed that melanoma cells respond to inhibition of their glycolytic metabolism by activating an ATF4 response, whose metabolic reconfiguration allows these cells to continue oncogenic growth, together arguing that ATF4 can provide cancer cells with a metabolic flexibility that allows them to tolerate hypoxic and nutritional stress or cancer therapy aimed at metabolism. Among the many conditions activating ATF4, recent work with cultured cells showed that inhibition of mitochondrial function is linked to ATF4 translation and activity. However, from these and other studies, both the mechanistic basis and the in vivo implications of this response remain to be elucidated (Sorge, 2020).

This study shows that in the fruit fly, Drosophila melanogaster, genetic perturbation of the electron transport chain (ETC), which induces a Warburg-like metabolism, activates a transcriptional stress response mediated through the eIF2α-kinase PERK and ATF4 in eye progenitor cells of Drosophila larvae. Importantly, this in vivo stress response is activated under ETC knockdown conditions, in the absence of obvious mitochondrial dysfunction. Interestingly, these results show that the ATF4 transcriptional response, which by itself causes reduced fitness of progenitor cells, is hijacked by growth-promoting pathways like Notch or Ras, leading to increased cellular fitness and enhanced proliferation. The data furthermore suggest that the pH changes associated with ETC impairment resulting in a switch of the metabolism to aerobic glycolysis play an important role in progenitor over-proliferation. In sum, this study shows that ATF4-mediated transcriptional adaptation provides a cell-autonomous response to ETC defects, altering cellular behavior through metabolic adaptation (Sorge, 2020).

Genetically induced disturbance of ETC complex assembly resultrd in a metabolic shift typical for mitochondrial impairment and activated an ATF4-dependent stress response. The in vivo transcriptional adaptation presented in this study confirmed the regulation of LDH and glycolytic enzymes, as shown in Drosophila cultured cells, and further includes several targets shown to be ATF4 target genes in mammalian models. The results showed that the eIF2α-kinase PERK, so far only described for its role in mediating one branch of the unfolded protein response of the endoplasmic reticulum (UPRER), is the upstream kinase phosphorylating eIF2α, thereby inducing ATF4 translation in response to mitochondrial ETC disturbance. Mitochondrial ETC disturbance specifically activated PERK, while other branches of the UPRER were non-responsive. PERK activation upon mitochondrial defects was recently observed in Drosophila models of Parkinson's disease and was explained by the authors by its preferential localization to mitochondria-associated ER membranes, which might make PERK more susceptible to a local stress signal. ROS (reactive oxygen species) released by mitochondria have been suggested to mediate mitochondrial retrograde signaling. While this study observed an attenuation of Delta overexpression (Dl^OE), COX7RNAi-induced over-proliferation upon overexpression of either cytoplasmic catalase or GPx (but not mitochondrial catalase), this study failed to detect increased ROS levels in the larval eye disc. A possible scenario to explain these observations is that ROS are generated locally in the cytoplasm or ER in response to ETC disturbance, thereby triggering PERK activation. Importantly, Drosophila PERK isoform B contains a potential mitochondrial signal peptide, which is not found in mammalian PERK isoforms. Although no evidence for this has been found, Drosophila PERK could reside in the mitochondrial membrane and sense the folding status of mitochondrial complexes. This hypothesis could explain the evolutionary difference between mitochondrial defects and ATF4 induction, as this appears to require GCN2 but not PERK in mammals or to be triggered independently of a single eIF2α-kinase. In addition to canonical ATF4 target genes, ATF4-dependent upregulation of mitochondrial chaperones, a response classically referred to as the mitochondrial UPR (UPRmt) was observed. In C. elegans, mitochondrial chaperone induction upon stress is mediated by ATF4-like transcription factor Atfs-1, while the mammalian UPRmt has been shown to be regulated by another evolutionary-related transcription factor, ATF5. The current data now showed that Drosophila ATF4 is required cell autonomously for the induction of mitochondrial chaperones upon ETC subunit knockdown, implying that Drosophila might represent the evolutionary ancestral ISR-UPRmt regulation through a single ATF4-like transcription factor (Sorge, 2020).

The cooperation between ATF4 target genes and the Notch or Ras pathways in Drosophila imaginal progenitors raised the intriguing possibility that these or other oncogenic pathways could benefit from ATF4 activity in human cancers. Over the last decades, it had been demonstrated that human cancer cells are exposed to several stresses, including hypoxia, ROS, or limitations in nutrient availability. In order to survive these conditions and maintain their growth capacity, tumor cells activate responses like the HIF1α transcription factor axis. Though less well studied, an involvement of ATF4 in cancer has been suggested mostly through work with cultured cells. This study analyzed gene expression in human cancer samples of The Cancer Genome Atlas (TCGA) datasets using Cancer-RNaseq-Nexus and the human protein pathology atlas and found that many of the well-characterized direct ATF4 targets are upregulated in a variety of cancer types. Most strikingly, transcriptomes of kidney renal clear cell carcinoma showed progressive induction of ATF4 and many of its direct targets (EIF4EBP1, ASNS, TRIB3, and VEGFA) on the transcriptional level, which strongly correlated with a poor prognosis in this type of cancer. These data suggest that the ATF4-mediated ISR is used by cancer cells to adapt their metabolic repertoire, thereby sustaining fast growth under increasingly unfavorable conditions (Sorge, 2020).

A novel finding presented in this study was the discovery that ATF4-mediated transcriptional adaptation due to ETC impairment allowed eye progenitors to increase their proliferation in response to signals from the Notch and Ras pathways. The primary questions arising from this genetic interaction is how these signaling pathways can overcome the apparent cellular stress and reduction in proliferation and induce the opposite effect, a massively increased rate of proliferation. Several lines of evidence suggest that over-proliferation in Dl^OE, COX7RNAi eye imaginal discs is controlled by pH changes induced by LDH that modify the activity of Notch downstream effectors. First, in COX7a-depleted cells, the metabolism is switched to aerobic glycolysis, leading to an increased production of lactate due to the activity of LDH. And, consistent with an accumulation of this metabolic acid, this study found the intracellular pH to be reduced in COX7RNAi cells. Second, Dl^OE, COX7RNAi-mediated over-proliferation was rescued by ATF4 knockdown and, to a lesser extent, pH buffering, showing that intracellular pH changes (downstream of ATF4 and LDH) play an important role in proliferation control. Third, LDH phenocopies COX7RNAi, indicating that most of the cooperative effects of Dl overexpression and COX7a knockdown are mediated by the ATF4 target LDH. In the same line, expression of LDH as one of the many ATF4 targets was sufficient to drive Dl-expressing cells into over-proliferation, strongly suggesting that the processes downstream of LDH-in particular, the changes in intracellular pH-lead to a modification of the Notch pathway. Finally, a cooperation of Notch and the COX7a nuclear effector ATF4 on the transcriptional level was not observed, showing that the Notch pathway is not hyper-activated, but arguing that over-proliferation is due to changes in the activity of Notch downstream effectors. The next obvious question is how changes in intracellular pH can modify the activity of signaling pathways. It is known that the intracellular pH can control the protonation of specific histidine residues in proteins acting as pH sensors, leading to changes in protein properties. Importantly, it has been shown recently in chicken embryos that intracellular pH changes induced by a Warburg-like metabolism control the acetylation of the Wnt effector &betsa;-catenin, thereby mediating Wnt signaling activation. Thus, this study envisions that pH changes induced by LDH expression lead to a (non-enzymatic) modification of Notch effectors, thereby increasing fitness and proliferation rates of eye progenitor cells (Sorge, 2020).

This is a very attractive model; however, one result was puzzling. Although LDH is sufficient to induce over-proliferation when combined with the Notch pathway, no rescue (but an increase in the severity) of the Dl^OE, COX7RNAi phenotype was observed when LDH was selectively depleted. This obvious discrepancy can be explained by different hypotheses. One of them is based on a recent study showing that LDHA inhibition in melanoma cell lines also failed to impact cell proliferation, survival, or tumor growth. In this context, LDHA inhibition engaged the GCN2-ATF4 signaling axis to initiate an expansive pro-survival response, including the upregulation of the glutamine transporter SLC1A5 and glutamine uptake, as well as mTORC1 activation. Another hypothesis is based on the finding that a major driver of over-proliferation is the intracellular pH. Since LDH catalyzes the conversion of pyruvate to lactate (and back), reducing LDH levels will affect the ratio of lactate to pyruvate, leading to an increase of the even stronger metabolic acid pyruvate. It has been shown that pyruvate as lactate induces a concentration-dependent intracellular acidification. Thus, it could be envisioned that an enhancement of proliferation rates beyond those observed in Dl^OE, COX7RNAi cells is a consequence of pyruvate accumulation in the absence of LDH, which enhances the decrease in the intracellular pH, resulting in the increase in proliferation rates (Sorge, 2020).

NAD kinase sustains lipogenesis and mitochondrial metabolism through fatty acid synthesis

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Downregulation of respiratory complex I mediates major signalling changes triggered by TOR activation

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