Akhmetova, K., Balasov, M. and Chesnokov, I. (2021). Drosophila STING protein has a role in lipid metabolism. Elife 10. PubMed ID: 34467853

Miao, T., Kim, J., Kang, P., Fujiwara, H., Hsu, F. F. and Bai, H. (2022). Acetyl-CoA-mediated autoacetylation of fatty acid synthase as a metabolic switch of de novo lipogenesis in Drosophila. Proc Natl Acad Sci U S A 119(49): e2212220119. PubMed ID: 36459649

• Hormones receptors and other membrane proteins
  • Adipokinetic hormone-like
  • Adipokinetic hormone receptor
  • bride of sevenless
  • CCHamide-2
  • convoluted
  • Diuretic hormone 44

• Intracellular Signaling Proteins and RNAs
  • Akt1 (also known as PKB)
  • chico
  • gigas (TSC2)
  • Glial Lazarillo
  • Lipid storage droplet-1
  • Lipid storage droplet-2
  • Lipophorin
  • Lnk
  • melted
  • mir-8 stem loop
  • mir-14
  • Neural Lazarillo
  • Pdk1
  • Pten
  • Phosphotidylinositol 3 kinase 59F
  • Phosphotidylinositol 3 kinase 92E (Pi3K catalytic subunit also known as Dp110 or Pi3K92E)
  • Pi3K21B (Pi3K regulatory subunit)
  • Ribosomal protein S6
  • RPS6-p70-protein kinase
  • scylla and charybde
  • small wing
  • spastin
  • steppke
  • Target of rapamycin
  • TSC1
  • Thor (4E-BP)
  • Tor
  • twins
  • unkempt
  • wilderborst

• Enzymes
  • brummer

• Transcription Factors
  • adipose
  • bigmax
  • CREB-regulated transcription coactivator
  • foxo
  • Helix loop helix protein 106
  • Hepatocyte nuclear factor 4
  • Hormone receptor-like in 96
  • Kruppel
  • mondo
  • mushroom body miniature
  • Myc (Diminutive or dMyc)
  • Spargel
  • Helix loop helix protein 106 (SREBP or Sterol regulatory element-binding protein)

Insulin-pathway and nutritional status

Studies in Drosophila have characterized Insulin receptor/Phosphoinositide 3-kinase (Inr/PI3K) signaling as a potent regulator of cell growth, but an understanding of its function during development has remained uncertain. Inhibiting Inr/PI3K signaling phenocopies the cellular and organismal effects of starvation, whereas activating this pathway bypasses the nutritional requirement for cell growth, causing starvation sensitivity at the organismal level. Consistent with these findings, studies using a pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion as an indicator for PI3K activity show that PI3K is regulated by the availability of dietary protein in vivo. It is surmised that an essential function of insulin/PI3K signaling in Drosophila is to coordinate cellular metabolism with nutritional conditions (Britton, 2002).

To test whether PI3K is required for larval growth, its activity was inhibited by expressing p60, Deltap60, or Pten in large domains of the larva using the Gal4/UAS system. p60 is an adaptor that couples Inr to Pi3K92E (Dp110), and Deltap60 is a deletion variant lacking part of the Dp110 binding domain. These molecules and their mammalian homologs have dominant-negative effects on PI3K activity when overexpressed, presumably because they compete with endogenous Dp110/p60 complexes for binding sites on upstream activators such as insulin receptors and IRSs. When p60 is expressed under the control of Act-Gal4 (expressed ubiquitously) or Adh-Gal4 (expressed predominantly in fat body), larvae remain growth arrested in the first instar for as long as 2 weeks. Deltap60 and Pten had similar effects. Dissection of these animals revealed that all of their tissues and organs were proportionally reduced. This developmental arrest is indistinguishable from the effects of starvation or inhibition of protein synthesis (Britton, 2002).

To test whether PI3K is autonomously required for growth in the different larval cell types, the Flp/Gal4 technique was used to express p60, Deltap60, or Pten in scattered cells throughout the larva. This method employs the Act>CD2>Gal4 and hs-Flp transgenes to activate UAS-linked target genes, including the cell marker UAS-GFPnls, in cell clones. Heat shock-independent activation of Gal4 occurs prior to the onset of larval growth and DNA endoreplication in 1%-10% of cells (depending on organ) in the fat body, gut, salivary glands, renal (Malpigian) tubules, and epidermis. Cells overexpressing p60, Deltap60, or Pten in the salivary glands and fat body are greatly reduced in size and have much smaller nuclei with far less DNA than adjacent control cells. Similar effects are observed in other larval tissues. Despite their reduced growth, p60-, Deltap60-, and Pten-expressing cells are found at approximately the same frequencies as control GFP-marked cells. Apoptotic cells are not observed. Thus, reductions in PI3K activity are not incompatible with cell viability. Overt effects on cell morphology that might reflect changes in cell adhesion, motility, or identity are also not observed. It is concluded that reducing Inr/PI3K activity in the differentiated tissues of the larva has cell-autonomous effects that are limited to reducing cell growth and DNA replication (Britton, 2002).

These observations suggest that PI3K activity might be responsive to nutritional conditions. To directly test this possibility, a fusion protein was made for use as an in vivo reporter for PI3K activity. The pleckstrin homology (PH) domain of the Drosophila homolog of general receptor for phosphoinositides-1 (GRP1) was fused to green fluorescent protein (GFP), generating a protein called GPH (GFP-PH domain). PH domains from mammalian GRP1 genes bind specifically to phosphatidylinositol-3,4,5-P₃ (PIP₃), the second messenger generated by class I PI3-kinases. Since PIP₃ generally resides in lipid membranes, particularly the plasma membrane, GRP1 is recruited to membranes when PI3-kinase activity raises cellular levels of PIP₃. Fusion proteins containing the GRP1 PH domain are likewise recruited to plasma membranes by binding PIP₃, and thus serve as in situ reporters for PI3K activity (Britton, 2002).

For in vivo studies, the GPH gene was placed under control of the Drosophila ß-tubulin promotor, generating a gene called 'tGPH' (tubulin-GPH), and introduced into Drosophila by P element-mediated transformation. Membrane localization of tGPH was observed in the larval epidermis, fat body, salivary glands, malpighian tubules, and wing imaginal discs. Cytoplasmic and nuclear tGPH was also visible in these cell types. The degree of membrane localization depends upon the developmental stage. Epidermal cells show little membrane localization of tGPH in embryos or newly hatched first instar (L1) larvae, but have strong membrane localization in second (L2) and early third (L3) instar larvae. Later, in wandering stage L3 larvae, membrane-associated tGPH is again diminished. Similar trends are observed in the fat body. These variations might reflect changes in the levels of Inr, PI3K, Pten, or insulin-like peptides (dILPs) in the larva as it feeds and grows (Britton, 2002).

To test whether tGPH localization was responsive to PI3K activity in vivo, Inr or Dp110 was overexpressed using the Gal4 system. Either gene causes a striking redistribution of tGPH to plasma membranes in cells of the fat body, epidermis, Malpighian tubules, gut, and imaginal discs. To determine whether endogenous PI3K is responsible for the membrane localization of tGPH, PI3K activity was suppressed by expressing p60 with the heat-inducible driver hs-Gal4. In the larval epidermis, partial loss of membrane-bound tGPH is apparent 1.5 hr post-heat shock (phs), and by 4 hr, phs tGPH is nearly completely lost from cell membranes. Similar results were obtained in the fat body when either p60 or dPten were expressed mosaically using the Flp/Gal4 method (Britton, 2002).

To determine whether PI3K activity is nutritionally modulated, tGPH localization was monitored after starvation. Early L2 larvae (48-60 hr AED) were deprived of dietary protein by culture on either 20% sucrose or Sang's defined media lacking casein. These treatments arrest cell growth in all of the differentiated larval tissues. L2 larvae fed on either protein-free diet survive for up to 14 days, but a marked shrinkage of cells in the epidermis and fat body is observed. In the epidermis of early L2 larvae, culture on either protein-free diet causes membrane-bound tGPH to be diminished after 24 hr, and to be nearly undetectable after 48 hr. Levels of total tGPH also decrease after protein deprivation, but nuclear and cytoplasmic tGPH remain detectable for more than 6 days. Loss of membrane-associated tGPH also occurs in fat body cells of protein-deprived L2 larvae, with similar kinetics. Starvation causes shrinkage of the nuclei and nonlipid cytoplasm in fat body cells, leaving large tGPH-negative lipid droplets that occupy most of the cell volume. To test whether membrane association of tGPH is reversible, L2 larvae were cultured on 20% sucrose for 8 days and then returned to whole food. In this experiment, tGPH levels rose and the protein reassociated with plasma membranes in epidermal cells between 24 and 48 hr after feeding. These results indicate that cellular levels of PIP₃ drop as a consequence of starvation for dietary protein (Britton, 2002).

Terminally differentiated endoreplicating tissues (ERTs) constitute most of a Drosophila larva, and the growth of these tissues accounts for virtually all of the ~200-fold mass increase sustained by the animal during the larval stages. During larval life, the ERTs provide a physiologically nurturing environment for undifferentiated imaginal cells and neuroblasts, which generate much of the reproductive adult stage. Most of the biomass accumulated in the ERTs is eventually recycled into these progenitor cells as they form the adult. This study provides evidence that Inr/PI3K signaling coordinates nutritional status with ERT cell metabolism and growth. To determine whether Inr/PI3K signaling can maintain cell growth in the face of starvation, Flp/Gal4 was used to express Dp110 or Inr in scattered ERT cells, and then changes in cell size and DNA replication were assessed at time points during a protein starvation regime. At larval hatching, prior to starvation, cells expressing Dp110 or Inr in the gut, fat body, Malpighian tubules, and epidermis are only slightly larger than nonexpressing cells. After several days of starvation on 20% sucrose, Inr- or Dp110-expressing cells in these organs are much larger than adjacent control cells, and have visibly increased DNA content. BrdU incorporation indicated that gut and fat body cells expressing Dp110 or Inr continue to replicate their DNA for at least 2 to 3 days under starvation conditions. Normally, DNA endoreplication in these cells ceases within 1 to 2 days of starvation (Britton, 1998). A catalytically inactive PI3K, Dp110^D945A, does not promote cell growth or DNA endoreplication, indicating that lipid kinase activity was required. These experiments indicate that active Inr/PI3K signaling is sufficient to bypass the nutritional requirement for cellular growth and DNA replication in many larval cell types, and that this effect is cell autonomous (Britton, 2002).

To explore the means by which Inr/PI3K signaling induces cell growth, an examination was carried out of the morphology of fat body cells in which PI3K activity had been manipulated. Fat body cells accumulate large stores of protein, carbohydrate, and lipids during larval life, and also produce growth factors. During the third larval instar, these accumulations of nutrients cause fat body cells to become opaque. These nutrients are normally utilized during metamorphosis, but if a larva is starved, they are precociously mobilized into the haemolymph to support the animal during the ensuing dietary crisis. This causes the fat body cells to shrink and become clear as they lose organelles by autophagy and deplete stored metabolites (Britton, 2002).

Expression of Inr or PI3K in fat body cells increases the opacity of the cytoplasm, and thus promotes nutrient storage. A similar cytoplasmic effect is observed in intestinal cells from L1 animals. Close inspection has revealed that in both cell types, ectopic Inr or PI3K decreases the size of prominent vesicles in the cytoplasm. Induction of p60 in early L3 larvae has opposite effects, causing fat body cells to become more translucent. A loss of opacity was observed in fat bodies from Dp110 mutants after their growth arrest at L3. In further tests, fat body cells were stained for lipids with Nile red or for protein with Texas red X succinimidyl ester. This revealed that the cytoplasm of Inr-expressing cells contains many more, but much smaller, lipid droplets than neighboring control cells. In summary, activation of the Inr/PI3K pathway has profound effects on cytoplasmic composition. These effects mimic changes in the fat body that normally take place late in the L3 stage when nutrient storage by these cells is maximal. Suppression of Inr/PI3K activity has opposite effects on cytoplasmic composition, and these appear to mimic the mobilization of nutrients that normally accompanies starvation (Britton, 2002).

Considering the above results, it should be advantageous to larvae to downregulate insulin/PI3K signaling when nutrients are limited, since this would suppress nutrient storage and cell growth and allow nutrient mobilization by tissues such as the fat body. This idea was tested by hyperactivating Inr/PI3K signaling and then tracking development under different nutritional conditions. Several Gal4 drivers were used to induce expression in large numbers of cells, including Adh-Gal4 (expressed in the fat body, trachea, and a few cells in the gut), en-Gal4 (expressed in posterior epidermal cells, the hindgut, and some neural cells), Act-Gal4 (expressed ubiquitously), and hs-Flp/Act>Cd2>Gal4 (induced by heat shock in all cells). In several cases, overexpressed Dp110 and Inr were tolerated in feeding animals. For instance, animals expressing Dp110 under Adh-Gal4 or en-Gal4 control developed without delay and eclosed at the same frequency as controls, giving viable fertile adults. Inr was more deleterious, but some animals expressing Inr under Adh-Gal4 control developed to the L3 stage and a few viable adults eclosed. Ubiquitous expression of Dp110 or Inr using the Act-Gal4 driver, however, was 100% lethal at prelarval stages (Britton, 2002).

In contrast, hyperactivating Inr/PI3K signaling under starvation conditions was catastrophic. When Adh-Gal4 was used to drive Dp110 or Inr expression, for instance, L1 larvae raised on the sucrose/PBS diet all perished within 3 to 4 days of hatching, whereas control animals survived 8 to 9 days. Animals expressing Dp110 under en-Gal4 control also perished within 2 to 3 days, 4 to 5 days before controls, when deprived of dietary protein. Suppressing PI3K activity by expressing p60, Deltap60, Pten, or Dp110^D945A using Adh-Gal4, en-Gal4, or even Act-Gal4 had no effect on viability under starvation conditions. These results suggest that the starvation sensitivity caused by high Inr/PI3K activity is specifically related to nutrient uptake and storage, functions that appear to be unique to Inr and PI3K (Britton, 2002).

These results demonstrate that downregulation of Inr/PI3K activity is critical to maintaining metabolic homeostasis under starvation conditions. The remarkable ability of Inr/PI3K-expressing cells to continue stockpiling nutrients and grow, even in starved animals, may account for the starvation sensitivity observed at the organismal level. This idea was supported by observations made in starved tGPH larvae that overexpressed Dp110 in posterior compartment epidermal cells (genotype, en-Gal4 UAS-Dp110 tGPH). In these animals, high levels of membrane-bound tGPH persisted in Dp110-expressing epidermal cells until 2 days after nutrient withdrawal, at which point the animals died. In anterior (A) cells, which did not overexpress Dp110, tGPH was completely lost from plasma membranes within 18 hr after nutrient deprivation, and tGPH protein became undetectable by 36 hr. This is a much more rapid starvation response than observed in animals that did not contain Dp110-expressing cells. Anterior epidermal cells also shrank rapidly during starvation, whereas posterior, Dp110-expressing cells maintained their large size. These effects might result from the rapid depletion of nutrients by the PI3K-expressing cells, and a consequent drop in levels of hemolymph insulins (Britton, 2002).

In performing these experiments, it was noticed that larvae that overexpress Inr or Dp110 wander away from their food. To more carefully analyze this phenotype, animals expressing various PI3K signaling components under Adh-Gal4 control were cultured on agar plates with red-colored food (yeast paste) in the center for ~24 hr after hatching. These animals were then scored for the presence of red food in the gut as well as their proximity to the food source. Animals overexpressing Inr, Dp110, or Dp110^CAAX feed poorly (i.e., often have no food in the gut) and frequently wander away from their food. Similar aberrant behaviors were observed when Dp110 was expressed ubiquitously using Flp/Gal4, in which case nearly all animals wandered out of the food and pupated precociously. Thus, elevated levels of Inr/PI3K signaling alter larval feeding behavior, perhaps by affecting the animal's perceived level of hunger (Britton, 2002).

Is nutrition sensed in Drosophila at the cell level or by the animal as a whole? Although animal cells can sense amino acids directly, these studies suggest that cells in Drosophila larvae sense and respond to changes in dietary protein indirectly, using secondary humoral signals -- most likely insulins -- long before they become acutely starved for amino acids. In support of this idea, some cells in the larva can continue to grow and replicate their DNA long after the animal is deprived of dietary protein. Imaginal cells and neuroblasts do this, as do ERT cells in which Inr or PI3K have been artificially switched on. This attests to the fact that starvation for dietary protein does not completely deplete the larval hemolymph of amino acids or cause a global shutdown of protein synthesis. This is probably possible because nutrients stored in ERTs such as the fat body are mobilized during starvation to maintain levels of hemolymph nutrients. Nevertheless, starvation does cause a rapid, global shutdown of ERT cell growth, and thus some essential signal is lost (Britton, 2002).

One factor that all animal cells use for nutritional sensing is TOR (target of rapamycin), a protein kinase that mediates diverse effects on cell metabolism including protein synthesis, amino acid import, ribosome biogenesis, and autophagy. What role might Drosophila TOR (dTOR) play in the nutrition response system addressed here? The mechanism by which TOR 'senses' nutrition remains uncertain, but cellular levels of amino acids, aminoacylated tRNAs, and ATP have been suggested as direct inputs. Drosophila dTOR mutations or the TOR-specific inhibitor rapamycin inactivate the TOR target S6-kinase and phenocopy starvation in fed larvae. Starvation, however, does not completely inactivate S6K, suggesting that dTOR retains some activity under starvation conditions). Consistent with this interpretation, overexpressed PI3K is a potent promotor of cell growth in starved larvae, but PI3K cannot drive cell growth in dTOR mutant larvae (J. Lande and T. Neufeld, personal communication to Britton, 2002). This suggests that although dTOR may act as a cell-autonomous nutrient-dependent checkpoint for metabolism, the larva's physiology is so effective in buffering cells against absolute starvation that this checkpoint is rarely if ever fully engaged. TOR is found in fungi and plants and so seems to be a metabolic regulator that was used prior to the advent of multicellularity. Insulin signaling, which is absent from fungi and plants, probably evolved later when multicellular animals required a system to coordinate and fine-tune metabolism in communities of cells. The insulin system is clearly advantageous, since animals in which it is 'short-circuited' by hyperactivation of Inr or PI3K are unable to tolerate even brief periods of starvation (Britton, 2002).

The most direct evidence that insulin signaling is nutritionally controlled was obtained using a cellular indicator of PI3K activity, tGPH, which is recruited to plasma membranes by the second messenger product of PI3K, PIP₃. Subcellular tGPH distributions indicate that PIP₃ levels are high in many cell types in fed larvae, but low in larvae that have been starved for protein. Although these changes in PIP₃ levels might be due to altered expression of Inr, PI3K, or Pten, expression profiling experiments using cDNA microarrays indicate that levels of p60 and Dp110 mRNA are not depressed in L2 larvae that had been deprived of protein for 4 days. Perhaps the most attractive explanation for the apparent loss of PIP₃ upon starvation is that some of the seven Drosophila insulin-like peptides (dILPs) are produced in a nutrition-dependent fashion. Several of the dilp genes are expressed in the larval gut which, as the conduit of nutritional influx, might be expected to mediate metabolic responses to feeding throughout the animal. Other dilps are expressed in the salivary glands, imaginal discs, and small numbers of cells in the central nervous system (Britton, 2002).

In mammals, insulin promotes the cellular uptake and storage of carbohydrates, proteins, and lipids, and is the strongest anabolic inducer known. Insulin-mediated responses are indirectly antagonized by the hormone glucagon, which stimulates catabolic reactions and the mobilization of stored nutrients. Insulin and glucagon are produced in the pancreas, and their relative levels are constantly adjusted to maintain proper blood sugar levels. The mammalian liver is also a key player in the regulation of metabolic homeostasis. In humans, most of the accessible glycogen, the principle form of stored carbohydrate, is found in the liver. This glycogen can be mobilized in response to exercise or starvation. In Drosophila larvae, hyperactivation of the Inr/PI3K pathway leads to increased accumulation of nutrients in the fat body, an organ that resembles the mammalian liver as the principal site of stored glycogen. Conversely, inhibition of PI3K activity depletes stored nutrients from the fat body, as does starvation. This suggests that like mammals, insects regulate storage of metabolites in response to changes in levels of Inr/PI3K signaling. Direct assays of the levels of carbohydrates, storage proteins, and lipids in the fat body after starvation or manipulation of Inr/PI3K activity should prove informative. While there are as yet no known Drosophila homologs of glucagons, there must be some mechanism by which stored resources can be mobilized during starvation or at the transition from feeding to metamorphosis (Britton, 2002).

Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells

Antagonistic activities of glucagon and insulin control metabolism in mammals, and disruption of this balance underlies diabetes pathogenesis. Insulin-producing cells (IPCs) in the brain of insects such as Drosophila also regulate serum glucose, but it remains unclear whether insulin is the sole hormonal regulator of glucose homeostasis and whether mechanisms of glucose-sensing and response in IPCs resemble those in pancreatic islets. This study shows, by targeted cell ablation, that Drosophila corpora cardiaca (CC) cells of the ring gland are also essential for larval glucose homeostasis. Unlike IPCs, CC cells express Drosophila cognates of sulphonylurea receptor (Sur) and potassium channel (Ir), proteins that comprise ATP-sensitive potassium channels regulating hormone secretion by islets and other mammalian glucose-sensing cells. They also produce adipokinetic hormone, a polypeptide with glucagon-like functions. Glucose regulation by CC cells is impaired by exposure to sulphonylureas, drugs that target the Sur subunit. Furthermore, ubiquitous expression of an akh transgene reverses the effect of CC ablation on serum glucose. Thus, Drosophila CC cells are crucial regulators of glucose homeostasis and they use glucose-sensing and response mechanisms similar to islet cells (Kim, 2004).

Insect corpora cardiaca (CC) are clusters of endocrine cells in the ring gland adjacent to the prothoracic gland and corpus allatum. A principal CC product is adipokinetic hormone (AKH), a polypeptide that mobilizes stored macromolecular energy reserves to sustain energy-consuming activities, such as crawling and flight. AKH is similar to mammalian glucagon; like glucagon in pancreatic islet α-cells, AKH is synthesized as a pre-prohormone, processed, and stored in dense core vesicles. Like mammalian glucagon activity in liver, AKH has been shown to bind a G-protein-coupled transmembrane receptor and to increase lipolysis, glycogenolysis and production of trehalose in the insect fat body, a storage organ for lipid and glycogen (Kim, 2004).

Previous studies of AKH microinjection and ring gland transplantation in locusts and other insects suggest that AKH is sufficient to increase haemolymph glucose concentrations, but have not yet shown a requirement for AKH in glucose homeostasis. To examine phenotypes resulting from CC cell ablation and AKH deficiency, 1,000-base-pair DNA segment derived from sequences immediately 5' of the Drosophila akh gene was used to drive the expression of the transcriptional trans-activator GAL4 in CC cells. The akh-GAL4 construct, when crossed with a UAS-mCD8GFP (membrane-tethered green fluorescent protein, mGFP) reporter line, directed a GFP expression pattern that reflected endogenous akh expression in the ring gland corpora cardiaca of third-instar larvae. Using in situ hybridizations, it was establised that embryonic akh messenger RNA expression initiates in cells of the presumptive CC anlage and that in later larval stages it is maintained only in CC cells. To assess the role of the CC as an endocrine regulator of haemolymph glucose concentrations, akh-GAL4 lines were used to express the cell death factor Reaper in akh-expressing CC cells. This resulted in the ablation of only CC cells at high efficiency: in more than 96% of newly hatched first-instar larvae harbouring akh-GAL4, UAS-Reaper and UAS-mCD8GFP, no mGFP-labelled CC cells were detected. In contrast, mGFP was detected in CC cells within all control larvae at the same stage, and at later stages. In Drosophila , haemolymph glucose is composed of trehalose (a disaccharide of glucose) and monomeric free glucose, and the combined circulating concentration of these (hereafter referred to as total haemolymph glucose) is maintained in a narrow range for a given feeding condition. Ablation of akh-expressing CC cells in larvae raised on dextrose-supplemented medium decreased the mean total haemolymph glucose and trehalose by 50%, an effect similar to that recently reported by others. CC cell deficiency did not result in discernible growth reduction, developmental delay or lethality, phenotypes that arise after the ablation of IPCs in the brain. Thus, like glucagon, AKH is an essential regulator of energy metabolism but might be dispensable for developmental growth control (Kim, 2004).

To test whether AKH activity alone could account for the glucose-regulating action of CC cells, the ability was tested of an akh transgene with ubiquitous expression from a heat shock promoter to reverse the effect of CC ablation. Lower haemolymph glucose concentrations resulting from CC ablation were partly restored by the ubiquitous expression of an akh transgene. Thus, bioactive AKH from the akh transgene might be produced in target tissues, as has been shown for transgene-encoded neuropeptides such as Drosophila insulin. These data indicate that AKH is an essential regulator of haemolymph carbohydrate concentrations in Drosophila . It is suggested that the hyperglycaemic effects of AKH counter-regulate the activity of other systemic hormones such as insulin and that these antagonistic activities might refine the levels of circulating energy to match systemic energy requirements. If so, it is postulated that the negative energy balance accompanying starvation might worsen the hypoglycaemic effects of AKH deficiency. In comparison with starved control larvae, total haemolymph glucose was decreased by 75% in starving larvae after CC cell ablation. Thus, starvation increased the severity of hypoglycaemia in animals lacking CC cells, indicating that AKH might be required for the compensatory mechanisms that maintain circulating glucose during periods of food deprivation in Drosophila larvae (Kim, 2004).

Labelling of CC cell processes with mGFP and an antibody against AKH revealed that AKH-producing cells extend processes that terminate on the heart and on the prothoracic gland compartment of the ring gland. On the surface of the heart, CC cell processes have extensive contact with axons that project from insulin-producing cells from the brain. Labelling of CC cell processes with mGFP and an antibody against AKH revealed localization of AKH within the processes that contact the IPCs, and AKH peptide on the processes contacting the heart. These results indicate that the heart surface is the principal site of AKH release into the circulating haemolymph. Thus, like glucagon-producing cells in mammalian islets and brain, AKH-producing CC cells in the Drosophila ring gland have direct systemic vascular access, consistent with their role as endocrine regulators of metabolism (Kim, 2004).

ATP-sensitive potassium (K_ATP) channels regulate neuroendocrine cell function in organs such as the mammalian pancreas and brain, and this study examined whether K_ATP functions regulate CC cell activity. K_ATP channels are heteromeric protein complexes composed of sulphonylurea receptor (Sur) and inward-rectifying potassium channel (Ir; also called Kir) subunits. An ATP-binding domain in the Ir subunit regulates K_ATP channel activity, allowing these channels to serve as cellular energy sensors, opening or closing in response to the intracellular ADP/ATP ratio, thus influencing membrane potential and subsequent calcium currents that regulate hormone secretion. Using mRNA in situ hybridization, it was showm that larval CC cells expressed Sur (Nasonkin, 1999) and Ir (Döring, 2002), which have sequence similarity to mammalian Sur1 and Kir6 proteins, respectively. Expression of Sur or Ir was not detected in the larval brain IPCs, another group of cells known to regulate haemolymph glucose. Drosophila Sur has been shown to be sufficient to allow K⁺ currents that polarize membrane potentials (Nasonkin, 1999). Drosophila Ir was demonstrated to evoke an inwardly rectifying K⁺ current (Kim, 2004).

Tests were performed to see whether increased haemolymph glucose concentrations might result from excess AKH secretion brought about by sulphonylurea inhibition of the Sur and K⁺-dependent depolarization of CC cells. Glyburide and tolbutamide are representative members of the two major classes of sulphonylureas. These drugs promote the closure of K_ATP channels and cellular depolarization, thereby regulating secretion in mammalian neuroendocrine cells. For example, sulphonylureas stimulate glucagon secretion in diabetic patients. Glyburide has previously been shown to inhibit Drosophila Sur-mediated outward K⁺ currents, resulting in the depolarization of cell potential. Exposure of feeding third-instar larvae to glyburide mixed in yeast paste (standard dextrose medium did not permit drug delivery) produced a 10% increase in mean total haemolymph glucose concentration compared with controls. Exposure of larvae to tolbutamide had a greater effect, producing a 40% increase in mean total haemolymph glucose, and tolbutamide was used in subsequent studies. Average haemolymph glucose concentrations were generally decreased in animals fed with yeast paste compared with animals fed with standard dextrose medium, and this might have accentuated the hyperglycaemic effect of sulphonylureas administered in yeast paste. Moreover, the hypoglycaemic effect induced by CC cell ablation (or hyperpolarization) seemed attenuated in yeast-fed animals, further supporting the hypothesis that requirements for AKH might be altered by manipulating feeding conditions (Kim, 2004).

To test the hypothesis that Sur and Ir function in the CC to regulate haemolymph glucose concentrations in Drosophila , CC cells were ablated in larvae fed with tolbutamide. Ablation of the CC cells using Akh-GAL4 and UAS-Reaper blocked the hyperglycaemic effect of tolbutamide, indicating that CC cells must be present to support the hyperglycaemic action of tolbutamide. To determine whether the hyperglycaemic effect of tolbutamide resulted from Sur and Ir-mediated depolarization of CC cells, membrane potential was hyperpolarized in CC cells, in the presence and absence of tolbutamide. Kir2.1 is a human K⁺ channel that evokes an outward K⁺ current, independently of ATP regulation, and has previously been used to impair cellular depolarization in vivo in Drosophila by inducing persistent outward K⁺ current and a hyperpolarized resting potential. One indication that AKH release by CC cells requires membrane depolarization and might be regulated by K⁺-channel-dependent membrane potential comes from the observation that, on standard dextrose medium, third-instar larvae expressing Kir2.1 in CC cells had a 23% decrease in mean haemolymph glucose concentration, compared with controls. Expression of the Kir2.1 channel in CC cells prevented the hyperglycaemic effect of tolbutamide, indicating that K⁺-channel-dependent CC cell depolarization resulted from exposure to sulphonylurea. Together, these pharmacological and genetic data support the view that K_ATP channel activity in CC cells governs AKH release, thereby controlling concentrations of circulating glucose in Drosophila (Kim, 2004).

In pancreatic α-cells, hypoglycaemia stimulates increased intracellular calcium concentrations promoting glucagon secretion, whereas hyperglycaemia inhibits these responses. To test whether Drosophila CC cells sense glucose changes and, like pancreatic α-cells, modulate intracellular calcium concentrations, CC cells were mared with fluorescent transgene-encoded calcium sensors ('camgaroos'). The fluorescence intensity of camgaroos increases in response to elevated intracellular calcium concentration, an effect used previously to measure cytoplasmic calcium transients in depolarized Drosophila neurons. Elevation of cytoplasmic calcium concentration after CC cell depolarization stimulates AKH secretion; thus, in these experiments elevated intracellular calcium concentration in CC cells was used as an indicator of AKH secretion. Fluorescence of camgaroo-2 (cg-2) in cultured CC cells increased as extracellular trehalose or glucose concentration decreased. Direct CC cell depolarization with increased extracellular potassium concentration similarly led to increased cg-2 fluorescence. In contrast, fluorescence in cg-2-labelled CC cells decreased as extracellular trehalose concentration increased. These results corroborate previous studies of locust CC cells showing that decreases in extracellular trehalose or glucose concentration stimulated AKH secretion. Drosophila CC cells express the enzyme trehalase, raising the possibility that the sensing of trehalose by CC cells involves the hydrolysis of trehalose to glucose, a view also supported by similar effects of trehalose and glucose in in vitro studies. Thus, hypoglycaemic sensing in CC cells leads to increased concentrations of the intracellular second messenger calcium, a signal for subsequent regulated exocytosis of AKH -- a mechanism similar to those regulating glucagon secretion by mammalian pancreatic α-cells (Kim, 2004).

Thus, there are remarkable parallels in endocrine cell functions that ensure the supply of circulating glucose in Diptera and in mammals. On the basis of these parallels, it is speculated that insect CC cells and mammalian neuroendocrine cells that regulate metabolism might have arisen from an ancestral energy-sensing cell. If so, it is further speculated that pancreatic islet cells, including β-cells, might have evolved from an ancient α-cell. Similarly to pancreatic islets, insect CC cells might delaminate from embryonic epithelial cells that give rise to both gut and neuroendocrine structures. Thus, common mechanisms might regulate the development of CC and pancreatic islet cells. Understanding CC cell development could therefore accelerate the discovery of cell-replacement therapies for type 1 diabetes mellitus. This Drosophila model might also serve to elucidate the mechanisms that control stimulus-secretion coupling in CC cells, and hence the biology of hypoglycaemia. Moreover, the sensitivity of CC cells to drugs commonly prescribed for disorders such as type 2 diabetes indicates that Drosophila might provide a model system for the discovery of pharmacological agents to treat human endocrine diseases (Kim, 2004).

COPI complex is a regulator of lipid homeostasis

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. This study identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system. These regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets (Beller, 2008).

Lipid homeostasis is critical in health and disease, but remains poorly understood. Non-esterified free fatty acid (NEFA) is used for energy generation in beta-oxidation, membrane phospholipid synthesis, signaling, and in regulation of transcription factors such as the peroxisome proliferator-activated receptors (PPARs). Essentially all cells take up excess NEFA and convert it to energy-rich neutral lipids in the form of triglycerides (TG). TG is packaged into specialized organelles called lipid droplets. NEFA is regenerated from lipid droplet stores to meet metabolic and energy needs, and lipid droplets protect cells against lipotoxicity by sequestering excess NEFA. Lipid droplets are the main energy storage organelles and are thus central to the understanding of energy homeostasis. Despite their importance, little is known about the ontogeny and regulation of these organelles (Beller, 2008).

Lipid droplets are believed to form in the ER membrane by incorporating a growing TG core between the leaflets of the bilayer, and ultimately are released surrounded by a phospholipid monolayer. Cytosolic lipid droplets possess a protein coat and grow by synthesis of TG at the lipid droplet surface and by fusion with other lipid droplets. Formation of nascent droplets and aggregation of existing droplets is likely to require a dynamic exchange of lipids and proteins from and to the droplet. Indeed, the range of proteins identified in lipid droplet proteomic studies suggests extensive trafficking between lipid droplets and other cellular compartments, including the endoplasmic reticulum (ER). Additionally, lipid droplet-associated proteins translocate between the cytosol and lipid droplets. For example, tail interacting protein of 47 kDa (TIP47) associates with small, putative nascent, lipid droplets, but is not found on larger droplets, which are coated by other members of the perilipin, adipocyte differentiation related protein (ADRP), TIP47 (PAT) protein family. Intriguingly, TIP47 mediates mannose 6-phosphate receptor trafficking between the lysosome and Golgi, raising the possibility that trafficking is involved in lipid droplet ontogeny or fate. However, unlike the well-studied Golgi trafficking system, the routes to and from the lipid droplet are unknown (Beller, 2008).

Once lipid droplets are formed, stored TG is mobilized in a regulated manner. Triglyceride, diglyceride (DG), and monoglyceride lipases convert TG back into NEFA. Most of the knowledge concerning lipolysis is based on extensively studied adipocytes in which at least two lipolytic enzymes have been identified: adipocyte triglyceride lipase (ATGL; Drosophila brummer) and hormone sensitive lipase (HSL). Due to the hydrophobic properties of the lipid droplet TG core, lipases are likely to act at the surface of lipid droplets, where members of the PAT protein family regulate lipase access to the TG core. Mammalian genomes encode at least five PAT-proteins. Whereas perilipin is the dominant PAT protein in adipocytes, ADRP is the dominant PAT protein in nonadipose tissues in which it is tightly associated with the lipid droplet surface. PAT members appear to have a hierarchical affinity for the lipid droplet surface. In nonmammalian genomes, there are fewer PAT proteins. For example, two PAT proteins termed lipid storage droplet 1 and 2 (LSD-1 and LSD-2) are found in Drosophila (Miura, 2002). The crucial role of PAT proteins is evolutionary conserved as the absence of perilipin in mice, or LSD-2 in flies results in lean animals. Overexpression of LSD-2 results in obese flies. These data indicate the conserved PAT proteins at the lipid droplet surface are important regulators of energy storage (Beller, 2008 and references therein).

It seems likely that PAT proteins protect lipid from lipolysis, but the role of PAT proteins may not be limited to passive steric hindrance of lipase access to the TG core, as illustrated by perilipin. Unphosphorylated perilipin protects the lipid droplet from lipase activity. Following stimulation by protein kinase A (PKA), however, phospho-perilipin acts as a docking site for HSL, which translocates from the cytosol to the droplet surface. Whereas phospho-perilipin promotes massive NEFA release from the droplet, this is not mediated exclusively by HSL, as mice lacking HSL function show a relatively mild phenotype marked by the accumulation of DG, thus demonstrating that HSL acts as a DG lipase in vivo. The TG lipase functioning in HSL null mice is ATGL. In the current view of adipocyte lipolysis, ATGL is responsible for the first step in TG hydrolysis, liberating DG and NEFA, whereas HSL acts as a DG lipase. Very little is known about how ATGL is targeted to the lipid droplet (Beller, 2008).

In contrast to the lean phenotype in animals lacking perilipin (mouse) or LSD-2 (fly), both mice and flies lacking ATGL are obese. In mice, the absence of ATGL results in excessive TG accumulation in liver and muscle. Similarly, human patients suffering from neutral lipid storage disease carry mutations resulting in truncated ATGL isoforms. ATGL function is evolutionary conserved, as flies lacking the Drosophila ATGL ortholog, Brummer, accumulate copious amounts of body fat. The lipid droplet-associated protein Comparative Gene Identification-58 (CGI-58) acts as an ATGL colipase (Lass, 2006). Mutations in the CGI-58 gene result in ectopic fat accumulation in patients suffering from Chanarin Dorfman Syndrome (CDS), supporting the idea that both ATGL and CGI-58 are required for mobilizing lipid stores in nonadipose tissue. Interestingly, CGI-58 physically interacts with perilipin as demonstrated by both coimmunoprecipitation and fluorescence resonance energy transfer (FRET) studies. In addition, there are other lipases and probably many more cofactors encoded in the genome. Understanding which ones act at the lipid droplet surface and how their localization is regulated will be important (Beller, 2008).

Drosophila is a powerful model for pathway discovery due to well-developed genetics. Additionally, greater than 60% of the genes associated with human disease have clear orthologs in Drosophila. Drosophila is highly relevant to lipid droplet study, as lipid droplets in Drosophila and mammals are associated with many of the same proteins. Finally, the emerging model of lipid storage and endocrine regulation are similar in humans and Drosophila, suggesting that Drosophila will be a good genetic model for lipid storage and lipid storage diseases in humans. This study therefore utilized genome-wide RNA interference (RNAi) screening in Drosophila tissue culture cells to identify and characterize novel regulators of lipid storage. The function of these regulators was tested in mouse lipid droplet regulation by directed RNAi studies. 318 Drosophila genes were identified that were required to limit lipid storage and 208 Drosophila genes were identified that were required to promote lipid storage. These genes encode known regulators of lipid storage as well as genes not previously associated with lipid storage regulation (Beller, 2008).

Positive regulation of lipolysis by the COPI retrograde-vesicle trafficking pathway was the most striking and unexpected result of the screen. Interference with COPI function, either by RNAi or compounds, in Drosophila Kc₁₆₇ or S3 cells, or in mouse 3T3-L1 or AML12 cells, results in increased lipid storage. Furthermore, recent and parallel studies in yeast and Drosophila S2 cells (Guo, 2008) also suggested a role of COPI function in lipid droplet regulation. Interestingly, only the epsilon-subunit of the COPI complex failed to result in a lipid droplet deposition phenotype on knockdown. Although limited RNAi efficacy or increased protein stability cannot be ruled out, epsilonCOP was the only canonical COP subunit not resulting in a lipid storage phenotype in a parallel study using different cells and reagents (Guo, 2008), and targeting of epsilonCOP transcripts by RNAi in AML12 cells had a weak effect on lipid storage at best. Finally, epsilonCOP is the only dispensable subunit in a recent study identifying COPI activity coupled with fatty acid biosynthesis as a host factor important for Drosophila C virus replication (Cherry, 2006). This is especially interesting, since certain enveloped viruses, including Hepatitis C virus, assemble on lipid droplets. Taken together, these results indicate that six out of the seven wild-type COPI subunits mediate lipid storage by positively regulating lipolysis (Beller, 2008).

COPI could have a direct or indirect effect on lipid storage. The indirect mechanism is poorly defined, but if the Golgi is a 'sink' for phospholipids derived from TG stores, then decreased Golgi function could simply decrease demand for TG substrate. If non-esterified free fatty acid (from the media in fed cells, and from biosynthesis in unfed cells) conversion to TG continues, then increased lipid droplet volume would occur. It is also possible that canonical COPI function transporting lipids and proteins from the Golgi to the ER is ultimately responsible for lipid droplet utilization and protein composition at the lipid droplet surface. For example, COPI might be required for the particular phospholipid composition in hemimembranes formed on nascent droplets, which secondarily alter TIP47 and ATGL localization in mature lipid droplets (Beller, 2008).

However, evidence that Golgi function per se is not linked to lipid storage phenotypes, as well as direct association of COPI members and regulators with the lipid droplet or PAT proteins supports a more direct model. The COPI and COPII pathways have established roles as constitutive vesicle transport systems that cycle proteins as well as lipid from the Golgi to the ER (COPI), or vice versa (COPII). Interference with either of the COP trafficking systems results in disturbed ER and Golgi function. The lipid overstorage phenotype was seen only in the case of interference with COPI trafficking. This indicates that the lipid overstorage phenotype is not a simple consequence of ER and Golgi function. Finally, in an indirect model in which COPI shuttles only between the Golgi and the ER, COPI should not be lipid droplet associated. However, COPI subunits are directly associated with the lipid droplet surface as shown by proteomics. Additionally, Arf1 binds to ADRP, which is exclusively associated with the lipid droplet surface. Arf79F, the Drosophila homolog of mammalian Arf1, also localizes to lipid droplets in Drosophila S2 cells (Beller, 2008 and references therein).

It is proposed that COPI is likely to function directly at the lipid droplet surface and not indirectly through the Golgi. Perhaps COPI is a destination-specific transporter returning lipid droplet surface hemimembrane and Golgi membrane to the ER. The transport system that brings nascent lipid droplets from the ER to the lipid droplet has not been elucidated, but it is intriguing that the transport/PAT protein TIP47 is found preferentially on small lipid droplets. Small lipid droplets derived from the ER are thought to help build larger droplets by fusion. TIP47-coated droplets might form in the ER, and then COPI could return TIP47 to the ER after the lipid cargo is deposited. In this model, TIP47 becomes trapped at the lipid droplet surface in the absence of COPI (Beller, 2008).

Although increased TIP47 was observed on ADRP-positive droplets by both western blot and cell staining, the cell staining result was more dramatic. The current model might also explain why. The punctate staining of TIP47 in untreated cells could be due to TIP47 on nascent droplets that might also cofractionate with the larger ADRP-positive droplets in the western blots, leading to a less dramatic enrichment for TIP47 relative to ADRP in that experiment. However, other explanations cannot be ruled out, such as nonlinear detection of antigen concentration or epitope masking in the cell staining experiments (Beller, 2008).

COPI perturbation increases stored TG by decreasing the lipolysis rate indicating that the wild-type COPI complex promotes lipolysis. COPI directly or indirectly removes TIP47 from the lipid droplet surface and promotes ATGL localization to the droplet surface, where lipolysis occurs. ATGL has a key role in lipid droplet utilization, and ATGL association with the droplet is reduced by ADRP and Tip47 (Bell, 2008). The epistasis experiments combining siRNA-mediated ATGL knockdown and BFA or Exo1 compound treatment demonstrated that the decrease in lipolysis rate is due to loss of ATGL activity. COPI activity specifically alters lipid droplet surface composition by increasing the amount of TIP47 and reducing the amount of ATGL at ADRP-coated lipid droplets. It is suggested that COPI negatively regulates localization of TIP47. TIP47 in turn prevents ATGL localization. The rescue of the double-knockdown phenotype of TIP47 and ADRP by BFA or Exo1 suggests that COPI has an independent feed-forward effect on ATGL levels at the lipid droplet surface (Beller, 2008).

Although this study focused attention here on COPI, systematic and genome-wide exploration of gene functions required for lipid storage in Drosophila significantly increases experimental access to the complex molecular processes regulating lipid storage and utilization. Further, the use of multiple screens using different cell types and different organisms greatly increases confidence in the genes in the intersection. Given widespread concerns about RNAi screening efficacy and off-target effects, as well as the time and effort required for downstream analysis, systematic use of multiple species and libraries to address a single biological question might be cost effective in addition to resulting in more durable datasets. Primary screens in Drosophila cells followed by secondary screens in mouse cells are much less expensive than a similar genome-wide screen in mammalian cells. Additionally, the availability of mutants in most Drosophila genes, along with demonstrated translation to mammalian systems, provides a valuable entry point for in-depth analyses in both fly and mouse; and eventually for the selection of therapeutic targets for emerging problems associated with obesity and other metabolic disorders (Beller, 2008).

Functional genomic screen reveals genes involved in lipid-droplet formation and utilization

Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets enclosed in a monolayer of phospholipids and associated proteins. These dynamic organelles serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. This study shows, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells, that about 1.5% of all genes function in lipid-droplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1-COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to the understanding of human diseases involving excessive lipid storage (Guo, 2008).

Lipid-droplet formation was induced by incubation with 1 mM oleate for 24 h. Staining with 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY493/503) showed that droplet size, number and overall volume were increased; cellular triacylglycerol content increased sevenfold. Imaging this process by time-lapse microscopy of BODIPY-labelled cells after oleate addition showed that droplet formation occurred in steps. First, increased numbers of small droplets formed in dispersed locations throughout the cell. Next, droplets increased in size and finally aggregated into one or several large clusters, resembling grapes. Electron microscopy confirmed the tight clustering of the droplets, which were often near the nucleus (Guo, 2008).

To unravel the molecular mechanisms governing this progression of changes during lipid-droplet formation, a genome-wide RNAi screen was performed in S2 cells. Images were acquired and examined by two independent observers, who scored them for alterations in droplet number, size and dispersion. The same data were analysed computationally. From visual screening, both observers identified 847 candidate genes with altered lipid-droplet morphology. To verify these genes and to minimize the misidentification of genes from off-target effects of RNAi treatments, RNAi experiments for these genes were repeated with a second, distinct set of double-stranded (ds)RNAs. Visual analyses identified 132 genes whose knockdown consistently and repeatedly altered lipid-droplet morphology and an additional 48 genes for which knockdown phenotypes were scored in two of three rounds. Computational analysis confirmed 86 of these 180 genes and added 47 genes with altered lipid-droplet morphology. Thus, 227 genes (about 1.5% of the genome) were identified that affect lipid-droplet morphology (Guo, 2008).

The 132 genes with striking phenotypes were categorized into five distinct phenotypic classes, which were validated for selected knockdowns by electron microscopy. Class I genes showed reduced numbers of droplets and included midway (encoding a diacylglycerol acyl-transferase), subunits of the proteasome and the spliceosome, and several uncharacterized open reading frames. Class II genes gave smaller, more dispersed, droplets and included subunits of the COP9-signallosome complex, dynein, and RNA polymerase II subunits. Class III genes showed more dispersed droplets of slightly larger size and were members of the Arf1-COPI vesicular transport machinery. Class IV genes yielded highly condensed clusters of droplets and included members of the translational machinery. Class V genes contained one or a few very large droplets and included an orthologue of sterol regulatory element binding-protein (SREBP), a master transcriptional regulator of lipid metabolism, and SREBP cleavage activating protein (SCAP). In Drosophila, the SREBP pathway is sensitive to and regulates phospholipid biosynthesis. This class also included Cct1 and Cct2, which encode isoforms of phosphocholine cytidylyltransferase, the enzyme that catalyses the rate-limiting step in phosphatidylcholine synthesis, and CG2201, which is predicted to have choline kinase activity that phosphorylates and activates choline. Thus, most class V genes were linked directly or indirectly to phospholipid biosynthesis (Guo, 2008).

To further explore how phospholipid metabolism regulates lipid-droplet formation, the Cct1 and Cct2 knockdowns were characterized. Larger droplets in Cct knockdowns could arise from a failure to form new droplets, forcing newly synthesized neutral lipids into a few large droplets, or from the fusion of independently formed droplets. To distinguish between these possibilities, the dynamics of lipid-droplet formation were observed by time-lapse microscopy, and evidence was found that the droplets fuse (Guo, 2008).

Then, where CCT proteins act was examined. In untreated cells, mCherry-tagged Cct1 localized exclusively to the nucleus, similar to mammalian cytidylyltransferase-α (CT-α). After treatment with oleate, a significant portion of Cct1 localized to the lipid-droplet surface. By contrast, similarly tagged Cct2 localized to the cytoplasm but was also concentrated on droplet surfaces after treatment with oleate. This marked translocation of CCT enzymes to the droplet surface may serve to provide adequate phosphatidylcholine to the phospholipid monolayers of growing lipid droplets. If so, the ratio of surface phospholipids to core neutral lipids may regulate lipid-droplet morphology: when phospholipids are limiting (as in Cct1 or Cct2 knockdowns), fusion is induced to decrease the surface-to-volume ratio of droplets. In fact, the content of phosphatidylcholine in cells with Cct1 knockdown was decreased by about 60%, and the triacylglycerol content was increased by about 40%. The increase in triacylglycerol may reflect compensatory channelling of diacylglycerol into neutral lipids. A decreased phosphatidylcholine content would increase the relative amount of phosphatidylethanolamine in the droplet monolayer (as observed in flies lacking Cct1, which itself may directly promote droplet fusion. The results suggest a model in which phosphatidylcholine availability is a crucial regulator of lipid-droplet size and number (Guo, 2008).

Class III genes, whose knockdowns showed slightly larger and more dispersed droplets, were investigated. All class III genes were members of the Arf1-COPI machinery, including Arf79F, encoding an Arf1 family member, a gene encoding guanine nucleoside exchange factor (GEF), garz, and genes encoding components of the COPI coat. Similar effects were obtained by incubating cells with brefeldin A, a specific inhibitor of Arf1 exchange factors, and by expressing a dominant-negative version of Arf79F, encoding the T31N mutant, analogously to dominant-negative mutants for Ras or Ran. To test the specificity of this phenotype, RNAi knockdowns were separately repeated with dsRNAs for Drosophila genes encoding six ARF proteins, three GEFs, two GTPase-activating proteins, and all COPI subunits. Other coat proteins were also tested, such as clathrin subunits and components of the COPII coat. Only Arf79f, garz and six of eight members of the COPI coat (α-, β-, β′-, δ-, γ- and ζ-Cop) exhibited the class III phenotype, indicating that the screen identifies a highly specific subset of vesicular transport components. Arf102F knockdown gave a partial phenotype. This function of the Arf1-COPI machinery in lipid-droplet formation seems to have been evolutionarily conserved; similar phenotypes were found in yeast and human cells (Guo, 2008).

Next, attempts sere made to determine whether Arf79F acts directly on lipid droplets. ARF proteins exchange rapidly between active (GTP-bound) and inactive (GDP-bound) forms, making it difficult to localize only the active form. However, Arf79F(T31N) binds its exchange factor tightly, and the distribution of the exchange factor is predicted to reflect the localization of active ARF protein. Expressed Arf79F(T31N) appeared diffusely in the cytosol but was enriched at the droplet surface. Thus, Arf79F may act at the lipid-droplet surface where, as for other ARF proteins, it interacts with its GEF (presumably encoded by garz) and recruits COPI components. A recent in vitro study showed that Arf1 and several subunits of the COPI complex are recruited from the cytosol to purified lipid droplets in the presence of GTP-γS (Bartz, 2007). Although the recruitment of Arf1 to lipid droplets was reported to activate phospholipase D, no effect of phospholipase D knockdown on lipid-droplet formation was found in Drosophila cells (Guo, 2008).

The well-established functions of class III genes in Arf1-COPI-mediated vesicular transport implicate this machinery in a similar budding mechanism at the surface of lipid droplets, possibly to promote the budding-off of droplets during lipid mobilization. Lipolysis is associated with the break-up of larger droplets into smaller ones, presumably to provide more surface area for lipases. The effect of the Arf79F knockdown on lipolysis was examined by inducing lipid-droplet formation and then inducing lipid mobilization by incubation with serum-free medium lacking oleate. After 24 h, control cells had few droplets. By contrast, many droplets remained when Arf79F was inactivated. In addition, much less glycerol, a product of lipolysis, was released by cells lacking the Arf1-COPI machinery. Supporting a function of the Arf1-COPI machinery in lipolysis, more glycerol was released by cells expressing a dominant-active form of Arf79F encoding Arf79F(Q71L). These data indicate that the Arf1-COPI machinery is required for efficient lipolysis. The data agree with a report showing that lipolysis in murine adipocytes is accompanied by a brefeldin A-sensitive process that is required for the mobilization of cholesterol from storage pools in droplets (Guo, 2008).

Increased droplet surface area during lipolysis would require more phospholipids in the surrounding monolayer. Because Cct1 knockdown limits phosphatidylcholine amounts, its effect on lipolysis were tested. As predicted, Cct1 knockdown markedly decreased the efficiency of lipolysis, as seen by lipid-droplet staining. The effects of knockdowns of Arf79F and Cct1 on lipolysis were additive, suggesting that these genes function independently (Guo, 2008).

Arf1-COPI complexes mediate retrograde vesicular trafficking of membranes and proteins from the Golgi apparatus to the endoplasmic reticulum and are also involved in vesicular transport processes from the trans-Golgi network and endosomes. Notably, the members of the Arf1-COPI complex this study identified were recently found in a Drosophila screen for genes involved in protein secretion and Golgi organization (Bard, 2006). Although the primary defect in class III knockdowns is as yet unknown, the phenotype on lipid-droplet formation is not likely to be an indirect consequence of inhibition of protein secretion. The effects are highly specific and are not observed with knockdowns of other proteins mediating secretory transport (endoplasmic reticulum translocation, COPII and clathrin). In addition, Arf79F is recruited to the lipid-droplet surface, where it is presumably activated by the loading of GTP on its exchange factor (Guo, 2008).

This study provides an initial systematic examination of the Drosophila genome to identify genes involved in lipid-droplet formation and utilization. Many genes identified sort to distinct classes of morphological changes, with each class containing functionally related proteins. These classes potentially link diverse processes, such as protein synthesis and degradation, the cell cycle, and organelle movement, with lipid-droplet biology. The variety of genes identified lends support to the emerging view of lipid droplets as dynamic organelles that are functionally connected to a variety of organelles and cellular processes, including the replication of intra-cellular pathogens such as Chlamydia trachomatis and hepatitis C. Many components of these processes are likely to be highly conserved across species. These studies in S2 cells may therefore be directly relevant to cellular lipid storage in general, holding the promise of identifying pathways and mechanisms central to human diseases involving excessive lipid storage and to the engineering of cellular lipid storage in organisms for the improved production of oils and biofuels (Guo, 2008).

Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate

Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. A systems-level genetic dissection of adiposity regulation was performed using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo; hedgehog signaling was the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, fat-specific hedgehog-activation mutant mice were generated. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals (Pospisilik, 2010).

To assess the in vivo relevance of hedgehog signaling in mammalian adipogenesis, fat-specific Sufu knockout animals (aP2-SufuKO) were generated. Sufu is a potent endogenous inhibitor of hedgehog signaling in mammals. Sufu^flox/flox mice were crossed to the adipose tissue deleting aP2-Cre transgenic line, and the resulting aP2- SufuKO animals were born healthy and at Mendelian ratios. PCR amplification revealed target deletion in both white adipose tissue (WAT) and brown adipose tissue (BAT). aP2-SufuKO mice displayed an immediate and obvious lean phenotype. MRI analysis revealed a significant and global reduction in white adipose tissue mass, including subcutaneous, perigonadal, and mesenteric depots. Intriguingly, though, in contrast to the gross loss of WAT, cross-sectional examination of the interscapular region revealed fully developed BAT depots of both normal size and lipid content. Direct measurement of WAT and BAT depot weights corroborated the divergent WAT/BAT phenotype, with an ~85% reduction in perigonadal fat pad mass in aP2-SufuKO mice concomitant with unaltered BAT mass. Tissue weight and histological analyses confirmed lack of any remarkable phenotype in multiple other tissues including pancreas and liver (no indication of steatosis), and muscle mass was unaffected. Cutaneous adipose was also markedly diminished. Whereas the morphology of Sufu-deficient BAT depots was largely indistinguishable from that of control animals, examination of multiple WAT pads revealed marked and significant reductions in both adipocyte size and total numbers in mutant animals. Of note, qPCR showed elevated Gli1, Gli2, and Ptch2 expression in both WAT and BAT verifying the intended pathway activation in both tissues. Thus, deletion of Sufu in fat tissue results in a markedly decreased white fat cell number and, remarkably, in normal brown adipose tissue (Pospisilik, 2010).

When the literature was cross-referenced focusing on adipogenesis, an impressive 18 of 65 key regulators of adipogenesis were found to be described as Gli targets in other systems. Intriguingly, when examined in 3T3-L1 preadipocytes, hedgehog activation induced a coordinated downregulation of the proadipogenic targets Bmp2, Bmp4, Egr2/Krox20, Sfrp1, and Sfrp2 by an average of ~50% after only 24 hr. In contrast, quantification of the antiadipogenic target set showed upregulation of the multiple critical repressors (Nr2f2, Gilz, Hes1, and Ncor2); the negative regulators Jag1 and Pref1 remained unchanged at this time point. Analysis of the master regulatory machinery downstream of these effectors revealed critical reductions in Pparg, Cebpb, and Cebpd and increases in the antiadipogenic factors Cebpg and Ddit3. Outside of this dramatic antiadipogenic profile, elevated levels of Cebpa were observed. Importantly, a similar coordinate downregulation of Pparg, Cebpb, Cebpd, as well as Cebpa was observed in WAT-derived primary murine adipocyte progenitors (stromal vascular cell, SVC, preparations) following genetic activation of hedgehog signaling (Pospisilik, 2010).

To establish a direct link between hedgehog activation and adipogenic block in white adipose, in silico predictions were used to identify clusters of probable Gli-binding sites in the highly SAG-responsive genes Ncor2, Nr2f2, Sfrp2, and Hes1. To assess the functionality of these putative binding sites, the relevant promoter fragments were cloned and luciferase reporter assays were performed. Gli1 and Gli2 induced activation of all Ncor2 and Nr2f2 reporter constructs, with the binding site clusters Ncor2_B, Nr2f2_A, and Nr2f2_B showing responses comparable to the hallmark target Ptch. Further, chromatin immunoprecipitations on 3T3-L1 preadipocytes using Gli2- and Gli3-specifc antibodies revealed increases in Gli2 and Gli3 binding within the endogenous Ncor2, Hes1, Nr2f2, and Sfrp2 regulatory regions following SAG treatment. Together, these findings demonstrate endogenous Gli2/Gli3 binding to multiple adipogenic loci and implicate direct modulation of Ncor2 and Nr2f2 in the dysregulation of adipogenesis (Pospisilik, 2010).

The power of D. melanogaster RNAi transgenics to probe gene function on a genome-wide scale has allowed screening of ~78% coverage of the Drosophila genome. One significant advantage of this inducible approach is the ability to interrogate the fat regulatory potential of the ~30% of the Drosophila genome that is developmentally lethal under classic mutation conditions. Indeed, the result that cell differentiation genes scored as the most enriched ontology subcategory substantiates the inducible strategy employed and identifies a large number of developmentally lethal genes with strong lipid storage regulatory potential. Consistent with a previous feeding-induced RNAi C. elegans screen, the fraction of candidate genes resulting in decreased fat content upon knockdown (360 of 516; 70%) exceeded that of obesity-causing candidates (216 of 516; 30%), which is consistent with the hypothesis that the major evolutionary pressures for animals have been to favor nutrient storage. The screen identified a large number of genes already known to play a key role in mammalian fat or lipid metabolism, including enzymes of membrane lipid biosynthesis, fatty acid and glucose metabolism, and sterol metabolism. Further, the whole-genome screen has uncovered a plethora of additional candidate genes of adiposity regulation, a large proportion of which had no previous annotated biological function. Moreover, multiple genes were identified that either positively or negatively regulate whole fly triglyceride levels when targeted specifically in neurons, the fly liver (oenocyte), the fat body, or muscle cells. Analyses of the hits allowed definition of either gene sets that function globally in all these tissues or others that display coordinate regulation of adiposity when targeted in metabolically linked organs such as the fat and the liver. Since >60% of the candidate genes are conserved across phyla to humans, this data set is a unique starting point for the elucidation of novel regulatory modalities in mammals (Pospisilik, 2010).

The top-scoring signal transduction pathway in the GO-based enrichment analysis was the hedgehog pathway. Tissue-specificity assessment revealed further that this enrichment was primarily derived from a pronounced fat-body restriction in function. Hedgehog signaling has been previously implicated in adipose tissue biology. In Drosophila larvae, hedgehog activation reduces lipid content consistent with what was found in adult flies and the fat-specific fly knockdown lines (Suh, 2006). Similarly, knockdown of the C. elegans equivalent of the inhibitory hedgehog receptor Ptch results in a prominent adiposity reducing phenotype in a feeding-based RNAi screen. Therefore this study homed into the hedgehog pathway to provide proof of principle for the fly screen and to translate Drosophila results directly into the mammalian context (Pospisilik, 2010).

Several reports exist describing systemic manipulation of hedgehog signaling, either by injection of ligand-depleting antibody or through examination of a systemic hypomorphic mutant, the Ptchmes/mes mouse. Indeed Ptchmes/mes mice display largely normal white adipose tissue depots albeit reduced in size (Li, 2008). Hedgehog signaling plays a crucial role in multiple organs systems including at least one intimately involved in nutrient storage and the etiologies of obesity and insulin resistance, namely, the pancreatic islet. In vitro and in vivo data using the adipose-specific Sufu mutant mice clearly show that hedgehog activation results in a complete and cell-autonomous inhibition of white adipocyte differentiation. The residual white adipose tissue observed in aP2-SufuKO mice is most likely due to late inefficient deletion and/or is due to developmental timing effects. Indeed, aP2 (and thus aP2-Cre) are expressed relatively late during adipocyte differentiation. The remarkable finding was that genetic activation of hedgehog signaling in vivo and in vitro blocks only white but not brown adipocyte differentiation (Pospisilik, 2010).

Fat is mainly stored in two cell types: WAT, which is the major storage site for triglycerides, and BAT, which, through the burning of lipids to heat (through uncoupling of mitochondrial oxidative phosphorylation), serves to regulate body temperature. Recent PET-CT data have revealed that adult humans contain functional BAT and that the amount of BAT is inversely correlated with body mass index. These new data in humans rekindle the notion that a functional BAT depot in humans could represent a potent therapeutic target in the context of obesity control. Lineage tracking and genetic studies have shown that WAT and interscapular BAT cells derive from two different but related progenitor pools. The current genetic data now demonstrate both in vitro and in vivo that hedgehog activation results in a virtually complete block of WAT development but leaves the differentiation process of brown adipocytes wholly intact. These data further support the concept that white and brown adipocytes are derived from distinct precursor cells (Pospisilik, 2010).

aP2-SufuKO mice are the first white adipose-specific lipoatrophic mice with a fully functional BAT depot over the long-term and normal glucose tolerance and insulin sensitivity. The capacity of an intact BAT depot to burn energy in aP2-SufuKO mice likely underlies, at least in part, their lack of ectopic lipid accumulation and insulin resistance. This largely normal metabolic picture highlights the potent regulatory capacity of brown adipose tissue and should prove invaluable in understanding the distinct roles of brown and white adipose tissues (Pospisilik, 2010).

Obesity-blocking neurons in Drosophila

In mammals, fat store levels are communicated by leptin and insulin signaling to brain centers that regulate food intake and metabolism. By using transgenic manipulation of neural activity, the isolation is reported of two distinct neuronal populations in flies that perform a similar function, the c673a-Gal4 and fruitless-Gal4 neurons. When either of these neuronal groups is silenced, fat store levels increase. This change is mediated through an increase in food intake and altered metabolism in c673a-Gal4-silenced flies, while silencing fruitless-Gal4 neurons alters only metabolism. Hyperactivation of either neuronal group causes depletion of fat stores by increasing metabolic rate and decreasing fatty acid synthesis. Altering the activities of these neurons causes changes in expression of genes known to regulate fat utilization. These results show that the fly brain measures fat store levels and can induce changes in food intake and metabolism to maintain them within normal limits (Al-Anzi, 2009).

This paper describes the isolation of two distinct populations of Drosophila brain neurons that regulate fat deposition. These populations, denoted as c673a-Gal4 and Fru-Gal4, were identified by using Gal4 driver lines to express neuronal silencing or hyperactivating genes. For both neuronal populations, silencing produces obesity, defined as excess fat deposition, and hyperactivation produces leanness, defined as a reduction in fat store levels. Silencing and hyperactivation affect the expression of genes that are likely to be regulators of fat storage. However, the observed phenotypes are unlikely to be mediated by signaling through receptors for NPY-like or insulin-like peptides, which are important regulators of growth, feeding, and fat deposition (Al-Anzi, 2009).

The two populations have only a few neurons in common, and the analysis suggests that the shared neurons are not responsible for the observed phenotypes. Metabolic analysis shows that the two populations affect fat deposition by different mechanisms. The obesity phenotype produced by silencing is reversible (Al-Anzi, 2009).

Reduced use of fat stores and increases in de novo fatty acid synthesis correlate with the obesity phenotype when either Fru-Gal4 or c673a-Gal4 neurons are silenced, and c673a-Gal4-silenced animals also consume excess food. Conversely, the depletion of fat stores that occurs when either neuronal population is hyperactivated is likely to be caused by increased metabolism and decreases in de novo fatty acid synthesis. Interestingly, when Fru-Gal4 neurons, but not c673a-Gal4 neurons, are hyperactivated, the animals enter a state in which they use protein precursors to synthesize carbohydrates, and probably catabolize their own proteins via autophagy. This suggests that Fru-Gal4 hyperactivated animals are in a state of perceived starvation, despite the fact that they consume a normal amount of food (Al-Anzi, 2009).

Fru-Gal4 is expressed in a large number of brain neurons. Fru-Gal4-silenced flies accumulate excess fat despite consuming less food and are less obese than c673a-Gal4 silenced flies, which consume more food. These facts suggest that driving the silencing gene with Fru-Gal4 might have two opposing effects. Neurons that are positive regulators of feeding might be silenced as well as neurons that sense fat store levels. Because of this, the flies might reduce food intake, which would decrease the severity of the obesity phenotype that would have been produced by silencing only the fat-sensing subset of the Fru-Gal4 neurons (Al-Anzi, 2009).

Alternatively (or in addition), Fru-Gal4-silenced flies might still be able to detect an increase in their fat stores and respond to it by decreasing feeding. However, the decrease would be insufficient to prevent the accumulation of excess fat that is driven by the metabolic changes occurring when Fru-Gal4 neurons are silenced. c673a-Gal4-silenced flies probably cannot sense fat store levels at all, since they consume more food despite having an excess of energy reserves (Al-Anzi, 2009).

The different defects underlying the obesity phenotype when the two neuronal populations are silenced and the observation that there is very little overlap between these populations suggest that they are parts of two independent neural circuits. It is speculated that c673a-Gal4 and Fru-Gal4 neurons may have different roles in the wild, regulating fat stores in response to different environmental or internal stimuli. Since silencing of c673a-GAL4 neurons increases food intake, the activity of these neurons might be turned down under unfavorable environmental conditions in order to increase the ability of the flies to accumulate additional energy stores. For Fru-Gal4 neurons, whose hyperactivation induces an autophagic state, it is speculated that activity might be increased under severe starvation conditions to allow the utilization of cellular protein as an energy source (Al-Anzi, 2009).

Lipid metabolism is essential for generating much of the energy needed during periods of starvation. In Drosophila, stored fats are released from the fat body through the activity of lipases such as Bmm lipase. This is in turn causes the accumulation of fat molecules in the oenocytes, where they will be further metabolized through the activity of cytochrome P450 proteins such as Cyp4g1. It was observed that altering the activity of Fru-Gal4 neurons affected the expression levels of the cyp316a1, cyp4g1, and bmm lipase genes, while neural activity of c673a-Gal4 only affects cyp316a1 levels. Cyp316a1 is a cytochrome P450 that is closely related to Cyp4g1, and although its role in fat metabolism has not been studied, the fact that it belongs to the same cytochrome c family as Cyp4g1 indicates that it might have similar functions. Perturbation of both neuronal groups affects fatty acid synthesis by inducing changes in the expression of acetyl CoA-carboxylase, the main regulatory enzyme of the de novo fatty acid synthesis pathway (Al-Anzi, 2009).

In mammals, hypothalamic brain centers such as the ventromedial nuclei (VMN), paraventricular nuclei (PVN), and the lateral hypothalamic area (LHA) are informed about the status of body fat storage by the leptin and insulin pathways. These centers respond by inducing changes in food intake and metabolism that maintain constant body weight. Electrical stimulation of VMN or PVN neurons suppresses food intake, while bilateral lesions of VMN or PVN cause hyperphagia and obesity (Al-Anzi, 2009).

Leptin and insulin circulating in the bloodstream affect the activity of neurons in the arcuate nucleus of the hypothalamus (ARN). ARN is located in an area with a reduced blood-brain barrier, thus endowing it with the ability to sense leptin, insulin, and circulating nutrient levels. A subset of ARN neurons express the leptin receptor. ARN axons project to VMN, PVN, and LHA, and thereby communicate the status of fat stores to these feeding centers (Al-Anzi, 2009).

It is unknown whether the fly brain has feeding centers with equivalent roles to these mammalian hypothalamic nuclei. The 673a-Gal4 and Fru-Gal4 populations are dispersed throughout the brain, so the locations of neurons expressing these drivers do not indicate that any particular region of the brain is central to regulation of fat storage. However, the phenotypes produced by silencing and hyperactivation of these populations suggest that, like mammalian hypothalamic nuclei, they respond to humoral signals made by adipocytes that report on fat store levels. In particular, the fact that the obesity phenotype caused by silencing is reversible and that previously silenced flies dramatically reduce food consumption in order to reduce fat stores back to normal levels suggest that adipocytes alter release of a humoral factor when their fat content changes. The levels of this humoral factor are interpreted by the c673a-Gal4 and Fru-Gal4 neurons and used to control food consumption. Thus, flies that have accumulated excess fat stores during the silencing period communicate this fact to the brain, and brain neurons respond by reducing caloric intake when the activity block is released (Al-Anzi, 2009).

The isolation of c673a-Gal4 and Fru-Gal4 neurons in Drosophila should allow the future identification of genes involved in brain/fat store communication, possibly including those encoding the putative adipocyte humoral factor(s). This might be done by examining the consequences of transgenic expression of components of candidate signaling pathways in these neurons on flies' fat stores or by finding transcripts selectively expressed in them. The role of such genes in regulating fat storage could then be tested by RNAi or overexpression. The expression patterns of functionally validated genes could, in turn, more precisely identify which neurons within both populations are required for regulation of fat storage and what receptors they use to detect circulating humoral regulators that convey information about fat store levels (Al-Anzi, 2009).

Remote control of insulin secretion by fat cells in Drosophila

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

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

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

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

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

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

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

Dopaminergic modulation of sucrose acceptance behavior in Drosophila

For an animal to survive in a constantly changing environment, its behavior must be shaped by the complex milieu of sensory stimuli it detects, its previous experience, and its internal state. Although taste behaviors in the fly are relatively simple, with sugars eliciting acceptance behavior and bitter compounds avoidance, these behaviors are also plastic and are modified by intrinsic and extrinsic cues, such as hunger and sensory stimuli. This study shows that dopamine modulates a simple taste behavior, proboscis extension to sucrose. Conditional silencing of dopaminergic neurons reduces proboscis extension probability, and increased activation of dopaminergic neurons increases extension to sucrose, but not to bitter compounds or water. One dopaminergic neuron with extensive branching in the primary taste relay, the subesophageal ganglion, triggers proboscis extension, and its activity is altered by satiety state. These studies demonstrate the marked specificity of dopamine signaling and provide a foundation to examine neural mechanisms of feeding modulation in the fly (Marella, 2012).

Invertebrate models with less complex nervous systems and robust sensory-motor behaviors may illuminate simple neural modules that regulate behavior. This study examined flexibility in a gustatory-driven behavior and found that a dopaminergic neuron is a critical modulator. Loss-of-function studies involving dopamine receptor mutants and gain-of-function studies argue that increased dopaminergic activity promotes proboscis extension to sucrose, and decreased dopaminergic activity inhibits it. These studies show that a single dopaminergic neuron in the SOG, TH-VUM, can drive proboscis extension. TH-VUM does not respond to sugars, arguing that it is not directly in the pathway from taste detection to behavior, but instead acts over a longer timescale or in response to other cues to modulate proboscis extension to sucrose. Consistent with this idea, satiety state influences TH-VUM activity, promoting activity when the animal is food deprived and the probability of proboscis extension is increased. These studies suggest that dopaminergic activity regulates the probability of extension according to an animal's nutritional needs (Marella, 2012).

The finding that dopamine neural activity affects proboscis extension to sucrose, but not water, argues that dopamine regulation occurs upstream of shared motor neurons involved in proboscis extension. The pathway selectivity also argues that different molecular mechanisms modulate food and water intake independently in the fly, with parallels to hunger and thirst drives in mammals. Where dopamine acts in the sugar pathway is not known. Experiments to test for proximity between sugar sensory neurons and TH-VUM using the GRASP approach suggested that a few fibers are in close proximity, but the significance is unclear. The broad arborizations of TH-VUM suggest it may have many targets (Marella, 2012).

Dopamine is a potent modulator of a variety of behaviors in mammals and flies. In mammals, functions of dopamine include motor control, reward, arousal, motivation, and saliency. Dopamine also critically regulates feeding behavior. Mice mutant for tyrosine hydroxylase fail to initiate feeding, although they distinguish sucrose concentrations and have the motor ability to consume. Dopamine pathways that regulate feeding are complex, with the tuberoinfundibular, nigrostriatal, and mesolimbic and mesocortical pathways implicated in different aspects of feeding regulation. Although several studies show that dopamine promotes positive aspects of feeding, there is debate over whether dopamine is involved in pleasure ('liking'), motivation or salience ('wanting'), associative learning, or sensory-motor activation. With 20,000-30,000 TH-positive neurons in mice and 400,000-600,000 in humans, the complexity of dopaminergic regulation makes it difficult to parse the function of different neurons (Marella, 2012).

In Drosophila, as in mammals, dopamine participates in conditioning and arousal, and this study highlights a shared role in feeding regulation. There are only a few hundred TH-positive neurons in Drosophila, and recent studies have begun to elucidate the function of different dopaminergic neural subsets. This work demonstrates that a single dopaminergic neuron in the SOG potently modulates proboscis extension behavior. Other dopaminergic neurons have cell bodies near TH-VUM and extensive projections in the SOG, yet activation of these neurons is not associated with proboscis extension. It is possible that additional dopaminergic neurons regulate other aspects of taste behavior, but they are insufficient to drive proboscis extension (Marella, 2012).

In mammals, dopamine levels in the nucleus accumbens, the target of the mesolimbic pathway, increase upon sugar detection in the absence of consumption or upon nutrient consumption in the absence of detection, suggesting that dopamine encodes multiple rewarding aspects of sugar: intensity on the tongue and nutritional value. Recent studies in Drosophila also show that they sense nutritional content independent of taste detection, and this influences ingestion. It will be interesting to determine whether dopamine plays a role in sensing internal nutritional state and regulates other aspects of ingestion in addition to its role in proboscis extension (Marella, 2012).

The anatomical location of the dopaminergic interneuron highlights the central role of the SOG in taste processing and suggests that local SOG circuits may control proboscis extension behavior. Future studies identifying the downstream targets of TH-VUM will ultimately enable a deeper understanding of how dopamine achieves spatial and temporal modulation of extension probability. This study identifies an essential role for dopamine in gain control of proboscis extension to sucrose and underscores the exquisite specificity of single neurons as thin threads to behavior (Marella, 2012).

Coordination between Drosophila Arc1 and a specific population of brain neurons regulates organismal fat

The brain plays a critical yet incompletely understood role in regulating organismal fat. This study performed a neuronal silencing screen in Drosophila larvae to identify brain regions required to maintain proper levels of organismal fat. When used to modulate synaptic activity in specific brain regions, the enhancer-trap driver line E347 elevated fat upon neuronal silencing, and decreased fat upon neuronal activation. Unbiased sequencing revealed that mRNA levels of CCHC zinc finger transcription factor Arc1 increase upon E347 activation. This study revealed metabolic changes in Arc1 mutants consistent with a high-fat phenotype and an overall shift toward energy storage. Arc1-expressing cells neighbor E347 neurons, and manipulating E347 synaptic activity alters Arc1 expression patterns. Elevating Arc1 expression in these cells decreased fat, a phenocopy of E347 activation. Finally, loss of Arc1 prevented the lean phenotype caused by E347 activation, suggesting that Arc1 activity is required for E347 control of body fat. Importantly, neither E347 nor Arc1 manipulation altered energy-related behaviors. These results support a model wherein E347 neurons induce Arc1 in specific neighboring cells to prevent excess fat accumulation (Mosher, 2015).

The brain plays a critical yet incompletely understood role in regulating organismal fat. This study performed a neuronal silencing screen in Drosophila larvae to identify brain regions required to maintain proper levels of organismal fat. When used to modulate synaptic activity in specific brain regions, the enhancer-trap driver line E347 elevated fat upon neuronal silencing, and decreased fat upon neuronal activation. Unbiased sequencing revealed that mRNA levels of CCHC zinc finger transcription factor Arc1 increase upon E347 activation. This study revealed metabolic changes in Arc1 mutants consistent with a high-fat phenotype and an overall shift toward energy storage. Arc1-expressing cells neighbor E347 neurons, and manipulating E347 synaptic activity alters Arc1 expression patterns. Elevating Arc1 expression in these cells decreased fat, a phenocopy of E347 activation. Finally, loss of Arc1 prevented the lean phenotype caused by E347 activation, suggesting that Arc1 activity is required for E347 control of body fat. Importantly, neither E347 nor Arc1 manipulation altered energy-related behaviors. These results support a model wherein E347 neurons induce Arc1 in specific neighboring cells to prevent excess fat accumulation (Mosher, 2015).

MEF2 is an in vivo immune-metabolic switch

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

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

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

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

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

The control of lipid metabolism by mRNA splicing in Drosophila

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

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

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

Suppression of insulin production and secretion by a Decretin hormone

Decretins, hormones induced by fasting that suppress insulin production and secretion, have been postulated from classical human metabolic studies. From genetic screens, this study identified Drosophila Limostatin (Lst), a peptide hormone that suppresses insulin secretion. Lst is induced by nutrient restriction in gut-associated endocrine cells. limostatin deficiency leads to hyperinsulinemia, hypoglycemia, and excess adiposity. A conserved 15-residue polypeptide encoded by limostatin suppresses secretion by insulin-producing cells. Targeted knockdown of CG9918, a Drosophila ortholog of mammalian Neuromedin U receptors (NMURs), in insulin-producing cells phenocopied limostatin deficiency and attenuated insulin suppression by purified Lst, suggesting CG9918 encodes an Lst receptor. Human NMUR1 is expressed in islet β cells, and purified NMU suppressed insulin secretion from human islets. A human mutant NMU variant that co-segregates with familial early-onset obesity and hyperinsulinemia failed to suppress insulin secretion. The study proposes Lst as an index member of an ancient hormone class called decretins, which suppress insulin output (Alfa, 2015).

The coupling of hormonal responses to nutrient availability is fundamental for metabolic control. In mammals, regulated secretion of insulin from pancreatic b cells is a principal hormonal response orchestrating metabolic homeostasis. Circulating insulin levels constitute a dynamic metabolic switch, signaling the fed state and nutrient storage (anabolic pathways) when elevated, or starvation and nutrient mobilization (catabolic path ways) when decreased. Thus, insulin secretion must be precisely tuned to the nutritional state of the animal. Increased circulating glucose stimulates b cell depolarization and insulin secretion. In concert with glucose, gut-derived incretin hormones amplify glucose-stimulated insulin secretion (GSIS) in response to ingested carbohydrates, thereby tuning insulin output to the feeding state of the host (Alfa, 2015).

While the incretin effect on insulin secretion during feeding is well-documented, counter-regulatory mechanisms that suppress insulin secretion during or after starvation are incompletely understood. Classical starvation experiments in humans and other mammals revealed that sustained fasting profoundly alters the dynamics of insulin production and secretion, resulting in impaired glucose tolerance, relative insulin deficits, and 'starvation diabetes'. Remarkably, starvation-induced suppression of GSIS was not reverted by normalizing circulating glucose levels, suggesting that the dampening effect of starvation on insulin secretion perdures and is uncoupled from blood glucose and macronutrient concentrations. Based on these observations, it has been postulated that hormonal signals induced by fasting may actively attenuate insulin secretion suggested that enteroendocrine 'decretin' hormones may constrain the release of insulin to prevent hypoglycemia. This concept is further supported by recent studies identifying a G protein that suppresses insulin secretion from pancreatic b cell. Thus, after nutrient restriction, decretin hormones could signal through G protein-coupled receptors (GPCRs) to attenuate GSIS from b cells (Alfa, 2015).

The discovery of hormonal pathways regulating metabolism in mammals presents a formidable challenge. However, progress has revealed conserved mechanisms of metabolic regulation by insulin and glucagon-like peptides in Drosophila, providing a powerful genetic model to address unresolved questions relevant to mammalian metabolism. Similar to mammals, secretion of Drosophila insulin-like peptides (Ilps) from neuroendocrine cells in the brain regulates glucose homeostasis and nutrient stores in the fly. Ilp secretion from insulin-producing cells (IPCs) is responsive to circulating glucose and macronutrients and is suppressed upon nutrient withdrawal. Notably, recent studies have identified hormonal and GPCR-linked mechanisms regulating the secretion of Ilps from IPCs, suggesting further conservation of pathways regulating insulin secretion in the fly (Alfa, 2015).

In mammals, the incretin hormones gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by enteroendocrine cells following a meal and enhance glucose-stimulated insulin production and secretion from pancreatic b cells. Thus, It was postulated that a decretin hormone would have the 'opposite' hallmarks of incretins. Specifically, a decretin (1) derives from an enteroendocrine source that is sensitive to nutrient availability, (2) is responsive to fasting or carbohydrate deficiency, and (3) suppresses insulin production and secretion from insulin-producing cells. However, like incretins, the action of decretins on insulin secretion would be manifest during feeding, when a stimulus for secretion is present (Alfa, 2015).

This study identifed a secreted hormone, Limostatin (Lst), that suppresses insulin secretion following starvation in Drosophila. lst is regulated by starvation, and flies deficient for lst display phenotypes consistent with hyperinsulinemia. Lst production was shown to be localized to glucose-sensing, endocrine corpora cardiaca (CC) cells associated with the gut, and show that lst is suppressed by carbohydrate feeding. Using calcium imaging and in vitro insulin secretion assays, a 15-aa Lst peptide (Lst-15) was identified that is sufficient to suppress activity of IPCs and Ilp secretion. An orphan GPCR was identified in IPCs as a candidate Lst receptor. Moreover, Neuromedin U (NMU) is likely a functional mammalian ortholog of Lst that inhibits islet b cell insulin secretion. These results establish a decretin signaling pathway that suppresses insulin output in Drosophila (Alfa, 2015).

Limostatin is a peptide hormone induced by carbohydrate restriction from endocrine cells associated with the gut that suppresses insulin production and release by insulin-producing cells. Thus, Drosophila Lst fulfills the functional criteria for a decretin and serves as an index member of this hormone class in metazoans. Results here also show that Lst signaling from corpora cardica cells may be mediated by the GPCR encoded by CG9918 in insulin-producing cells. In addition, the results reveal cellular and molecular features of a cell-cell signaling system in Drosophila with likely homologies to a mammalian entero-insular axis (Alfa, 2015).

Reduction of nutrient-derived secretogogues, like glucose, is a primary mechanism for attenuating insulin output during starvation in humans and flies. Consistent with this, it was found that circulating Ilp2HF levels were reduced to a similar degree in lst mutant or control flies during prolonged fasting. Therefore, lst was dispensable for Ilp2 reduction during fasting. However, lst mutants upon refeeding or during subsequent ad libitum feeding had enhanced circulating Ilp2HF levels compared to controls, findings that demonstrate a requirement for Lst to restrict insulin output in fed flies. Thus, while induced by nutrient restriction, Lst decretin function was revealed by nutrient challenge. This linkage of feeding to decretin regulation of insulin output is reminiscent of incretin regulation and action (Alfa, 2015 and references therein).

Recent studies have demonstrated functional conservation in Drosophila of fundamental hormonal systems for metabolic regulation in mammals, including insulin, glucagon, and leptin. This study used Drosophila to identify a hormonal regulator of insulin output, glucose, and lipid metabolism without an identified antecedent mammalian ortholog -- emphasizing the possibility for work on flies to presage endocrine hormone discovery in mammals. Gain of Lst function in these studies led to reduced insulin signaling, and hyperglycemia, consistent with prior work. By contrast, loss of Lst function led to excessive insulin production and secretion, hypoglycemia, and elevated triglycerides, phenotypes consistent with the recognized anabolic functions of insulin signaling in metazoans, and with the few prior metabolic studies of flies with insulin excess (Alfa, 2015).

Prior studies show that somatostatin and galanin are mammalian gastrointestinal hormones that can suppress insulin secretion. Somatostatin-28 (SST-28) is a peptide derivative of the pro-somatostatin gene that is expressed widely, including in gastrointestinal cells and pancreatic islet cells. Islet somatostatin signaling is thought to be principally paracrine, rather than endocrine, and serum SST-28 concentrations increase post-prandially. Galanin is an orexigenic neuropeptide produced throughout the CNS and in peripheral neurons and has been reported to inhibit insulin secretion. Unlike enteroendocrine-derived hormones that act systemically, galanin is secreted from intrapancreatic autonomic nerve terminals and is thought to exert local effects. In addition, Galanin synthesis and secretion are increased by feeding and dietary fat. Thus, like incretins, output of SST- 28 and galanin are induced by feeding, but in contrast to incretins, these peptides suppress insulin secretion. Further studies are needed to assess the roles of these peptide regulators in the modulation of insulin secretion during fasting (Alfa, 2015).

While sequence-based searches did not identify vertebrate orthologs of Lst, this study found that the postulated Lst receptor in IPCs, encoded by CG9918, is most similar to the GPCRs NMUR1 and NMUR2. In rodents, NMU signaling may be a central regulator of satiety and feeding behavior, and this role may be conserved in other organisms. In addition, NMU mutant mice have increased adiposity and hyperinsulinemia, but a direct role for NMU in regulating insulin secretion by insulin-producing cells was not identified. In rodents, the central effects of NMU on satiety are thought to be mediated by the receptor NMUR2; however, hyperphagia, hyperinsulinemia, and obesity were not reported in NMUR2 mutant mice. Together, these studies suggest that a subset of phenotypes observed in NMU mutant mice may instead reflect the activity of NMU on peripheral tissues like pancreatic islets, but this has not been previously shown. Notably, humans harboring the NMU R165W allele displayed obesity and elevated insulin C-peptide levels, without evident hyperphagia -- further suggesting that the central and peripheral effects of NMU reflect distinct pathways that may be uncoupled. This study has shown that NMU is produced abundantly in human foregut organs and suppresses insulin secretion from pancreatic b cells, supporting the view that NMU has important functions outside the CNS in regulating metabolism. Thus, like the incretin GLP-1, NMU may have dual central and peripheral signaling functions in the regulating metabolism. Demonstration that NMU is a mammalian decretin will require further studies on NMU regulation and robust methods to measure circulating NMU levels in fasting and re-feeding. In summary, these findings should invigorate searches for mammalian decretins with possible roles in both physiological and pathological settings (Alfa, 2015).

Energy homeostasis control in Drosophila adipokinetic hormone mutants

Maintenance of biological functions under negative energy balance depends on mobilization of storage lipids and carbohydrates in animals. In mammals, glucagon and glucocorticoid signaling mobilizes energy reserves, whereas Adipokinetic hormones (AKHs) play a homologous role in insects. Numerous studies based in AKH injections and correlative studies in a broad range of insect species established the view that AKH acts as master regulator of energy mobilization during development, reproduction, and stress. In contrast to AKH, the second peptide, which is processed from the Akh encoded prohormone - termed Adipokinetic hormone precursor related peptide (APRP) - is functionally orphan. APRP is discussed as ecdysiotropic hormone or as scaffold peptide during AKH prohormone processing. However, as in the case of AKH, final evidence for APRP functions requires genetic mutant analysis. This study employed CRISPR/Cas9-mediated genome engineering to create AKH and AKH plus APRP-specific mutants in the model insect Drosophila melanogaster. Lack of APRP did not affect any of the tested steroid-dependent processes. Similarly, Drosophila AKH signaling is dispensable for ontogenesis, locomotion, oogenesis, and homeostasis of lipid or carbohydrate storage until up to the end of metamorphosis. During adulthood, however, AKH regulates body fat content and the hemolymph sugar level as well as nutritional and oxidative stress responses. Finally, evidence is provided for a negative auto-regulatory loop, in Akh gene regulation (Galikova, 2015).

Neuronal energy-sensing pathway promotes energy balance by modulating disease tolerance

The starvation-inducible coactivator cAMP response element binding protein (CREB)-cAMP-regulated transcription coactivator (Crtc) has been shown to promote starvation resistance in Drosophila by up-regulating CREB target gene expression in neurons, although the underlying mechanism is unclear. This study found that Crtc and its binding partner CREB enhance energy homeostasis by stimulating the expression of short neuropeptide F (sNPF), an ortholog of mammalian neuropeptide Y, which was shown to be a direct target of CREB and Crtc. Neuronal sNPF was found to promote energy homeostasis via gut enterocyte sNPF receptors, which appear to maintain gut epithelial integrity. Loss of Crtc-sNPF signaling disrupts epithelial tight junctions, allowing resident gut flora to promote chronic increases in antimicrobial peptide (AMP) gene expression that compromised energy balance. Growth on germ-free food reduces AMP gene expression and rescues starvation sensitivity in Crtc mutant flies. Overexpression of Crtc or sNPF in neurons of wild-type flies dampens the gut immune response and enhances starvation resistance. These results reveal a previously unidentified tolerance defense strategy involving a brain-gut pathway that maintains homeostasis through its effects on epithelial integrity (Shen, 2016).

Disruptions in energy balance are a component of the collateral damage associated with mounting an immune response. In addition to regulating the magnitude of an immune response, energy allocation must be properly regulated to minimize physiological damage during infection. This study found that Drosophila sNPF, a mammalian NPY homolog, is regulated by CrebB/Crtc within the CNS, where it promotes energy balance by maintaining epithelial integrity and thereby attenuating overexuberant immune activation in the gut. The effects of sNPF were unexpected, given its role in food-seeking behavior. Indeed, food intake appears comparable between Crtc mutants and control flies (Shen, 2016).

The effects of sNPF are mediated by enterocyte sNPF-Rs, suggesting that the sNPF brain-gut signal is released by a subset of the sNPF+ neurons that directly innervate the gut. Although neuronal activity is known to contribute to energy homeostasis, the results suggest that the modulation of the gut immune system by CrebB/Crtc is a critical component in this process (Shen, 2016).

Epithelial tissues are typically colonized by both commensal and invasive microbes. sNPF appears to be actively expressed and released from the CNS in times of stress, providing nonautonomous control of gut immunity from the brain. Based on its widespread expression in the midgut, sNPF-R may provide ubiquitous attenuation of the innate immune response. Consistent with observations in Drosophila, activation of the NPY receptor ortholog (NPR-1) in Caenorhabditis elegans also down-regulates inflammatory gene expression. The current studies extend these findings by showing how a neuronal fasting-inducible pathway modulates energy balance via its effects on the gut immune system (Shen, 2016).

Following their activation, sNPF-Rs appear to promote energy balance by enhancing epithelial integrity. Although the mechanism underlying these effects is unclear, it is noted that disruption of the tight junction protein Bbg in flies also causes constitutive up-regulation of innate immunity genes. Future studies should reveal whether sNPF-R modulates the activity of Bbg or related proteins in enterocytes (Shen, 2016).

In mammals, inflammatory bowel diseases, such as ulcerative colitis, are often associated with profound weight loss, due, in part, to the chronic activation of the immune system. By reducing inflammatory gene expression and enhancing energy homeostasis, gut neuropeptides, such as NPY, may provide therapeutic benefit in this setting (Shen, 2016).

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

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

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

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

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

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

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

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

The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila

Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers (Singh, 2016)

To investigate the molecular mechanism behind the resistance of CSCs to therapeutics, the death of stem cells with different degrees of quiescence was studied in the adult Drosophila digestive system, including intestinal stem cells (ISCs). Expression of the proapoptotic genes rpr and p53 effectively ablated differentiated cells but had little effect on stem cells (Singh, 2016).

In mammals, treatment-resistant leukaemic stem cells (LSCs) can be eliminated by a two-step protocol involving initial activation by interferon-α (IFNα) or colony-stimulating factor (G-CSF), followed by targeted chemotherapy. In Drosophila, activation of the hopscotch (also known as JAK)-Stat92E signalling pathway induces hyperplastic stem cells, which are overproliferating, but retain their apico-basal polarity and differentiation ability. A slightly different two-step protocol was conducted in Drosophila stem cells by overexpressing the JAK-Stat92E pathway ligand unpaired (upd) and rpr together. The induction of upd + rpr using the temperature-sensitive (ts) mutant esg-Gal4 (esg^ts > upd + rpr effectively ablated all of the ISCs and RNSCs through apoptosis within four days. Consistent with this result, expressing a gain-of-function Raf mutant (Raf^gof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

Expressing a constitutively active form of Ras oncogene at 85D (also known as Ras^V12) in RNSCs and the knockdown of Notch activity in ISCs can transform these cell types into CSC-like neoplastic stem cells, which were not only overproliferating, but also lost their apico-basal polarity and differentiation abilit. It ws found that expressing rpr in Ras^V12-transformed RNSCs or in ISCs expressing a dominant-negative form of Notch (N^DN) caused the ablation of only a proportion of the transformed RNSCs and few transformed ISCs and it did not affect differentiated cells; substantial populations of the neoplastic stem cells remained even seven days after rpr induction (Singh, 2016).

These results suggest that the activation of proliferation can accelerate the apoptotic cell death of hyperplastic stem cells, but that a proportion of actively proliferating neoplastic RNSCs and ISCs are resistant to apoptotic cell death. Neoplastic tumours in Drosophila are more similar to high-grade malignant human tumours than are the hyperplastic Drosophila tumours (Singh, 2016).

Vesicle-mediated COPI and COPII are essential components of the trafficking machinery for vesicle transportation between the endoplasmic reticulum and the Golgi. In addition, the COPI complex regulates the transport of lipolysis enzymes to the surface of lipid droplets for lipid droplet usage. In a previous screen, it was found that knockdown of COPI components (including Arf79F, the Drosophila homologue of ADP-ribosylation factor 1 (Arf1)) rather than COPII components resulted in stem-cell death, suggesting that lipid-droplet usage (lipolysis) rather than the general trafficking machinery between the endoplasmic reticulum and Golgi is important for stem-cell survival (Singh, 2016)

To further investigate the roles of these genes in stem cells, a recombined double Gal4 line of esg-Gal4 and wg-Gal4 was used to express genes in ISCs, RNSCs, and HISCs (esg^ts wg^ts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79F^RNAi in enterocytes or in differentiated stellate cells in Malpighian tubules did not cause similar marked ablation. These results suggest that Arf79F knockdown selectively kills stem cells and not differentiated cells (Singh, 2016).

It was also found that expressing Arf79F^RNAi in Ras^V12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79F^RNAi in N^DN-transformed ISCs ablated all of the cells within one week, but restored differentiated cells to close to their normal levels within one week (Singh, 2016).

δ-COP- and γ-COP-mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique, and it was found that the COPI complex cell-autonomously regulated stem cell survival. In summary, knockdown of the COPI-Arf79F complex effectively ablated normal and transformed stem cells but not differentiated enterocytes or stellate cells (Singh, 2016)

In the RNAi screen acyl-CoA synthetase long-chain (ACSL), an enzyme in the Drosophila lipolysis-β-oxidation pathway, and bubblegum (bgm), a very long-chain fatty acid-CoA ligase, were also identified. RNAi-mediated knockdown of Acsl and bgm effectively killed ISCs and RNSCs, but killed HISCs less effectively. Expressing Acsl^RNAi in Ras^V12-transformed RNSCs also ablated almost all of the transformed RNSCs in one week (Singh, 2016).

Brummer (bmm) is a triglyceride lipase, the Drosophila homologue of mammalian ATGL, the first enzyme in the lipolysis pathway. Scully (scu) is the Drosophila orthologue of hydroxy-acyl-CoA dehydrogenase, an enzyme in the β-oxidation pathway. Hepatocyte nuclear factor 4 (Hnf4) regulates the expression of several genes involved in lipid mobilization and β-oxidation. To determine whether the lipolysis-β-oxidation pathway is required for COPI-Arf79F-mediated stem cell survival, upstream activating sequence (UAS)-regulated constructs (UAS-bmm, UAS-Hnf4, and UAS-scu) were also expressed in stem cells that were depleted of Arf79F, β-COP, or ζ-COP. Overexpressing either scu or Hnf4 significantly attenuated the stem cell death caused by knockdown of the COPI-Arf79F complex. Expressing UAS-Hnf4 MARCM clones also rescued the stem cell death phenotype induced by γ-COP knockdown. However, bmm overexpression did not rescue the stem-cell death induced by Arf79F knockdown. Since there are several other triglyceride lipases in Drosophila in addition to bmm, another lipase may redundantly regulate the lipolysis pathway (Singh, 2016)

To further investigate the function of lipolysis in stem cells, the expression of a lipolysis reporter (GAL4-dHFN4; UAS-nlacZ which consisted of hsp70-GAL4-dHNF4 combined with a UAS-nlacZ reporter gene was investigated. The flies were either cultured continuously at 29°C or heat-shocked for 30 min at 37°C, 12 h before dissection. Without heat shock, the reporter was expressed only in ISCs and RNSCs of mature adult flies, but not in enteroendocrine cells, enterocytes, quiescent HISCs or quiescent ISCs of freshly emerged young adult flies (less than 3 days old. Expressing δ-COP^RNAi almost completely eliminated the reporter expression, suggesting that the reporter was specifically regulated by the COPI complex. After heat shock or when a constitutively active form of JAK (hop^Tum-l) was expressed, the reporter was strongly expressed in ISCs, RNSCs and HISCs, but not in enteroendocrine cells or enterocytes. These data suggest that COPI-complex-regulated lipolysis was active in stem cells, but not in differentiated cells, and that the absence of the reporter expression in quiescent HISCs at 29°C was probably owing to weak hsp70 promoter activity rather than to low lipolysis in these cells (Singh, 2006).

Lipid storage was futher investigated, and it was found that the size and number of lipid droplets were markedly increased in stem cells after knockdown of Arf79F (Singh, 2016).

Arf1 inhibitors (brefeldin A, golgicide A, secin H3, LM11 and LG8) and fatty-acid-oxidation (FAO) inhibitors (triacsin C, mildronate, etomoxir and enoximone) were used, and it was found that these inhibitors markedly reduced stem-cell tumours in Drosophila through the lipolysis pathway but had a negligible effect on normal stem cells (Singh, 2016)

These data together suggest that the COPI-Arf1 complex regulates stem-cell survival through the lipolysis-β-oxidation pathway, and that knockdown of these genes blocks lipolysis but promotes lipid storage. Further, the transformed stem cells are more sensitive to Arf1 inhibitors and may be selectively eliminated by controlling the concentration of Arf1 inhibitors (Singh, 2016)

These data suggest that neither caspase-mediated apoptosis nor autophagy-regulated cell death regulates the stem-cell death induced by the knockdown of components of the COPI-Arf79F complex. Therefore whether necrosis regulates the stem-cell death induced by knockdown of the COPI-Arf79F complex was investigated. Necrosis is characterized by early plasma membrane rupture, reactive oxygen species (ROS) accumulation and intracellular acidification. Propidium iodide detects necrotic cells with compromised membrane integrity, the oxidant-sensitive dye dihydroethidium (DHE) indicates cellular ROS levels and LysoTracker staining detects intracellular acidification. The membrane rupture phenotype was detected only in esg and the propidium iodide signal was observed only in ISCs from flies that had RNAi-induced knockdown of expression of COPI-Arf79F components, and not in cells from wild-type flies. In the esg^ts wg^ts > Acsl^RNAi flies, all of the ISCs and RNSCs were ablated after four days at 29°C, but a fraction of the HISCs remained, and these were also propidium iodide positive, indicating that the HISCs were dying slowly. This slowness may have been due to either a lower GAL4 (wg-Gal4) activity in these cells compared to ISCs and RNSCs (esg-Gal4) or quiescence of the HISCs. Furthermore, strong propidium iodide signals were detected in transformed ISCs from esg^ts > N^DN + Arf79F^RNAi but not esg^ts flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016)

Similarly, DHE signals were detected only in ISCs from esg^ts > Arf79F^RNAi flies, indicating that the dying ISCs had accumulated ROS and were intracellularly acidified. Overexpressing catalase (a ROS-chelating enzyme) rescued the stem-cell death specifically induced by the γ-COP mutant clone, and the ROS inhibitor NAC blocked the Arf1 inhibitor-induced death of Ras^V12-induced RNSC tumours. These data together suggest that knockdown of the COPI-Arf1 complex induced the death of stem cells or of transformed stem cells (Ras^V12-RNSCs, N^DN-ISCs) through ROS-induced necrosis. Although ISCs, RNSCs, and HISCs exhibit different degrees of quiescence, they all rely on lipolysis for survival, suggesting that this is a general property of stem cells (Singh, 2016)

Cases were noticed where the GFP-positive material of the dying ISCs was present within neighbouring enterocytes, suggesting that these enterocytes had engulfed dying ISCs (Singh, 2016)

The JNK pathway, autophagy and engulfment genes are involved in the engulfment of dying cells. Therefore, whether these genes are required for COPI-Arf79F-regulated ISC death was investigated. The following was found: (1) ISC death activated JNK signalling and autophagy in neighbouring enterocytes; (2) knockdown of these genes in enterocytes but not in ISCs rescued ISC death to different degrees; (3) the drpr-mbc-Rac1-JNK pathway in enterocytes is not only necessary but also sufficient for ISC death; and (4) inhibitors of JNK and Rac1 could block Arf1-inhibitor-induced cell death of the Ras^V12-induced RNSC tumours. These data together suggest that the drpr-mbc-Rac1-JNK pathway in neighbouring differentiated cells controls the engulfment of dying or transformed stem cells (Singh, 2016)

The finding that the COPI-Arf79F-lipolysis-β-oxidation pathway regulated transformed stem-cell survival in the fly led to an investigation of whether the pathway has a similar role in CSCs. WTwo Arf1 inhibitors (brefeldin A and golgicide A) and two FAO inhibitors (triascin C and etomoxir) were tested on human cancer cell lines, and it was found that the growth, tumoursphere formation and expression of tumour-initiating cell markers of the four cancer cell lines were significantly suppressed by these inhibitors, suggesting that these inhibitors suppress CSCs. In mouse xenografts of BSY-1 human breast cancer cells, a novel low-cytotoxicity Arf1-ArfGEF inhibitor called AMF-26 was reported to induce complete regression in vivo in five days. Together, this report and the current results suggest that inhibiting Arf1 activity or blocking the lipolysis pathway can kill CSCs and block tumour growth (Singh, 2016)

Stem cells or CSCs are usually localized to a hypoxic storage niche, surrounded by a dense extracellular matrix, which may make them less accessible to sugar and amino acid nutrition from the body's circulatory system. Most normal cells rely on sugar and amino acids for their energy supply, with lipolysis playing only a minor role in their survival. The current results suggest that stem cells and CSCs are metabolically unique; they rely mainly on lipid reserves for their energy supply, and blocking COPI-Arf1-mediated lipolysis can starve them to death. It was further found that transformed stem cells were more sensitive than normal stem cells to Arf1 inhibitors. Thus, selectively blocking lipolysis may kill CSCs without severe side effects. Therefore, targeting the COPI-Arf1 complex or the lipolysis pathway may prove to be a well-tolerated, novel approach for eliminating CSCs (Singh, 2016)

Cross-phenotype association tests uncover genes mediating nutrient response in Drosophila

Obesity-related diseases are major contributors to morbidity and mortality in the developed world. Molecular diagnostics and targets of therapies to combat nutritional imbalance are urgently needed in the clinic. Invertebrate animals have been a cornerstone of basic research efforts to dissect the genetics of metabolism and nutrient response. This study used fruit flies reared on restricted and nutrient-rich diets to identify genes associated with starvation resistance, body mass and composition, in a survey of genetic variation across the Drosophila Genetic Reference Panel (DGRP). Starvation resistance, body weight and composition were measured in DGRP lines on each of two diets, and several association mapping strategies were used to harness this panel of phenotypes for molecular insights. DNA sequence variants were tested for a relationship with single metabolic traits and with multiple traits at once, using a scheme for cross-phenotype association mapping; association tests focused on homologs of human disease genes and common polymorphisms; and gene-by-diet interactions were tested. The results revealed gene and gene-by-diet associations between 17 variants and body mass, whole-body triglyceride and glucose content, or starvation resistance. Focused molecular experiments validated the role in body mass of an uncharacterized gene, CG43921 (which was rename heavyweight), and previously unknown functions for the diacylglycerol kinase rdgA, the huntingtin homolog htt, and the ceramide synthase schlank in nutrient-dependent body mass, starvation resistance, and lifespan. The findings implicate a wealth of gene candidates in fly metabolism and nutrient response, and ascribe novel functions to htt, rdgA, hwt and schlank (Nelson, 2016).

Dietary L-arginine accelerates pupation and promotes high protein levels but induces oxidative stress and reduces fecundity and life span in Drosophila melanogaster

L-Arginine, a precursor of many amino acids and of nitric oxide, plays multiple important roles in nutrient metabolism and regulation of physiological functions. In this study, the effects of L-arginine-enriched diets on selected physiological responses and metabolic processes were assessed in Drosophila melanogaster. Dietary L-arginine at concentrations 5-20 mM accelerated larval development and increased body mass, and total protein concentrations in third instar larvae, but did not affect these parameters when diets contained 100 mM arginine. Young (2 days old) adult flies of both sexes reared on food supplemented with 20 and 100 mM L-arginine possessed higher total protein concentrations and lower glucose and triacylglycerol concentrations than controls. Additionally, flies fed 20 mM L-arginine had higher proline and uric acid concentrations. L-Arginine concentration in the diet also affected oxidative stress intensity in adult flies. Food with 20 mM L-arginine promoted lower protein thiol concentrations and higher catalase activity in flies of both sexes and higher concentrations of low molecular mass thiols in males. When flies were fed on a diet with 100 mM L-arginine, lower catalase activities and concentrations of protein thiols were found in both sexes as well as lower low molecular mass thiols in females. L-Arginine-fed males demonstrated higher climbing activity, whereas females showed higher cold tolerance and lower fecundity, compared with controls. Food containing 20 mM L-arginine shortened life span in both males and females. The results suggest that dietary L-arginine shows certain beneficial effects at the larval stage and in young adults. However, the long-term consumption of L-arginine-enriched food had unfavorable effects on D. melanogaster due to decreasing fecundity and life span (Bayliak, 2017).

Adaptation to dietary conditions by trehalose metabolism in Drosophila

Trehalose is a non-reducing disaccharide that serves as the main sugar component of haemolymph in insects. Trehalose hydrolysis enzyme, called trehalase, is highly conserved from bacteria to humans. However, understanding of the physiological role of trehalase remains incomplete. This study analyzed the phenotypes of several Trehalase (Treh) loss-of-function alleles in a comparative manner in Drosophila. The previously reported mutant phenotype of Treh affecting neuroepithelial stem cell maintenance and differentiation in the optic lobe is caused by second-site alleles in addition to Treh. It is further reported that the survival rate of Treh null mutants is significantly influenced by dietary conditions. Treh mutant larvae are lethal not only on a low-sugar diet but also under low-protein diet conditions. A reduction in adaptation ability under poor food conditions in Treh mutants is mainly caused by the overaccumulation of trehalose rather than the loss of Treh, because the additional loss of Trehalose-6-phosphate synthase 1 mitigates the lethal effect of Treh mutants. These results demonstrate that proper trehalose metabolism plays a critical role in adaptation under various environmental conditions (Yasugi, 2017).

Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger

Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. This study identified a dopamine circuit that encodes protein-specific hunger in Drosophila. The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis (Liu, 2017).

Beyond satisfying caloric needs, animals must also ingest nutrients that cannot be biosynthesized. The 'specific appetite' hypothesis posits that nutrient-specific hunger drives homeostatic consumption of substances such as protein and salt. Protein is an indispensable macronutrient and is particularly required in anabolic states, such as infancy and pregnancy. Recent studies in Drosophila have identified signaling mechanisms regulating protein hunger, particularly in the context of postmating effects. However, specific circuits encoding protein hunger remain unknown (Liu, 2017).

Yeast is an ethologically relevant protein food source for Drosophila, containing a negligible amount of sugars. Studies in fruit flies demonstrated that mated females have greater drive for protein and amino acid intake. This study used this potent physiological drive for protein in mated females as an entry point to investigate the neural basis of protein hunger. Yeast-feeding in mated females were assayed after conditional silencing of different neuromodulatory cell groups. Dopamine (DA) neurons were specifically required for yeast consumption and preference. To identify the specific DA neurons mediating this effect, two restricted Gal4 drivers (TH-C-Gal4 and TH-D-Gal4) containing regulatory sequences of the tyrosine hydroxylase gene (TH) were used, and it was found that conditional silencing of the neurons labeled with either driver led to significantly reduced protein preference. These drivers label largely nonoverlapping DA cells, aside from two cells in each protocerebral posterior medial 2 (PPM2) subgroup (Liu, 2017).

To isolate these PPM2 neurons, an intersectional approach (TH-C-FLP with TH-D-Gal4, was used where FLP encodes flippase) to drive expression of FRT-stop-FRT-mCD8-GFP (a membrane tethered green fluorescent protein). Two PPM2 neurons in each hemisphere were identifed that project ventrally to the 'wedge' neuropil. On the basis of this projection pattern, these two PPM2 neurons were named DA-WED cells. Next, using the FLP-induced intersectional GAL80/GAL4 repression (FINGR) system [TH-D-Gal4, TH-C-FLP, tub-FRT-Gal80-FRT (WED1-Gal4)] to express dendritic versus terminal markers, it was found that the wedge area contains the dendritic field, whereas the two dorsally bifurcating branches contain presynaptic terminals. Intersection between TH-C-Gal4 and another restricted driver TH-F3-Gal4 (15) (WED2-Gal4) also revealed the two DA-WED neurons. These cells were specifically inactivated with the inward-rectifying potassium channel Kir2.1, and substantial inhibition was found of yeast preference and consumption. General hunger, thirst, and salt appetite were not affected when DA-WED neurons were silenced (Liu, 2017).

It was next asked whether the DA-WED neurons play a general role in homeostatic regulation of protein intake, beyond mated females. Although male flies did not prefer yeast at baseline, they exhibited significant protein preference and consumption after yeast deprivation for 8 days, which was fully suppressed if an amino acid mix was provided during the deprivation period. Inactivating the DA-WED neurons significantly reduced yeast preference and consumption in protein-deprived male flies. Conversely, conditional activation of these cells with the heat-activated cation channel dTrpA1 induced yeast preference and consumption in males. Similar data were obtained for virgin females. Conditional silencing or activation of DA-WED neurons reduced or increased protein consumption, respectively, over a range of internal protein-hunger states in both mated female and male flies, it was decided to focus on male flies for subsequent experiments to avoid postmating effects. Reducing DA levels in DA-WED neurons by knocking down the neuronal specific isoform of TH in these cells decreased yeast intake in protein-starved male flies (Liu, 2017).

To investigate whether the activity of the DA-WED neurons correlates with protein need, perforated patch-clamp recordings were performed from these neurons. After yeast deprivation, the spontaneous action potential (AP) firing rate of DA-WED neurons increased about fourfold compared to controls. Moreover, evoked AP firing rates were higher at all measured depolarizing currents after yeast deprivation. Protein starvation did not significantly alter the resting membrane potential or input resistance of these cells. Cytosolic Ca2+ concentrations in these cells (but not in nearby PPM3 DA neurons) was substantially increased after yeast deprivation when monitored with the CaLexA (calcium-dependent nuclear import of LexA) activity reporter system. This increase in intracellular Ca2+ concentration was suppressed when an amino acid mix was provided in the protein-deficient diet. This effect was specific for protein, as general starvation did not affect CaLexA signal in the DA-WED neurons. Similar data were obtained with GCaMP imaging, as an alternative method to measure cytosolic Ca2+ levels (Liu, 2017).

What component of protein serves to signal protein satiety to the DA-WED neurons? It was hypothesized that a specific amino acid may play this role. Flies were first subjected to protein deprivation, but provided individual amino acids in the diet, and then monitored the activity of DA-WED neurons using CaLexA. Tryptophan and glutamine (Gln) supplementation suppressed the enhanced activity of DA-WED neurons induced by protein deprivation, and proline and glutamate supplementation showed a trend toward this effect. Next, whether supplementation of these four individual amino acids modulated homeostatic regulation of yeast intake was tested. Yeast intake was significantly inhibited when Gln was added back to a protein-deficient diet. Conversely, an amino acid mix lacking only Gln failed to suppress the increase in DA-WED activity and was less effective at inhibiting protein consumption after yeast deprivation. It was also found that Gln levels in the hemolymph were significantly reduced after protein deprivation. Together, these data suggest that Gln in particular may be important for signaling protein deficiency (Liu, 2017).

Despite strong preference for sucrose at baseline, yeast deprivation caused flies to decrease their consumption of this nutrient. Because DA-WED neurons are activated after substantial protein deprivation, it was hypothesized that they also play a direct role in reducing sucrose intake under these conditions. Silencing DA-WED neurons with Kir2.1 significantly enhanced sugar intake after protein deprivation, whereas activating these neurons reduced sucrose consumption (Liu, 2017).

The mechanisms underlying the opposing effects of DA-WED neurons on sucrose and yeast intake was investigated. There are four DA receptors in Drosophila encoded by DopR1, DopR2, D2R, and DopEcR. Both DopR1 and DopR2 mutants exhibited a reduced preference for yeast after protein deprivation in a two-choice assay, which could reflect either a reduced preference for protein or a greater preference for sucrose. Thus, focus was placed on these two receptors as potential targets acting downstream of the DA-WED neurons. In one-choice assays, DopR2 mutants exhibited significantly reduced yeast intake, whereas DopR1 mutants displayed a robust increase in sucrose feeding. Mutations in DopR2 and DopR1 also suppressed the increase in yeast consumption and decrease in sucrose feeding triggered by activating DA-WED neurons, respectively. These findings were confirmed by pan-neuronal knockdown of DopR1 or DopR2 (nsyb-Gal4>UAS-DopR1-miR or nsyb-Gal4>UAS-DopR2-miR), which yielded similar results. It was therefore hypothesized that DopR2 and DopR1 act in discrete neuronal targets of the DA-WED cells to regulate protein and sugar feeding, respectively (Liu, 2017).

To identify putative candidate circuits acting downstream of the DA-WED cells, a GRASP (GFP reconstitution across synaptic partners)-based screen was performed with Rubin Gal4 drivers derived from DopR2 and DopR1 enhancer sequences to assess connectivity with the DA-WED neurons. Among DopR2-derived driver lines, only the R70G12-Gal4 line exhibited GRASP signal at the DA-WED terminal branches (specifically, the medial branch). Moreover, knockdown of DopR2 with R70G12-Gal4 significantly reduced yeast intake after protein starvation, without affecting general hunger or sucrose consumption in yeast-deprived animals. This reduction in protein appetite in R70G12-Gal4>UAS-DopR2-miR flies was observed over a range of protein-hunger states in both mated female and male flies. To identify the specific cells in this driver that contact the DA-WED neurons, a multicolor flip out (MCFO) experiment was performed. A neuron whose cell body is located in the posterior medial protocerebrum and sends its projections first to the fan-shaped body (FB), before extending more anteriorly to the lateral accessory lobe (LAL) area was identified where the GRASP signal was observed. These neurons were named FB-LAL cells based on this projection pattern. An independent driver R75B10-Gal4 was identified that also labeled the FB-LAL neuron and displayed GRASP signal at the medial branch of the DA-WED neurons. Knockdown of DopR2 with R75B10-Gal4 decreased yeast intake in protein-starved flies. As expected, intersection between R70G12-Gal4 and R75B10-Gal4 drivers (R70G12-Gal4, R75B10-LexA>LexAop-FLP, UAS-FRT-stop-FRT-mCD8-GFP) revealed two pairs of FB-LAL neurons. Silencing these cells reduced yeast intake in protein-starved flies, whereas activating these cells induced elevated yeast consumption in male flies. Activation of DA-WED neurons expressing ATP (adenosine 5'-triphosphate)-gated P2X2 receptors induced a substantial increase in GCaMP signal in the cell bodies of downstream FB-LAL neurons (Liu, 2017).

By contrast, among the DopR1-derived lines, the R72B03-Gal4 driver demonstrated GRASP signal at the DA-WED lateral branches. Knockdown of DopR1 with the R72B03-Gal4 driver triggered an increase in sucrose intake in protein-deprived flies, without affecting general hunger or yeast intake after protein starvation. MCFO analysis using R72B03-Gal4 revealed a small subset of neurons in the posterior lateral protocerebrum (PLP) that send their projections locally to the ventrolateral and superior neuropils, so these cells were named PLP neurons. PLP neurons were also labeled by another driver line R32A06-Gal4. Similar to R72B03-Gal4, R32A06-Gal4 exhibited GRASP signal at the lateral branch of DA-WED neurons. MCFO analysis with the new R32A06-Gal4 driver revealed cells whose cell body location and projection pattern match that seen with the PLP neuron identified in the R72B03-Gal4 driver. Knockdown of DopR1 with R32A06-Gal4 increased sucrose intake in protein-starved flies. Silencing or activating these PLP neurons with R72B03-Gal4 or R32A06-Gal4 led to a reduction or an increase, respectively, of sucrose intake (Liu, 2017).

Following substantial deprivation of an essential nutrient, animals must engage in persistent behavior aimed at replenishing it. Plasticity of relevant neural circuits may underlie the persistent drive for motivated behaviors. To address whether the DA-WED neurons undergo protein hunger-dependent plastic changes, the morphology of their terminal branches was assessed using mCD8-GFP and a GFP-tagged version of the synaptic vesicle-associated protein synaptotagmin (Syt-GFP). The medial, but not the lateral, branches were significantly elongated following protein deprivation, which was suppressed when Gln was selectively restored in the protein-deficient diet. Similar data were obtained for the number and total volume of Syt-GFP+ puncta. To address whether the number of active zones also changed in the medial branches of DA-WED neurons after protein starvation, a truncated fragment of the active-zone marker Bruchpilot (Brp-short) was expressed in these cells. The number of BRP+ puncta in the medial, but not lateral, branch was significantly elevated when flies were protein-deprived, and this increase showed a trend toward being inhibited by Gln in the protein-deficient diet. GRASP was used to determine whether protein starvation altered the connectivity between the DA-WED cells and their downstream targets. GRASP signal between the medial branch of the DA-WED neurons and the FB-LAL cells, but not the lateral branch and PLP neurons, was substantially elevated with protein starvation, and this increase was again suppressed if Gln was provided in the protein-deficient diet (Liu, 2017).

To characterize the functional consequences of the plastic changes in the DA-WED neurons, sharp intracellular current-clamp recordings of the FB-LAL cells were performed to measure frequency and amplitude of postsynaptic potentials (PSPs). Protein deprivation induced a substantial increase in the frequency of PSPs in the FB-LAL neurons. Moreover, examination of the distribution of PSP amplitudes revealed the presence of high-amplitude PSPs solely in yeast-deprived animals. This population of high-amplitude PSPs likely reflects evoked responses seen only with protein deprivation and suggests that downstream signaling is markedly enhanced after protein starvation. Knockdown of DopR2 in FB-LAL cells significantly reduced the number of high-amplitude PSPs in yeast-deprived animals, consistent with a model wherein the DA-WED neurons signal via DopR2 on the FB-LAL cells to promote protein consumption (Liu, 2017).

Whether the plastic changes in the medial branches of the DA-WED cells led to a change in feeding behavior was examined. dTrpA1 was used to activate the DA-WED cells and yeast or sucrose consumption was assessed at different time points after cessation of heat treatment. dTrpA1 activation of the DA-WED neurons resulted in a prolonged increase in yeast consumption lasting at least 6 hours and induced persistent plastic changes in the medial, but not lateral, branches as revealed by Syt-GFP staining. In contrast, although sucrose intake was reduced immediately after dTrpA1 activation, this response did not persist and was no longer present within 1 hour. After 24 hours, both yeast consumption and terminal morphology of DA-WED neurons were indistinguishable from control flies. Together, these findings demonstrate branch-specific plastic changes of the DA-WED neurons in response to substantial protein deprivation, providing a mechanism for the persistent hunger for protein, but not sugar, under these conditions (Liu, 2017).

Appropriate regulation of food consumption is essential for the survival of organisms that must navigate environments with variable and uncertain food availability and quality. A number of studies have investigated how animals reject diets devoid of essential amino acids and how monoaminergic and TOR-S6K (target of rapamycin-S6 kinase) signaling in Drosophila regulates mating-induced protein feeding. However, little is known about the circuit mechanisms mediating the homeostatic regulation of protein intake. This study suggests that protein hunger is encoded by the DA-WED neurons, providing a path toward dissecting these mechanisms. The findings suggest that glutamine, directly or indirectly, regulates the activity of these neurons, thus modulating behavioral responses to protein deprivation (Liu, 2017).

The data suggest that the DA-WED neurons play a crucial role in homeostatic control of protein intake, functioning on multiple time scales to restore protein balance. Shortly after substantial protein deprivation, the DA-WED neurons act to mediate the behavioral switch between consumption of a food source preferred at baseline (sucrose) and the deprived nutrient (protein), by activating a dedicated 'protein' feeding circuit, while simultaneously inhibiting a 'sugar' feeding circuit. Over a longer time frame, the DA-WED cells undergo branch-specific plastic changes that underlie the selective and persistent hunger for protein under these conditions. In the wild, these actions may correspond to promoting greater selectivity for protein in an initial foraging response, followed by maintenance of protein consumption after the protein food source has been identified (Liu, 2017).

Given that branch-specific plasticity has generally been described in postsynaptic dendrites, the finding that the presynaptic terminals of the DA-WED neurons exhibit this phenomenon is highly unusual. The mechanisms underlying this process are currently unclear but may depend on extrinsic neuromodulatory influences that differentially regulate potential for plasticity of the distinct presynaptic terminals of the DA-WED cells. Further characterization of these and related circuit mechanisms should help delineate the fundamental principles governing protein-specific hunger. A better understanding of how animals choose to consume protein may also have implications for the treatment of obesity (Liu, 2017).

Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory

Efficient energy use has constrained the evolution of nervous systems. However, it is unresolved whether energy metabolism may resultantly regulate major brain functions. The observation that Drosophila flies double their sucrose intake at an early stage of long-term memory formation initiated the investigation of how energy metabolism intervenes in this process. Cellular-resolution imaging of energy metabolism reveals a concurrent elevation of energy consumption in neurons of the mushroom body, the fly's major memory centre. Strikingly, upregulation of mushroom body energy flux is both necessary and sufficient to drive long-term memory formation. This effect is triggered by a specific pair of dopaminergic neurons afferent to the mushroom bodies, via the D5-like DAMB dopamine receptor. Hence, dopamine signalling mediates an energy switch in the mushroom body that controls long-term memory encoding. These data thus point to an instructional role for energy flux in the execution of demanding higher brain functions (Placais, 2017).

Fat storage in Drosophila suzukii is influenced by different dietary sugars in relation to their palatability

The peripheral sensitivity and palatability of different carbohydrates was evaluated and their nutritional value assessed in adult females of D. suzukii by means of an electrophysiological, behavioural and metabolic approach. The electrophysiological responses were recorded from the labellar "l" type sensilla stimulated with metabolizable mono- and disaccharides (glucose and maltose) and a non-metabolizable sugar (sucralose); the response rating and the palatability to the same sugars, evaluated by recording the proboscis extension reflex (PER), was maltose>glucose>sucralose. The nutritional value of carbohydrates was assessed by means of survival trials and fatty acids profile. Flies fed on a diet containing maltose had a longer lifespan than flies on monosaccharides, while flies fed on a diet containing sucralose had a shorter one. In addition, the ability to store fat seems to be influenced by the different sugars in the diet and is in relationship with their palatability. In fact, data showed a higher synthesis of palmitic and palmitoleic acids, most likely derived from de-novo lipogenesis with glucose as precursor, in flies fed with maltose and glucose than with non-metabolizable sucralose. In conclusion, these results suggest that the ability to select different sugars on the basis of their palatability may favour the storage of energy reserves such as fat by de-novo lipogenesis, determining a longer survival capability during prolonged periods of fasting (Biolchini, 2017).

Acetyl-CoA-mediated autoacetylation of fatty acid synthase as a metabolic switch of de novo lipogenesis in Drosophila

De novo lipogenesis is a highly regulated metabolic process, which is known to be activated through transcriptional regulation of lipogenic genes, including fatty acid synthase (FASN). Unexpectedly, this study found that the expression of FASN protein remains unchanged during Drosophila larval development from the second to the third instar larval stages (L2 to L3) when lipogenesis is hyperactive. Instead, acetylation of FASN is significantly upregulated in fast-growing larvae. This study further showed that lysine K813 residue is highly acetylated in developing larvae, and its acetylation is required for elevated FASN activity, body fat accumulation, and normal development. Intriguingly, K813 is autoacetylated by acetyl-CoA (AcCoA) in a dosage-dependent manner independent of acetyltransferases. Mechanistically, the autoacetylation of K813 is mediated by a novel P-loop-like motif (N-xx-G-x-A). Lastly, this study found that K813 is deacetylated by Sirt1, which brings FASN activity to baseline level. In summary, this work uncovers a previously unappreciated role of FASN acetylation in developmental lipogenesis and a novel mechanism for protein autoacetylation, through which Drosophila larvae control metabolic homeostasis by linking AcCoA, lysine acetylation, and de novo lipogenesis (Miao, 2022).

De novo lipogenesis (DNL) is a complex yet highly regulated metabolic process which converts excess carbohydrates into fatty acids that are then esterified to storage triglyceride (TAG). Abnormal upregulation of DNL is a vital contributor to increased fat mass in the pathogenesis of various metabolic disorders involving non-alcoholic fatty liver disease and diabetes and in the progression of tumors. DNL is known to be transcriptionally regulated via sterol regulatory element-binding protein 1 (SREBP1) and carbohydrate-responsive element-binding protein (ChREBP) in response to metabolic and hormonal cues. However, it has been recently proposed that allosteric regulation and post-translational modifications (PTMs) are indispensable to control metabolic flux since the timescale of the gene expression is too long to balance the quick turnover of metabolites. Lipid anabolism is active during Drosophila larval development, and the excessive TAG storage in larvae is an essential reservoir for surviving from starvation in the post-feeding pupa stage of metamorphosis. The fatty acids stockpiled in TAG are either derived from the diet or DNL. Regulated by hormonal and transcriptional programs, the mRNA expressions of multiple enzymes in DNL pathways, including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN), correlate with the dynamic changes in TAG levels in fly embryonic and larval development. However, whether PTMs are involved in the regulation of developmental DNL is poorly studied (Miao, 2022).

Lysine acetylation has recently risen as a novel player that links metabolites [e.g., acetyl coenzyme A (acetyl-CoA)], cell signaling, and gene regulation. Previous acetylome studies found that almost all metabolic enzymes are acetylated, including FASN . FASN, an essential cytosolic enzyme in DNL pathway, catalyzes the biosynthesis of saturated fatty acids from acetyl-CoA (AcCoA) and malonyl coenzyme A (malonyl-CoA). Recently, FASN has emerged as a novel therapeutic target for the treatment of obesity, diabetes, fatty liver diseases, and cancers. Although FASN is known to be regulated through SREBP1-mediated transcriptional activation, several conflicting results show little correlation between FASN expression and its enzymatic activity. These findings suggest a possible involvement of PTMs in the regulation of FASN function. Phosphorylation and acetylation have been proposed as alternative mechanisms of FASN regulation. Nevertheless, how PTMs regulate FASN activity and lipogenesis remains largely unknown (Miao, 2022).

Although protein acetylation is mainly catalyzed by lysine acetyltransferases (KATs), it has been recently reported that acetylation also arises from a nonenzymatic reaction with AcCoA in eukaryotes. AcCoA is the acetyl donor for protein acetylation and is a reactive metabolic intermediate involved in various metabolic pathways. The levels of AcCoA fluctuate in response to both intracellular and extracellular cues (e.g., growth signals and nutrient conditions), which consequently impacts chromatin modifications and transcriptional reprogramming. Previously, it was thought that nonenzymatic acetylation only occurs to mitochondria proteins, as high concentrations of AcCoA and alkaline environment inside mitochondrial matrix favor lysine nucleophilic attack on the carbonyl carbon of AcCoA. In recent years, the capability of enzyme-independent acetylation of cytosolic proteins was also determined. Yet, the mechanism of nonenzymatic acetylation, especially of cytosolic proteins, remains elusive (Miao, 2022).

This study shows that the expression of Drosophila FASN (dFASN or FASN1) protein remains unchanged during larval development, the stages when lipogenesis is hyperactive. In contrast, acetylation of dFASN at K813 is significantly induced in response to increased cellular AcCoA levels, which elevates dFASN enzymatic activity and lipogenesis in fast-growing larvae. Strikingly, acetylation of K813 is controlled through a unique KAT-independent mechanism that involves a novel motif "N-xx-G-x-A." In summary, these findings uncover a novel AcCoA-mediated self-regulatory module that regulates developmental lipogenesis via autoacetylation of dFASN (Miao, 2022).

Metabolic homeostasis plays an important role in animal development and growth. One novel mechanism underlying the coordination of metabolic homeostasis and growth is the interplay between metabolic intermediates and PTMs. This study uncover a novel role of AcCoA-mediated autoacetylation of dFASN in lipogenesis during Drosophila larval development. On the one hand, AcCoA fuels dFASN as the carbon donor for the growing fatty acid chain. On the other hand, AcCoA, as the acetyl-group donor, directly modulates dFASN enzymatic activity through acetylation of the critical lysine residue K813. Surprisingly it was found that acetylation of dFASN does not require KATs; instead, it is mediated by a conserved P-loop-like motif N-xx-G-x-A neighboring K813. Lastly, Sirt1 was identied as the primary deacetylase for dFASN, which acts as a negative regulatory mechanism (Miao, 2022).

TAG levels are tightly controlled during Drosophila development, and the massive buildup of TAG storage is a feature of larval growth. TAG synthesis is catalyzed by isoenzymes and competes with pathways that consume fatty acids, such as fatty acid oxidation and membrane lipid synthesis. During the entire embryonic and larval development, the mRNA expressions of multiple lipogenic enzymes, including FASN, correlate with TAG levels. Moreover, flies with mutations in FASN1 and FASN2 store less TAG in both larval and adult stages of Drosophila, suggesting that DNL is a vital contributor to TAG storage throughout development (Miao, 2022).

Under conditions like excess nutrition, growth factor stimulation, obesity, diabetes, fatty liver diseases, or cancer, DNL is significantly elevated, and the mRNA expression of FASN positively correlates with elevated lipogenesis. However, the protein levels of FASN are rarely characterized. Interestingly, several conflicting results show little correlation between FASN protein expression and its enzymatic activity. Consistently, it was found that the protein levels of dFASN remain unchanged from L2 to L3 and do not match the pattern of its enzymatic activity. In contrast, acetylation of dFASN at lysine K813 is positively associated with dFASN activity, developmental lipogenesis, and TAG accumulation. However, although dFASN level is not upregulated with elevated lipogenesis and TAG accumulation at L3 larvae, it correlates with TAG levels when the entire embryonic and larval development stages are considered. These findings suggest that despite the well-established transcriptional program controlling the expression of dFASN at different stages of development, lysine acetylation of dFASN plays a crucial role in accelerating dFASN activity and fine-tuning dFASN-mediated lipogenesis in fast-growing L3 animals (Miao, 2022).

Indeed, genetic and biochemical analysis further demonstrates that K813R substitution reduces dFASN enzymatic activity and lipogenesis, while AcCoA-mediated acetylation of recombinant dFASN proteins increases its enzyme activity. Although acetylation of FASN has been reported in several previous global acetylome studies, the functional roles of FASN acetylation remain largely unknown. A recent study investigated the role of FASN acetylation in DNL in human cell culture. The study shows that treatment of KDAC inhibitors induces the acetylation of hFASN, promotes FASN degradation, and reduces lipogenesis. However, it remains to be determined whether the regulation of lipogenesis by KDAC inhibition is due to global acetylation, or if it is directly through FASN acetylation. In addition, the functional lysine residues of hFASN that are responsible for altered lipogenesis are not identified. Because of the high conservation between K813 of dFASN and K673 of hFASN, it is possible that K673 is the key lysine residue mediating DNL in human (Miao, 2022).

Apart from K813, other three lysine residues of dFASN (K926, K1800, and K2466) are highly conserved among animal species, and their homologs are also found to be acetylated in other animal species. Since these lysine residues are located on different domains, it is not hard to imagine that acetylation of each lysine may play distinct roles related to their associated domains. The present study shows that acetylation of K813, but not K926, modulates dFASN activity, body fat accumulation, and Drosophila developmental timing. It is likely that acetylation of K926 affects other aspects of enzyme properties that are less important for larval development. K813 is at the substrate docking pocket of the MAT domain. This unique localization suggests that acetylation of K813 might introduce conformational changes to the docking site and modulate dFASN catalytic activity in response to substrate availability during larval development (Miao, 2022).

Another surprising finding from this study is that acetylation of dFASN at K813 does not require a KAT; rather, it is autoacetylated by AcCoA in a dosage-dependent manner. The cytosolic pool of AcCoA increases under feeding or excess nutrient conditions. Consistently, these studies reveal that the amount of AcCoA elevates in fast-growing larvae, which could modulate dFASN activity by promoting both the biosynthesis of MalCoA and autoacetylation of K813 for the conformational changes of MalCoA docking pocket (Miao, 2022).

It was previously thought that only mitochondria proteins were nonenzymatically acetylated since no KATs have been identified in mitochondria. Besides, the high AcCoA concentration and relatively high pH of the mitochondrial matrix facilitate the lysine nucleophilic attack on the carbonyl carbon of AcCoA. Recently, KAT-independent acetylation of cytosolic proteins has been reported. Yet, the underlying mechanism for nonenzymatic acetylation remains largely unknown. When investigating how dFASN is autoacetylated by AcCoA, this study uncovered a novel motif N-xx-G-x-A near acetylated K813. Substituting any of the three key amino acids largely blocks AcCoA-mediated dFASN autoacetylation. The N-xx-G-x-A motif resembles the signature P-loop sequence (Q/R-xx-G-x-A/G) of KATs, which is required for AcCoA recognition and binding. It is predicted that the N-xx-G-x-A motif of dFASN performs a similar function as the P-loop of KATs for AcCoA binding. Moreover, the N-xx-G-x-A motif is highly conserved, pointing out a conserved mechanism for autoacetylation of FASN (Miao, 2022).

In addition to the well-established KATs of the MYST, p300/CBP, and GCN5 families, there are over 15 proteins that have been reported to possess KATs activity, such as CLOCK and Eco1. Since FASN may contain an AcCoA binding motif of KATs, it is possible that FASN, particularly MAT domain, possesses KATs activity and acetylates other proteins, especially those in DNL pathways. This possibility may be further explored through acetylome analysis in the future. In summary, this study uncovered a previously unappreciated role of FASN acetylation in developmental lipogenesis and a novel mechanism for lysine autoacetylation. These findings provide new insights into AcCoA-mediated metabolic homeostasis during animal development. In addition, rhwaw studies underscore a promising therapeutic strategy to combat metabolic disorders by targeting autoacetylation of FASN (Miao, 2022).

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).

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Zygotically transcribed genes

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