The Interactive Fly

Zygotically transcribed genes

Growth response - The Insulin receptor signaling pathway


Control of Insulin Signaling
  • Suppression of insulin production and secretion by a Decretin hormone
  • The neuropeptide Allatostatin A regulates metabolism and feeding decisions in Drosophila
  • Direct sensing of nutrients via a LAT1-like transporter in Drosophila insulin-producing cells
  • Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis
  • The nutrient-responsive hormone CCHamide-2 controls growth by regulating insulin-like peptides in the brain of Drosophila melanogaster
  • Cold-sensing regulates Drosophila growth through insulin-producing cells
  • The Drosophila ortholog of TMEM18 regulates insulin and glucagon-like signaling
  • Drosophila insulin release is triggered by adipose Stunted ligand to brain Methuselah receptor
  • The neuropeptide Allatostatin A regulates metabolism and feeding decisions in Drosophila
  • Drosophila neprilysins control insulin signaling and food intake via cleavage of regulatory peptides
  • Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells
  • Cbt modulates Foxo activation by positively regulating insulin signaling in Drosophila embryos

    Insulin Signaling to fat cells
  • Remote control of insulin secretion by fat cells in Drosophila

    Insulin Signaling to the gut
  • Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut

    Insulin Signaling and Nutrition
  • Insulin-pathway and nutritional status
  • Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila
  • An investigation of nutrient-dependent mRNA translation in Drosophila larvae
  • Genetic dissection of nutrition-induced plasticity in insulin/insulin-like growth factor signaling and median life span in a Drosophila multiparent population

    Insulin Signaling, Growth and Development
  • The cytohesin Steppke is essential for insulin signalling in Drosophila
  • Genome-wide microRNA screening reveals that the evolutionary conserved miR-9a regulates body growth by targeting sNPFR1/NPYR
  • Insulin receptor-mediated signaling via phospholipase C-γ regulates growth and differentiation in Drosophila
  • A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila
  • Insulin signaling regulates neurite growth during metamorphic neuronal remodeling
  • Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis
  • Dally proteoglycan mediates the autonomous and nonautonomous effects on tissue growth caused by activation of the PI3K and TOR pathways
  • A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3
  • Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
  • Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila
  • Intra-organ growth coordination in Drosophila is mediated by systemic ecdysone signaling

    Insulin Signaling and Lifespan
  • Both overlapping and independent mechanisms determine how diet and insulin-ligand knockouts extend lifespan of Drosophila melanogaster
  • Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies
  • A proteomic atlas of insulin signalling reveals tissue-specific mechanisms of longevity assurance

    Insulin Signaling and Behavior
  • Feeding regulates sex pheromone attraction and courtship in Drosophila females
  • Insulin signalling mediates the response to male-induced harm in female Drosophila melanogaster
  • Insulin signaling in the peripheral and central nervous system regulates female sexual receptivity during starvation in Drosophila
  • Tissue-specific insulin signaling mediates female sexual attractiveness

    Insulin Signaling and Disease
  • Drosophila as a model for human diseases: Diabetes
  • The obesity-linked gene Nudt3 Drosophila homolog Aps is associated with insulin signalling
  • High sugar-induced insulin resistance in Drosophila relies on the lipocalin Neural Lazarillo
  • Systemic organ wasting induced by localized expression of the secreted Insulin/IGF antagonist ImpL2
  • Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting

    Miscellaneous effects of the Insulin Pathway
  • Transgenerational inheritance of diet-induced genome rearrangements in Drosophila
  • An integrative analysis of the InR/PI3K/Akt network identifies the dynamic response to insulin signaling


  • 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, Dp110D945A, 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 Dp110D945A 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 Dp110CAAX 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 (Raught, 2001; Schmelzle, 2000). What role might Drosophila TOR (dTOR) play in the nutrition response system addressed here. The mechanism by which TOR 'senses' nutrition remains uncertain (Kleijn and Proud, 2000), but cellular levels of amino acids, aminoacylated tRNAs, and ATP have been suggested as direct inputs (Dennis, 2001; Iiboshi, 1999). Drosophila dTOR mutations or the TOR-specific inhibitor rapamycin inactivate the TOR target S6-kinase and phenocopy starvation in fed larvae (Zhang, 2000; Oldham, 2000). Starvation, however, does not completely inactivate S6K, suggesting that dTOR retains some activity under starvation conditions (Oldham, 2000). 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 (Brogiolo, 2001) 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 (Brogiolo, 2001; 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).

    Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila

    Animals use the insulin/TOR signaling pathway to mediate their response to fluctuations in nutrient availability. Energy and amino acids are monitored at the single-cell level via the TOR branch of the pathway and systemically via insulin signaling to regulate cellular growth and metabolism. Using a combination of genetics, expression profiling, and chromatin immunoprecipitation, this study examined nutritional control of gene expression and identified the transcription factor Myc as an important mediator of TOR-dependent regulation of ribosome biogenesis. myc was also identified as a direct target of FOXO, and genetic evidence is provided that Myc has a key role in mediating the effects of TOR and FOXO on growth and metabolism. FOXO and TOR also converge to regulate protein synthesis, acting via 4E-BP and Lk6, regulators of the translation factor eIF4E. This study uncovers a network of convergent regulation of protein biosynthesis by the FOXO and TOR branches of the nutrient-sensing pathway (Teleman, 2008).

    The global transcriptional analysis reported in this study has revealed a surprising degree of interconnectedness between the two branches of the nutrient-sensing pathway. Insulin, acting through PI3K and Akt, feeds into the FOXO and TORC1 branches of the pathway, whereas energy levels (AMP/ATP) and amino acids act directly on the TORC1 branch. How are these inputs integrated to maintain energy balance? It was previously known that 4E-BP is transcriptionally regulated by FOXO and posttranslationally regulated by TOR. This study has identified the protein kinase Lk6 as a second direct FOXO target. Thus, there appear to be two parallel, independent mechanisms by which the TOR and FOXO branches of the insulin signaling pathway converge to regulate eIF4E activity and hence cellular protein translation. This 'belt and suspenders' approach to translational control might be important to make the system robust (Teleman, 2008).

    A key finding of this study is the identification of Myc as a point of convergent regulation by the FOXO and TOR branches of the pathway. myc mRNA levels are controlled by FOXO in a tissue-specific manner. In addition, Myc protein levels are dependent on TORC1. Why use two independent means to control Myc levels? Transcription alone would limit the speed with which the system can respond to changing nutritional conditions. This might be detrimental, particularly as conditions worsen. Regulation of Myc activity by TORC1 permits a rapid response to changes in energy levels or amino acid availability and could serve to fine tune the nutritional response in the cell by controlling translational outputs. This parallels the situation with 4E-BP, albeit with a slightly different logic. Reduced insulin signaling allows FOXO to enter the nucleus and increase 4E-BP expression and at the same time alleviates TORC1-mediated inhibition of the existing pool of 4E-BP. A subsequent increase in energy or amino acid levels would permit rapid reinhibition of 4E-BP and thus allow a flexible response during the time needed for the pool of protein elevated in response to reduced insulin levels to decay (Teleman, 2008).

    In yeast, TORC1 is known to regulate ribosome biogenesis through different nuclear RNA polymerases. It has been shown that yeast TORC1 can bind DNA directly at the 35S rDNA promoter and activate Pol I-mediated transcription in a rapamycin-sensitive manner. Moreover, yeast TORC1 is known regulate Pol II-dependent RP gene expression by controlling the nuclear localization of the transcription factor SFP1 and CRF1, a corepressor of the forkhead transcription factor FHL1. In Drosophila, TORC1 has recently been reported to regulate a set of protein-coding genes involved in ribosome assembly. This study has identified Myc as the missing link mediating TORC1-dependent regulation of this set of genes. Indeed, the fact that more than 90% of TORC1-activated genes contain E boxes suggests that Myc might be the main mediator of this transcriptional program. This connection suggests that expression of Myc targets as a whole should be responsive to nutrient conditions. Indeed, this study found that 33% of direct Myc targets -- defined as genes reported to be bound by Myc when assayed by DNA adenine methyltransferase ID (DamID) in Kc cells and to be regulated by myc overexpression in larvae -- are downregulated upon nutrient deprivation. This is a significant enrichment of 4-fold relative to all genes in the genome, despite the comparison being based on correlating data from different tissue types (Teleman, 2008).

    It seems reasonable that cellular translation rates need to be dampened if the TOR branch of the pathway senses low amino acid levels. As ribosome biogenesis is energetically expensive, it may be advantageous to link ribosome biogenesis and translational control via TORC1. This dual regulation is well reflected in tissue growth, since this study observed that Myc, the regulator of ribosome biogenesis, is essential for tissue growth driven by the TOR pathway but not sufficient to drive growth in the absence of TOR activity. The FOXO branch of the pathway senses reduced insulin or mitogen levels. FOXO is also highly responsive to oxidative and other stresses and would integrate this information into the cellular control of translation. The data support the notion of a network in which TOR and FOXO regulate protein biosynthesis by converging on Myc to regulate ribosome biogenesis and on eIF4E activity via 4E-BP and Lk6 to regulate translation initiation (Teleman, 2008).

    The work presented in this study complements a previous study in which larvae were either starved completely or starved for amino acids only, while having a supply of energy in the form of sugar. A significant and positive correlation (~0.4) indicates general agreement between the two data sets, but they differ in two ways. The current goal was to explore the regulatory network by which insulin controls cellular transcription. Individual tissues were isolated rather than assaying the whole animal. Genes found to be regulated in a previous but not in the current assays may be regulated in tissues other than muscle or adipose tissue. Conversely, genes identified only by the current study might be regulated oppositely in different tissues or might only be regulated in a subset of tissues and so be missed in a whole-animal analysis.

    Is Myc also involved in nutritional signaling networks in mammals? No similar rapid downregulation of c-myc was seen in response to rapamycin in human cell lines, suggesting that the mechanism by which TOR signaling controls gene expression may differ between phyla. This is further supported by the fact that the sets of genes reported to be rapamycin regulated also appear to be largely distinct in Drosophila and mammalian cells, with the caveat that different cell types were used in the two analyses. Although the mechanism does not appear to be identical in mammals, there are several suggestions in the literature of a connection between c-Myc and nutritional signaling. For example, dMyc and c-Myc share the ability to regulate ribosome biogenesis, although the specific target genes through which they do so are different. There is also evidence that mammalian c-myc expression in liver is regulated by nutrition and that transgenic expression of c-myc in liver affects metabolism, i.e., glucose uptake and gluconeogenesis. Furthermore, it has been reported that FOXO3 represses Myc activity in colon cancer cells by inducing members of the Mad/Mxi family, which are known to antagonize Myc. The current data suggest that Max and Mnt are not transcriptionally regulated by insulin or FOXO in Drosophila, whereas myc is. This is similar to what has been reported in murine lymphoid cells, in which c-myc expression is regulated by the FOXO homolog FKHRL1. These parallels between the fly and mammalian systems suggest a broader connection between insulin signaling and activity of the Myc/Mnt/Max network. Although some features may be different in the two systems, the similarities merit further investigation (Teleman, 2008).

    Finally, this work has revealed a surprising amount of tissue specificity in the transcriptional response to insulin signaling. Roughly half of the genes regulated by insulin in adipose tissue or in muscle were not significantly regulated in the other tissue. Furthermore, 155 genes were differentially regulated in the two tissues (i.e., upregulated in one tissue and downregulated in the other). This likely reflects the roles of the different tissues in the organism's response to nutrient deprivation. Further work will elucidate the underlying molecular mechanisms (Teleman, 2008).

    The cytohesin Steppke is essential for insulin signalling in Drosophila

    In metazoans, the insulin signalling pathway has a key function in regulating energy metabolism and organismal growth. Its activation stimulates a highly conserved downstream kinase cascade that includes phosphatidylinositol-3-OH kinase (PI(3)K) and the serine-threonine protein kinase Akt. This study identifies a new component of insulin signalling in Drosophila, the steppke gene (step). step encodes a member of the cytohesin family of guanine nucleotide exchange factors (GEFs), which have been characterized as activators for ADP-ribosylation factor (ARF) GTPases. In step mutant animals both cell size and cell number are reduced, resulting in decreased body size and body weight in larvae, pupae and adults. step acts upstream of PI(3)K and is required for the proper regulation of Akt and the transcription factor FOXO. Temporally controlled interference with the GEF activity of the Step protein by feeding the chemical inhibitor SecinH3 causes a block of insulin signalling and a phenocopy of the step mutant growth defect. Step represses its own expression and the synthesis of growth inhibitors such as the translational repressor 4E-BP. These findings indicate a crucial role of an ARF-GEF in insulin signalling that has implications for understanding insulin-related disorders, such as diabetes and obesity (Fuss, 2006).

    All animals coordinate growth to reach their final size and shape. The insulin–insulin-like growth factor signalling pathway, which is genetically conserved from flies to humans, has been identified as a key regulator of cell growth in response to extrinsic signals such as growth factors and nutrient availability. In mammals, loss of the ability to respond to insulin, a phenomenon known as insulin resistance, is associated with pathological manifestations such as type 2 diabetes. In Drosophila, activation of a unique insulin-like receptor (InR) stimulates a conserved downstream cascade that includes PI(3)K and Akt. This signalling cascade controls organismal growth directly by regulating cell size and cell number (Fuss, 2006).

    In a search for genes controlling larval growth in Drosophila, a genetic locus was identified that was named steppke (step). Molecular analysis and genetic rescue experiments show that the lethality of the P element alleles is linked to the step gene function. The step gene encodes a protein that belongs to the highly conserved cytohesin protein family of GEFs that consists of four family members in humans and one family member in invertebrates such as the nematode, mosquito and fly. GEFs mediate the exchange of GDP for GTP on the ARFs, which belong to the Ras superfamily of small GTPases. Like other Ras-related GTP-binding proteins, the ARF proteins cycle between their active GTP-bound and inactive GDP-bound conformations. In concert with ARFs, cytohesin proteins regulate vesicle trafficking, cell adhesion, migration and structural organization at the cell surface (Fuss, 2006).

    Cytohesin proteins contain two characteristic motifs: a Sec7 domain responsible for the GEF activity, and a pleckstrin homology domain (PH) required for plasma membrane recruitment as a result of specific binding to phosphatidylinositol-3,4,5-trisphosphate, the second messenger generated by class I PI(3)Ks. The Sec7 and PH domains of Step are highly conserved compared with the corresponding protein domains of mammalian cytohesins (Fuss, 2006).

    Phenotypic analysis of homozygous stepk08110 and stepSH0323 mutants and transheterozygous allelic combinations indicate an essential role of step in regulating growth and body size at all stages of the Drosophila life cycle. Both males and females of stepk08110/stepSH0323 transheterozygous adults are significantly smaller than control animals; however, the body proportions of these animals are not changed. Consistently, larval and pupal development are also slowed down in step mutants and body size is reduced. The observed growth defects mimic a starvation phenotype that is not caused by a failure of food intake, as verified by feeding coloured yeast and by the analysis of a metabolic marker gene (Fuss, 2006).

    It is known that larval growth is largely based on an increase in cell size in all terminally differentiated tissues that is accomplished by endoreplication, a modified cell cycle, consisting of successive rounds of DNA synthesis without intervening mitoses. To examine the cause for the growth defects of step mutant larvae, cell cycle activity was investigated in the midgut and the salivary glands, which are representative endoreplicating tissues in the larval stage. A general decrease in endoreplication activity was found, indicated by a slowing down of the S phase of the cell cycle. Both the size and the total number of salivary gland cells are decreased, resulting in a smaller organ (Fuss, 2006).

    Because embryonic lethality was observed in a small proportion of the homozygous stepk08110 mutants, it was important to exclude the possibility that the growth defects observed in the mutant larvae derive from a defect laid down during embryogenesis. For this purpose an assay was established to analyse step function exclusively in the larval stage, in which the growth rate is maximal. Use was made of the small molecule SecinH3, which was recently identified as an inhibitor of the Drosophila Step protein and the vertebrate cytohesin family members. SecinH3 binds to the Sec7 domain of Step, thereby inhibiting the guanine nucleotide exchange of interacting ARF proteins. Feeding SecinH3 to wild-type larvae induced a phenocopy of the growth defects observed in step mutants and led to a marked decrease in body size. It is concluded from the phenotypic analysis of step mutants and from the experiments inhibiting Step protein function directly by using the chemical inhibitor SecinH3 that Step is essential for organismal growth of Drosophila larvae, pupae and adults (Fuss, 2006).

    In step mutants, organismal growth is strongly reduced and development is delayed, which is also a hallmark of mutants affecting the insulin signalling pathway. To investigate whether step has a function in insulin signalling, the expression of two known target genes of the pathway was analysed in step mutants, namely 4E-BP, encoding a translational repressor, and InR, encoding the insulin receptor, by using quantitative reverse-transcriptase-mediated polymerase chain reaction (RT–PCR); both 4E-BP and InR transcription are upregulated in response to repressed insulin signalling. Lipase3 (Lip3) expression was used as a starvation marker in these experiments. In step mutant larvae and also in wild-type larvae treated with the Step inhibitor SecinH3, 4E-BP and InR transcription is activated, whereas Lip3 expression is unaffected. This indicates that the growth phenotype observed in step mutant larvae is not caused by a complete block of nutrition but is associated with a specific downregulation of insulin signalling activity. Similarly, interfering with Step function by feeding SecinH3 to transheterozygous step mutant flies or applying SecinH3 in S2 tissue culture cells also results in an activation of 4E-BP and InR transcription (Fuss, 2006).

    It has been shown previously that 4E-BP and InR are target genes of the transcription factor FOXO (forkhead box, sub-group ‘O’). In Drosophila cells, insulin receptor signalling results in a high activity of PI(3)K and phosphorylation of Akt. Akt phosphorylates FOXO and causes cytoplasmic retention of FOXO, whereas low activities of PI(3)K and Akt allow FOXO to enter the nucleus, where it promotes the expression of factors such as 4E-BP that retard cell growth and proliferation. In step mutant larvae or in S2 tissue culture cells in which Step protein function is inhibited with SecinH3, a nuclear localization of FOXO was found, indicating that step is required for insulin-signalling-dependent cytoplasmic localization of FOXO. Because this is regulated by phosphorylation by means of Akt, whether step is necessary for Akt phosphorylation was tested, and it was found that under conditions in which the step function is affected, the amount of phosphorylated Akt protein is significantly decreased (Fuss, 2006).

    It has been shown that activation of Akt during growth in Drosophila is regulated by the class I PI(3)K Dp110. Overexpression of Dp110-CAAX, a constitutively active form of PI(3)K, in wing or eye imaginal discs enhances cellular growth, resulting in enlarged cells and organs, whereas mutations in Dp110 are lethal and result in a larval growth arrest in the third instar. It has been shown previously that Dp110 interacts with key components of the insulin signalling pathway including Chico, PTEN and Akt to control insulin-signalling-dependent cell and organ growth in Drosophila. To test whether step acts together with PI(3)K in a common pathway involved in Akt and FOXO regulation and, if so, to address whether step is genetically upstream or downstream of PI(3)K in the insulin pathway, Dp110-CAAX was expressed in heterozygous and transheterozygous step mutant animals (Fuss, 2006).

    step mutant adults are greatly decreased in size and weight in comparison with wild-type animals. In control flies in which Dp110-CAAX has been overexpressed, body size and weight are greatly increased in comparison with wild-type flies. If step were positioned downstream of PI(3)K, the oversize phenotype induced by the expression of Dp110-CAAX should be suppressed or at least strongly reduced, whereas if step were positioned upstream of PI(3)K, Dp110-CAAX expression would rescue the growth phenotype of step mutants. The latter was found, providing in vivo evidence that the cytohesin family member step is upstream of PI(3)K (Fuss, 2006).

    Tight regulation of insulin signalling activity has been shown to be crucial for cell and organ growth in Drosophila and for numerous growth-related and homeostasis-related diseases such as cancer and type 2 diabetes in humans. It is known from recent studies in Drosophila that InR represses its own synthesis by a feedback mechanism directed by the transcription factor FOXO. To test whether step is also part of a negative feedback control mechanism, step transcription was analysed at different levels of insulin signalling activity in vivo by using quantitative RT–PCR experiments. Similarly to the 4E-BP and InR genes, step transcription was found to be upregulated under conditions promoting FOXO activity such as starvation or in mutants of the insulin signalling pathway, such as chico mutants. Consistently, step transcription is induced 24-fold in response to a brief pulse of ectopic FOXO expression during larval development. These results indicate a FOXO-dependent transcription of step, which may be direct, presumably through several FOXO consensus binding motifs present in the step promoter, or indirect (Fuss, 2006).

    It is therefore proposed that Step is a previously unrecognized and essential component of the insulin signalling cascade in Drosophila that regulates organismal growth. These results are consistent with the findings of a parallel study on the role of mammalian cytohesins. Both papers provide independent evidence for the central involvement of cytohesins in the insulin pathway upstream of PI(3)K and show a functional conservation of these proteins for at least 900 million years (Fuss, 2006).

    The obesity-linked gene Nudt3 Drosophila homolog Aps is associated with insulin signalling

    Several genome wide association studies have linked the Nudix hydralase family member Nucleoside Diphosphate-Linked Moiety X Motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown and NUDT3 expression, regulation and signalling in the central nervous system (CNS) has not been studied. This study performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward and feeding related regions of the hypothalamus and amygdala; while Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brain stem of food-deprived mice. Knocking down Aps in the Drosophila CNS, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels, as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype, while inducing hyperphagia. Finally, loss of IPC Aps lowered the expression of Akh, Ilp6 and Ilp3, genes known to be inhibited by insulin signalling. These results point towards a role for this gene in the regulation of insulin signalling which could explain the robust association to obesity in humans (Williams, 2015).

    Remote control of insulin secretion by fat cells in Drosophila

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

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

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

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

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

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

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

    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).

    The neuropeptide Allatostatin A regulates metabolism and feeding decisions in Drosophila

    Coordinating metabolism and feeding is important to avoid obesity and metabolic diseases, yet the underlying mechanisms, balancing nutrient intake and metabolic expenditure, are poorly understood. Several mechanisms controlling these processes are conserved in Drosophila, where homeostasis and energy mobilization are regulated by the glucagon-related adipokinetic hormone (AKH) and the Drosophila insulin-like peptides (DILPs). This study provides evidence that the Drosophila neuropeptide Allatostatin A (AstA) regulates AKH and DILP signaling. The AstA receptor gene, Dar-2, is expressed in both the insulin and AKH producing cells. Silencing of Dar-2 in these cells results in changes in gene expression and physiology associated with reduced DILP and AKH signaling and animals lacking AstA accumulate high lipid levels. This suggests that AstA is regulating the balance between DILP and AKH, believed to be important for the maintenance of nutrient homeostasis in response to changing ratios of dietary sugar and protein. Furthermore, AstA and Dar-2 are regulated differentially by dietary carbohydrates and protein and AstA-neuronal activity modulates feeding choices between these types of nutrients. These results suggest that AstA is involved in assigning value to these nutrients to coordinate metabolic and feeding decisions, responses that are important to balance food intake according to metabolic needs (Hentze, 2015).

    Imbalance between the amount and type of nutrients consumed and metabolized can cause obesity. It is therefore important to understand how animals maintain energy balancing, which is determined by mechanisms that guide feeding decisions according to the internal nutritional status. The fruit fly Drosophila melanogaster has become an important model for studies of feeding and metabolism, as the regulation of metabolic homeostasis is conserved from flies to mammals. In Drosophila, hormones similar to insulin and glucagon regulate metabolic programs and nutrient homeostasis. Adipokinetic hormone (AKH) is an important metabolic hormone and considered functionally related to human glucagon and a key regulator of sugar homeostasis. The release of AKH promotes mobilization of stored energy from the fat body, the equivalent of the mammalian liver and adipose tissues. Neuroendocrine cells in the corpus cardiacum (CC) express and release AKH3 that binds to the AKH receptor (AKHR), a G-protein coupled receptor (GPCR) expressed mainly in the fat body, and promotes mobilization of stored sugar and fat. Insulin and glucagon have opposing effects important to maintain balanced blood glucose levels. The Drosophila genome contains 7 genes coding for insulin-like peptides (DILPs), called dilp1-7, which are homologous to the mammalian insulin and insulin-like growth factors (IGFs). The seven DILPs are believed to act through one ortholog of the human insulin receptor that activates conserved intracellular signaling pathways. The DILPs are important regulators of metabolism, sugar homeostasis and cell growth. DILP2, 3 and 5 are produced in 14 neurosecretory cells in the brain; the insulin producing cells (IPCs). Genetic ablation of the IPCs results in a diabetic phenotype, increased lifespan and reduced growth. Because of the growth promoting effects, the activity of the DILPs is tightly linked to dietary amino acid concentrations (Hentze, 2015).

    Although metabolism has been extensively studied, the mechanisms that coordinate metabolism and feeding decisions to maintain energy balancing are poorly understood. Neuropeptides are major regulators of behavior and metabolism in mammals and insects making them obvious candidates to coordinate these processes. Peptides with a FGL-amide carboxy terminus, called type A allatostatins, have previously been related to feeding and foraging behavior. Four Drosophila Allatostatin A (AstA) peptides have been identified that are ligands for two GPCRs, the Drosophila Allatostatin A receptors DAR-1 and DAR-2. AstA peptides were originally identified as inhibitors of juvenile hormone (JH) synthesis from the corpora allata (CA) of the cockroach Diploptera punctata. However, recently it was shown that AstA does not regulate JH in Drosophila. Moreover, DAR-1 and DAR-2 are homologs of the mammalian galanin receptors, known to be involved in both feeding behavior and metabolic regulation (Hentze, 2015).

    The function of AstA in Drosophila was examined in an effort to determine whether it is involved in the neuroendocrine mechanisms coupling feeding behavior to metabolic pathways that manage energy supplies. The data suggest that AstA is a modulator of AKH and DILP signaling. Dar-2 is expressed in both the IPCs and the AKH producing cells (APCs) of the CC. Silencing of AstA receptor gene Dar-2 in the APCs or IPCs resulted in changes in expression of genes associated with reduced AKH or DILP signaling, respectively. Moreover, loss of AstA is associated with increased fat body lipid levels, resembling the phenotype of mutants in the DILP and AKH pathways. The connection between nutrients and AstA signaling was also investigated, and AstA and Dar-2 were found to be regulated differently in response to dietary carbohydrates and protein, and activation of AstA-neurons was found to increase the preference for a protein rich diet, while AstA loss enhances sugar consumption. The results suggest that AstA is a key coordinator of metabolism and feeding behavior (Hentze, 2015).

    In order to adjust energy homeostasis to different environmental conditions, feeding-related behavior needs to be coordinated with nutrient sensing and metabolism. The current data suggest that AstA is a modulator of AKH and DILP signaling that control metabolism and nutrient storage, but also affects feeding decisions. The positive effect of AstA on AKH signaling indicated by these observations is supported by the recent finding that expression of a presumably constitutive active mu opioid receptor, a mammalian GPCR which is also closely related to DAR-2, stimulates AKH release from the APCs in Drosophila. Moreover, AstA-type peptides have also been shown to stimulate AKH release in Locusta migratoria. AKH is primarily regulated at the level of secretion to allow a rapid response to metabolic needs. Considering that only a minor effect of Dar-2 silencing in the APCs on Akh transcription was detected, it is likely that AstA primarily acts at the level of AKH release in Drosophila (Hentze, 2015).

    The data suggest regulation of both the DILPs and AKH by AstA indicating a close coupling between the activity of these two hormones. Consistent with this notion, the results also indicate a feedback relationship between the IPCs and APCs. The IPCs have processes that contact the corpora cardiaca (CC) cells of the ring gland and it is possible that DILPs released from these affect AKH release. The current findings are supported by a previous study that identifies a tight association between DILPs and AKH secretion in Drosophila. Furthermore, it was recently found that AKH regulates DILP3 release from the IPCs, and that sugar promotes DILP3 release, while DILP2 release is amino acid dependent. Interestingly, the data, which suggest that AstA is involved in the cross-talk between DILPs and AKH related specifically to sugar and protein, also indicate that AstA has a strong influence on dilp3 expression. Why is the relationship between the DILPs and AKH so tight? Even though insulin-like peptides reduce hemolymph sugar, they also reduce the content of stored glycogen and lipids, like AKH. Consistent with this, both AKH and the DILPs stimulate expression of tobi, which encodes a glycosidase believed to be involved in glycogen breakdown. However, since AKH and the DILPs have opposing effect on hemolymph sugar levels, a balance between these hormones is presumably required to maintain homeostasis. It is likely that different sources of AstA affect these two hormones, since the IPCs are located in the brain in proximity of AstA-positive neurites, while AstA-positive processes do not innervate the CC. Thus, it is likely that neuronal-derived AstA affects DILP secretion from the IPCs, while circulating AstA, which may be released from the endocrine cells of the gut, may be the source of AstA that acts on the APCs to regulate AKH. AstA regulation of DILP and AKH release may therefore not occur simultaneously and could also depend on the type of nutrient ingested, or be sequential. Since the data suggest feedback regulation between AKH and DILP, the overall outcome of simultaneous AstA induced activation of both cell types will not necessarily be a strong and equal increase in both hormones in the hemolymph. It is possible that AstA is involved in metabolic balancing, adjusting the ratio between AKH and DILPs in response to different dietary conditions. In mammals, glucagon and insulin are secreted simultaneously when the animal feeds on a protein-rich diet, to prevent hypoglycemia and promote cellular protein synthesis, since insulin is strongly induced after ingestion of amino acids. A similar mechanism has been proposed to explain the relationship between DILPs and AKH in Drosophila. The balance between DILP and AKH therefore may be important for resource allocation into growth and reproduction (Hentze, 2015).

    Several differences in the expression of genes involved in energy mobilization were observed between males and females, which possibly reflects sex-specific strategies for energy mobilization and allocation of resources towards reproduction. Interestingly, 4EBP expression was significantly decreased in females with reduced AKH signaling, but upregulated in males. This suggests that in females AKH has a strong negative influence on DILP signaling that is not present in males. Why does the interaction between AKH and DILPs differ between sexes? An interesting possibility is that this sexually dimorphic interaction is related to the different preferences and requirements for sugar and protein in males and females. Males generally have a higher preference for sugar compared to females that prefer more dietary protein and show strong correlation between amino acid uptake, insulin and reproduction. In both mammals and Drosophila the balance between insulin and glucagon/AKH is important for nutrient homeostasis in response to high-protein versus high-sugar diets. This balance ensures that insulin promotes protein synthesis in response to dietary amino acids, while maintaining sugar levels stable, a function possibly important in females to allocate the high consumption of amino acids into reproduction. Thus, the sex-specific interplay between DILPs and AKH likely reflects difference in the metabolic wiring of males and females that underlie the sexually dimorphic reproductive requirements for dietary sugar and protein (Hentze, 2015).

    Interestingly, AstA expression showed a general increase after feeding with a stronger transcriptional response of both AstA and Dar-2 to the carbohydrate rich diet compared to the protein rich diet. AstA may therefore be important for coordinating carbohydrate and protein dependent metabolic programs. The strong response to carbohydrates indicates that AstA may be involved in signaling related to carbohydrate feeding, although increased transcription may not necessarily result in elevated release of the mature AstA peptide. Nonetheless, the data indicate that feeding regulates AstA-signaling and that the response is influenced by the food composition. Consistent with the notion that AstA is involved in different responses to dietary carbohydrate and protein, this study found that flies with increased AstA neuronal activity increase their protein preference on the expense of their natural preference for sucrose. The AstA regulated circuitry may therefore be important for guiding the decision to feed on protein or sugar, a decision influenced by metabolic needs. The AstA neurons have projections that may contact the Gr5a sugar sensing neurons and AstA>NaChBac flies with increased activity of the AstA neurons display reduced feeding and responsiveness to sucrose under starvation. Thus, the increased preference for dietary protein in AstA>NaChBac flies observed in this study may be caused by reduced sucrose responsiveness. If AstA signaling is high after feeding on carbohydrates as indicated by the data showing increased expression of AstA and Dar-2, then an increase in AstA signaling might mimic carbohydrate satiety. In line with this view, the data show that animals lacking AstA enhance their intake of dietary sugar. AstA signaling may therefore increase the animals preference for essential amino acids, as suggested by a recent study indicating that amino acid depleted flies increased their taste sensitivity for amino acids, even when they were replete with glucose. Based on the current data, it is therefore proposed that AstA plays a central role in a circuitry important for encoding nutritional value related to these distinct nutrients and the regulation of feeding decisions and metabolic programs. Excess dietary sugar is associated with obesity, and this study found that flies lacking AstA enhance intake of sugar and have increased lipid storage droplets in their fat bodies, like animals lacking AKH or its receptor. Thus, the data implicate AstA in regulation of appetite and food intake related to sugar, which is relevant for understanding obesity (Hentze, 2015).

    This study suggests that AstA affects metabolism through its action on two key players, the DILPs and AKH. AstA expression is induced by feeding, but exhibits a differential nutritional response to dietary sugar and protein and influence metabolic programs and feeding choices associated with the intake of these nutrients. Interestingly, the homolog of AstA, galanin, regulates both feeding and metabolism in mammals and in Caenorhabditis elegans loss of the Allatostatin/galanin-like receptor npr9 affects foraging behavior and nutrient storage. Altogether the data suggest that AstA is part of a conserved mechanism involved in coordinating nutrient sensing, feeding decisions and metabolism to ensure adequate intake of amino acids and sugar to maintain nutrient homeostasis under different feeding conditions (Hentze, 2015).

    Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut

    Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. This study presents evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions (Foronda, 2014).

    Genome-wide microRNA screening reveals that the evolutionary conserved miR-9a regulates body growth by targeting sNPFR1/NPYR

    MicroRNAs (miRNAs) regulate many physiological processes including body growth. Insulin/IGF signalling is the primary regulator of animal body growth, but the extent to which miRNAs act in insulin-producing cells (IPCs) is unclear. This study generated a UAS-miRNA library of Drosophila stocks, and a genetic screen was performed to identify miRNAs whose overexpression in the IPCs inhibits body growth in Drosophila. Through this screen, miR-9a was identified as an evolutionarily conserved regulator of insulin signalling and body growth. IPC-specific miR-9a overexpression reduces insulin signalling and body size. Of the predicted targets of miR-9a, loss of miR-9a was found to enhance the level of short neuropeptide F receptor (sNPFR1). An in vitro binding assay showed that miR-9a binds to sNPFR1 mRNA in insect cells and to the mammalian orthologue NPY2R in rat insulinoma cells. These findings indicate that the conserved miR-9a regulates body growth by controlling sNPFR1/NPYR-mediated modulation of insulin signalling (Suh, 2015).

    An investigation of nutrient-dependent mRNA translation in Drosophila larvae

    The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. This work addresses this issue. Using polysome analyses, it was observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6-18 hours. By analyzing individual genes, it was observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, this study found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, this study found that genetic activation of PI3K and TOR signaling, or regulation of two effectors - 4EBP and S6K - could not prevent the starvation-mediated translation inhibition. Similarly, it was shown that the nutrient stress-activated eIF2α kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling (Nagarajan, 2014: PubMed).

    Direct sensing of nutrients via a LAT1-like transporter in Drosophila insulin-producing cells

    Dietary leucine has been suspected to play an important role in insulin release, a hormone that controls satiety and metabolism. The mechanism by which insulin-producing cells (IPCs) sense leucine and regulate insulin secretion is still poorly understood. In Drosophila, insulin-like peptides (DILP2 and DILP5) are produced by brain IPCs and are released in the hemolymph after leucine ingestion. Using Ca(2+)-imaging and ex vivo cultured larval brains, IPCs were shown to directly sense extracellular leucine levels via minidiscs (MND), a leucine transporter. MND knockdown in IPCs abolished leucine-dependent changes, including loss of DILP2 and DILP5 in IPC bodies, consistent with the idea that MND is necessary for leucine-dependent DILP release. This, in turn, leads to a strong increase in hemolymph sugar levels and reduced growth. GDH knockdown in IPCs also reduced leucine-dependent DILP release, suggesting that nutrient sensing is coupled to the glutamate dehydrogenase pathway (Maniere, 2016).

    Insulin receptor-mediated signaling via phospholipase C-γ regulates growth and differentiation in Drosophila

    Coordination between growth and patterning/differentiation is critical if appropriate final organ structure and size is to be achieved. Understanding how these two processes are regulated is therefore a fundamental and as yet incompletely answered question. This study shows through genetic analysis that the phospholipase C-γ (PLC-γ) encoded by small wing (sl) acts as such a link between growth and patterning/differentiation by modulating some MAPK outputs once activated by the insulin pathway; particularly, sl promotes growth and suppresses ectopic differentiation in the developing eye and wing, allowing cells to attain a normal size and differentiate properly. sl mutants have previously been shown to have a combination of both growth and patterning/differentiation phenotypes: small wings, ectopic wing veins, and extra R7 photoreceptor cells. This study shows that PLC-γ activated by the insulin pathway participates broadly and positively during cell growth modulating EGF pathway activity, whereas in cell differentiation PLC-γ activated by the insulin receptor negatively regulates the EGF pathway. These roles require different SH2 domains of PLC-γ, and act via classic PLC-γ signaling and EGF ligand processing. By means of PLC-γ, the insulin receptor therefore modulates differentiation as well as growth. Overall, these results provide evidence that PLC-γ acts during development at a time when growth ends and differentiation begins, and is important for proper coordination of these two processes (Murillo-Maldonado, 2011).

    By measuring cell density, this study shows that sl mutant wings have a reduction in cell growth but not cell proliferation. This defect is qualitatively similar to mutations in MAPK signaling; cells with homozygous mutations for members of this pathway have higher cell densities, suggesting smaller cells. Of the several signaling pathways known to be involved in Drosophila wing growth, only the MAPK and insulin pathways are triggered by tyrosine kinase receptors that are likely to activate Sl. The results show that indeed both pathways are genetically linked to Sl in promoting cell growth, probably acting in a concerted fashion; further molecular studies will be required to reveal the molecular mechanisms and physical interactions that allow this link. Sl signaling thus provides a means for coordinating growth by forming a regulatory link between the MAPK and insulin pathways. In this scenario, Sl activated by the insulin pathway would function by modulating MAPK output; that is to say, to reduce somewhat the levels of MAPK activity, but not to stop it, as no MAPK activity leads to no growth and cell death, and too much MAPK activity leads to ectopic differentiation and reduced growth (Murillo-Maldonado, 2011).

    Sl regulates cellular growth in the eye. Whole eyes are smaller, and the difference in size can be largely explained by the presence of fewer ommatidia. This means that sl mutant eyes very likely contain fewer cells, despite the fact that some ommatidia sport one or two extra R7 cells, as the number of cells missing due to reduced numbers of ommatidia is bigger than the number of extra R7 cells present. This suggests either reduced proliferation or increased cell death in differentiating sl mutant eyes, and is different from the growth defect found in wings, yet consistent with a moderate requirement of MAPK output to promote growth and cellular survival (Murillo-Maldonado, 2011).

    Not only is cell size reduced to a similar extent in both the eye and wing of sl homozygotes; the adult animal as a whole has reduced mass. Given that the reduction in mass (8%) is of a similar magnitude to the reduction in cell size in the eye (15%) and wing (20%), the most parsimonious explanation for this change in mass is that the same Sl functions found in the eye and wing are required more generally throughout the animal, suggesting that cell size may be reduced in many tissues. However, it was found that the reduced growth observed in the adult was not reflected by a reduction in length of sl mutant pupae. This is in contrast to mutations of other genes involved in growth control, such as the neurofibromin 1 gene, which shows a significant reduction in pupal length. This might be because sl has a relatively small effect on growth, varying between 5% and 20% in different contexts, so this sample may not have been large enough to observe a small change in mean length. Given that Sl does not appear to affect the length of appendages other than the wing, it may be that there are other compensatory effects resulting from lost Sl function that maintain the pupal case at an approximately wild-type length (Murillo-Maldonado, 2011).

    Another complementary explanation for the reduction in adult mass is via a role for Sl on nutrient sensing. As Sl is clearly involved in insulin signaling, and as insulin is required for integrating nutrient sensation in Drosophila, the effect on mass might be a combination of impacts on both growth signaling and nutrient sensing (Murillo-Maldonado, 2011).

    It is proposed that the overall role for Sl is to act as a pro-growth agent, allowing cells and tissues to attain normal numbers and sizes. This is achieved by dampening MAPK output in growth control in a non-cell autonomous manner, by restricting processing of EGFR ligand(s), as shown previously for R7 cell differentiation. Since both the MAPK and insulin pathways initially act to favor proliferation and growth, it is proposed that Sl functions here under insulin pathway control, allowing growth to continue, preventing ectopic differentiation. There are several ways in which it could do so: by directing activated MAPK to a different cellular compartment (cytosolic versus nuclear or by controlling overall strength and duration of signaling, examples of which have been shown to elicit such changes in developing wing cells in both Drosophila and PC12 cells (Murillo-Maldonado, 2011).

    A central function of all phospholipase C enzymes is hydrolysis of PIP2. In this study has shown that regulation of growth and differentiation by Sl must depend on PIP2 hydrolysis to some extent, because of the interaction between sl and mutations in IP3R, PKC53E and Rack1. Also, by means of genetic tests, it was found that Sl requires the Spi processing machinery (S, Rho) to regulate growth and differentiation. It has previously been shown that Sl acts on Spi processing during R7 differentiation, by favoring Spi retention in the endoplasmic reticulum. In order to rationalize Sl function in all the phenotypes studied, it was reasoned that by inhibiting Spitz processing, Sl could delay initiation of differentiation, allowing still undifferentiated cells to grow and attain a normal size before the onset of differentiation. Sl modes of action in growth and differentiation may be different; sl alleles affecting the wing but not the eye is strong evidence for this assertion (Murillo-Maldonado, 2011).

    In general, during growth, Sl activated by the insulin pathway acts as a liaison regulating MAPK pathway ligand processing, to promote MAPK activation to a level permitting growth. In agreement with a well-characterized case in mammalian cells, it is proposed that this level of activity of MAPK is different from the level required for differentiation; either it is of a different duration, or of an overall different stimulation level, or happening at a different time. Alternatively it occurs in a different subcellular compartment from that required for differentiation, acting thru Sl regulation of Spi processing. This scenario also requires both the MAPK and the insulin pathways to be active for cellular growth. Conversely, for differentiation, reduced insulin receptor signaling leads to altered (lower) levels of Sl activation and augmented Spi processing, and this in turn allows MAPK activation in a manner consistent with promotion of differentiation. This could either be caused by longer or stronger MAPK stimulation, as documented for PC12 cells, since lower Sl activity now allows higher levels of MAPK ligand processing, and/or by compartmentalization of the activated MAPK pathway, as shown for the Drosophila wing, besides happening at different times during development. In this second case, only the MAPK pathway is required to be fully active. Finally, loss-of-function mutant conditions for sl lead to ectopic differentiation at the expense of growth (Murillo-Maldonado, 2011).

    Taken together, these results indicate that Sl participates in fine coordination of growth and differentiation during development. Although Sl is not essential for wing or eye growth and development, it is necessary to achieve appropriate final structure and size. In the absence of Sl function, these tissues arrest growth prematurely and probably initiate differentiation earlier, resulting in ectopic differentiation while attaining smaller cellular sizes. As such, Sl can be seen as exerting a kind of 'parental control' that protects cells from differentiating before attaining a normal size. This function requires Sl to change cellular behavior from growth (or possibly inhibition of differentiation) to differentiation in a short period of time (Murillo-Maldonado, 2011).

    PLC-γ1 has been demonstrated to be a phosphorylation target of MAPK, and some PKC isoforms can phosphorylate PLC-γ without affecting PIP2 hydrolysis so it is clear that there is a complex interplay of signaling among this set of molecules following RTK activation. Further study of the dynamics of Sl-regulated EGF/MAPK signaling in space and time during wing and eye development in Drosophila may help to expose more of this network (Murillo-Maldonado, 2011).

    High sugar-induced insulin resistance in Drosophila relies on the lipocalin Neural Lazarillo

    In multicellular organisms, insulin/IGF signaling (IIS) plays a central role in matching energy needs with uptake and storage, participating in functions as diverse as metabolic homeostasis, growth, reproduction and ageing. In mammals, this pleiotropy of action relies in part on a dichotomy of action of insulin, IGF-I and their respective membrane-bound receptors. In organisms with simpler IIS, this functional separation is questionable. In Drosophila IIS consists of several insulin-like peptides called Dilps, activating a unique membrane receptor and its downstream signaling cascade. During larval development, IIS is involved in metabolic homeostasis and growth. This study has used feeding conditions (high sugar diet, HSD) that induce an important change in metabolic homeostasis to monitor possible effects on growth. Unexpectedly it was observed that HSD-fed animals exhibited severe growth inhibition as a consequence of peripheral Dilp resistance. Dilp-resistant animals present several metabolic disorders similar to those observed in type II diabetes (T2D) patients. By exploring the molecular mechanisms involved in Drosophila Dilp resistance, a major role was found for the lipocalin Neural Lazarillo (NLaz), a target of JNK signaling. NLaz expression is strongly increased upon HSD and animals heterozygous for an NLaz null mutation are fully protected from HSD-induced Dilp resistance. NLaz is a secreted protein homologous to the Retinol-Binding Protein 4 involved in the onset of T2D in human and mice. These results indicate that insulin resistance shares common molecular mechanisms in flies and human and that Drosophila could emerge as a powerful genetic system to study some aspects of this complex syndrome (Pasco, 2012).

    One particularity of the insect IIS is the presence of a unique receptor for multiple insulin-like peptides. This raises the possibility that the multiple functions assigned to IIS might not be independently regulated following an acute variation in environmental conditions (the 'coupling hypothesis'). This was tested experimentally during larval development, where IIS controls both systemic growth and carbohydrate homeostasis. Previous results showed that a limitation in dietary amino acids reduces circulating Dilps, which impacts both growth and carbohydrate homeostasis. This study used experimental conditions where carbohydrate metabolism is challenged by a high sugar diet and its effect on growth is monitored. HSD induced an increase in glycemia followed by increased insulinemia (high Dilp expression and accumulation in the IPCs, elevated Dilp2 concentrations in the hemolymph), which was anticipated to induce overgrowth. In contrast, HSD fed larvae gave rise to small flies due to Dilp resistance in peripheral tissue. This indicates that Dilp resistance in flies impacts both metabolic and growth functions. This raises the possibility that Dilps and IIS are not used to maintain glucose homeostasis in normal physiological conditions. Previous work has demonstrated that the fly glucagon AKH has a selective action on carbohydrate and lipid homeostasis without influencing growth. Therefore, using AKH and not Dilps to control energy homeostasis would prevent larvae from accidental coupling between metabolism and growth. This possibility finds support in the fact that AKH cells, but not Dilp cells, couple secretion to variations in glucose and internal ATP levels. The current experiments did not did not reveal noticeable changes in AKH expression or accumulation in the AKH-producing cells in response to HSD. Moreover, there is strong experimental evidence that, in addition to their growth-promoting function, circulating Dilps can influence metabolic homeostasis. This overall indicates that despite a conservation of its multiple functional outputs, the hard wiring of IIS in Drosophila does not allow a clear discrimination of growth and metabolic regulations during larval development. What are the respective contributions of Dilps and AKH to energy homeostasis in the adult fly are questions awaiting further investigation (Pasco, 2012).

    In human studies, the link between dietary carbohydrates and the development of insulin resistance and type II diabetes has long been elusive, mainly because of the difficulty to evaluate glycemic loads and indexes from food questionnaires. An increasing number of epidemiological studies now point to a role of carbohydrates in the emergence of T2D in human. In this study, in less than four days of feeding on HSD, larval tissues become strongly resistant to the effect of Dilps in vivo and to human insulin ex-vivo. This insulin-resistant state is characterized by: (1) high glycemia despite increased insulinemia, (2) increased lipid storage and circulating lipids, (3) rescue by forced Dilps secretion, (4) lack of response of peripheral tissues to stimulation by exogenous insulin. This last point was tested in different larval tissues including the fat body, which carries both hepatic and adipose functions in the larva. HSD-fed animals accumulate high lipid levels in the fat body, which becomes resistant to the action of exogenous insulin. This is reminiscent of metabolic alterations seen in response to over-nutrition in mammals, where lipid metabolites accumulate in the liver leading to liver steatosis, a hallmark of insulin resistance and T2D. In line with this, it was found that ACC expression is strongly increased in the fat body of HSD-fed larvae. This enzyme transforms acetyl-CoA into malonyl-CoA, a precursor for lipogenesis and an inhibitor of CPT-1, which imports long chain acyl CoA in the mitochondria for beta-oxydation. Suppression of ACC2 activity in mice induces beta-oxidation and was shown sufficient to reverse hepatic insulin resistance. Therefore, the fat body of HSD-fed animals is subjected to metabolic alterations similar to those taking place in the fatty liver of T2D or obese patients. These observations parallel those of Musselman (2011) who recently published a state of sugar-induced insulin resistance in Drosophila (Pasco, 2012).

    One striking finding is the fact that heterozygous NLaz/+ animals are fully protected of insulin resistance when exposed to a HSD. NLaz is a Drosophila lipocalin that is strongly up-regulated upon HSD feeding. NLaz was previously shown to act downstream of JNK to maintain metabolic homeostasis, in part by controlling lipid biogenesis and circulating carbohydrate levels. NLaz expression in the larval fat body reduces general IIS levels, whereas NLaz mutant larvae present elevated IIS. It was also found that silencing NLaz in fat cells protects larvae from HSD-induced Dilp resistance. The role of NLaz as a potential adipokine antagonizing IIS for metabolic regulation is remarkably similar to the role of its mammalian orthologs, Lipocalin 2 and the Retinol-Binding Protein 4 (RBP4). Serum concentration of both lipocalins correlate with obesity, T2D and insulin resistance in human and mice, although some of these associations have been disputed in human patients in the case of RBP4. The reduction of RBP4 concentration in diet-induced obese mice was shown to improve insulin sensitivity whereas injection of recombinant RBP4 decreases insulin sensitivity in normal mice, a phenotype associated with a strong induction of the neoglucogenic enzyme PEPCK (Yang, 2005). In addition, a functional polymorphism in the RBP4 gene associated with increased serum RBP4 was found in a Mongolian population suffering rapid increase of diabetes. These observations are functionally related to the present findings in Drosophila showing that heterozygosity for NLaz is sufficient to protect animals from diet-induced insulin resistance. In addition, the level of expression of the Drosophila PEPCK gene is strongly reduced in Nlaz mutant animals, even if ectopic expression of Nlaz is not sufficient to drive PEPCK expression (a result in line with the absence of PEPCK induction upon HSD). These data collectively suggest a common molecular basis for the mechanism of insulin resistance in organisms as distant as insects and mammals. Further work using both vertebrate and invertebrate models should help understand the role of circulating lipocalins in reducing insulin sensitivity in peripheral tissues (Pasco, 2012).

    In summary, the present study recapitulates in a highly genetically amenable system some of the interactions observed between genetic factors and environmental factors leading to T2D as pinpointed by epidemiological studies in patients. This is the demonstration that the fly can be used to screen for genes that predispose to insulin resistance with conserved functions in mammals. The clinical progression towards TD2 is still not well understood and the use of genetic models might prove useful to decipher some of its underlying mechanisms (Pasco, 2012).

    A secreted decoy of InR antagonizes insulin/IGF signaling to restrict body growth in Drosophila

    Members of the insulin peptide family have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. Drosophila insulin-like peptides (Dilps) promote tissue growth through the single insulin-like receptor (InR). Despite the important role of Dilps in nutrient-dependent growth control, the molecular mechanism that regulates the activity of circulating Dilps is not well understood. This study reports the function of a novel Secreted decoy of InR (SDR) as a negative regulator of insulin signaling. SDR is predominantly expressed in surface glia of the larval CNS and is secreted into the hemolymph. Larvae lacking SDR grow at a faster rate, thereby increasing adult body size. Conversely, overexpression of SDR reduces body growth non-cell-autonomously. SDR is structurally similar to the extracellular domain of InR and interacts with several Dilps in vitro independent of Imp-L2, the ortholog of the mammalian insulin-like growth factor-binding protein 7 (IGFBP7). It was further demonstrated that SDR is constantly secreted into the hemolymph independent of nutritional status and is essential for adjusting insulin signaling under adverse food conditions. It is proposed that Drosophila uses a secreted decoy to fine-tune systemic growth against fluctuations of circulating insulin levels (Okamoto, 2013).

    The insulin/insulin-like growth factor (IGF) signaling (IIS) pathway is an evolutionarily conserved endocrine signaling pathway that controls a wide variety of processes, including growth and development. The central players in this pathway are insulin-like peptides, which include insulin, IGF-1, and IGF-II in mammals; 40 insulin-like peptides in worms and the seven canonical Drosophila insulin-like peptides (Dilps) in flies that can promote body growth. These secreted ligands transmit intercellular signals through the activation of insulin receptor tyrosine kinase (insulin-like receptor [InR] in Drosophila), leading to the activation of the PI3-kinase (PI3K) signaling pathway (Okamoto, 2013).

    In mammals, six classic IGF-binding proteins (IGFBPs) bind to IGF-I and IGF-II with high affinity in serum and modulate IGF activity. Less than 5% of the IGFs in the circulation are free, and most IGFs are bound in the complex, which consists of IGF-I or IGF-II, IGFBP3, and an acid-labile subunit (ALS). This complex is believed to be the principle carrier form of IGFs. These proteins either enhance or dampen the IIS pathway by extending the half-life of IGFs, by altering the local and systemic availability of IGFs, or by preventing them from binding to the receptor (Hwa, 1999). In addition, an IGFBP-related protein, IGFBP7, binds to IGFs with comparatively low affinity and has been demonstrated to be a potent tumor suppressor in a wide variety of cancers (Okamoto, 2013).

    Insects also express an IGFBP-like protein, referred to as Imp-L2, that resembles IGFBP7. Imp-L2 binds to Dilp-2 and Dilp-5 and acts as a non-cell-autonomous inhibitor of IIS during development. However, it remains unknown whether other factors besides Imp-L2 regulate the seven Dilps in the extracellular space (Okamoto, 2013).

    This study characterizes a secreted decoy of InR (SDR) that binds to Dilps and antagonizes IIS during development. Biochemical and genetic analyses suggest that SDR belongs to a novel class of functional insulin-binding proteins and that it acts in a manner complementary to Imp-L2 in Drosophila (Okamoto, 2013).

    Drosophila has seven Dilps and one IGFBP-type protein (Imp-L2), whereas mammals have seven IGFBPs. An ongoing challenge has been to resolve how these proteins cooperate in the control of systemic growth via IIS. The biochemical experiments described in this study revealed that Imp-L2 binds to several Dilps, including Dilp1, Dilp2, Dilp4, Dilp5, and Dilp6. In contrast, SDR binds most strongly to Dilp3, indicating that each Dilp has distinct binding preferences for either Imp-L2 or SDR. Interestingly, in addition to expression in brain insulin-producing cells (IPCs), dilp3 is expressed in a subset of glia and midgut muscles where SDR is highly expressed. Although SDR can be detected in the hemolymph and regulates systemic growth, SDR may also act locally in the tissues where it is expressed. The slight up-regulation of the SDR transcripts during the third instar likely reflects SDR expression in imaginal discs. It is possible that SDR expression is actively regulated in a stage- and tissue-specific manner to fulfill such a local function (Okamoto, 2013).

    Recent reports have revealed that fluctuations in ligand levels have more significant biological impacts on downstream signaling events than was previously appreciated. The dynamics of insulin levels and their specific temporal pattern can elicit a unique physiological response through different kinetic behavior and network connectivity of Akt. Therefore, the function of SDR and Imp-L2 in the regulation of Dilps may be more complex than the interference of a ligand–receptor interaction. The ability to maintain constant Dilp-binding protein levels in the hemolymph would provide a robust system for growth regulation in combination with dynamic Imp-L2 levels (Okamoto, 2013).

    There are functional similarities between SDR and Imp- L2 in the sense that both act as negative regulators of IIS. However, phenotypic and biochemical analyses revealed important differences between SDR and the Imp-L2–ALS complex. First, heterozygous SDR mutants exhibit approximately normal body size, whereas loss of one copy of Imp-L2 leads to a moderate increase in body size. The partial knockdown of SDR by a weak ubiquitous Gal4 driver, arm-Gal4, consistently showed no phenotype. Second, overexpression of Imp-L2 causes lethality, whereas moderate overexpression of Imp-L2 significantly impairs body growth, resulting in a delay of larval development. In contrast, SDR overexpression leads to moderate reduction in body weight with no apparent developmental delay or lethality, even though the vast excess of SDR proteins was achieved compared with endogenous levels. Consistently, the lethality induced by ectopic Dilp2 expression can be rescued by overexpression of Imp-L2 but not by overexpression of SDR. Third, both Imp-L2 and ALS are widely expressed in a number of different tissues. Fat body-derived Imp-L2–ALS, however, seems to be critical for the systemic regulation of IIS, whereas glia-derived SDR is important in this respect. Last, Imp-L2 and ALS expression responds to nutritional status, whereas the production of SDR is constant (Okamoto, 2013).

    It is interesting to consider the analogous case in mammals, which exhibit distinct alterations in IGFBP protein levels after fasting; IGFBP1 is up-regulated by fasting, whereas IGFBP2 and IGFBP3 remain constant in circulating blood. SDR seems to act as a constitutive regulator of Dilps in the hemolymph, whereas Imp-L2 is a dynamic regulator that inhibits IIS in response to nutrient levels. It is equally possible, however, that the function of SDR is regulated post-translationally in the hemolymph; secreted SDR is inactive, and modifications and/or binding partners allow SDR to bind to Dilps. In contrast, Imp-L2 is likely active once secreted into the hemolymph. Further analysis will be required to understand the regulatory mechanism of SDR in the hemolymph and the functional relationship between SDR and the Imp-L2–ALS complex. Together, these observations suggest that Drosophila uses two different regulators that have distinct molecular activities to fine-tune the activity of circulating Dilps (Okamoto, 2013).

    In mammals, antagonistic soluble decoys have been described for many receptors, including receptor tyrosine kinases, immune receptors, and seven-pass transmembrane receptors. Although the SDR-like gene is found only in dipterous insects, including flies and mosquitoes, similar decoy systems for IIS are likely present in other species. The C. elegans insulin receptor Daf-2 contains an alternative splice variant that encodes a putative secreted protein. Similarly, the mammalian insulin receptor can potentially produce a soluble decoy by alternative intronic polyadenylation. In both cases, the physiological function of the putative secreted protein has not been addressed. The type II IGF receptor, also known as mannose-6-phosphate receptor, is thought to be cleaved to produce a soluble form (sIGF2R) that binds to IGF-II with high affinity in vivo. Indeed, ectopic expression of sIGF2R inhibits cellular growth and reduces organ size. In Drosophila, however, a soluble form of InR that is produced by alternative splicing or ectodomain shedding has not been described. Instead, SDR may have arisen by a gene duplication event in Drosophila (Okamoto, 2013).

    It remains unknown whether SDR can form a nonfunctional heterodimer with InR on the plasma membrane and thereby directly antagonize signaling through InR. Based on sequence similarity, SDR and InR are expected to show similar binding affinities for each Dilp. It is therefore hypothesized that receptor-like decoy molecules function to fine-tune receptor signaling by sequestering multiple ligands. The constitutive production of such decoys may be beneficial to adapt endocrine signals in response to environmental changes, including the availability of food (Okamoto, 2013).

    Insulin signaling regulates neurite growth during metamorphic neuronal remodeling

    Although the growth capacity of mature neurons is often limited, some neurons can shift through largely unknown mechanisms from stable maintenance growth to dynamic, organizational growth (e.g. to repair injury, or during development transitions). During insect metamorphosis, many terminally differentiated larval neurons undergo extensive remodeling, involving elimination of larval neurites and outgrowth and elaboration of adult-specific projections. This study shows in the fruit fly that a metamorphosis-specific increase in insulin signaling promotes neuronal growth and axon branching after prolonged stability during the larval stages. FOXO, a negative effector in the insulin signaling pathway, blocks metamorphic growth of peptidergic neurons that secrete the neuropeptides CCAP and bursicon. RNA interference and CCAP/bursicon cell-targeted expression of dominant-negative constructs for other components of the insulin signaling pathway (InR, Pi3K92E, Akt1, S6K) also partially suppresses the growth of the CCAP/bursicon neuron somata and neurite arbor. In contrast, expression of wild-type or constitutively active forms of InR, Pi3K92E, Akt1, Rheb, and TOR, as well as RNA interference for negative regulators of insulin signaling (PTEN, FOXO), stimulate overgrowth. Interestingly, InR displays little effect on larval CCAP/bursicon neuron growth, in contrast to its strong effects during metamorphosis. Manipulations of insulin signaling in many other peptidergic neurons revealed generalized growth stimulation during metamorphosis, but not during larval development. These findings reveal a fundamental shift in growth control mechanisms when mature, differentiated neurons enter a new phase of organizational growth. Moreover, they highlight strong evolutionarily conservation of insulin signaling in neuronal growth regulation (Gu, 2014).

    It is well established that insulin/insulin-like signaling (IIS) is crucial for regulating cell growth and division in response to nutritional conditions in Drosophila. However, most studies have focused on growth of the body or individual organs, and comparatively little is known about the roles of IIS during neuronal development, particularly in later developmental stages. Drosophila InR transcripts are ubiquitously expressed throughout embryogenesis, but are concentrated in the nervous system after mid-embryogenesis and remain at high levels there through the adult stage. This suggests that IIS plays important roles in the post-embryonic nervous system. Recently, analysis of Drosophila motorneurons, mushroom body neurons, and IPCs has revealed important roles of PI3K and Rheb in synapse growth or axon branching. These studies revealed some growth regulatory functions of IIS in the CNS, but they have not explored whether the control of neuronal growth by IIS is temporally regulated (Gu, 2014).

    This study has shown that IIS strongly stimulates organizational growth of neurons during metamorphosis, whereas the effects of IIS on larval neurons are comparatively modest. Recently, similar results have been reported in mushroom body neurons, in which the TOR pathway strongly promoted axon outgrowth of γ-neurons after metamorphic pruning. Expression of FOXO or reduction of InR function had no significant effect on larval growth of the CCAP/bursicon neurons, or on the soma size of many other larval neurons. Thus, while IIS has been shown to regulate motorneuron synapse expansion in larvae, the current findings indicate that IIS may not play a major role in regulating structural growth in many larval neurons. This is consistent with a recent report that concluded that the Drosophila larval CNS is insensitive to changes in IIS (Gu, 2014).

    When InRact was used to activate IIS without ligand, a modest but significant increase was seen in the soma size of the more anterior CCAP/bursicon neurons during larval development. This result indicates that the IIS pathway is present and functional in these larval neurons, but the ligand for InR is either absent or inactive. During metamorphosis, unlike in larvae, downregulation of IIS by altering the level of InR or downstream components of the pathway significantly reduced CCAP/bursicon neuron growth. Thus, the results suggest that IIS is strongly upregulated during metamorphosis to support post-embryonic, organizational growth of diverse peptidergic neurons, and this activation may at least in part be due to the presence of as yet unidentified InR ligands during metamorphosis (Gu, 2014).

    Attempts were to identify this proposed InR ligand source by eliminating, in turn, most of the known sources of systemic DILPs. None of these manipulations had any effect on metamorphic growth of the CCAP/bursicon neurons. These results are consistent with three possible mechanisms. First, there may be a compensatory IIS response to loss of some dilp genes. For example, a compensatory increase in fat body DILP expression has been observed in response to ablation of brain dilp genes. Second, the growth may be regulated by another systemic hormone (e.g. DILP8) that was not tested, or by residual DILP peptides in the RNAi knockdown animals. Third, a local insulin source may be responsible for stimulating metamorphic outgrowth of the CCAP/bursicon neurons. Consistent with this view, a recent report showed that DILPs secreted from glial cells were sufficient to reactivate neuroblasts during nutrient restriction without affecting body growth, while overexpression of seven dilp genes (dilp1-7) in the IPCs had no effect on neuroblast reactivation under the same conditions. It seems likely that glia or other local DILP sources play an important role in regulating metamorphic neuron growth, but further experiments will be needed to test this model (Gu, 2014).

    When IIS was manipulated in the CCAP/bursicon neurons, changes were observed in cell body size (and sometimes shape) and in the extent of branching in the peripheral axon arbor. Although this study focused analysis of neurite growth on the peripheral axons, which are easily resolved in fillet preparations, corresponding changes were also observed in the size and complexity of the central CCAP/bursicon neuron arbor. These IIS manipulations (both upregulation or downregulation) resulted in the above structural changes as well as wing expansion defects, suggesting that the normal connectivity of the CCAP/bursicon neurons was required for proper functioning of this cellular network. This model is consistent with the observation of two subsets of morphologically distinct bursicon-expressing neurons (the BSEG and BAG neurons), which are activated sequentially to control central and peripheral aspects of wing expansion. The BSEG neurons project widely within the CNS to trigger wing expansion behavior as well as secretion of bursicon by the BAG neurons. In turn, the BAG neurons send axons into the periphery to secrete bursicon into the hemolymph to control the process of tanning in the external cuticle. Therefore, manipulation of IIS within these neurons, and the changes in morphology that result, may disrupt the wiring and function of this network. However, because the possibility cannot be ruled out that these IIS manipulations also altered neuronal excitability, synaptic transmission, or neuropeptide secretion, this study relied on measurements of cellular properties (and not wing expansion rates) when assessing the relative effects of different IIS manipulations on cell growth (Gu, 2014).

    The results indicate that IIS is critical for organizational growth, which occurs during insect metamorphosis but is also seen during neuronal regeneration in other systems. However, the regenerative ability of many neurons is age-dependent and context-dependent; immature neurons possess a more robust regenerative capacity, while the regenerative potential of many mature neurons is largely reduced. In particular, the adult vertebrate CNS displays very limited regeneration, in marked contrast to the regeneration abilities displayed by the peripheral nervous system. Recent studies in mice suggest that age-dependent inactivation of mTOR contributes to the reduced regenerative capacity of adult corticospinal neurons, and activation of mTOR activity through PTEN deletion promoted robust growth of corticospinal tract axons in injured adult mice. The current genetic experiments demonstrate a requirement for activity of TOR, as well as several other IIS pathway components both upstream and downstream of TOR, in controlling organizational growth of many peptidergic neurons. This suggests that under certain conditions, the activation of IIS may be a crucial component of the conversion of mature neurons to a more embryonic-like state, in which reorganizational growth either after injury or as a function of developmental stage is possible. Given the strong evolutionary conservation of these systems and the powerful genetic tools available to identify novel regulatory interactions in Drosophila, studies on the control of organizational growth in this species hold great promise for revealing factors that are crucial for CNS regeneration (Gu, 2014).

    Delivery of circulating lipoproteins to specific neurons in the brain regulates systemic insulin signaling

    The Insulin signaling pathway couples growth, development and lifespan to nutritional conditions. This study demonstrates a function for the Drosophila lipoprotein LTP (FlyBase term: Apolipoprotein lipid transfer particle) in conveying information about dietary lipid composition to the brain to regulate Insulin signaling. When yeast lipids are present in the diet, free calcium levels rise in blood brain barrier (BBB) glial cells. This induces transport of LTP across the Blood Brain Barrier by two LDL receptor-related proteins: LRP1 and Megalin. LTP accumulates on specific neurons that connect to cells that produce Insulin-like peptides, and induces their release into the circulation. This increases systemic Insulin signaling and the rate of larval development on yeast-containing food compared with a plant-based food of similar nutritional content (Brankatschk, 2014).

    Nutrient sensing by the central nervous system is emerging as an important regulator of systemic metabolism in both vertebrates and invertebrates. Little is known about how nutrition-dependent signals pass the blood brain barrier to convey this information. Like the vertebrate BBB, the BBB of Drosophila forms a tight barrier to passive transport, and is formed by highly conserved molecular components. Its simple structure and genetic accessibility make it an ideal model to study how nutritional signals are communicated to the CNS. Insulin and Insulin-like growth factors are conserved systemic signals that regulate growth and metabolism in response to nutrition. Although fruit flies do not have a single pancreas-like organ, they do produce eight distinct Drosophila Insulin/IGF-like peptides (Dilps) that are expressed in different tissues. A set of three Dilps (dILP2,3,5), released into circulation by Dilp-producing cells (IPCs) in the brain, have particularly important functions in regulating nutrition-dependent growth and sugar metabolism; ablation of IPCs in the CNS causes diabetes-like phenotypes, slows growth and development, and produces small, long-lived adult flies. Systemic Insulin/IGF signaling (IIS) increases in response to dietary sugars, proteins and lipids. Sugars act on IPCs directly to promote Dilp release, but other nutrients are sensed indirectly through signals from the fat body (an organ analogous to vertebrate liver/adipose tissue) (Brankatschk, 2014).

    The Drosophila fat body produces two major types of lipoprotein particles: Lipophorin (LPP; Retinoid- and fatty acid-binding glycoprotein), the major hemolymph lipid carrier, and Lipid Transfer Particle (LTP). LTP transfers lipids from the intestine to LPP. These lipids include fatty acids from food, as well as from endogenous synthesis in the intestine. LTP also unloads LPP lipids to other cells (Van Heusden, 1989; Canavoso, 204; Parra-Peralbo, 2011). LPP crosses the BBB and accumulates throughout the brain. It is required for nutrition-dependent exit of neural stem cells from quiescence (Brankatschk, 2010). This study investigated possible functions of LTP in the brain (Brankatschk, 2014).

    This work demonstrates a key requirement for lipoproteins in conveying nutritional information across the BBB to specific neurons in the brain. As particles that carry both endogenously synthesized and diet-derived lipids, lipoproteins are well-positioned to perform this function. The data suggest that transport of LTP across the BBB to Dilp2-recruiting neurons (DRNs) influences communication between DRNs and the Dilp-producing IPCs, increasing the release of Dilp2 into circulation. Since the IPCs also deliver Dilp2 to the DRNs, this indicates that these two neuronal populations may communicate bidirectionally. How might LTP affect the function of DRNs? One possibility is that it acts to deliver a signaling lipid to the DRNs. It could do so either directly, or indirectly by promoting lipid transfer from LPP, which is present throughout the brain. LTP enrichment on specific neurons may increase lipid transfer to these cells (Brankatschk, 2014).

    This work highlights a key function for BBB cells in transmitting nutritional information to neurons within the brain. Feeding with yeast food increases free calcium in BBB glia, which then increases transport of LTP to DRNs. How might BBB cells detect the difference between yeast and plant food? The data suggest differences in the lipid composition of yeast and plant-derived foods are responsible. Previous work has shown that the lipids in these foods differ in their fatty acid composition. Yeast food has shorter and more saturated fatty acids than plant food (24). How could these nutritional lipids affect the activity of BBB glia? Interestingly, differences in food fatty acid composition are directly reflected in the fatty acids present in membrane lipids of all larval tissues including the brain. Thus, it is possible that the bulk membrane properties of BBB glia are different on these two diets. Membrane lipid composition is known to affect a variety of signaling events. Alternatively, yeast food may influence the specific fatty acids present in signaling lipids that activate BBB glia (Brankatschk, 2014).

    This study demonstrates an unexpected functional specialization of the BBB glial network, which permits specific and regulated LTP transport to particular neurons. How this specificity arises is an important question for the future. It is noted that a subset of glial cells within the brain also accumulates LTP derived from the fat body. Could these represent specific transport routes from the BBB (Brankatschk, 2014)?

    An alternative possibility is that transport depends on neuronal activity. Mammalian LRP1 promotes localized transfer of IGF in response to neuronal activity. Could LTP delivery by LRP1 and LRP2 (Megalin) in the Drosophila brain depend on similar mechanisms? The remarkable specificity of LTP trafficking in the Drosophila CNS provides a novel framework for understanding information flow between the circulation and the brain (Brankatschk, 2014).

    To what extent might this be relevant to vertebrate systems? While it is clear that the vertebrate brain (unlike that of Drosophila) does not depend on lipoproteins to supply it with bulk sterols, this does not rule out possible functions for these particles in nutrient sensing. The vertebrate cerebrospinal fluid is rich in many types of HDL particles, including those containing ApoA-1, which is not expressed in the brain - this suggests that at least some lipoprotein particles in the brain may derive from the circulation. Consistent with this idea, ApoA-I can target albumin-containing nanoparticles across the BBB in rodents. Recent work suggests that lipoproteins may be the source of specific Long Chain Fatty Acids that signal to the hypothalamus to regulate glucose homeostasis, since neuronal lipoprotein lipase is required for this process. Thus, it would be interesting to investigate whether circulating mammalian lipoproteins might reach a subset of neurons in the hypothalamus (Brankatschk, 2014).

    It has been known for some time that increasing the amount of yeast in the diet of lab grown Drosophila melanogaster increases the rate of development and adult fertility, but reduces lifespan. This study shows that flies have evolved specific mechanisms to increase systemic IIS in response to yeast, independently of the number of calories in the diet or its proportions of sugars proteins and fats. What pressures could have driven the evolution of such mechanisms? In the wild, Drosophila melanogaster feed on rotting plant material and their diets comprise both fungal and plant components. Drosophila disperse yeasts and transfer them to breeding sites during oviposition improving the nutritional resources available to developing larvae. Yeast that are able to induce more rapid development of the agents that disperse them may propagate more efficiently. On the other hand, it has been noted that Drosophila species that feed on ephemeral nutrient sources like yeasts or flowers have more rapid rates of development than other species. It may be that, even within a single species, the ability to adjust developmental rate to the presence of a short-lived resource is advantageous. Humans subsist on diets of both plant and animal materials that during most of evolution have differed in their availability. It would be interesting to investigate whether Insulin/IGF signaling in humans might respond to qualitative differences in the lipid composition of these nutritional components (Brankatschk, 2014).

    Insulin signalling mediates the response to male-induced harm in female Drosophila melanogaster

    Genetic manipulations in nutrient-sensing pathways are known to both extend lifespan and modify responses to environmental stressors (e.g., starvation, oxidative and thermal stresses), suggesting that similar mechanisms regulate lifespan and stress resistance. However, despite being a key factor reducing female lifespan and affecting female fitness, male-induced harm has rarely been considered as a stressor mediated by nutrient sensing pathways. This study explored whether a lifespan-extending manipulation also modifies female resistance to male-induced harm. To do so, long-lived female Drosophila melanogaster were used that had their insulin signalling pathway downregulated by genetically ablating the median neurosecretory cells (mNSC). The level of exposure to males was varied for control and ablated females, and tests were performed for interacting effects on female lifespan and fitness. As expected, lifespan significantly declined with exposure to males. However, mNSC-ablated females maintained significantly increased lifespan across all male exposure treatments. Furthermore, lifespan extension and relative fitness of mNSC-ablated females were maximized under intermediate exposure to males, and minimized under low and high exposure to males. Overall, these results suggest that wild-type levels of insulin signalling reduce female susceptibility to male-induced harm under intense sexual conflict, and may also protect females when mating opportunities are sub-optimally low (Sepil, 2016).

    Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis

    In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). This study shows that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulated ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. The study proposes that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant (Ohhara, 2015).

    In many animal species, the developmental transition is a well-known biological process in which the organism alters its body morphology and physiology to proceed from the juvenile growth stage to the adult reproductive stage. For example, in mammals, puberty causes a drastic change from adolescent to adulthood, whereas in insects, metamorphosis initiates alteration of body structures to produce sexually mature adults, a process accompanied by changes in habitat and behavior. These developmental transitions are primarily regulated by steroid hormones, production of which is regulated coordinately by developmental timing and nutritional conditions. How these processes are precisely regulated in response to developmental and environ mental cues is a longstanding question in biology (Ohhara, 2015).

    In holometabolous insects, the steroid hormone ecdysone plays a pivotal role in metamorphosis. In Drosophila, metamorphic development from the third-instar larva into the adult, through the prepupa and pupa, initiates 90-96 h after hatching (hAH) at 25°C under a standard culture condition. At the onset of the larval-prepupal transition, ecdysone is produced in the prothoracic gland (PG) and then converted into its active form, 20-hydroxyecdysone (20E), in the peripheral organs. The activities of 20E terminate larval development and growth and initiates metamorphosis. Ecdysone biosynthesis is regulated in the PG by neuropeptides, enabling modulation of the timing of 20E pulses during development. The best-known stimulator of ecdysone biosynthesis is prothoracico-tropic hormone (PTTH), which is produced by neurons in the CNS. PTTH activates the receptor tyrosine kinase Torso in the PG to stimulate expression of ecdysone biosynthetic genes through the Ras85D/Raf/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Insulin-like peptides (Ilps), members of another class of neuron-derived factors, activate PI3K in the PG, resulting in production of ecdysone biosynthetic proteins. The Activin/transforming growth factor-β (TGF-β) signaling pathway is also required in the PG for the expression of PTTH and Ilps receptors, although to date it remains unclear which organ produces the ligand that acts on the PG (Ohhara, 2015).

    In addition to these neuropeptides, the larval-prepupal transition is modulated by environmental cues such as nutritional conditions that influence larval body size. For example, at 56 hAH, early third-instar larvae attain the minimal viable weight (MVW), at which sufficient nutrition is stored in larvae to ensure their survival through metamorphosis. After attaining MVW, larvae pass another checkpoint, critical weight (CW), defined as the minimum larval size at which starvation no longer delays the larval-prepupal transition. In Drosophila, both checkpoints occur almost simultaneously, making it difficult to distinguish them. However, CW is regarded as a body size checkpoint that initiates metamorphosis and is therefore believed to ultimately modulate ecdysone production in the PG. However, its downstream effectors and signaling pathway remain elusive (Ohhara, 2015).

    Based on data obtained in Manduca and Bombyx, a G protein-coupled receptor (GPCR) has long been postulated to be essential for ecdysone biosynthesis in the PG. However, this GPCR and its ligand have not yet been identified. This study shows that monoaminergic autocrine signaling through a GPCR, β3-octopamine receptor (Octβ3R), plays an essential role in ecdysone biosynthesis to execute the larval-prepupal transition. Octβ3R is also required for activation of PTTH and Ilps signaling. It is proposed that this autocrine system acts downstream of the CW checkpoint to allow the larval-prepupal transition. It is speculated that monoamines play an evolutionarily conserved role in the regulation of steroid hormone production during developmental transitions (Ohhara, 2015).

    Previously studies have shown that the GPCR Octβ3R is expressed in multiple larval tissues, including the PG. To determine whether Octβ3R is involved in ecdysone biosynthesis and metamorphosis, RNAi was used to knock down Octβ3R function specifically in the PG, using the Gal4-upstream activation sequence (UAS) system. Two different UAS-Octβ3RRNAi constructs targeting distinct regions of the Octβ3R mRNA (Octβ3RRNAi-1 and Octβ3RRNAi-2) were used to knock down Octβ3R in the PG with the help of a phantom (phm)-22-Gal4 driver. Strikingly, larvae expressing Octβ3RRNAi in the PG never developed into adult flies, and 96% of phm>Octβ3RRNAi-1 animals and 34% of phm>Octβ3RRNAi-2 animals arrested at the larval stage. When UAS-dicer2 was introduced into phm>Octβ3RRNAi-2 larvae (phm>Octβ3RRNAi-2+dicer2) to increase RNAi activity, all of these animals arrested at the larval stage. Using in situ hybridization, a significant reduction was confirmed in the Octβ3R mRNA levels in the PG of knockdown animals relative to control larvae. These data suggest that Octβ3R expression in the PG is essential for executing the larval-prepupal transition in metamorphosis (Ohhara, 2015).

    Because a similar defect in the larval-prepupal transition occurs in ecdysone-deficient larvae, it was hypothesized that the Octβ3R knockdown phenotype was due to lack of ecdysone production. Consistent with this idea, the 20E titer was much lower in phm>Octβ3RRNAi-1 larvae than in control larvae just before the larval-prepupal transition (90 hAH). Moreover, administration of 20E by feeding rescued the defect in the larval- prepupal transition caused by Octβ3R knockdown. When phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae were cultured on media containing 20E (1 mg/mL) from 48 hAH onward, approximately half of them developed to the prepupal stage, compared with only 2-3% of larvae not fed 20E. Thus, PG-specific loss of Octβ3R activity causes an arrest in the larval-prepupal transition due to lack of ecdysone (Ohhara, 2015).

    Ecdysone is synthesized in the PG from dietary cholesterol through the action of seven ecdysone biosynthetic genes (neverland, spookier, shroud, Cyp6t3, phantom, disembodied, and shadow). Quantitative RT- PCR (qPCR) was performed to investigate whether loss of Octβ3R function affects expression of these genes in the PG. In control larvae, expression of these genes increased dramatically between 72 and 96 hAH, when the larval-prepupal transition occurs. By contrast, in phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae, the expression of all of these genes was significantly reduced relative to control larvae at 96 hAH. The reduced expression of ecdysone biosynthetic genes in the PG was confirmed by in situ hybridization. Furthermore, immunostaining revealed that Neverland, Shroud, Phantom, Disembodied, and Shadow protein levels were reduced in the PG of phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae. Taken together, these data show that Octβ3R function is required in the PG for proper expression of ecdysone biosynthetic genes (Ohhara, 2015).

    Octβ3R is thought to be activated by octopamine and tyramine binding. Octopamine is synthesized from tyramine by tyramine β-hydroxylase (Tbh), and tyramine is synthesized from tyrosine by tyrosine decarboxylase (Tdc). In Drosophila, two Tdc genes (Tdc1 and Tdc2) and one Tbh gene have been identified, and all of them are expressed in the larval CNS. Tdc1, Tdc2, and Tbh are also expressed in the PG. Furthermore, octopamine and tyramine were detected in the PG by immunostaining. Thus, octopamine and/or tyramine synthesized in the PG may activate Octβ3R in an autocrine manner to induce ecdysone production (Ohhara, 2015).

    To test this, PG-specific knockdowns of Tdc1, Tdc2, and Tbh were generated. To knock down Tdc2, two constructs targeting distinct regions of the Tdc2 transcript (Tdc2RNAi-1 and Tdc2RNAi-2) were expressed along with dicer2 in the PG under the control of the phm-22-Gal4 driver (phm > Tdc2RNAi-1+dicer2 and phm > Tdc2RNAi-2+dicer2). All phm > Tdc2RNAi-1+dicer2 larvae arrested at the larval stage, and phm > Tdc2RNAi-2+dicer2 larvae were significantly delayed at the larval-prepupal transition, relative to control animals. Tdc2 mRNA level was reduced in the ring gland (RG) containing the PG in both sets of knockdown animals, as demonstrated by qPCR. Moreover, octopamine and tyramine production in the PG was impaired by Tdc2 knockdown. By contrast, Tdc1 knockdown (phm > Tdc1RNAi+dicer2) caused only a subtle delay in the larval-prepupal transition and had no detectable effect on octopamine or tyramine production. These results suggest that Tdc2 is the predominant Tdc regulating octopamine and tyramine biosynthesis in the PG and the larval-prepupal transition. Contrary to these findings, a null mutation in Tdc2 does not affect metamorphosis, and these mutant flies are viable. Thus, PG-specific knockdown causes a stronger phenotype than complete loss of Tdc2 activity in whole animals. A similar situation has been reported in regulation of metamorphosis by Activin signaling. These phenomena can be explained by a model in which some compensatory changes in other mutant tissues rescue the PG-specific knockdown phenotype in null-mutant animals (Ohhara, 2015).

    PG-specific Tdc2 knockdown caused a reduction in larval 20E concentration. Therefore, whether feeding 20E to Tdc2 knockdown larvae would rescue the larval- prepupal transition defect was examined. To this end, phm > Tdc2RNAi-1+ dicer2 and phm > Tdc2RNAi-2+dicer2 larvae were cultured in media with or without 20E (1 mg/mL) from 48 hAH onward. Approximately 40% of the 20E-fed phm > Tdc2RNAi-1+dicer2 larvae developed to the prepupal stage, whereas none of those larvae grown on control media progressed beyond the larval stage. Furthermore, the delay in the larval-prepupal transition in phm > Tdc2RNAi-2+dicer2 larvae was rescued by 20E feeding. These results indicate that the defect in the larval-prepupal transition in Tdc2 knockdown animals results from a lack of 20E production. Thus, octopamine/ tyramine synthesized in the PG appears to activate Octβ3R in an autocrine manner to execute the larval-prepupal transition by regulating ecdysone production (Ohhara, 2015).

    To determine which Octβ3R ligand is responsible for this autocrine signaling, Tbh was knocked down in the PG to prevent conversion of tyramine into octopamine. To knock down Tbh, two constructs targeting distinct regions of the Tbh transcript (TbhRNAi-1 and TbhRNAi-2) were expressed along with dicer2 under the control of phm-22-Gal4 (phm > TbhRNAi-1+ dicer2 and phm > TbhRNAi-2+dicer2). Although the Tbh knockdown caused a reduction in octopamine production in the PG, these larvae did not exhibit any obvious defects in the larval-prepupal transition or subsequent metamorphosi. These data suggest that tyramine, rather than octopamine, is the Octβ3R ligand that activates ecdysone production in the PG (Ohhara, 2015).

    Because ecdysone biosynthesis in the PG is under the control of Ilps and PTTH signaling, it was next examined whether Octβ3R function is required to activate these signaling pathways. To detect Ilps signaling activity, a pleckstrin-homology domain fused to GFP (PH-GFP), which is recruited to the plasma membrane when insulin signaling is activated, was used. In the PG cells of control larvae, PH-GFP was only weakly localized to the plasma membrane at 48 hAH, whereas its membrane localization became increasingly evident at 60, 84, and 90 hAH. By contrast, in PG cells of phm>Octβ3RRNAi-1 larvae, the tight localization of PH-GFP to the plasma membrane was no longer detectable, indicating that activation of Ilps signaling had been disrupted. Moreover, overexpression of a constitutively active form of the Ilps receptor InR (InRCA) was able to rescue the larval arrest in phm>Octβ3RRNAi-1 animals. Next, immunostaining was performed of the diphosphorylated form of ERK (dpERK), a downstream signaling component of the PTTH pathway. dpERK expression was found to be very weak at 48 hAH, but was activated in the PG of control larvae at 60, 84, and 90 hAH; by contrast, this activation was reduced in the PG of phm>Octβ3RRNAi-1 larvae. Expression of a constitutively active form of another downstream PTTH signaling component, Ras (RasV12), rescued the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. These results show that Octβ3R function is required to activate Ilps and PTTH signaling in the PG and that these signaling pathways execute the larval-prepupal transition. Although activation of both the Ilps and PTTH signaling pathways requires Activin/TGFβ signaling in the PG, expression of a constitutively active form of the Activin/ TGFβ receptor Baboon (BaboCA) failed to rescue the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. This observation suggests that Octβ3R acts downstream or independent of Activin/TGFβ signaling to regulate Ilps and PTTH signaling in the PG (Ohhara, 2015).

    The observations described above demonstrate that phm>Octβ3RRNAi affects Ilps and PTTH signaling in the PG as early as 60 hAH, raising the question of when Octβ3R function is required in the PG for execution of the larval-prepupal transition. To address this issue, the Gal80ts and Gal4/UAS system, which restricts expression of Octβ3R dsRNA in the PG at 18oC, but allows its expression at 28oC, was used. The results of temperature upshift and downshift experiments revealed that the larval-prepupal transition was impaired only when Octβ3R dsRNA was expressed in the PG at around 60 hAH. Notably, 60 hAH is the critical period during which larvae attain CW under nutrient-rich conditions. As noted above, when larvae are starved before attainment of CW, they are unable to transit into the prepupal stage. By contrast, starved larvae can successfully transit to prepupal/pupal stage without developmental delay once they have attained CW by growing beyond the critical period (~56 hAH) under nutrient-rich conditions in standard Drosophila medium. Thus, it is hypothesized that Octβ3R signaling acts downstream of the body-size checkpoint, or attainment of CW, to allow the larval-prepupal transition (Ohhara, 2015).

    Several lines of evidence support this hypothesis. First, Octβ3R function is required for activation of Ilps and PTTH signaling detected in the PG at 60 hAH. By contrast, at 48 hAH, before the attainment of CW, neither signaling pathway is active in the PG. Second, Ilps and PTTH signaling was not activated in the PG when the larvae were starved from 48 hAH onward (early starvation), whereas these signaling pathways were active when the larvae were starved after 60 hAH (late starvation). Finally, a ligand for Octβ3R, tyramine, was detectable in the PG at 60 hAH, but decreases after this stage under a nutrient-rich condition. This decrease in tyramine was abrogated by early starvation but not by late starvation. Assuming that this decrease in tyramine in the PG is due to its secretion from PG cells, it is reasonable to propose that attainment of CW causes tyramine secretion from the PG at around 60 hAH, which in turn activates Octβ3R to regulate the Ilps and PTTH pathways, leading to the larval-prepupal transition (Ohhara, 2015).

    This study demonstrates that monoaminergic regulation plays a pivotal role in ecdysone biosynthesis to induce metamorphosis and that Octβ3R acts as an upstream regulator essential for the Ilps and PTTH signaling. In addition, the data indicate that Octβ3R ligands are produced in the PG to stimulate ecdysone biosynthesis in an autocrine manner. Autocrine signaling has been proposed to mediate the community effect, in which identical neighboring cells are coordinated in their stimulation and maintenance of cell type-specific gene expression and their differentiation, as observed in muscle development of amphibian embryos. Thus, it is proposed that monoaminergic autocrine signaling among PG cells acts to increase their responsiveness to Ilps and PTTH, thereby allowing coordinated ex- pression of ecdysone biosynthetic genes within a time window following exposure to neuropeptides (Ohhara, 2015).

    These findings raise the larger question of whether monoamine acts as part of an evolutionarily conserved mechanism of steroid hormone production. In vertebrates, there is limited evidence of monoaminergic regulation of steroid hormone biosynthesis. For example, in cultured adrenal glands, catecholamine stimulates the biosynthesis of the steroid hormone cortisol in a paracrine manner to elicit a stress reaction. Another example is the Leydig cells of the mammalian testes, in which the steroid hormone testosterone is produced mainly in response to pituitary gonadotropin. However, catecholamine signaling through β-adrenergic receptors, the orthologs of Octβ3R, also promotes the production of testosterone from cultured fetal Leydig cells, which may be the site of catecholamine synthesis in the fetal and mature human testes. Thus, monoamines may play a conserved role in modulating and/or stimulating steroid hormone production during physiological and developmental transitions (Ohhara, 2015).

    Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis

    Despite their fundamental importance for body size regulation, the mechanisms that stop growth are poorly understood. In Drosophila melanogaster, growth ceases in response to a peak of the molting hormone ecdysone that coincides with a nutrition-dependent checkpoint, critical weight. Previous studies indicate that insulin/insulin-like growth factor signaling (IIS)/Target of Rapamycin (TOR) signaling in the prothoracic glands (PGs) regulates ecdysone biosynthesis and critical weight. This study elucidates a mechanism through which this occurs. This study shows that Forkhead Box class O (FoxO), a negative regulator of IIS/TOR, directly interacts with Ultraspiracle (Usp), part of the ecdysone receptor. While overexpressing FoxO in the PGs delays ecdysone biosynthesis and critical weight, disrupting FoxO-Usp binding reduces these delays. Further, feeding ecdysone to larvae eliminates the effects of critical weight. Thus, nutrition controls ecdysone biosynthesis partially via FoxO-Usp prior to critical weight, ensuring that growth only stops once larvae have achieved a target nutritional status (Koyama, 2014).

    Dally proteoglycan mediates the autonomous and nonautonomous effects on tissue growth caused by activation of the PI3K and TOR pathways

    How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The PTEN and TOR pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. This study used the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic Dpp signaling molecule. The findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. This study presents evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size (Ferreira, 2015).

    Evidence is presented that targeted deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways, known to promote tissue overgrowth by increasing the number and/or size of cells, induces a nonautonomous reduction in tissue size of adjacent cell populations. This nonautonomous effect is a consequence of a reduction in both cell size and proliferation rates (cell number), and it is not a consequence of programmed cell death or the withdrawal of nutrients from neighboring tissues, as reducing the levels of proapoptotic genes or subjecting larvae to different amino-acid diets does not have any impact on the size reduction of neighboring cell populations. The glypican Dally, which plays a major role in regulating the spread of Dpp in Drosophila tissues, is up-regulated upon deregulation of these tumor suppressor pathways, and the increase in Dally expression levels contributes to the autonomous effects on tissue size and to the nonautonomous reduction in cell number. Whereas the autonomous effects on tissue size caused by deregulation of these tumor suppressor pathways are most probably due, as least in part, to the capacity of Dally to facilitate Dpp spreading throughout the tissue, it is proposed that the nonautonomous effects on cell number are a consequence of withdrawal of Dpp from neighboring tissues. This proposal is based on a number of observations. First, the width of the Dpp activity gradient as well as the total amount of Dpp activity was reduced in adjacent cell populations upon targeted depletion of tumor suppressor pathways. Second, the nonautonomous effects on tissue size were fully rescued by Dally depletion, which has a rather specific role on the spread of Dpp when overexpressed. Third, the nonautonomous effects on tissue size, growth and proliferation rates, and/or Dpp availability and signaling can be phenocopied by overexpression of Dally or the Dpp receptor Tkv (Ferreira, 2015).

    Different strengths of the autonomous and nonautonomous effects were observed upon deregulation of these tumor suppressor pathways or overexpression of Dally in either the A or P compartments. Despite the mild autonomous induction of tissue growth caused by the ci-gal4 driver in A cells, it caused a relatively strong nonautonomous reduction of the neighboring compartment. On the contrary, the en-gal4 driver caused a strong autonomous induction of tissue growth in P cells but a relatively weak nonautonomous reduction of the neighboring compartment. The differential autonomous response might simply reflect different strengths of these Gal4 drivers. By contrast, the strongest nonautonomous effects caused by the ci-gal4 driver (when compared to the en-gal4 driver) might be because Dpp expression is restricted to the A compartment and increased levels of Dally in Dpp expressing cells are more efficient at titrating out the levels of this growth factor from the neighboring compartment. It was noticed that the nonautonomous effects on cell size observed upon deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways are Dally independent, as overexpression of Dally did not cause a nonautonomous reduction in cell size. Moreover, depletion of Dally did not rescue the nonautonomous reduction in cell size caused by activation of these pathways. These results are consistent with the fact that changes in Dpp signaling do not cause any effect on cell size and indicate that Dally and Dpp are regulating cell number but not cell size. Somatic mutations in tumor suppressor genes such as PTEN or TSC are frequently accumulated in early events of tumor development, and these mutations are thought to contribute to the selection of tumorigenic cells. Competition for available growth factors, by modulating the levels of glypicans, such as Dally, might contribute to the outcompetition of wild-type cells and to the selection of malignant mutation-carrying cells in human cancer (Ferreira, 2015).

    The PI3K/PTEN and TSC/TOR signaling pathways play a role not only in disease but also during normal development. These two pathways modulate the final size of the developing organism according to nutrient availability. The current results also identify, in this context, Dally as a molecular bridge between nutrient sensing and wing scaling in Drosophila. In a condition of high nutrient availability, which leads to the activation of the nutrient-sensing PI3K/PTEN and TSC/TOR pathways, increased levels of Dally facilitate the spread of Dpp throughout the growing tissue and contribute to the generation of larger but well-proportioned and scaled adult structures. Depletion of Dally expression levels rescues the tissue growth caused by high levels of nutrients or activation of the nutrient-sensing pathways and gives rise to smaller and, again, well-proportioned and scaled adult structures. Of remarkable interest is the capacity of Dally to induce tissue overgrowth when overexpressed or to mediate tissue growth upon deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways. Interestingly, deregulation of these pathways, and the resulting tissue overgrowth, leads to the expansion of the Dpp gradient without affecting the total levels of Dpp signaling (Ferreira, 2015).

    These results imply that Dpp activity levels do not play an instructive role in promoting tissue growth but rather that it is the range of the Dpp gradient that regulates final tissue size. Consistent with this proposal, depletion of Dally levels in one compartment (which might lead to increased levels of available Dpp in the neighboring cell population) does not cause any visible nonautonomous effect in tissue size. These results are reminiscent of the capacity of Dpp to restrict its own spreading through the repression of Pentagone, a diffusible protein that interacts with Dally and contributes to the expansion of the Dpp gradient. The graded distribution of Dpp leads, via the interaction with its receptor complex, to the graded activation of Mad/Medea, which in turn represses the transcription of brinker (brk). This creates a gradient of Brk expression that is reciprocal to the Dpp gradient. Brk is a transcriptional repressor that acts negatively to establish, in a dose-dependent manner, the expression domain of Dpp target genes like spalt. Thus, Dpp regulates the expression of target genes by repressing brinker. Remarkably, the reduced size of the wing primordium observed in hypomorphic alleles of dpp is restored when combined with brk mutants. This experimental evidence indicates that Dpp controls wing growth entirely via repression of brk. The Dally-mediated increase in the width of the Dpp gradient observed upon deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways might contribute to restrict the expression domain of brk to the lateral sides of the wing primordium. Similarly, the nonautonomous decrease in the width of the Dpp gradient might cause an expansion of the brkdomain, which is known to repress growth. Interestingly, Dally-mediated spreading of other secreted growth factors might also contribute to the autonomous effects on tissue growth caused by deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways. This is revealed by the fact that Dally depletion rescues both the autonomous and the nonautonomous effects, whereas deregulation of these pathways are still able to induce some growth upon knocking down Dpp (Ferreira, 2015).

    Compartments have been proposed to be units of growth control. In other words, the size of each compartment is controlled independently. The results on the lack of nonautonomous effects on tissue growth upon depletion of Dally or Sfl, the enzyme needed for the modification of HS chains within glypicans, indicate that this is the case. Targeted depletion of glypican expression or activity in the developing compartments gave rise to an autonomous reduction in tissue size without affecting the neighboring compartment. However, independent lines of evidence support the view that adjacent compartments buffer local variations in tissue growth caused by different means, including a nonautonomous reduction in tissue size upon depletion of the protein biosynthetic machinery or reduced epidermal growth factor receptor (EGFR) activity. The current results on the capacity of overgrowing compartments to withdraw Dpp from neighboring tissues upon targeted deregulation of the PI3K/PTEN, TSC/TOR, or hippo/Yorkie pathways and to cause a nonautonomous reduction in growth and proliferation rates reinforce the view that compartments are susceptible to modulate their growth rates upon different types of stress, including depletion of tumor suppressor genes. Interestingly, the halteres and wings of Drosophila are homologous thoracic appendages, and the activity of the Ultrabithorax (Ubx) Hox gene in the haltere discs contributes to defining its reduced size. Remarkably, it does so by reducing the expression levels of Dally, thus reinforcing the role of Dally in modulating tissue growth in epithelial organs (Ferreira, 2015).

    Transgenerational inheritance of diet-induced genome rearrangements in Drosophila

    Ribosomal RNA gene (rDNA) copy number variation modulates heterochromatin formation and influences the expression of a large fraction of the Drosophila genome. This discovery, along with the link between rDNA, aging, and disease, highlights the importance of understanding how natural rDNA copy number variation arises. Pursuing the relationship between rDNA expression and stability, this study discovered that increased dietary yeast concentration, emulating periods of dietary excess during life, results in somatic rDNA instability and copy number reduction. Modulation of Insulin/TOR signaling produced similar results, indicating a role for known nutrient sensing signaling pathways in this process. Furthermore, adults fed elevated dietary yeast concentrations produced offspring with fewer rDNA copies demonstrating that these effects also occurred in the germline, and were transgenerationally heritable. This finding explains one source of natural rDNA copy number variation revealing a clear long-term consequence of diet (Aldrich, 2015).

    This work has established that ribosomal DNA (rDNA) copy number polymorphisms can be created by manipulating the diet of wild-type flies. By directly altering insulin-like signaling and phenocopying nucleolar instability in culture using recombinant insulin, normal IIS signaling can be a significant source of rDNA copy number variation in the soma. Diet-induced rDNA copy number changes occur in both the soma and germline. As a result, they are both permanent within an organism and are capable of being transmitted to subsequent generations, hence may act as a codex for dietary history of an individual or for a population (Aldrich, 2015).

    Dietary modulation can account for loss of rDNA, but some unknown factor must be responsible for establishing some limit to the loss. The mechanism for this maintenance is unknown, although it could be an as-yet unobserved intentional regulated processes that assures minimal rDNA copy number, or it could be by normal selective pressures exerted by the Minute or bobbed phenotypes that result from very low ribosome number. Alternatively, loss may be balanced by gain of rDNA through unequal sister chromatid exchange, gene conversion, re-replication, or cycles of excision, rolling-circle replication, and re-integration. Meiotic magnification and somatic pseudo-magnification at the rDNA have long been known in Drosophila, although the identification of a mechanism has eluded researchers for over 40 years. Part of the asymptotic limit to loss may be the natural ecology of Drosophila, wherein older males (with greater loss) may be less likely to mate, produce fewer offspring, or produce an altered sex ratio; ecological experiments would be needed to address these possible contributions (Aldrich, 2015).

    The rDNA is the major site of nuclear energy utilization-transcription, processing, packaging, and export-and was known to be responsive to the energy status of the cell. This response by rDNA to diet, and its fortuitous cleavage by I-CreI, will allowed identification of rDNA copy number as a factor which stabilizes the genome. This observation is now confirmed in Drosophila and similar hypotheses have been proposed for yeast rDNA. However it seems unlikely that the rDNA is alone in this ability. Half of the genome of Drosophila is composed of interspersed or tandem repeats -- the transposable elements, highly-repetitive DNAs, expressed repeat gene clusters -- and these sequences may account for some of the remaining regulatory variation that as yet has been unmapped. It will require the ability to alter and measure copy numbers of the other repeated DNAs of the genome to ascertain if complex or quantitative traits map to these large blocks of 'junk.' (Aldrich, 2015).

    The observation of diet-induced rDNA loss integrates with previous results which indicate that rDNA copy number polymorphisms account for a large fraction of Y-linked gene regulatory variation (termed 'YRV'), including the ability of heterochromatin to induce gene silencing (position effect variegation). Ecological phenotypic variation implied by gene expression differences may be quite significant in competitive, food-rich or food-scarce natural environments. The current observations may directly explain why food and culture conditions alter the extent of position effect variegation, and may further explain why chromosomes from different strains -- natural isolates or mutant stocks -- differ in their ability to suppress position effect variegation (Aldrich, 2015).

    It is believed that the diet- and IIS-induced rDNA instability this study observed is a general, or at least common, feature of Y-linked rDNA because it has been measurable in males of many strains used in this study. For instance, two other Y chromosomes were specifically tested: a wild-type male from a laboratory Canton-S stock and a freshly wild-caught ('Texas-B') male by backcrossing males from these strains to a strain to genetically isolate the Y chromosome. rDNA copy number of flies raised on SY10 or SY30 was compared and otherwise-isogenic males bearing the Canton-S exhibited a 38% decrease in rDNA copy number, while the Texas-B chromosome exhibited an 8% decrease. Thus, while diet-induced loss appears to be a common feature of Y-linked rDNA genes, there are likely other genetic factors that influence the rate or bounds of loss. Additionally, the two presumably-unrelated transgenic lines (the Y from the UAS-InR strain and the Y from the Fibrillarin-RFP strain) both showed nucleolar instability under conditions with increased IIS signaling. The same phenomenon of rDNA loss was less clear in females, who appeared to exhibit small amounts of loss that was not statistically robust. Because the biology of X-linked rDNA arrays differs from that of the Y-linked arrays, and the consequence of X-X exchange at the rDNA is very different from that of X-Y exchange, there is no reason to believe that the phenomenon was related and it was pursued no further (Aldrich, 2015).

    rDNA instability is observed in a number of eukaryotes and is associated with a variety of complex phenotypes including position effect variegation in Drosophila, replicative lifespan in yeast, plant size in flax, cancer progression in humans, and the aforementioned 'hidden variation' of Y-linked Regulatory Variation. The current findings provide a mechanism for the influence of diet on all of these processes. These findings are likely generally relevant to many organisms due to the conserved structure of ribosomal DNA arrays, the common copy number polymorphisms at that locus and the common modes of rDNA regulation. While this study focused on diet, other processes that influence rRNA transcription (e.g., cell proliferation, DNA damage, determination and differentiation, stress, aging, temperature, etc.) would presumably also affect rDNA stability via similar mechanisms, and thus, the rDNA may be a common mediator of induced and heritable effects. It is not expected that induced changes to the genome are limited to the rDNA, in fact satellite sequences show copy number polymorphisms that are only now being investigated. In terms of epigenetic inheritance, it is unclear whether diet-induced rDNA copy number polymorphisms may act as an inducible and heritable modifying mutation that subsequently destabilizes epigenetic silencing (Aldrich, 2015).

    Systemic organ wasting induced by localized expression of the secreted Insulin/IGF antagonist ImpL2

    Organ wasting (see Drosophila as a Model for Human Diseases: Cachexia or Wasting Disease), related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. A model for organ wasting in adult Drosophila is described, whereby overproliferation induced by activation of Yorkie, the Yap1 oncogene ortholog, in intestinal stem cells leads to wasting of the ovary, fat body, and muscle. These organ-wasting phenotypes are associated with a reduction in systemic insulin/IGF signaling due to increased expression of the secreted insulin/IGF antagonist ImpL2 from the overproliferating gut. Strikingly, expression of rate-limiting glycolytic enzymes and central components of the insulin/IGF pathway is upregulated with activation of Yorkie in the gut, which may provide a mechanism for this overproliferating tissue to evade the effect of ImpL2. Altogether, this study provides insights into the mechanisms underlying organ-wasting phenotypes in Drosophila and how overproliferating tissues adapt to global changes in metabolism (Kwon, 2015).

    This study describes the unexpected observation that the overproliferating midgut due to aberrant Yki activity in ISCs induces the bloating syndrome and systemic organ wasting. Additionally, the overproliferating midgut perturbs organismal metabolism, resulting in an increase of hemolymph trehalose and depletion of glycogen and triglyceride storage. Strikingly, it was shown that the accumulation of hemolymph trehalose and organ-wasting processes are dependent on the antagonist of insulin/IGF signaling, ImpL2, which is specifically upregulated in the proliferating midgut. This study provides strong genetic evidence supporting that systemic organ wasting associated with the aberrant activation of Yki in ISCs cannot be explained solely by the perturbation of general gut function. Based on these findings, it is proposed that ImpL2 is a critical factor involved in systemic organ wasting in Drosophila (Kwon, 2015).

    An accompanying paper (Figueroa-Clarevega, 2015) shows that transplantation of scrib1/RasV12 disc tumors into wild-type flies induces the bloating syndrome phenotype and systemic organ wasting, affecting ovaries, fat bodies, and muscles. That study also identified ImpL2 as a tumor-driven factor that plays a critical role in the organ-wasting process. These results are consistent with earlier findings and indicate that the bloating syndrome and organ-wasting phenotypes are not associated specifically with perturbation of gut function. Interestingly, Figueroa-Clarevega and Bilder observe that disc tumors derived by the expression of ykiS/A (an active form of yki that is less potent than ykiact used in this study) did not cause organ wasting, which can be explained by the low level of ImpL2 induction in the ykiS/A tumors as compared to scrib1/RasV12 tumors (Kwon, 2015).

    The current results do not rule out the existence of an additional factor(s) contributing to the bloating syndrome and organ-wasting phenotypes. Indeed, the partial rescue of the bloating syndrome and organ-wasting phenotypes by depletion of ImpL2 in esgts>ykiact midguts suggests the existence of an additional factor(s). Moreover, this study observed that ectopic expression of ImpL2 in ECs was not sufficient to reduce whole-body triglyceride and glycogen levels, although it caused hyperglycemia, reduction of Akt1 phosphorylation, and increase of hemolymph volume. Thus, given the involvement of diverse factors in the wasting process in mammals, it is likely that in addition to ImpL2, another factor(s) contributes to systemic organ wasting in Drosophila (Kwon, 2015).

    This study shows that the bloating syndrome caused by esgts>ykiact is associated with ImpL2, as depletion of ImpL2 from esgts>ykiact midguts significantly rescues the bloating phenotype. Given the observation that elevated expression of ImpL2 from esgts>ykiact midgut induces hyperglycemia, it is speculated that the accumulation of trehalose in hemolymph is a factor involved in bloating, because a high concentration of trehalose can cause water influx to adjust hemolymph osmolarity to physiological levels. Interestingly, recent findings have shown that disruption of l(2)gl in discs activates yki, suggesting that the bloating syndrome observed in flies with transplanted l(2)gl mutant discs may be due to aberrant yki activity (Kwon, 2015).

    The current findings are reminiscent of a previous study showing that in Drosophila, humoral infection with the bacterial pathogen Mycobacterium marinum (closely related to Mycobacterium tuberculosis) causes a progressive loss of energy stores in the form of fat and glycogen—a wasting-like phenotype. Similar to the current observation, the previous study found that infection with M. marinum caused a downregulation of Akt1 phosphorylation. Given the observation that ImpL2 produced from esgts>ykiact affects systemic insulin/IGF signaling, it will be of interest to test whether ImpL2 expression is increased upon infection with M. marinum and mediates the effect on the loss of fat and glycogen storage (Kwon, 2015).

    yki plays critical roles in tissue growth, repair, and regeneration by inducing cell proliferation, a process requiring additional nutrients to support rapid synthesis of macromolecules including lipids, proteins, and nucleotides. In particular, increased aerobic glycolysis metabolizing glucose into lactate is a characteristic feature of many cancerous and normal proliferating cells. Interestingly, the aberrant activation of yki in ISCs caused a disparity in the gene expression of glycolytic enzymes and the activity of insulin/IGF signaling between the proliferating midgut and other tissues, such as muscle and ovaries. Thus, it is speculated that this disparity favors Yki-induced cell proliferation by increasing the availability of trehalose/glucose to the proliferating midgut, which presumably requires high levels of trehalose/glucose. Additionally, it will be of interest to test whether activation of Yki during tissue growth, repair, and regeneration alters systemic metabolism in a similar manner (Kwon, 2015).

    Malignant Drosophila tumors interrupt insulin signaling to induce cachexia-like wasting

    Tumors kill patients not only through well-characterized perturbations to their local environment but also through poorly understood pathophysiological interactions with distant tissues. This study uses a Drosophila tumor model to investigate the elusive mechanisms underlying such long-range interactions. Transplantation of tumors into adults induced robust wasting of adipose, muscle, and gonadal tissues that were distant from the tumor, phenotypes that resembled the cancer cachexia seen in human patients. Notably, malignant, but not benign, tumors induced peripheral wasting (see Drosophila as a Model for Human Diseases: Cachexia or Wasting Disease). The study identified the insulin growth factor binding protein (IGFBP) homolog ImpL2, an antagonist of insulin signaling, as a secreted factor mediating wasting. ImpL2 was sufficient to drive tissue loss, and insulin activity was reduced in peripheral tissues of tumor-bearing hosts. Importantly, knocking down ImpL2, specifically in the tumor, ameliorated wasting phenotypes. The study proposes that the tumor-secreted IGFBP creates insulin resistance in distant tissues, thus driving a systemic wasting response (Figueroa-Clarevega, 2005).

    Cachexia remains a major obstacle to cancer treatment, in part because the molecular mechanisms that drive it remain uncertain. This study describes a fly model that mimics certain aspects of human cachexia and utilize this model to identify a specific cachectic mediator. The tumor-induced wasting describe in flies resembles cancer cachexia in its independence from food consumption, its target tissues, its progressive nature, and its induction by certain but not all types of tumors. The fly model does not parallel all features associated with the human condition; for instance, only slight upregulation of putative fly orthologs of mammalian regulators implicated in muscle catabolism. Human cancer cachexia is clearly a heterogeneous and multifactorial condition, and this complexity has impeded progress in its understanding. This work used a reductionist system to identify a single tumor-derived factor that can drive the robust deterioration of peripheral tissues (Figueroa-Clarevega, 2015).

    Insulin signaling is a central regulator of tissue mass in both flies and humans. These data demonstrate that ImpL2, a secreted insulin antagonist produced by malignant tumors, is a major mediator that is both necessary and sufficient for wasting. In an accompanying paper in the issue of Developmental Cell, Kwon, (2015) shows that ImpL2 is also a systemic wasting factor in a different fly tumor model. Reduced insulin signaling is further responsible for wasting induced by mycobacterial infection of flies; whether ImpL2 is the relevant mediator in this case is not known. ImpL2 is the single fly homolog of mammalian IGFBPs and can bind to systemic insulin-like ligands to antagonize insulin signaling. By this mechanism, the tumor effectively induces insulin resistance in peripheral tissues (Figueroa-Clarevega, 2015).

    Insulin resistance is a frequent feature of both cachectic patients and rodent cachexia models; indeed, some evidence suggests that exogenous insulin can ameliorate tissue loss in these contexts. The seven mammalian IGFBPs are variously upregulated or downregulated in different tumors, but they have been evaluated in cancer, primarily with respect to their affects on tumor growth. These data motivate assessments of whether highly cachectogenic human tumors, such as pancreatic and gastric cancers, display elevated expression of IGFBPs and how therapies designed to correct insulin resistance might be used to treat such tumors (Figueroa-Clarevega, 2015).

    ImpL2 joins the list of effectors induced by neoplastic transformation in fly tumors, including mitogens and pro-invasive factors. Recent work shows that the Upd3 mitogen is upregulated by dual activity of JNK and Hippo signaling. The ImpL2 regulatory region, like that of Upd3, contains evolutionarily conserved binding sites for AP-1 and Sd transcription factors, suggesting that it may also be synergistically regulated by these pathways that monitor epithelial integrity. Despite the reduced insulin signaling in neoplastic tumors themselves (e.g., 4EBP levels are elevated ∼21-fold, and they are hypersensitive to PI3K reduction, the tumors nevertheless robustly proliferate. How ImpL2-upregulating tumors escape insulin resistance remains an unanswered question, although metabolic changes suggested by transcriptome alterations may be a possible mechanism (Figueroa-Clarevega, 2015).

    While tumor-specific inhibition of ImpL2 causes a significant amelioration of the wasting phenotype, rescue is not complete, suggesting that other aspects of tumor-host interaction remain to be uncovered. A fly homolog of IL-6 was found, a molecule implicated in several rodent cachexia models, was not sufficient to induce wasting, while partial ablation of host innate immune cells did not qualitatively alter wasting phenotypes; however, contributing roles for these factors have not been ruled out. Future work will analyze other tumor-produced factors, including metabolites generated by anabolic and catabolic alterations in the tumor, to evaluate their involvement as well. The manipulability of the simple model developed here, including the ability to rapidly assess fully defined combinations of host and tumor genotypes, opens the door to candidate as well as forward genetic approaches to identify additional factors mediating tumor-host interactions (Figueroa-Clarevega, 2015).

    The nutrient-responsive hormone CCHamide-2 controls growth by regulating insulin-like peptides in the brain of Drosophila melanogaster

    The coordination of growth with nutritional status is essential for proper development and physiology. Nutritional information is mostly perceived by peripheral organs before being relayed to the brain, which modulates physiological responses. Hormonal signaling ensures this organ-to-organ communication, and the failure of endocrine regulation in humans can cause diseases including obesity and diabetes. In Drosophila melanogaster, the fat body (adipose tissue) has been suggested to play an important role in coupling growth with nutritional status. This study shows that the peripheral tissue-derived peptide hormone CCHamide-2 (CCHa2) acts as a nutrient-dependent regulator of Drosophila insulin-like peptides (Dilps). A BAC-based transgenic reporter revealed strong expression of CCHa2 receptor (CCHa2-R) in insulin-producing cells (IPCs) in the brain. Calcium imaging of brain explants and IPC-specific CCHa2-R knockdown demonstrated that peripheral-tissue derived CCHa2 directly activates IPCs. Interestingly, genetic disruption of either CCHa2 or CCHa2-R caused almost identical defects in larval growth and developmental timing. Consistent with these phenotypes, the expression of dilp5, and the release of both Dilp2 and Dilp5, were severely reduced. Furthermore, transcription of CCHa2 is altered in response to nutritional levels, particularly of glucose. These findings demonstrate that CCHa2 and CCHa2-R form a direct link between peripheral tissues and the brain, and that this pathway is essential for the coordination of systemic growth with nutritional availability. A mammalian homologue of CCHa2-R, Bombesin receptor subtype-3 (Brs3), is an orphan receptor that is expressed in the islet β-cells; however, the role of Brs3 in insulin regulation remains elusive. This genetic approach in Drosophila melanogaster provides the first evidence that bombesin receptor signaling with its endogenous ligand promotes insulin production (Sano, 2015)

    Organisms need to coordinate growth and metabolism with their nutritional status to ensure proper development and the maintenance of homeostasis. In multicellular animals, nutritional information is mostly perceived by peripheral organs. It is subsequently relayed to other peripheral organs or to the central nervous system (CNS), which generates appropriate physiological and behavioral responses. Endocrine systems ensure this type of organ-to-organ communication via hormonal signals secreted from specialized glandular cells. For example, mammalian insulin is secreted from pancreatic β-cells in response to high blood glucose levels; insulin is then received by its receptor in the liver as well as in many other tissues to promote glucose uptake and anabolism, thereby reducing blood sugar levels. In a similar manner, leptin secreted from adipose tissues is received by the hypothalamus, where it acts to alter energy expenditure and food intake. Caloric restriction reduces the secretion of leptin, leading to both an increase in appetite and a decrease in energy expenditure, which is known to be an adaptive response to starvation. These findings demonstrate the significance of peripheral tissues in the maintenance of homoeostasis. However, only a few peripheral hormones have been identifie, and the mechanisms by which they regulate an organism's development or physiology in response to external stimuli remain elusive (Sano, 2015)

    It has been reported that the endocrine system of Drosophila allows adipose tissue, known as the fat body, to communicate with the CNS in a manner similar to that observed in mammals. This signaling depends on nutritional conditions and ultimately couples growth and metabolism with nutritional status. To date, two pathways have been described. In one pathway described from larvae, the fat body-specific down-regulation of either the Slimfast (Slif) amino acid transporter or the Target of Rapamycin (TOR) nutrient-sensing pathway affects systemic growth, suggesting that a hitherto unidentified amino acid-dependent signal(s) is secreted by the fat body for proper growth control. In a second pathway that was identified in adults, Unpaired-2 (Upd2), which is a functional analogue of leptin, was identified as another fat body-derived growth regulator. The expression of upd2is both sugar- and lipid-sensitive and is apparently independent of the amino acid-activated TOR pathway. Although no signaling molecules that act downstream of the Slif/TOR pathway have been identified yet, these fat body-derived signals ultimately regulate the production of insulin-like peptides (Drosophila insulin-like peptides; Dilps) secreted from the brain (Sano, 2015)

    Dilps are evolutionarily conserved peptide hormones with functions similar to those of mammalian insulin/insulin-like growth factor (IGF), including the control of tissue growth and blood sugar levels in response to nutritional conditions. Eight dilp genes exist in the Drosophila melanogaster genome. Unlike mammalian insulin, which is secreted from the pancreas, the major Dilps (Dilp2, -3, and -5) are specifically expressed in bilateral clusters of neurosecretory cells [insulin-producing cells (IPCs)] located in the anteromedial region of the brain hemispheres. With regard to the regulation of insulin-like peptides, the knockdown of the Slif/TOR pathway or upd2 in the larval fat body results in the down-regulation of Dilp2 secretion. Upd2, a type-I cytokine, activates the JAK/STAT pathway through its receptor Domeless (Dome). Dome is expressed in the GABAergic neurons juxtaposed to the IPCs in the adult brain. Activation of Dome by Upd2 blocks GABAergic inhibition of the IPCs and thereby facilitates Dilp secretion. Therefore, signaling from peripheral tissues to the brain appears to be essential for the regulation of organismal growth and metabolism in response to nutrition availability in Drosophila melanogaster (Sano, 2015)

    This study has investigated the roles of CCHa2 and its receptor in growth control in Drosophila. CCHa2 was identified as a bioactive peptide that activates a G protein-coupled receptor (GPCR) encoded by CG14593 (now named CCHa2-R). Strong expression of CCHa2 in the larval fat body and gut motivated an examination of the roles of CCHa2 and its receptor in nutrient sensing and growth control. By generating mutants of CCHa2 and CCHa2-R, this study has shown that CCHa2/CCHa2-R signaling from the periphery to the CNS can control the synthesis and secretion of Dilps. These results demonstrate that CCHa2 is a novel hormone derived from peripheral tissues and that CCHa2/CCHa2-R form an additional afferent hormonal signaling pathway that coordinates systemic growth with nutrition availability (Sano, 2015)

    A previous study suggested the existence of an amino acid-sensitive Dilp regulator(s) in larvae. This as-yet-unidentified Dilp regulator(s) is regulated by the Slif/TOR pathway, and leucine and isoleucine, positive regulators of TOR signaling, are sufficient to promote the secretion of Dilp2 in both in vivo and ex vivo co-cultures of brain and fat bodies. The current results demonstrate that the TOR pathway is required for CCHa2 expression during the larval stages. However, feeding with amino acids, including leucine and isoleucine, was insufficient to promote CCHa2 expression. CCHa2 expression was, however, induced by feeding with glucose. Therefore, unlike predicted amino acid-dependent Dilp regulator(s), CCHa2 was found to be primarily sensitive to glucose. Some biological substances are produced by the metabolism of specific nutrients. For example, pyrimidine or purine bases are synthesized from amino acids. Therefore, it is possible that CCHa2 is down-regulated when glucose is abundant but other nutrients are not available, to limit growth in inhospitable environments. The reduction of CCHa2 mRNA in TOR-pathway knockdown larvae may recapitulate this scenario (Sano, 2015)

    In addition to CCHa2, Upd2 was reported to be a glucose-sensitive Dilp regulator expressed in the fat body. The expression of upd2 in adult flies is up-regulated by feeding with a high-glucose or high-lipid diet. CCHa2 and Upd2, however, responded differently when the TOR pathway was disturbed: whereas CCHa2 expression was down-regulated in TOR-pathway-knockdown larvae, upd2 was up-regulated by the inhibition of the TOR pathway in adults. Furthermore, the time course of CCHa2/CCHa2-R signaling is distinct from that of Upd2/Dome signaling. Disruption of upd2 down-regulated animals' growth from larval to adult stages, whereas CCHa2-R mutations reduced growth until late-L3 stages, after which growth was recovered, leading to adults of normal size. This growth recovery resulted from up-regulation of dilp6 expression, which appears to be a consequence of dysregulated brain Dilps. The lack of growth recovery in upd2 -knockdown animals in spite of abnormal Dilp production remains unexplained. Nevertheless, these results indicate that Drosophila melanogaster possesses multiple insulin regulators that have different nutrient sensitivities. Multi-input Dilp regulation might be advantageous under the imbalanced nutritional conditions that arise in the wild, and this could represent a general strategy for animal growth regulation (Sano, 2015)

    In mammals, different hormones are secreted in response to long-term or short-term metabolic changes. For instance, gut-derived cholecystokinin, glucagon-like peptide-1, and PYY3-36, as well as stomach-derived ghrelin, all of which control feeding behavior, are secreted in response to food ingestion. These hormones respond to acute metabolic changes and immediately signal to the feeding center in the brain. On the other hand, the synthesis or secretion of leptin and adiponectin is affected by the amount of lipid stored in adipocytes, suggesting that leptin and adiponectin respond to long-term changes in metabolic status. The expression of CCHa2 responds to yeast and glucose within 6 hours, indicating that CCHa2 mediates relatively rapid changes in metabolic status. Thus, it appears that CCHa2 functions as a short-acting metabolic regulator analogous to the mammalian gut- or stomach-derived hormones described above, and that Drosophila melanogaster CCHa2 might have an important role in the maintenance of energy homeostasis under volatile nutritional conditions (Sano, 2015)

    The results from the calcium imaging experiments using brain explants and IPC-specific CCHa2-R knockdown strongly suggest that CCHa2 crosses the blood-brain barrier (BBB) to regulate the IPCs, although the underlying mechanism remains elusive. The Drosophila BBB consists of two different glial cell layers composed of either the perineurial glia (PG) or the subperineurial glia (SPG). The SPG cell layer, which is adjacent to the neurons of the brain, forms septate junctions, which function as a barrier to separate the humoral space and the brain, analogously to the mammalian tight junctions formed between endothelial cells. Although several studies have identified important molecules involved in the formation of these septate junctions, little is known about functional aspects of the BBB. CCHa2 could provide an ideal model for the study of BBB function as well as drug delivery across the BBB (Sano, 2015)

    These experiments also show that peripheral tissue-derived CCHa2 directly activates IPCs in the brain. In mammals, direct sensing of blood glucose levels by pancreatic β-cells is a major trigger for insulin secretion. In these cells, glucose metabolism inhibits the ATP-dependent potassium channel (KATP channel) and opens voltage-dependent calcium channels (VDCCs), resulting in the exocytosis of insulin-containing granules. The KATP channel also seems to be involved in insulin secretion in Drosophila IPCs. Interestingly, a group of Gαs- and Gαq/11-coupled GPCRs can also activate the insulin secretion pathway in mammals. The closest mammalian homologues of CCHa2-R-the Bombesin-related receptor subtypes 3, 1, and 2 (also known as gastrin-releasing-peptide receptor)-signal through Gαq/11. The slow rise in [Ca2+] in the IPCs in response to CCHa2 application is consistent with CCHa2-R's mediation of Dilp release through the same pathway (Sano, 2015)

    In contrast to Dilp2, dilp5 is also regulated by CCHa2/CCHa2-R signaling at the transcriptional level. Although the expression of dilp5 in the IPCs is activated by the conserved transcription factors Dachshund and Eyeless, whether CCHa2-R regulates these factors in IPCs remains unknown (Sano, 2015)

    Overexpression of CCHa2-R in IPCs using the GAL4/UAS system displayed inhibitory effects on dilp5 expression, which prevented investigation of whether direct CCHa2-R activation in IPCs is sufficient for Dilp regulation. CCHa2-R expression in the brain is not specific to IPCs but occurs in other central neurons. Therefore, although it was shown that CCHa2-R expression in the IPCs is required for full dilp5 expression, it is possible that there may also be additional indirect pathways by which CCHa2 may up-regulate the Dilps. Although BBB glial cells are proposed to receive as-yet-unidentified signal(s) from the fat body and re-activate neural stem cells in the brain by secreting Dilp6],CCHa2-R nlsGFP was undetectable in the BBB glial cells. Thus BBB cells are unlikely to receive CCHa2 signals or to relay the signals to the IPCs (Sano, 2015)

    The closest mammalian homologue of CCHa2-R is Brs3, an orphan GPCR, which is a member of the bombesin-like peptide receptor family. Brs3-deficient mice develop obesity in association with a reduced metabolic rate and elevated feeding activity. Interestingly, Brs3 is expressed in pancreatic β-cells both in mice and humans. However, its involvement in insulin regulation has been controversial. Only if Brs3 knockout adult mice become obese (especially after 23 weeks old) do their plasma insulin levels increase. Since hyper-insulinemia is generally observed in genetically obese mice, the elevation of insulin is most likely the consequence of the obesity rather than the loss of Brs3 function. On the other hand, a Brs3 agonist promoted insulin secretion in both rodent insulinoma cell lines and in islets isolated from wild-type but not Brs3 mutants. This vigorous genetic approach combined with direct observations of Dilp production in IPCs has provided the first evidence that Bombesin-related receptor signaling activated by its endogenous ligand promotes insulin production (Sano, 2015)

    A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3

    Body size constancy and symmetry are signs of developmental stability. Yet, it is unclear exactly how developing animals buffer size variation. Drosophila insulin-like peptide Dilp8 is responsive to growth perturbations and controls homeostatic mechanisms that co-ordinately adjust growth and maturation to maintain size within the normal range. This study shows that Lgr3 is a Dilp8 receptor. By functional and cAMP assays, a pair of Lgr3 neurons were found to mediate the homeostatic regulation. These neurons have extensive axonal arborizations, and genetic and GFP reconstitution across synaptic partners (GRASP) show these neurons connect with the insulin-producing cells and PTTH-producing neurons to attenuate growth and maturation. This previously unrecognized circuit suggests how growth and maturation rate are matched and co-regulated according to Dilp8 signals to stabilize organismal size (Vallejo, 2015).

    The impressive consistency and fidelity in size of developing organisms reflects both the robustness of genetic programs and the developmental plasticity necessary to counteract the variations in size arising from genetic noise, erroneous morphogenesis, disease, or injury. To counterbalance growth abnormalities, systemic homeostatic mechanisms are implemented that delay the onset of the reproductive stage of adulthood until a correct size of the individual and its body parts has been reached. Indeed, most animals initiate a pubertal transition only once a critical size and body mass has been achieved and generally, in the absence of tissue damage or growth abnormalities. However, the mechanisms underlying such homeostatic regulation have yet to be fully defined (Vallejo, 2015).

    Recently, the secreted peptide Dilp8, a member of the insulin/relaxin-like family has been identified as a factor mediating homeostatic control in Drosophila melanogaster. During the larval (growth) stage, the expression of dilp8 declines as maturation proceeds, whereas its expression is activated when growth is disturbed. Hence, fluctuating Dilp8 levels provides a reliable read-out of overall growth status (e.g., deficit) and of the time needed to complete growth and Dilp8 also orchestrates hormonal responses that stabilize body size. This includes inhibiting the production of the steroid hormone ecdysone by the prothoracic gland (PG) until the elements or organs affected are recomposed and also slowing down growth rates of undamaged tissues to ensure affected organs catch up with normal tissues in order to the adult flies reach a normal body size, maintain body proportions and symmetry. Accordingly, in the absence of dilp8, mutant flies are incapable of maintaining such strict control over their size, as reflected by the exaggerated variation in terms of overall proportionality and imperfect bilateral symmetry. However, the receptor that transduces Dilp8 signals and its site of action remained unknown (Vallejo, 2015).

    Two models can be envisioned to establish such homeostatic regulation: a 'central' mechanism that dictates coordinated adjustments in both the duration and rate of growth, and an 'endocrine' mechanism that involves sensing and processing Dilp8 signals directly by hormone-producing cells. In Drosophila, several anatomically separate neural populations regulate growth and maturation time by impinging directly on the ring gland (comprising the PG and the juvenile hormone-producing corpus allatum, CA). Thus, the receptors that transduce the Dilp8 signals of growth status may act directly or communicate with neurons that produce the prothoracicotropic hormone (PTTH) and/or the neurons of the pars intercerebralis, including the insulin-producing cells (IPCs), that synthesize and release insulin-like peptides Dilp2, Dilp3 and Dilp5. Insect PTTH neurons, which are analogous to the gonadotropin-releasing hormone (GnRH) neurons in mammals, signal the commitment to sexual reproduction by stimulating the production of ecdysone in the PG in order to terminate growth. The IPCs in the pars intercerebralis, a functional equivalent of the mammalian hypothalamus, integrate nutritional signals and modulate tissue growth accordingly. Manipulation of IPCs by genetic ablation, starvation, or mutations in the single insulin receptor leads to the generation of animals with smaller size. Similarly manipulations of the PTTH neuropeptide and neurons result in variations in size of the adult flies, leading to larger or smaller than normal flies due to an extension or acceleration of the larval period and delayed pupariation. The insulin receptor also directly activates synthesis of the juvenile hormone (JH) in the CA, a hormone that promotes growth and the juvenile programs, and of ecdysone production in the PG, again augmenting the variation in normal adult size. These observations may explain how environmental and internal influences by operating through individual IPCs or PTTH neurons enable body size variation and plasticity in developmental timing that can be vital for survival in changing environments. However, the origin of developmental stability and invariant body size may require different or more complex neural mechanisms from those involved in adaptive size regulation (Vallejo, 2015).

    By employing a candidate approach and biochemical assays, this study demonstrates that the orphan relaxin receptor Lgr3 acts as a Dilp8 receptor. This study identifies the neuronal population molecularly defined by the lgr3 enhancer fragment R19B09 (Jenett, 2012) and shows it is necessary and sufficient to mediate such homeostatic regulation. Using a cyclic AMP sensor as an indicator of Lgr3 receptor activation in vivo and tools for circuit mapping, it was determined that a pair of these Lgr3 neurons is highly sensitive to Dilp8. These neurons display extensive axonal arborizations and appear to connect with IPCs and PTTH neurons to form a brain circuit for homeostatic body size regulation. These data identify the insulin genes, dilp3 and dilp5, the JH, and ecdysone hormone as central in developmental size stability. Collectively, these findings unveil a homeostatic circuit that forms a framework for studying how the brain stabilize body size without constraining the adaptability of the system to reset body size in response to changing needs (Vallejo, 2015).

    The data presented provide strong evidence that Dilp8 signals for organismal and organ homeostatic regulation of size are transduced via the orphan relaxin receptor Lgr3 and that activation of Lgr3 in molecularly defined neurons mediates the necessary hormonal adjustments for such homeostasis. Human insulin/relaxin-like peptides are transduced through four GPCRs, RXFP1 to 4. RXFP1 and 2 are characterized by large extracellular domains containing leucine-rich repeats similar to fly Lgr3 and Lgr4 receptors, and like Lgr3 (this study), their activation by their cognate ligand binding results in an increase in cAMP production. RXFP3 is distinctly different in structure from fly Lgr3 and its biochemical properties are also distinct, but RXPF3 is analogous to fly Lgr3 in the sense that it is found in highest abundance in the brain, suggesting important central functions for relaxin 3/RXFP3. However, a function in pubertal development and/or growth control for vertebrate relaxin receptors is presently unknown (Vallejo, 2015).

    The neuronal populations that regulate body size and, in particularly, how their regulation generate variations in body size (plasticity) in response to internal and environmental cues such as nutrition have been intensely investigated. Less is known about how the brain stabilizes body size to ensure developing organisms reach the correct, genetically determined size. In particular, it remains unknown how limbs, and other bilaterally symmetric traits, grow to match precisely the size of the contralateral limb and maintain proportion with other parts even when they are faced with perturbations. Paired organs are controlled by an identical genetic program and grow in the same hormonal environment, and yet, small deviations in size can happen as result of developmental stress, genetic noise, or injury. Imperfections in symmetry thus reflect the inability of an individual to counterbalance variations and growth abnormalities (Vallejo, 2015).

    This study shows that without lgr3, the brain is unable to detect growth disturbances and more importantly, it is not able to adjust the internal hormonal environment to allocate additional time during development to restore affected parts or catch-up on growth. Without lgr3, the brain also cannot slow down the growth rate to compensate for the extra time for growth so that unaffected and affected tissues can grow in a harmonious manner so as to sustain normal size, proportionality and symmetry. Using a cAMP sensor, this study has been able to define a pair of neurons that are highly sensitive to Dilp8 (Vallejo, 2015).

    Communication in neuronal networks is essential to synchronize and perform efficiently. Notably, although most neurons have only one axon, Lgr3 responding neurons display extensive axonal arborizations reminiscent of hub neurons (Bonifazi, 2009). GRASP analyses show that Lgr3 neurons are broadly connected with the IPCs, and to a lesser extent with PTTH neurons, linking (Dilp8) inputs to the neuronal populations that regulate the key hormonal outputs that modulate larval and imaginal disc growth. Furthermore, the information flow from Lgr3 neurons to IPCs and to PTTH may explain how the brain matches growth with maturation in response to Dilp8. This brain circuit provides the basis for studying how the brain copes with genetic and environmental perturbations to stabilize body size, proportions and symmetry that is vital for the animal's survival (Vallejo, 2015).

    Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing

    How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. This study mutated an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3) and found body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, this Lgr3 activity was mapped to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. This work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations (Garelli, 2015).

    Different organs need to sense growth perturbations in distant tissues to coordinate their size and differentiation status during development. This study has determined that the sensing of peripheral growth perturbations requires a novel population of CNS neurons expressing the Lgr3 relaxin receptor. Neuronal Lgr3 is required for the transmission of the peripheral growth aberration signal, Dilp8, to the prothoracic gland, which controls the onset of metamorphosis and thereby the cessation of imaginal disc growth. This work reveals a new Dilp8-Lgr3 pathway that is critical to ensure developmental stability in Drosophila. This study opens many questions for further research, such as the determination of which of the eight bilateral Lgr3-positive interneuron populations are critical during Dilp8 expression, whether or not the interaction between Lgr3 and Dilp8 is direct and how Lgr3-positive neurons relay information to the ring gland (Garelli, 2015).

    Of the eight bilateral Lgr3-positive interneuron populations identified in in this study, the cholinergic PIL neurons both require Lgr3 for the Dilp8-dependent developmental delay activity and respond to Dilp8 by increasing cAMP levels. Therefore, PIL neurons are the best candidates to mediate the Dilp8-dependent developmental delay. Further research is necessary to determine if PIL neurons are sufficient to regulate developmental timing in the absence of growth aberrations or ectopic Dilp8 signals (Garelli, 2015).

    While the results clearly indicate that Dilp8 and Lgr3 act on the same pathway, their biochemical relationship is less clear. As Dilp8 is an Ilp and Lgr3 is a homologue of a vertebrate receptor for an Ilp (relaxin), it is tempting to propose a direct ligand-receptor interaction between them. This possibility is supported by the strong genetic interaction between dilp8 and Lgr3 and the finding of Dilp8-responsive Lgr3-positive neurons. However, this study also raises at least three possible issues with this interpretation of the data. First, the neuroanatomy of the CNS neuronal populations requiring Lgr3 activity suggests that Dilp8 could have to traverse the blood-brain barrier to activate Lgr3-positive interneurons deep in the brain, something which is presently unclear if it can be achieved. Alternatively, the data can also be explained if the Dilp8 signal is received by other cells (if by the CNS, these can be either glial cells or other neurons with projections exposed to the haemolymph), and relayed through one or more steps before reaching the Lgr3-positive cells. A similar route through blood-brain barrier glial cells has been proposed to explain the relay into the CNS of a fat-body-derived signal that controls neuroblast reactivation (Garelli, 2015).

    Second, Lgr3 was not identified among candidate Dilp8-binding cell surface receptors/co-receptors. Clearly, the biochemical identification of alternative cell surface-binding proteins such as the InR, Nrg and the RYK-like Drl does not rule out the possibility of a direct interaction between Dilp8 and Lgr3 in vivo, nevertheless it strongly indicates that Dilp8 can consistently interact with a likewise strong receptor candidate for an Ilp, such as the InR. The LRC technique that was used can identify receptors of interest with affinities spanning 4 orders of magnitude at expression levels as low as 2,000 receptors per cell (www.dualsystems.com). However, it is not yet clear how quantitative it can be relative to affinity constants. Dilp8 has been previously shown to modulate growth in vivo often in opposite ways depending on the observed tissue. Namely, Dilp8 ectopic expression throughout development leads to heavier adults and to reduced expression of the translational inhibitor and FOXO-target Thor (4E-BP) in the larval fat body, which is consistent with a local increase in insulin/IGF-like signalling. In contrast, Thor levels are higher in imaginal discs in the same animal. These results show that understanding the relationship between Dilp8, InR and Lgr3 will be a challenge for further studies. One possibility, if Dilp8 can indeed interact with Lgr3 in other contexts, is that there is a crosstalk between Lgr3 and InR receptors. The other possibility is that Dilp8 has a low-affinity interaction with the InR, which could be potentiated in certain physiological conditions. Affinity profiling of the Dilp8 and InR interaction, as well as that of Lgr3, should bring insight into this scenario. As regards the other Dilp8-specific candidate receptor, Drl, it also binds to Wnt5 to control aspects of axonal guidance, raising the possibility that the interaction between Dilp8 and Drl, if confirmed, can interfere with circuit formation. Interestingly, Drl has been shown to be expressed in four large glial cells in the interhemispheric region of the brain, close to the PIL neurons, and to be dynamically regulated between the third instar larvae and early pupae. Therefore, the interaction between Dilp8 and Drl should be carefully followed-up and independently verified (Garelli, 2015).

    Third, the fact that ectopic expression of Dilp8 only leads to a detectable increase in cAMP signalling in PIL neurons, and not in other Lgr3-positive neurons, indicates that Lgr3 activation by Dilp8 requires other molecular and/or cellular players. Any of the factors identified biochemically in this study could participate in PIL neuron selectivity, for instance, as a differentially enriched co-receptor. Alternatively, PIL neurons could be selectively activated downstream of other cellular players, via a mechanism which could involve a signal relay by direct synapsis or proximity to other cells that participate in the transduction of the Dilp8 signal from the periphery to the ring gland. In this case, Dilp8 would probably activate Lgr3-positive neurons indirectly. Therefore, in the absence of further evidence suggesting a direct relationship between Dilp8 and Lgr3, the possibility cannot be ruled out that Lgr3-positive neurons are not a direct target of Dilp8, but rather intermediary players in the Dilp8 developmental stability pathway (Garelli, 2015).

    How the Dilp8 signal reaches the ring gland after having triggered activity in some of the eight bilateral Lgr3-positive neuronal groups remains to be determined. The fact that sfGFP::Lgr3 or GMR19B09>myr::tdTomato expression were not detected in the ring gland or in neurons innervating the ring gland, strongly suggests that the Lgr3-positive neurons that are required for the Dilp8-dependent delay do not connect directly to the ring gland. Hence, it is likely that the Lgr3 neurons also need to relay the tissue stress signal at least once to the ring gland, either by secreting a second factor or connecting to a ring gland-innervating neuron. Together, these results indicate that the peripheral Dilp8 tissue damage signal is transduced through multiple steps before it reaches the ring gland, revealing unprecedented complexity and providing both important functional insight into the transduction of the Dilp8-dependent aberrant tissue growth signalling pathway and opening fertile ground for further research (Garelli, 2015).

    The similarities between the neuroendocrine mechanisms controlling the larval-to-pupal transition in Drosophila and the hypothalamic-pituitary axis in vertebrates has been highlighted. The neurosecretory cell-rich pars intercerebralis, in which the Dilp8-responding and Lgr3-expressing PIL neurons are located, has anatomical, developmental and functional analogies to the hypothalamus, the structure that integrates the vertebrate CNS to the endocrine system via the pituitary gland. Similarly, the Drosophila pars intercerebralis connects the CNS to the endocrine ring gland complex via neurosecretory cells. Both systems have roles in stress response, energy metabolism, growth, water retention and reproduction. The neuroanatomy of Lgr3-positive neurons, such as the PIL neurons, suggests they are well-positioned to relay signals or to modulate the activity of ring gland-innervating neurons during tissue stress events that trigger Dilp8 secretion from the periphery. Candidate neurons that could interact with PIL neurons are the IPCs, PTTH neurons and DMA1 neurons. Apart from arborizing in the pars intercerebralis region, PIL neurons send projections via the median bundle to the subesophageal region. This region is known to harbour the serotonergic SE0-PG neurons, which directly innervate the PG, thereby regulating developmental timing as a response to nutritional cues. It will be interesting to test whether PIL and SE0-PG neurons synapse in the subesophageal region and whether the latter also have a role in the tissue damage response (Garelli, 2015).

    As the timing of vertebrate developmental transitions, such as puberty, can also be altered by intrinsic and extrinsic factors affecting body growth, such as inflammatory disease and nutritional status, the exploration of the role of relaxin signalling in modulating the hypothalamic-pituitary axis is a promising area for research. This is highlighted by the fact that the hypothalamus expresses relaxin receptors, including the Lgr3-homologue, RXFP1, in mammals and fish, suggesting that a central neuroendocrine role for relaxin receptors might have evolved before the vertebrate and invertebrate split. A candidate peptide to regulate hypothalamic-pituitary stress-responses via relaxin receptors is the neuropeptide Relaxin-3 (RLN3), which has been traditionally viewed as being the ancestor ligand for all vertebrate relaxins. RLN3 is strongly expressed in stress-responsive neurons from the nucleus incertus that directly innervate and modulate hypothalamic activity. The current results therefore reveal an unexpected and striking similarity between the Dilp8-Lgr3 pathway and the vertebrate relaxin signalling pathway and hint to an ancient stress-responsive pathway coordinating animal growth and maturation timing (Garelli, 2015).

    Cold-sensing regulates Drosophila growth through insulin-producing cells

    Across phyla, body size is linked to climate. For example, rearing fruit flies at lower temperatures results in bigger body sizes than those observed at higher temperatures. The underlying molecular basis of this effect is poorly understood. This study provides evidence that the temperature-dependent regulation of Drosophila body size depends on a group of cold-sensing neurons and insulin-producing cells (IPCs). Electrically silencing IPCs completely abolishes the body size increase induced by cold temperature. IPCs are directly innervated by cold-sensing neurons. Stimulation of these cold-sensing neurons activates IPCs, promotes synthesis and secretion of Drosophila insulin-like peptides and induces a larger body size, mimicking the effects of rearing the flies in cold temperature. Taken together, these findings reveal a neuronal circuit that mediates the effects of low temperature on fly growth (Li, 2015).

    Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila

    In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). This paper reports studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, dramatic increase was observed in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

    Wing and haltere are the dorsal appendages of second and third thoracic segments, respectively, of adult Drosophila. They are homologous structures, although differ greatly in their morphology. The homeotic gene Ultrabithorax (Ubx), which is required and sufficient to confer haltere fate to epithelial cells, is known to regulate many wing patterning genes to specify haltere, but the mechanism is still poorly understood (Singh, 2015).

    There are a number of differences between wing and haltere at the cellular and organ levels. Wing is a large, flat and thin structure, while haltere is a small globular structure, although both are made up of 2-layered sheet of epithelial cells. Space between the two layers of cells in haltere is filled with haemocytes. Cuticle area of each wing cell is 8 fold more than a haltere cell. Haltere has smaller and fewer cells than the wing. Trichomes of wing cells are long and thin, while haltere trichomes are short and stout in morphology. The ratio of anterior to posterior compartment size in the haltere (~2.5:1) is much different from that in the wing (~1.2:1). Haltere also lacks wing-type vein and sensory bristles. Haltere cells are more cuboidal compared to flatter wing cells (Roch, 2000). Thus, cell number, size and shape all add to the differences in the size and shape of the two organs (Singh, 2015).

    However, cells of the third instar larval wing and haltere discs are similar in size and shape. The difference between cell size and shape becomes evident at late pupal stages. Wing cells become much larger, compared to haltere cells. At pupal stages, they also exhibit differences in the organization of actin cytoskeleton elements viz. F-actin levels are much higher in haltere cells compared to wing cells (Singh, 2015).

    In the context of final shape of wings and halteres, one needs to understand the mechanism by which Ubx influences cell size, shape and arrangement. It is possible that Ubx regulates overall shape of the haltere by regulating either cell size and/or shape. The current understanding of mechanisms by which wing and haltere differ at cellular, tissue and organ level is ambiguous (Sanchez-Herrero, 2013). For example, while removal of Ubx from the entire haltere, or at least from one entire compartment, leads to haltere to wing transformation with increased growth of Ubx minus tissues, mitotic clones of Ubx (using the null allele Ubx6.28) show similar sized twin spot in small clones. Only when very large clones of Ubx6.28Ubx6.28 are generated, one can see increased growth compared to their twin spots. This suggests that unless a certain threshold level of growth factors is de-repressed, the haltere does not show any overgrowth phenotype (Singh, 2015).

    There have been several efforts to identify functional and molecular mechanisms by which Ubx regulates genes/pathways to provide haltere its distinct morphology. Various approaches have been used to identify targets of Ubx that are expected to differentially express between wing and haltere, e.g., loss-of-function genetics, deficiency screens, enhancer-trap screening and genome wide approaches such as microarray analysis and chromatin immunoprecipitation (ChIP). Targets include genes involved in diverse cellular functions like components of the cuticle and extracellular matrix, genes involved in cell specification, cell proliferation, cell survival, cell adhesion, or cell differentiation, structural components of actin and microtubule filaments, and accessory proteins controlling filament dynamics (reviewed in Sanchez-Herrero, 2013; Singh, 2015).

    Decapentaplegic (Dpp), Wingless (Wg), and Epidermal growth factor receptor (EGFR) are some of the major growth and pattern regulating pathways that are repressed by Ubx in the haltere. However, over-expression of Dpp, Wg, Vestigial (Vg) or Vein (Vn) provides only marginal growth advantage to haltere compared to the wildtype. In this context, additional growth regulating pathways amongst the targets of Ubx were examined. Genome wide studies have identified many components of Fat/Hippo and Insulin-insulin like/dAkt signalling (IIS/dAkt) pathways as potential targets of Ubx. The Fat/Hippo pathway is a crucial determinant of organ size in both Drosophila and mammals. It regulates cell proliferation, cell death, and cell fate decisions and coordinates these events to specify organ size. In contrast, the IIS/dAkt pathway is known to regulate cell size (Singh, 2015).

    Recent studies have revealed that the Fat/Hippo pathway networks with other signalling pathways. For example, during wing development, Fat/Hippo pathway activities are dependent on Four-jointed (Fj) and Dachous (Ds) gradients, which are influenced by Dpp, Notch, Wg and Vg. Glypicans, which play a prominent role in morphogen signalling, are regulated by Fat/Hippo signalling (Baena-Lopez, 2008). EGFR activates Yorkie (Yki; effector of Fat/Hippo pathway) through its EGFR-RAS-MAPK signalling by promoting the phosphorylation of Ajuba family protein WTIP (Reddy, 2013). However, EGFR negatively regulates events downstream of Yki. The Fat/Hippo pathway is also known to inhibit EGFR signalling, which makes the interaction between the two pathways very complex and context-dependent. IIS/dAkt pathway is also known to activate Yki signalling and vice-versa. Thus, Fat/Hippo pathway may specify organ size by regulating both cell number (directly) and cell size (via regulating IIS/dAkt pathway) (Singh, 2015).

    This study reports studies on the functional implication of regulation of Fat/Hippo and IIS/dAkt pathways by Ubx in specifying haltere development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded (ex), induced considerable increase in haltere size, mainly due to increase in cell number. Although activation of dAkt alone did not affect the organ size or the cell size, activation of Yki or down regulation of ex in the background of over-expressed dAkt caused dramatic increase in haltere size, much severe than Yki or ex alone. In this background, increase was observed in both cell size and cell number. The resulted haltere appeared flatter than wildtype haltere and the morphology of trichomes and their spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

    The findings suggest that, downstream of Ubx, the Fat/Hippo pathway is critical for haltere specification. It is required for Ubx-mediated specification of organ size, sensory bristle repression, trichome morphology and arrangement. The Fat/Hippo pathway cooperates with the IIS/dAkt pathway, which is also a target of Ubx, in specifying cell size and compartment size in developing haltere. The fact that over-expression of Yki or downregulation of ex show haltere-to-wing transformations at the levels of organ size and shape, and trichome morphology and arrangement, suggest that regulation of the Fat/Hippo pathway by Ubx is central to the modification of wing identity to that of the haltere (Singh, 2015).

    The observations made in this study pose new questions and suggest various interesting possibilities to study the Fat/Hippo pathway with a new perspective.

    (1) It was observed that while Yki is nuclear in haltere discs, it appears to be non-functional. Yki is a transcriptional co-activator protein, which requires other DNA-binding partners for its activity. In this context, understanding the precise relationship between Yki and Ubx may provide an insight into mechanism of haltere specification (Singh, 2015).

    (2) The Fat/Hippo pathway (along with the IIS/dAkt pathway) may be involved in the specification of cell size, trichome morphology and their arrangement, all of which are important parameters in determining organ morphology. Recent studies indicate that the Fat/Hippo pathway regulates cellular architecture and the mechanical properties of cells in response to the environment. It would be interesting to study the role of the Fat/Hippo pathway in regulating the cytoskeleton of epithelial cells during development. Haltere cells at pupal stages exhibit higher levels of F-actin than wing cells. One possible mechanism that is currently being investigated is lowering of F-actin levels in transformed haltere cells due to over-expression of Yki or down regulation of ex. This may cause flattening of cells during morphogenesis leading to larger organ size (Singh, 2015).

    (3) Reversing cell size and number was sufficient to induce homeotic transformations at the level of haltere morphology. This suggests the importance of negative regulation of genetic mechanisms that determine cell size and number, in specifying an organ size and shape. As a corollary, Ubx-mediated regulation of Fat/Hippo and IIS/dAkt pathways provides an opportunity to study cooperative repression of cell number and cell size during organ specification (Singh, 2015).

    (4) Certain genetic backgrounds investigated in this study showed severe effect on cell proliferation in haltere discs than in wing discs. This could be due to the fact that, the wing disc has already attained a specific size by the third instar larval stage (the developmental stage examined in this study), which is controlled by several pathways. Any change to this size may need more drastic alteration to the controlling mechanisms. As Ubx specifies haltere by modulating various wing-patterning events, there may still exist a certain degree of plasticity in mechanisms that determine the size of the haltere. However, in absolute terms, the haltere is also resistant to changes in growth control due to regulation by Ubx at multiple levels. Thus, differential development of wing and haltere provides a very good assay system to study not only growth control, but also to dissect out function of important growth regulators (tumour suppressor pathways) such as the Fat/Hippo pathway using various genome-wide approaches (Singh, 2015).

    The Drosophila ortholog of TMEM18 regulates insulin and glucagon-like signaling

    Transmembrane protein 18 (TMEM18) is an ill-described, obesity-related gene, but few studies have explored its molecular function. This study found SNP data suggesting TMEM18 may be involved in the regulation/physiology of metabolic syndrome based on associations with insulin, HOMAb, triglycerides, and blood sugar. An ortholog, Drosophila Tmem18, was found in the Drosophila genome, was knocked down specifically in insulin-producing cells, and was tested for effects on metabolic function. Data suggest that TMEM18 affects substrate levels through insulin and glucagon signaling, and its downregulation induces a metabolic state resembling type-II diabetes. This work is the first to experimentally describe the metabolic consequences of TMEM18 knockdown, and further supports its association with obesity (Wiemerslage, 2016).

    An integrative analysis of the InR/PI3K/Akt network identifies the dynamic response to insulin signaling

    Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and metabolism. To expand understanding of the insulin pathway, biochemical, genetic, and computational approaches were applied to build a comprehensive Drosophila InR/PI3K/Akt network. First, the dynamic protein-protein interaction network surrounding the insulin core pathway was mapped using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, it was found that 47% of interacting proteins affect pathway activity, and, using quantitative phosphoproteomics, it was demonstrates that approximately 10% of interacting proteins are regulated by insulin stimulation at the level of phosphorylation. Next, these orthogonal datasets were integrated to characterize the structure and dynamics of the insulin network at the level of protein complexes, and this method was validated by identifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, this study represents a comprehensive resource for the study of the evolutionary conserved insulin network (Vinayagam, 2016).

    Intra-organ growth coordination in Drosophila is mediated by systemic ecdysone signaling

    In developing Drosophila, perturbing the growth of one imaginal disc - the parts of a holometabolous larva that become the external adult organs - has been shown to retard growth of other discs and delays development, resulting in tight inter-organ growth coordination and the generation of a correctly proportioned adult. This study used the wing imaginal disc in Drosophila to study and identify mechanisms of intra-organ growth coordination. Larvae were generated in which the two compartments of the wing imaginal disc have ostensibly different growth rates (wild-type or growth-perturbed). Tightly coordinated growth was found between the wild-type and growth-perturbed compartments, where growth of the wild-type compartment is retarded to match that of the growth-perturbed compartment. Crucially, this coordination is disrupted by application of exogenous 20-hydroxyecdysone (20E), which accelerates growth of the wild-type compartment. The role of 20E signaling in growth coordination was further elucidate by showing that in wild-type discs, compartment-autonomous up-regulation of 20E signaling accelerates compartment growth and disrupts coordination. Interestingly, growth acceleration through exogenous application of 20E is inhibited with suppression of the Insulin/Insulin-like Growth Factor Signaling (IIS) pathway. This suggests that an active IIS pathway is necessary for ecdysone to accelerate compartment growth. Collectively, these data indicate that discs utilize systemic mechanisms, specifically ecdysone signaling, to coordinate intra-organ growth (Gokhale, 2016).

    The results reveal that growth among developmental compartments in an organ is tightly coordinated, such that even if the growth of one compartment is perturbed, both compartments grow at more-or-less the same relative rate as observed in wild-type flies. This growth coordination between compartments is disrupted by exogenously feeding 20E to growth-perturbed larvae, resulting in acceleration in the growth rate of the unperturbed compartment. This growth acceleration upon feeding 20E is dependent on IIS in the unperturbed compartment. Collectively these data support a model of imaginal disc growth regulation whereby growth perturbation in one compartment causes a systemic reduction in circulating ecdysteroids, which results in reduction in growth rate of the adjacent compartment (Gokhale, 2016).

    These data are surprising in light of previous studies that suggest that imaginal discs and individual compartments within imaginal discs can autonomously grow to their target size. A previous study cultured WT imaginal discs in the abdomen of adults hosts and found that these discs grow autonomously to their normal size. Another study generated 'fast' discs and compartments in M-/+ larvae and demonstrated that these compartments have higher growth rates relative to the body as a whole and to adjacent compartments. It was further demonstrated that the 'fast' compartments and discs are developmentally advanced as compared to M-/+controls. Collectively, these data support the hypothesis that imaginal disc possesses an autonomous mechanism for arresting growth once they reach a target size, and that this mechanism operates at the level of developmental compartments. Whilst compartments may possess a target size, the current data suggest that they do not grow independently to this size, at least in vivo. Rather growth between developmental compartments is coordinated even when one compartment is growth perturbed, and this growth coordination appears to be regulated by systemic rather than disc-autonomous mechanisms, at least in part (Gokhale, 2016).

    The conclusions are supported by data from Mesquita (2010), who also looked at inter-compartmental growth in the Drosophila wing imaginal disc. They observed that slowing the growth of one compartment non-autonomously slowed the growth of the adjacent compartment. They further demonstrate that the signal from the growth-perturbed compartment is dependent on Drosophila p53. However, they do not elucidate what the signal is. The current results suggest that the signal involves ecdysone. This is surprising given the current understanding of wing imaginal disc growth. Recent models of disc growth suggest that growth of the wing imaginal disc is driven mainly by morphogen gradients formed by the patterning genes Wg, Dpp, and Vg, which drive cellular proliferation within the disc. Recent studies further implicate disc-autonomous mechanisms in regulating the relative size of different compartments within the wing (Ferreira, 2015). The current data show that systemic signaling, mediated by ecdysone, is also critical for regulating growth rates among different parts of the disc (Gokhale, 2016).

    The involvement of ecdysone in intra-organ growth coordination echoes its known role in inter-organ growth coordination. As noted above, growth among organs is tightly coordinated when one organ is growth perturbed-a consequence of the growth-perturbed organ suppressing ecdysone synthesis. Addition of ecdysone to these growth-perturbed larvae is able to rescue the growth rate of undamaged imaginal discs. Ecydsone is however not able to rescue the growth rate of the growth perturbed tissues, most likely because the inherent growth perturbation of these tissues prevents them from responding to ecdysone. Similar to these studies on inter-organ growth coordination, the current data suggest ecdysone is able to rescue the growth rate of wild-type compartments in M-/+larvae, and this is mediated by compartment-autonomous ecdysone signaling (Gokhale, 2016).

    While the current data indicate that ecdysone is an important growth-coordinating signal among developmental compartments, it is unclear precisely which tissue is influencing ecdysone synthesis. It is possible that in larvae with antfast:postslow discs the limitation on ecdysone synthesis might be an autonomous effect of the Minute mutation on the prothoracic gland, since the whole of the rest of the larvae is Minute. However, the data demonstrate that knock-down of RpS3 using engrailed-GAL4, which is not expressed in the prothoracic gland, still retards disc growth. This suggests that the growth coordination mechanism is regulated by a signal from the compartments themselves. As discussed above, in studies where systemic growth is retarded through localized tissue damage, including knock-down of ribosomal proteins, it is the damaged/growth-perturbed tissue itself that inhibits ecdysone synthesis by signaling via dILP8. Therefore, in larvae with antfast:postslow discs, ecdysteroidgenesis could be limited via a dILP8-dependent mechanism. Which compartment is generating a putative dILP8 signal is, however, unclear. dILP8 levels are highest at the L2-L3 transition and decline during L3, before increasing somewhat before pupariation. It is possible, therefore, that in larvae with antfast:postslow discs, it is the immature slow-growing posterior compartment that is secreting dILP8. Conversely, the residual generation and death of M-/- cells in the anterior compartment through mitotic recombination early in L3 may also drive dILP8 synthesis. Further experiments exploring the role of dILP8 in intra-organ growth coordination are clearly necessary (Gokhale, 2016).

    A key feature of growth coordination is that ecdysone acts as a promoter of growth for imaginal discs. This appears contrary to previous findings that show that ecdysone inhibits larval body growth by inhibiting IIS or Myc in the fat body. However, evidence from other insect species suggests that ecdysone can function as either a growth promoter or inhibitor, depending on its concentration. Specifically, in vitro evidence from Manduca shows that low concentrations of ecdysone can promote growth of imaginal tissues, while higher concentrations stimulate differentiation, and stop cell proliferation. Further evidence from Manduca suggests that ecdysone promotes mitosis by regulating the cell cycle, and thus acts as a mitogen. These data echo data from Drosophila that suggests that ecdysone regulates cell cycle progression and promotes imaginal disc growth via the ecdysone inducible gene crooked legs. Collectively, it is apparent, therefore, that ecdysone is a central regulator of larval and imaginal tissue growth, although the tissue-specific effects and molecular mechanisms involved have not yet been completely elucidated. Research from this and other labs supports the hypothesis that imaginal discs reduce their growth rates in response to low levels of ecdysone. At the same time, low levels of ecdysone increase body growth rate and final adult body size. Together these data suggest that ecdysone suppresses the growth of larval tissue (which comprises the majority of the larva) but promotes growth of imaginal tissues. This hypothesis has intuitive appeal in that a key function of ecdysone is to 'prepare' the larva for pupariation and metamorphosis, a process that involves breakdown and autophagy of the larval tissues to provide nutrients for final growth and differentiation of the imaginal discs (Gokhale, 2016).

    Research over the past decade has elucidated mechanisms by which ecdysone functions as a suppressor of larval growth. These studies demonstrate a role for IIS in ecdysone-mediated suppression of larval growth. Specifically, ecdysone signaling in the fat body suppresses IIS, which in turn inhibits systemic IIS and larval growth through repression of dILP2 release from the brain and promotes fat body autophagy. How ecdysone promotes imaginal disc growth is less clear, however. A recent paper by Herboso (2015), indicated that ecdysone promotes growth by suppressing Thor signaling in the imaginal discs. Discs from larvae with reduced ecdysone synthesis have elevated levels of Thor, a repressor of growth that is a target of the IIS pathway. The hypothesis that ecdysone regulates and coordinates growth via IIS/TOR signaling is further supported by the observation that down-regulation of Inr activity prevents the wild-type compartment of antfast:postslow discs from increasing its relative growth rate in response to ecdysone (Gokhale, 2016).

    However, additional data suggest a more nuanced role for IIS in coordinating growth among developmental compartments. In particular, changes in Inr activity in the anterior compartment do not affect relative compartment growth rate in larvae that are otherwise wholly wild-type. Rather, changes in Inr activity increase or decrease relative compartment size, presumably due to changes in compartment growth earlier in development. This is surprising, given that mutations of Inr reduce the growth and proliferation of clones in the wing imaginal disc during L3. In antfast:postslow discs, however, changes in Inr activity does alter growth coordination during L3, but in a counterintuitive way: reduced Inr activity increases relative growth rate, whilst increased Inr activity decreases relative growth rate. This is the opposite of what would be predicted if ecdysone promotes growth by directly upregulating IIS. One interpretation of these data is that the anterior compartments of the antfast:postslow disc adjust their relative growth rate to rescue the final anterior:posterior size ratio, presumably using a mechanism independent of ecdysone. Why this rescue is not evident in wild-type larvae is unclear, but suggest that the rescue mechanism is able to override the ecdysone-regulated mechanism that coordinates growth rates between compartments with different potential growth rates (Gokhale, 2016).

    From the current study and those of others, it seems unlikely that ecdysone promotes imaginal disc growth only through its effects on IIS. In particular, the role of ecdysone in the regulation of differentiation and patterning genes such as broad, senseless, and cut has been well elucidated. Patterning genes are known to regulate cell proliferation. It is therefore possible that ecdysone also regulates imaginal disc growth by regulating the expression of patterning genes in the imaginal disc. One of the challenges in elucidating the role of ecdysone signaling in imaginal disc development is that manipulating ecdysone-signaling organ-autonomously in imaginal discs is technically difficult. This study likely only subtly up-regulated ecdysone signaling by knocking down EcR compartment-autonomously and found that this mild knockdown accelerated compartment growth. It is seems likely that this effect is related to the degree of the knockdown, however, for two reasons. First, complete knockdown of EcR will ultimately block ecdysone signaling, even if it de-represses the expression of certain genes. Second, ecdysone levels can both promote and inhibit insect growth and development depending on its level. As discussed above, moderate level of ecdysone are sufficient to stimulate imaginal disc growth in vitro, while high levels suppress cell proliferation. More precise methods of manipulating ecdysone signaling at a cellular and tissue level are therefore needed (Gokhale, 2016).

    In summary, this study provides evidence for an ecdysone-dependent mechanism that coordinates growth between compartments in the wing imaginal disc of Drosophila. The data suggest that the control of cell proliferation across the imaginal disc is not an entirely autonomous process, but is coordinated through humoral signaling. This research also highlights the crosstalk between different systemic signaling mechanisms - insulin/IGF- and ecdysone-signaling - in the generation of correctly proportioned organs. The developmental mechanisms regulating organ size, while best studied in Drosophila, are conserved across all animals. There is considerable evidence that localized growth perturbation causes systemic growth retardation in humans. For example, children suffering from chronic inflammatory diseases such as Crohn's disease have systemic growth hormone insensitivity and experience severe growth retardation as a complication of the disease. The utilization of systemic signaling mechanisms to coordinate growth within and between organs may thus be a conserved mechanism across all animals (Gokhale, 2016).

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

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

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

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

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

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

    To directly quantify the amount of circulating Sun protein, Western blot experiments were performed on hemolymph using antibodies against Sun. A 6-kD band was detected in hemolymph collected from fed larvae, and size was confirmed using Schneider 2 (S2) cell extracts. The band intensity was reduced upon sun knockdown in fat body cells but not in gut cells. Therefore, circulating Sun peptide appears to be mostly contributed by fat cells, as suggested by functional experiments. The levels of circulating Sun are strongly reduced upon starvation. In line with this, sun transcripts are drastically reduced after 4 hours of protein starvation and start increasing after 1 hour of refeeding, whereas expression of the sun homolog CG31477 is not modified. sun transcription is not affected by blocking TORC1, the main sensor for amino acids in fat body cells (lpp>TSC1/2). However, adipose-specific TORC1 inhibition induces a dramatic reduction of circulating Sun, indicating that TORC1 signaling controls Sun peptide translation or secretion from fat cells. PGC1-Spargel is a transcription activator, the expression of which relies on nutritional input. PGC1 was found to be required for sun transcriptionand that fat body silencing of PGC1 and sun induce identical larval phenotypes. Although PGC1 expression is strongly suppressed upon starvation, blocking TORC1 activity in fat cells does not reduce PGC1 expression. Conversely, knocking down PGC1 does not inhibit TORC1 activity. This finding suggests that PGC1 and TORC1 act in parallel. Therefore, Sun production by fat cells in response to nutrition is controlled at two distinct levels by PGC1 and TORC1 (Delanoue, 2016).

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

    In conclusion, this study provides evidence for a molecular cross-talk between fat cells and brain IPCs involving the ligand Stunted and its receptor Methuselah. Stunted is a moonlighting peptide present both in the mitochondria as part of the F1F0-ATP synthase complex and as an insulinotropic ligand circulating in the hemolymph. The mechanism of Stunted release remains to be clarified. The beta subunit of the ectopic form of F1F0-ATP synthase is a receptor for lipoproteins, which serve as cargos for proteins and peptides. In addition, Drosophila lipid transfer particle-containing lipoproteins were shown to act on the larval brain to control systemic insulin signaling in response to nutrition. This suggests that Sun could be loaded on lipoproteins for its transport. Given the role of insulin-insulin-like growth factor (IGF) signaling in aging, these findings could help in understanding the role of Sun/Mth in aging adult flies. The same genetic screen previously identified the fly tumor necrosis factor α Eiger (Egr) as an adipokine necessary for long-term adaptation to protein starvation, and recent work pointed to other adipose factors, illustrating the key role of the larval fat body in orchestrating nutrient response. The multiplicity of adipose factors and their possible redundancy could explain the relatively mild starvation-like phenotype obtained after removal of only one of them. Overall, these findings suggest a model whereby partially redundant fat-derived signals account for differential response to positive and negative valence of various diet components, as well as acute versus long-term adaptive responses (Delanoue, 2016).

    Drosophila neprilysins control insulin signaling and food intake via cleavage of regulatory peptides

    Insulin and IGF signaling are critical to numerous developmental and physiological processes, with perturbations being pathognomonic of various diseases, including diabetes. Although the functional roles of the respective signaling pathways have been extensively studied, the control of insulin production and release is only partially understood. This study shows that in Drosophila expression of insulin-like peptides is regulated by neprilysin activity. Concomitant phenotypes of altered expression of the metallopeptidase neprilysin, included impaired food intake, reduced body size, and characteristic changes in the metabolite composition. Ectopic expression of a catalytically inactive mutant did not elicit any of the phenotypes, which confirms abnormal peptide hydrolysis as a causative factor. A screen for corresponding substrates of the neprilysin identified distinct peptides that regulate insulin-like peptide expression, feeding behavior, or both. The high functional conservation of neprilysins and their substrates renders the characterized principles applicable to numerous species, including higher eukaryotes and humans (Hallier, 2016).

    Neprilysins are highly conserved ectoenzymes that cleave and thereby inactivate many physiologically relevant peptides in the extracellular space, thus contributing considerably to the maintenance of peptide homeostasis in this compartment. Members of the neprilysin family generally consist of a short N-terminal cytoplasmic domain, a membrane spanning region, and a large extracellular domain with two highly conserved sequence motifs (HExxH; ExxA/GD) critical for zinc coordination, catalysis, and substrate or inhibitor binding. Because of these characteristics, neprilysins are classified as M13 zinc metallopeptidases. For human Neprilysin (NEP), the most well-characterized family member, identified substrates include endothelins, angiotensins I and II, enkephalins, bradykinin, atrial natriuretic peptide, substance P, and the amyloid-beta peptide. Because of this high substrate variability, NEP activity has been implicated in the pathogenesis of hypertension, analgesia, cancer, and Alzheimer's disease. Recent clinical trials have demonstrated significant efficacy of Neprilysin inhibitors in the treatment of certain indications. However, despite the clinical relevance of the neprilysins, the physiological function and in vivo substrates of most family members are unknown (Hallier, 2016).

    In Drosophila melanogaster, at least five neprilysin genes are expressed, two of the corresponding protein products, Nep2 and Nep4, were reported to be enzymatically active. With respect to Nep4, a critical function of the enzyme's non-catalytic intracellular N-terminus has been demonstrated: when present in excess, the domain induces severe muscle degeneration concomitant with lethality during late larval development. Because the intracellular domain interacts with a carbohydrate kinase, impaired energy metabolism has been proposed as the underlying cause of the phenotype. In addition, Nep2 has been implicated in the regulation of locomotion and geotactic behavior, and neprilysin activity in general appears to be critical to the formation of middle- and long-term memory, as well as to the regulation of pigment dispersing factor (PDF) signaling within circadian neural circuits. However, despite these experiments and recent findings that suggest a critical role of neprilysins in reproduction, the physiological functionality of these enzymes is still far from being understood. In this respect, the lack of identified substrates with in vivo relevance is a major hindrance (Hallier, 2016).

    This study describes the identification of numerous novel substrates of Drosophila Neprilysin 4 (Nep4) and provide evidence that Nep4-mediated peptide hydrolysis regulates insulin-like peptide (ILP) expression and food intake. These results establish a correlation between neprilysin activity and ILP expression and thus clarify understanding of the complex mechanisms that control the production and release of these essential peptides (Hallier, 2016).

    While the functional roles of insulin-like peptides (ILPs) and the corresponding insulin- and IGF-signaling have been intensively studied, the control of ILP production and release is not well understood. This study demonstrates that modulating the expression of a Drosophila neprilysin interferes with the expression of insulin-like peptides, thus establishing a correlation between neprilysin activity and the regulation of insulin signaling. A high physiological relevance is confirmed by the fact that altering nep4 expression phenocopies characteristic effects of IPC ablation, including reduced size and weight of corresponding animals, as well as increased levels of carbohydrates such as glucose and fructose. The result that the levels of these sugars are increased, although food intake rates are reduced presumably reflects the physiological impact of the diminished ilp expression that is also obvious in corresponding animals. In this respect, the impaired insulin signaling likely results in inefficient metabolization and thus accumulation of the sugars, which overcompensates the diametrical effects of reduced food intake. By identifying 16 novel peptide substrates of Nep4, the majority of which are involved in regulating dilp expression or feeding behavior, and by localizing the peptidase to the surface of body wall muscles and IPCs within the larval CNS, this study provides initial evidence that neprilysin-mediated hydrolysis of hemolymph circulating as well as CNS intrinsic peptides is the physiological basis of the described phenotypes. The finding that only the catalytically active enzyme affected dilp expression whereas the inactive construct did not, substantiates this evidence because it confirms aberrant enzymatic activity and thus abnormal peptide hydrolysis as a causative parameter. Interestingly, the strongest effects on size and dilp expression were observed with muscle-specific overexpression of Nep4; overexpression of the peptidase in the CNS was less detrimental. These results indicate that hemolymph circulating peptides accessible to muscle-bound Nep4 are mainly responsible for the observed effects, while CNS intrinsic peptide signaling is less relevant. The fact that all peptides cleaved by Nep4 could be released into the hemolymph, either from enteroendocrine cells or from neurohormonal release sites, substantiates this indication. Since the Drosophila midgut is the source of several neuropeptides, it is conceivable that a main reason for the observed phenotypes is aberrant cleavage of certain gut-derived peptides that are required for proper midgut-IPC communication. Allatostatin A, neuropeptide F, diuretic hormone 31, and some tachykinins are produced by endocrine cells of the gut. Interestingly, all have been implicated in regulating dilp expression and/or feeding behavior, and most of them, namely allatostatin A1-4, diuretic hormone 31, and tachykinin 1, 2, 4, and 5, were cleaved by Nep4, indicating enzyme-substrate relationships. Thus, these results suggest that Nep4 activity at the surface of muscle cells is necessary to maintain homeostasis of distinct hemolymph circulating signaling peptides, probably gut-derived, thereby ensuring proper midgut-IPC communication. On the other hand, fat body-IPC feedback may be affected as well. However, the only factors known to mediate this process, Unpaired 2, DILP6, and Stunted have molecular masses of more than 5 kDa, and thus exceed the maximum mass of a putative neprilysin substrate. Consequently, a direct regulatory influence of Nep4 on Unpaired 2, DILP6, or Stunted activity appears unlikely (Hallier, 2016).

    In addition to body wall muscles, nep4 is expressed in numerous cells of the central nervous system, predominantly in glial cells. Interestingly, compared to the muscle-specific effects, modulating nep4 expression in this tissue has distinct and less severe effects on dilp expression. This result suggests that CNS intrinsic Nep4 activity affects different neuropeptide regulatory systems than the corresponding muscle-bound activity. Considering the rather broad expression in glial cells, it is furthermore likely that the CNS regulation affects more than one system. However, localization at the IPC surface clearly supports a direct function in the regulation of dilp expression. In this context, spatial proximity of the peptidase may be necessary to ensure low ligand concentrations and thus tight regulation of specific neuropeptide receptors present at the surface of IPCs. Such receptors include an allatostatin A receptor (Dar-2), a tachykinin receptor (DTKR), and the short neuropeptide F receptor (sNPFR). All are essential to proper dilp expression . Interestingly, with respect to sNPFR, corresponding ligands (sNPF11-11, sNPF14-11, and sNPF212-19) exhibit very high-binding affinities, with IC50 values in the low nanomolar range, a finding that further emphasizes the need for effective ligand clearance mechanisms in order to prevent inadvertent receptor activation. Localization of Nep4 to the surface of IPCs and confirmation of Dar-2, DTKR, and sNPFR ligands as substrates of the peptidase strongly indicate that Nep4 participates in such clearance mechanisms (Hallier, 2016).

    Of note, sNPF species were detected in both, CNS and hemolymph preparations, with neuroendocrine functions of the respective peptides being suggested. The dual localization is interesting because both compartments are accessible to Nep4, either to the CNS resident or to the muscle-bound enzyme. Significantly, sNPF is a potent regulator of dilp expression. Increased sNPF levels result in upregulation of dilp expression, and decreased sNPF levels have the opposite effect. The fact that these results inversely correlate with the effects of modulating nep4 expression suggests a functional relationship between sNPF and the neprilysin. Nep4-mediated cleavage of distinct sNPF species represents further evidence for this relationship (Hallier, 2016).

    Besides sNPF, Nep4 also cleaves corazonin, drosulfakinins, and allatostatin A. Interestingly, corazonin promotes food intake, while allatostatin A and drosulfakinins inhibit it. This regulatory activity on peptides with opposing physiological functions indicates that Nep4 affects multiple aspects of feeding control, rather than promoting or inhibiting food intake in a mutually exclusive manner. The finding that both, nep4 knockdown and overexpression larvae exhibit reduced food intake supports this indication since it suggests that regular Nep4 activity adjusts the general peptide homeostasis in a manner that promotes optimal food intake, with deviations in either direction being deteriorative. The result that nep4 knockdown animals exhibit reduced food intake for only up to 20 min of feeding may reflect this complex regulation since it indicates that at the onset of feeding reduced cleavage of peptides inhibiting food intake (e.g., allatostatin A, drosulfakinins) is a dominant factor. With ongoing feeding, accumulation of peptides promoting food intake (e.g., corazonin) may become decisive, thus restoring intake rates (Hallier, 2016).

    In addition, Nep4 hydrolyzes numerous peptides that regulate dilp expression, including tachykinins, allatostatin A, and sNPF. However, AKH, a functional homolog of vertebrate glucagon that acts antagonistically to insulin, is also a substrate of Nep4. This finding indicates that the Nep4-mediated regulation of dilp expression and sugar homeostasis can also not be attributed to a single substrate or cleavage event. Rather, it is a result of the concerted hydrolysis of several critical peptides, including both, hemolymph circulating and CNS intrinsic factors. Taking into account that overexpression and knockdown of nep4 have discrete effects on dilp expression, but comparable effects on feeding, it furthermore appears likely that dysregulation of the Nep4-mediated peptide homeostasis affects both processes somewhat independently of each other. The fact that among the novel Nep4 substrates, peptides were identified that presumably affect either dilp signaling (e.g., DH31), or food intake (e.g., leucokinin, drosulfakinins) in a largely exclusive manner supports this indication (Hallier, 2016).

    Because neprilysins and many of the novel substrates identified in this study are evolutionarily conserved factors, neprilysin-mediated regulation of insulin-like peptide expression and feeding behavior may be relevant not only to the energy metabolism in Drosophila, but also to corresponding processes in vertebrates, including humans. Interestingly, a critical function of murine Neprilysin in determining body mass has already been reported. The regulation depended primarily on the catalytic activity of peripheral NEP, while the CNS-bound enzyme was less important. However, until now, the underlying physiology has been obscure, essentially because no causative hydrolysis event had been identified. The finding that also in Drosophila mainly peripheral (muscle-bound) Nep4 activity affected body mass, while CNS-specific modulations had only minor effects on size or weight, indicates that the neprilysin-mediated regulation of food intake, body size and insulin expression involves similar physiological pathways in both species. Furthermore, the fact that altered catalytic activity and thus abnormal peptide hydrolysis is a critical factor in miceand in Drosophila emphasizes the need to generate comprehensive, enzyme-specific lists of neprilysin in vivo substrates. In this context, the results of the current screen for novel Nep4 substrates may be a valuable resource in order to identify corresponding substrates in vertebrates and humans (Hallier, 2016).

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

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

    Genetic dissection of nutrition-induced plasticity in insulin/insulin-like growth factor signaling and median life span in a Drosophila multiparent population

    The insulin/insulin-like growth factor signaling (IIS) and target of rapamycin (TOR) pathways have been implicated in nutrition-dependent changes in metabolism and nutrient allocation. To characterize natural genetic variation in the IIS/TOR pathway, >250 recombinant inbred lines (RILs) derived from a multiparental mapping population, the Drosophila Synthetic Population Resource, was used to map transcript-level QTL of genes encoding 52 core IIS/TOR components in three different nutritional environments [dietary restriction (DR), control (C), and high sugar (HS)]. Nearly all genes, 87%, were significantly differentially expressed between diets, though not always in ways predicted by loss-of-function mutants. Cis (i.e., local) expression QTL (eQTL) were identified for six genes, all of which are significant in multiple nutrient environments. Further, trans (i.e., distant) eQTL were identified for two genes, specific to a single nutrient environment. The results are consistent with many small changes in the IIS/TOR pathways. A discriminant function analysis for the C and DR treatments identified a pattern of gene expression associated with the diet treatment. Mapping the composite discriminant function scores revealed a significant global eQTL within the DR diet. A correlation between the discriminant function scores and the median life span (r = 0.46) provides evidence that gene expression changes in response to diet are associated with longevity in these RILs (Stanley, 2017).

    Both overlapping and independent mechanisms determine how diet and insulin-ligand knockouts extend lifespan of Drosophila melanogaster

    Lifespan in many organisms, including Drosophila melanogaster, can be increased by reduced insulin-IGF-like signaling (IIS) or by changes in diet. Most studies testing whether IIS is involved in diet-mediated lifespan extension employ only a few diets, but recent data shows that a broad range of nutritional environments is required. This study presents lifespan data of long-lived Drosophila, lacking three of the eight insulin-like peptides [Drosophila insulin-like peptides 2,3,5 (dilp2-3,5)] on nine different diets that surround the optimum for lifespan. Their nutritional content was varied by manipulating sugar and yeast concentrations independently, and thus incorporated changes in both diet restriction and nutrient balance. The mutants were substantially longer-lived than controls on every diet, but the effects on the lifespan response to sugar and yeast differed. The data illustrates how a greater coverage of diet balance (DB) and restriction can unify differing interpretations of how IIS might be involved in the response of lifespan to diet (Zandveld, 2017).

    Reduced insulin signaling maintains electrical transmission in a neural circuit in aging flies

    Lowered insulin/insulin-like growth factor (IGF) signaling (IIS) can extend healthy lifespan in worms, flies, and mice, but it can also have adverse effects (the 'insulin paradox'). Chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber system (GFS), a simple escape response neuronal circuit, by increasing targeting of the gap junctional protein innexin shaking-B to gap junctions (GJs). Endosomal recycling of GJs was also stimulated in cultured human cells when IIS was reduced. Furthermore, increasing the activity of the recycling small guanosine triphosphatases (GTPases) Rab4 or Rab11 was sufficient to maintain GJs upon elevated IIS in cultured human cells and in flies, and to rescue age-related loss of GJs and of GFS function. Lowered IIS thus elevates endosomal recycling of GJs in neurons and other cell types, pointing to a cellular mechanism for therapeutic intervention into aging-related neuronal disorders (Augustin, 2017).

    A proteomic atlas of insulin signalling reveals tissue-specific mechanisms of longevity assurance
    Lowered activity of the insulin/IGF signalling (IIS) network can ameliorate the effects of ageing in laboratory animals and, possibly, humans. Although transcriptome remodelling in long-lived IIS mutants has been extensively documented, the causal mechanisms contributing to extended lifespan, particularly in specific tissues, remain unclear. This study has characterized the proteomes of four key insulin-sensitive tissues in a long-lived Drosophila IIS mutant and control, and detected 44% of the predicted proteome (6,085 proteins). Expression of ribosome-associated proteins in the fat body was reduced in the mutant, with a corresponding, tissue-specific reduction in translation. Expression of mitochondrial electron transport chain proteins in fat body was increased, leading to increased respiration, which was necessary for IIS-mediated lifespan extension, and alone sufficient to mediate it. Proteasomal subunits showed altered expression in IIS mutant gut, and gut-specific over-expression of the RPN6 proteasomal subunit, was sufficient to increase proteasomal activity and extend lifespan, whilst inhibition of proteasome activity abolished IIS-mediated longevity. This study thus uncovered strikingly tissue-specific responses of cellular processes to lowered IIS acting in concert to ameliorate ageing (Tain, 2017)

    Insulin signaling in the peripheral and central nervous system regulates female sexual receptivity during starvation in Drosophila

    Many animals adjust their reproductive behavior according to nutritional state and food availability. Drosophila females for instance decrease their sexual receptivity following starvation. Insulin signaling, which regulates many aspects of insect physiology and behavior, also affects reproduction in females. This study shows that insulin signaling is involved in the starvation-induced reduction in female receptivity. More specifically, females mutant for the insulin-like peptide (dilp5) were less affected by starvation compared to the other dilp mutants and wild-type flies. Knocking-down the insulin receptor, either in all fruitless-positive neurons or a subset of these neurons dedicated to the perception of a male aphrodisiac pheromone, decreased the effect of starvation on female receptivity. Disrupting insulin signaling in some parts of the brain, including the mushroom bodies even abolished the effect of starvation. In addition, Fruitless-positive neurons in the dorso-lateral protocerebrum and in the mushroom bodies co-expressing the insulin receptor were identified. Together, these results suggest that the interaction of insulin peptides determines the tuning of female sexual behavior, either by acting on pheromone perception or directly in the central nervous system (Lebreton, 2017).

    Drosophila females need nutrients to produce eggs and a nutrient rich substrate to lay their eggs. When food is scarce it would therefore be beneficial for flies to decrease their sexual behavior and to focus on food searching instead. On the other hand, female flies can store sperm and use it several days later when conditions are suitable. It could therefore be optimal for females to remain receptive for short periods of food deprivation. Several insulin peptides produced in specific spatiotemporal patterns acting through one single receptor enables a fine-scale regulation of behaviors in response to changes in physiology. The expression of the different dilps is differentially affected by food quality or food deprivation. For instance, both starvation and dietary restriction reduce the expression of dilp5 but increase the expression of dilp6, while the expression of dilp2 is not affected by either condition. The results suggest that DILP5 might be involved in the decrease of receptivity during non-feeding stages. Indeed, dilp5 mutant females were less affected by starvation than other dilp mutants. The effect of the lack of DILP5 was no longer observed in the simultaneous absence of DILP2 and DILP3. Although, background mutation effects cannot be completely ruled out, this suggests that DILP5 might interact with other DILPs to finely tune female sexual receptivity (Lebreton, 2017).

    Insulin is known to act on the olfactory system to modulate odor sensitivity after feeding. Moreover, normal InR expression in Or67d-expressing (Fruitless-positive) OSNs is necessary for fed females to be attracted to a blend of food odors and cVA, a pheromone promoting sexual receptivity. The results suggest that insulin signaling in Fruitless-positive neurons, and more specifically in Or67d OSNs may decrease sexual receptivity during starvation (Lebreton, 2017).

    Fruitless-positive cells other than pheromone-sensing neurons can also be involved. Different Fruitless-positive cells in the protocerebrum were found that strongly express InR. First of all, a large number of Kenyon cells in the calyx of the mushroom bodies express both Fruitless and the insulin receptor. Additionally, one pair of neurons was found with somata located in the anterior dorso-lateral protocerebrum. It was not possible to trace any processes from these somata, and thus it is not known what neuropils they innervate. However, the fact that InR immunostaining was observed in Fruitless neurons, most of which were Kenyon cells, corroborate the behavioral results. Indeed, the sexual receptivity of females in which insulin signaling was knocked down in the mushroom bodies was not affected by starvation. Interestingly, the mushroom bodies are not required for virgin females to be receptive, suggesting that these structures may regulate the activity of neuronal networks inducing sexual receptivity. However, this result must be take with caution, given the fact that the Gal4 line that were used to target the mushroom bodies also drive expression to some extent in other brain tissues. Further experiments will be necessary to confirm that the mushroom bodies are indeed responsible for this effect (Lebreton, 2017).

    Insulin signaling not only modulates neuronal activity in adults but also shapes neuronal networks during development. The effects observed in this study may therefore be the consequence of a developmental defect of specific neuronal circuitry rather than a direct effect of insulin on these neurons during starvation. However, Fruitless-positive neurons being required for females to be receptive, fed females would be expected to be unreceptive if the disruption of insulin signaling had altered the connectivity of these neurons during development, which was not the case. This suggests that insulin acts on these neurons during adult stage to modulate sexual receptivity. This is different for the mushroom bodies, which are not necessary for females to be receptive. Knocking down InR specifically during development or specifically in adults will be necessary to disentangle these two possible modes of action of insulin (Lebreton, 2017).

    In contrast with Fruitless neurons and the mushroom bodies, no effect was observed of the corpora allata in the insulin-dependent control of sexual receptivity, whereas these structures have been linked to the development of receptivity in virgin females. This result should however be taken with caution, considering the behavioral variability displayed by the different transgenic lines, which would have prevented observing of subtle changes. Nonetheless, the results suggest that the structures that generate behaviors (such as the corpora allata) and those modulating these behaviors (for example the mushroom bodies) can be different and the underlying mechanisms uncoupled (Lebreton, 2017).

    Taken together, Drosophila flies adjust their sexual behavior to match their nutritional state. Together with other hormonal pathways, insulin regulates some aspects of sexual activity, both after food intake and after a period of starvation. The results suggest that specific insulin peptides regulate female receptivity, possibly by acting on pheromone perception at the periphery or directly in the central nervous system. Indeed, the mushroom bodies probably play a major role in the insulin-dependent effect of starvation on female sexual receptivity. The next step will be to untangle the specific neuronal circuitry involved (Lebreton, 2017).

    Tissue-specific insulin signaling mediates female sexual attractiveness

    Global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in Drosophila. This study demonstrates that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, this study describes a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells (Fedina, 2017).

    Cbt modulates Foxo activation by positively regulating insulin signaling in Drosophila embryos

    In late Drosophila embryos, the epidermis exhibits a dorsal hole as a consequence of germ band retraction. It is sealed during dorsal closure (DC), a morphogenetic process in which the two lateral epidermal layers converge towards the dorsal midline and fuse. Previous work has demonstrated the involvement of the Cbt transcription factor in Drosophila DC. However its molecular role in the process remained obscure. This study used genomic approaches to identify genes regulated by Cbt as well as its direct targets during late embryogenesis. The results reveal a complex transcriptional circuit downstream of Cbt and evidence that it is functionally related with the Insulin/insulin-like growth factor signaling pathway. In this context, Cbt may act as a positive regulator of the pathway, leading to the repression of Foxo activity. The results also suggest that the DC defects observed in cbt embryos could be partially due to Foxo overactivation and that a regulatory feedback loop between Foxo and Cbt may be operating in the DC context (Munoz-Soriano, 2018).

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    Zygotically transcribed genes

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