The Interactive Fly
Zygotically transcribed genes
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).
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).
In Drosophila, changes to dietary protein elicit different body size responses between the sexes. Whether these differential body size effects extend to other macronutrients remains unclear. This study shows that lowering dietary sugar (0S diet) enhanced body size in male and female larvae. Despite an equivalent phenotypic effect between the sexes,sex-specific changes were detected to signalling pathways, transcription and whole-body glycogen and protein. In males, the low-sugar diet augmented insulin/insulin-like growth factor signalling pathway (IIS) activity by increasing insulin sensitivity, where increased IIS was required for male metabolic and body size responses in 0S. In females reared on low sugar, IIS activity and insulin sensitivity were unaffected, and IIS function did not fully account for metabolic and body size responses. Instead, a female-biased requirement for the Target of rapamycin pathway was detected in regulating metabolic and body size responses. Together, these data suggest the mechanisms underlying the low-sugar-induced increase in body size are not fully shared between the sexes, highlighting the importance of including males and females in larval studies even when similar phenotypic outcomes are observed (Millington, 2022).
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).
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).
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).
Insulin signaling is critical for developmental growth and adult homeostasis, yet the downstream regulators of this signaling pathway are not completely understood. Using the model organism Drosophila melanogaster, a genomic approach was undertaken to identify novel mediators of insulin signaling. These studies led to the identification of Meep, encoded by the gene CG32335. Expression of this gene is both insulin receptor- and diet-dependent. Meep was specifically required in the developing fat body to tolerate a high-sugar diet (HSD). Meep is not essential on a control diet, but when reared on an HSD, knockdown of meep causes hyperglycemia, reduced growth, developmental delay, pupal lethality, and reduced longevity. These phenotypes stem in part from Meep's role in promoting insulin sensitivity and protein stability. This work suggests a critical role for protein homeostasis in development during overnutrition. Because Meep is conserved and obesity-associated in mammals, future studies on Meep may help to understand the role of proteostasis in insulin-resistant type 2 diabetes (Pereira, 2020).
Glycolysis and fatty acid (FA) synthesis directs the production of energy-carrying molecules and building blocks necessary to support cell growth, although the absolute requirement of these metabolic pathways must be deeply investigated. This study used Drosophila genetics and focused on the TOR (Target of Rapamycin) signaling network that controls cell growth and homeostasis. In mammals, mTOR (mechanistic-TOR) is present in two distinct complexes, mTORC1 and mTORC2; the former directly responds to amino acids and energy levels, whereas the latter sustains insulin-like-peptide (Ilp) response. The TORC1 and Ilp signaling branches can be independently modulated in most Drosophila tissues. This study shows that TORC1 and Ilp-dependent overgrowth can operate independently in fat cells and that ubiquitous over-activation of TORC1 or Ilp signaling affects basal metabolism, supporting the use of Drosophila as a powerful model to study the link between growth and metabolism. Cell-autonomous restriction of glycolysis or FA synthesis in fat cells was shown to retrain overgrowth dependent on Ilp signaling but not TORC1 signaling. Additionally, the mutation of FASN (Fatty acid synthase) results in a drop in TORC1 but not Ilp signaling, whereas, at the cell-autonomous level, this mutation affects none of these signals in fat cells. These findings thus reveal differential metabolic sensitivity of TORC1- and Ilp-dependent growth and suggest that cell-autonomous metabolic defects might elicit local compensatory pathways. Conversely, enzyme knockdown in the whole organism results in animal death. Importantly, this study weakens the use of single inhibitors to fight mTOR-related diseases and strengthens the use of drug combination and selective tissue-targeting (Devilliers, 2021).
Drosophila Insulin-Producing Cells (IPCs) are the main production site of the Drosophila Insulin-like peptides or dilps which have key roles in regulating growth, development, reproduction, lifespan and metabolism. To better understand the signalling pathways and transcriptional networks that are active in the IPCs publicly available transcriptome data of over 180 highly inbred fly lines were queried for dilp expression, and dilp expression was used as the input for a Genome-wide association study (GWAS). This resulted in the identification of variants in 125 genes that were associated with variation in dilp expression. The function of 57 of these genes in the IPCs was tested using an RNAi-based approach. IPC-specific depletion of most genes was found to result in differences in expression of one or more of the dilps. Then on one of the candidate genes with the strongest effect on dilp expression, Homothorax, a transcription factor known for its role in eye development, was examined further. Homothorax and its binding partner Extradenticle were found to be involved in regulating dilp2, -3 and -5 expression; genetic depletion of both TFs shows phenotypes associated with reduced insulin signalling. Furthermore, evidence is provided that other transcription factors involved in eye development are also functional in the IPCs. In conclusion, this study showed that this expression level-based GWAS approach identified genetic regulators implicated in IPC function and dilp expression (Winant, 2022).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
West Nile virus (WNV) is the most prevalent mosquito-borne virus in the United States with approximately 2,000 cases each year. There are currently no approved human vaccines and a lack of prophylactic and therapeutic treatments. Understanding host responses to infection may reveal potential intervention targets to reduce virus replication and disease progression. The use of Drosophila melanogaster as a model organism to understand innate immunity and host antiviral responses is well established. Previous studies revealed that insulin-mediated signaling regulates WNV infection in invertebrates by regulating canonical antiviral pathways. Because insulin signaling is well-conserved across insect and mammalian species, this study sought to determine if results using D. melanogaster can be extrapolated for the analysis of orthologous pathways in humans. This study identified insulin-mediated endothelin signaling using the D. melanogaster model and evaluated an orthologous pathway in human cells during WNV infection. It was demonstrated that endothelin signaling reduces WNV replication through the activation of canonical antiviral signaling. Taken together, these findings show that endothelin-mediated antiviral immunity is broadly conserved across species and reduces replication of viruses that can cause severe human disease (Trammell, 2023).
Since, the S6K/4E-BP sub-pathway can be stimulated by various amino acids; this study examine if oral feeding of amino acids delivers rescue against human poly(Q) toxicity in Drosophila. Drosophila models of two different poly(Q) disorders were used to test this hypothesis. Glutamine was fed to the test flies orally mixed in the food. Control and treated flies were then tested for different parameters, such as formation of poly(Q) aggregates and neurodegeneration, to evaluate glutamine's proficiency in mitigating poly(Q) neurotoxicity. This study study, for the first time, reports that glutamine feeding stimulates the growth promoting S6K/4E-BP branch of insulin signalling pathway and restricts pathogenesis of poly(Q) disorders in Drosophila disease models. It is noted that glutamine treatment restricts the formation of neurotoxic poly(Q) aggregates and minimises neuronal deaths. Further, glutamine treatment re-establishes the chromatin architecture by improving the histone acetylation which is otherwise compromised in poly(Q) expressing neuronal cells. Since, the insulin signalling pathway as well as mechanism of action of glutamine are fairly conserved between human and Drosophila, this finding strongly suggests that glutamine holds immense potential to be developed as an intervention therapy against the incurable human poly(Q) disorders (Tandon, 2023).
Obesity significantly increases the risk of developing neurodegenerative disorders, yet the precise mechanisms underlying this connection remain unclear. Defects in glial phagocytic function are a key feature of neurodegenerative disorders, as delayed clearance of neuronal debris can result in inflammation, neuronal death, and poor nervous system recovery. Mounting evidence indicates that glial function can affect feeding behavior, weight, and systemic metabolism, suggesting that diet may play a role in regulating glial function. While it is appreciated that glial cells are insulin sensitive, whether obesogenic diets can induce glial insulin resistance and thereby impair glial phagocytic function remains unknown. In this study, using a Drosophila model, it is shown that a chronic obesogenic diet induces glial insulin resistance and impairs the clearance of neuronal debris. Specifically, obesogenic diet exposure downregulates the basal and injury-induced expression of the glia-associated phagocytic receptor, Draper. Constitutive activation of systemic insulin release from Drosophila Insulin-producing cells (IPCs) mimics the effect of diet-induced obesity on glial draper expression. In contrast, genetically attenuating systemic insulin release from the IPCs rescues diet-induced glial insulin resistance and draper expression. Significantly, this study shows that genetically stimulating Phosphoinositide 3-kinase (PI3K), a downstream effector of Insulin receptor signaling, rescues HSD-induced glial defects. This study established that obesogenic diets impair glial phagocytic function and delays the clearance of neuronal debris (Alassaf, 2023).
The insulin pathway is a crucial central system for metabolism and growth. The Nrf2 signaling pathway functions to counteract oxidative stress. This study examined the consequences of an oxidative stress challenge to insulin compromised and control adult flies of different ages, varying the activation state of the Nrf2 pathway in flies, the Cap'n'collar C pathway. For this, two different pro-oxidative conditions were employed: 3 % hydrogen peroxide or 20 mM paraquat laced in the food. In both cases, wild type (control) flies die within a few days, yet there are significant differences between males and females, and also within flies of different ages (seven versus thirty days old flies). The same conditions were repeated with young (seven days old) flies that were heterozygous for a loss-of-function mutation in Keap1. There were no significant differences. Two hypomorphic viable conditions of the insulin pathway were tested (heteroallelic combination for the insulin receptor and the S6 Kinase), challenged in the same way: Whereas they also die in the pro-oxidant conditions, they fare significantly better when heterozygous for Keap1, in contrast to controls. Locomotion was also monitored in all of these conditions, and, in general, significant differences were foundbetween flies without and with a mutant allele (heterozygous) for Keap1. The results point to altered oxidative stress conditions in diabetic flies. These findings suggest that modest activation of the Cap'n'collar C pathway may be a treatment for diabetic symptoms (Alvarez-Rendon, 2023).
Obesity is associated with various metabolic disorders, such as insulin resistance and adipose tissue inflammation (ATM), characterized by macrophage infiltration into adipose cells. This study presents a new Drosophila model to investigate the mechanisms underlying these obesity-related pathologies. Genetic manipulation was employed to reduce ecdysone levels to prolong the larval stage. These animals are hyperphagic and exhibit features resembling obesity in mammals, including increased lipid storage, adipocyte hypertrophy and high circulating glucose levels. Moreover, significant infiltration of immune cells (hemocytes) into the fat bodies, accompanied by insulin resistance. Attenuation of Eiger/TNFα signaling reduced ATM and improved insulin sensitivity. Furthermore, using metformin and the antioxidants anthocyanins, both phenotypes were ameliorated. The data highlight evolutionarily conserved mechanisms allowing the development of Drosophila models for discovering therapeutic pathways in adipose tissue immune cell infiltration and insulin resistance. This model can also provide a platform to perform genetic screens or test the efficacy of therapeutic interventions for diseases such as obesity, type 2 diabetes and non-alcoholic fatty liver disease (Mirzoyan, 2023).
Tauopathies such as Alzheimer's disease (AD), Frontotemporal dementia, and parkinsonism linked to chromosome 17 (FTDP-17), etc. are characterized by tau hyperphosphorylation and distinguished accumulation of paired helical filaments (PHFs)/or neurofibrillary tangles (NFTs) in a specific-neuronal subset of the brain. Among different reported risk factors, type 2 diabetes (T2D) has gained attention due to its correlation with tau pathogenesis. However, mechanistic details and the precise contribution of insulin pathway in tau etiology is still debatable. This study demonstrated that expression of human tau causes overactivation of insulin pathway in Drosophila disease models. It was subsequently noted that tissue-specific downregulation of insulin signaling or even exclusive reduction of its growth-promoting sub-branch effectively reinstates the overactivated insulin signaling pathway in human tau expressing cells, which in turn restricts pathogenic tau hyperphosphorylation and aggregate formation. It was further noted that restored tau phosphorylation was achieved due to a reestablished balance between the levels of different kinase(s) (GSK3β and ERK/P38 MAP kinase) and phosphatase (PP2A). Taken together, this study demonstrates a precise involvement of the insulin pathway and associated molecular events in the pathogenesis of human tauopathies in Drosophila, which will be immensely helpful in developing novel therapeutic options against these devastating human brain disorders. Moreover, this study reveals an interesting link between tau etiology and aberrant insulin signaling, which is a characteristic feature of Type 2 Diabetes (Pragati, 2023).
G(4)C(2) hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G(4)C(2) insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPRs) translated from G(4)C(2) transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPRs have shown that arginine-rich DPRs are most toxic, while polyGlycine-Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length or tagging affect the toxicity of GA has not been systematically assessed. Therefore, this study generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, these data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in patients (Moron-Oset, 2023).
The link between the biological clock and reproduction is evident in most metazoans. The fruit fly Drosophila melanogaster, a key model organism in the field of chronobiology because of its well-defined networks of molecular clock genes and pacemaker neurons in the brain, shows a pronounced diurnal rhythmicity in oogenesis. Still, it is unclear how the circadian clock generates this reproductive rhythm. A subset of the group of neurons designated "posterior dorsal neuron 1" (DN1p), which are among the ~150 pacemaker neurons in the fly brain, produces the neuropeptide allatostatin C (AstC-DN1p). This study reports that six pairs of AstC-DN1p neurons send inhibitory inputs to the brain insulin-producing cells, which express two AstC receptors, star1 and AICR2. Consistent with the roles of insulin/insulin-like signaling in oogenesis, activation of AstC-DN1p suppresses oogenesis through the insulin-producing cells. This study shows evidence that AstC-DN1p activity plays a role in generating an oogenesis rhythm by regulating juvenile hormone and vitellogenesis indirectly via insulin/insulin-like signaling. AstC is orthologous to the vertebrate neuropeptide somatostatin (SST). Like AstC, SST inhibits gonadotrophin secretion indirectly through gonadotropin-releasing hormone neurons in the hypothalamus. The functional and structural conservation linking the AstC and SST systems suggest an ancient origin for the neural substrates that generate reproductive rhythms (Zhang, 2021b).
Six pairs of DN1p neurons were discovered that are part of the circadian pacemaker neuron network in the brain and make functional inhibitory connections to the brain IPCs. The IPCs are endocrine sensors that link the organism's nutritional status with anabolic processes, such as those associated with growth in developmental stages and with reproduction in adults. In juvenile stages, activation of insulin and insulin-like growth factor (IGF) signaling (IIS) through the InR results in larger flies, whereas inhibition of this pathway produces smaller flies. Consistent with this, it was also found that forced activation of the AstC-DN1p (i.e., CNMa-Gal4/UAS-NaChBac) during development resulted in 12% smaller adults, confirming their role as a negative regulator of the IPCs. In adults, the IPCs are associated with many physiological and behavioral processes, such as feeding, glycaemic homeostasis, sleep, lifespan, and stress resistance. As such, the IPCs receive a variety of modulatory inputs from both central and peripheral sources, such as sNPF, corazonin, tachykinins, limostatin, allatostatin A, adipokinetic hormone, GABA, serotonin, and octopamine. Regarding reproduction, IIS directed by the IPCs stimulates GSC proliferation and vitellogenesis. The results also indicate that AstC from AstC-DN1p suppresses the secretory activity of the IPCs and juvenile hormone (JH)-dependent oocyte development (i.e., vitellogenesis). Indeed, it was found that the JH mimic methoprene can rescue the suppression of oogenesis induced by AstC-DN1p activation. From these results it is concluded that IPCs are inhibited by AstC released by AstC-DN1p. A similar link between IIS and the circadian clock has also been reported in mammals, but the mechanism remains unclear (Zhang, 2021b).
Although the genetic evidence supporting the inhibitory action of AstC-DN1p on IPCs is compelling, it is also puzzling because a previous study found forced activation of 8 to 10 pairs of DN1p neurons (i.e., Clk4.1-LexA+ neurons) induced Ca2+ transients in IPCs. This study also found that, under LD 12:12 conditions, the IPCs showed electrical activity early in the morning when DN1p neurons are also active. The same study, however, reported that, under DD conditions, the IPCs showed no bursting activity in the morning (i.e., CT0 to -4). Instead, they showed bursting activity in the late afternoon (i.e., CT8 to -12) when DN1 activity falls. Furthermore, DN1p activation evokes varying levels of Ca2+ transients from individual IPCs, some of which produce no detectable Ca2+ transient. Thus, like mammalian pancreatic β-cells, the IPCs in Drosophila seem to comprise a heterogeneous cell population. It is noted that individual IPCs show highly variable AstC-R1 expression, which would also lead to individual IPCs showing variable responses to AstC (Zhang, 2021b).
In D. melanogaster, the LD cycle generates an egg-laying rhythm by influencing oogenesis and oviposition. Oviposition depends on light cues, whereas oogenesis cycles with the circadian rhythm that itself continues to run in DD conditions. In live-brain Ca2+ imaging experiments, DN1 neurons show a circadian Ca2+ activity rhythm that peaks around CT19 and reaches its lowest point between CT6 and CT8. This DN1 activity rhythm correlates well with the rhythm of vitellogenesis initiation observed in this study. In this model, the lowest point in DN1 Ca2+ activity between CT6 and CT8 leads to a significant attenuation of AstC secretion. This leads to a derepression of IPC activity, which eventually induces JH biosynthesis and vitellogenesis initiation. The 6-h delay required for previtellogenic stage 7 follicles to develop into vitellogenic stage 8 follicles would result in a peak in the number of stage 8 follicles between CT12 and CT14. Notably, the ovaries of the AstC-deficient mutant showed similar numbers of stage 8 oocytes at all examined circadian time points, indicating that any other JH- or vitellogenesis-regulating factors play only minor roles in producing the circadian vitellogenesis rhythm (Zhang, 2021b).
Like the IPCs, the DN1p cluster is also heterogeneous. A subset of the DN1p neurons is most active at dawn and promotes wakefulness. Another subset of the DN1p cluster (also known as, spl-gDN1) promotes sleep. The DN1p cluster comprises two morphologically distinct subpopulations, a-DN1p and vc-DN1p. The a-DN1p subcluster promotes wakefulness by inhibiting sleep promoting neurons, whereas the vc-DN1p subcluster resembles the sleep-promoting spl-gDN1. These results indicate AstC-DN1p are a-DN1p neurons that project to the anterior optic tubercle (AOTU. Although the possibility cannot be ruled out that AstC-DN1p is also heterogeneous and includes some vc-DN1p neurons, the wake-promoting role of a-DN1p aligns well with the circadian vitellogenesis rhythm that requires the secretory activity of AstC-DN1p to be lowest in the afternoon and highest at dawn. Furthermore, AstC-DN1p neurons express Dh31. Dh31-expressing DN1 clock neurons are intrinsically wake-promoting and Dh31-DN1p activity in the late night or early morning suppresses sleep. Again, this is consistent with the observation that AstC-DN1p are also wake-promoting a-DN1p. It is speculated Dh31 plays a limited role in oogenesis regulation, because unlike AstC, RNAi-mediated knockdown of Dh31 had a negligible impact on female fecundity (Zhang, 2021b).
Besides AstC-DN1p, the female brain has many additional AstC neurons. However, it seems unlikely that other AstC neurons contribute to the circadian vitellogenesis rhythm. This is because restoring AstC expression specifically in AstC-DN1p almost completely restored the vitellogenesis rhythm in AstC-deficient mutants. It is feasible, however, that other AstC neurons contribute to different aspects of female reproduction. Indeed, a sizable difference was noted in the final oogenesis outcome between AstC-Gal4 neuron activation and brain-specific AstC-Gal4 neuron activation. This suggests AstC cells outside of the brain also regulate oogenesis probably in other physiological contexts, such as the postmating responses (Zhang, 2021b).
AstC receptors are orthologous to mammalian SST receptors (sstr1-5). SST is a brain neuropeptide that was originally identified as an inhibitor of growth hormone (GH) secretion in the anterior pituitary. Thus, the observation that AstC inhibits IIS from IPCs, a major endocrine signal that promotes growth in Drosophila, suggests remarkable structural and functional conservation between the invertebrate AstC and vertebrate SST systems. In addition, SST inhibits the hypothalamic neuropeptide GnRH, which stimulates the anterior pituitary's production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH stimulates clutches of immature follicles to initiate follicular development, while LH stimulates ovulation. Thus, both AstC and SST regulate the secretion of gonadotropins (JH in insects, FSH and LH in mammals) indirectly through the IPCs in insects and through the hypothalamic GnRH neurons in mammals. This functional conservation between AstC and SST is also evident in the immune system. AstC inhibits the innate immune system in insects, while SST inhibits inflammation in mammals (Zhang, 2021b).
In many seasonal breeders, the changing photoperiod as the seasons progress acts as an environmental cue for the biological clock system, which would then direct any necessary physiological changes. During the winter, Drosophila females enter a form of reproductive dormancy characterized by a pronounced suppression of vitellogenesis. A winter-like condition (i.e., short-day length, low temperature, and food shortage) down-regulates neural activity in the IPCs. But the IPCs are not equipped with a cell-autonomous clock, so they must receive seasonal information from the brain clock neuron network. Indeed, two clock related neuropeptides~pigment dispersing factor and short neuropeptide F~from circadian morning pacemaker or M-cells have been implicated in regulating reproductive dormancy. Intriguingly, AstC-DN1p neurons are the DN1p subset that receives pigment dispersing factor signals from these M-cells. Furthermore, DN1p can process light and temperature information for the circadian regulation of behavior. Finally, the finding that AstC-DN1p generates the circadian vitellogenesis rhythm via the IPCs makes AstC-DN1p neurons the prime candidates for integrating the seasonal cues that control the entrance, maintenance, or exit from reproductive dormancy. Considering the functional and structural conservation between the AstC and SST systems, the SST system may also link the brain clock, GnRH, and/or its downstream reproductive pathways in controlling seasonal reproductive patterns in vertebrates (Zhang, 2021b).
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).
Nutrient-dependent body size plasticity differs between the sexes in most species, including mammals. Previous work in Drosophila showed that body size plasticity was higher in females, yet the mechanisms underlying increased female body size plasticity remain unclear. This study discovered that a protein-rich diet augments body size in females and not males because of a female-biased increase in activity of the conserved insulin/insulin-like growth factor signaling pathway (IIS). This sex-biased upregulation of IIS activity was triggered by a diet-induced increase in stunted mRNA in females, and required Drosophila insulin-like peptide 2, illuminating new sex-specific roles for these genes. Importantly, this study shows that sex determination gene transformer promotes the diet-induced increase in stunted mRNA via transcriptional coactivator Spargel to regulate the male-female difference in body size plasticity. Together, these findings provide vital insight into conserved mechanisms underlying the sex difference in nutrient-dependent body size plasticity (Millington, 2021).
In many animals, body size plasticity in response to environmental factors such as nutrition differs between the sexes. While past studies have identified mechanisms underlying nutrient-dependent growth in a mixed-sex population, and revealed factors that promote sex-specific growth in a single nutritional context, the mechanisms underlying the sex difference in nutrient-dependent body size plasticity remain unknown. This study showed that females have higher phenotypic plasticity compared with males when reared on a protein-rich diet, and elucidated the molecular mechanisms underlying the sex difference in nutrient-dependent body size plasticity in this context. The data suggests a model in which high levels of dietary protein augment female body size by stimulating an increase in IIS activity, where a requirement was identified for dilp2 and stunted (sun) in promoting this nutrient-dependent increase in IIS activity. Importantly, it was discovered that tra is the factor responsible for stimulating sun mRNA levels and IIS activity in a protein-rich context, revealing a novel role for sex determination gene tra in regulating phenotypic plasticity. Mechanistically, tra enhanced sun mRNA levels and body size in protein-rich conditions via transcriptional coactivator Srl, identifying Srl as one link between tra and the nutrient-dependent regulation of gene expression. Together, these findings provide new insight into how Drosophila females achieve increased nutrient-dependent body size plasticity compared with males (Millington, 2021).
One key feature of this increased phenotypic plasticity in females was a female-biased increase in IIS activity in a protein-rich context. This reveals a previously unrecognized sex difference in the coupling between IIS activity and dietary protein. In females, there was tight coupling between increased nutrient input and enhanced IIS activity across a wide protein concentration range in all control genotypes. In males, this close coordination between dietary protein and IIS activity was weaker in a protein-rich context. The data shows that sex-biased nutrient-dependent change to IIS activity during development is physiologically significant, as it supports an increased rate of growth and consequently larger body size in females but not in males raised on a protein-rich diet. In future studies, it will be important to determine whether the sex difference in coupling between nutrients and IIS activity exists in other contexts. For example, previous studies on the extension of life span by dietary restriction have shown that male and female flies differ in the concentration of nutrients that produces the maximum life span extension, and in the magnitude of life span extension produced by dietary restriction. Similar sex-specific effects of dietary restriction and reduced IIS on life span have also been observed in mice and humans. Future studies will be needed to determine whether a male-female difference in coupling between nutrients and IIS activity account for these sex-specific life span responses to dietary restriction. Indeed, given that sex differences have been reported in the risk of developing diseases associated with overnutrition and dysregulation of IIS activity such as obesity and type 2 diabetes, more detailed knowledge of the male-female difference in coupling between nutrients and IIS activity in other models may provide insights into this sex-biased risk of disease (Millington, 2021).
In addition to revealing a sex difference in the nutrient-dependent upregulation of IIS activity, the data identified a female-specific requirement for dilp2 and sun in mediating the diet-induced increase in IIS activity in a protein-rich context. While previous studies have shown that both dilp2 and sun positively regulate body size, this study describes new sex-specific roles for dilp2 and sun in nutrient-dependent phenotypic plasticity. Elegant studies have shown that sun is a secreted factor that stimulates Dilp2 release from the IPCs. Together with the current data, this suggests a model in which females are able to achieve a larger body size in a protein-rich diet because they have the ability to upregulate sun mRNA levels, whereas males do not. Indeed, this study shows that higher sun mRNA levels are sufficient to augment body size. This model aligns well with findings from two previous studies on Dilp2 secretion in male and female larvae. The first study, which raised larvae on a protein-rich diet equivalent to the 2Y diet (high protein), found increased Dilp2 secretion in females compared to males. The second study, which raised larvae on a diet equivalent to the 1Y diet (low protein), found no sex difference in Dilp2 secretion and no effects of dilp2 loss on body size. Thus, while these previous studies differed in their initial findings on a sex difference in Dilp2 secretion, the current data reconcile these minor differences by identifying context-dependent effects of dilp2 on body size. It is important to note that absolute confirmation of a sex difference in hemolymph Dilp2 levels will be needed in future studies because the body size plasticity defects in the dilp2-HF strain precluded its use as a tool to quantify circulating Dilp2 levels in this study. Future studies will also need to determine whether these sex-specific and context-dependent effects of dilp2 are observed in other phenotypes regulated by dilp2 and other dilp genes. For example, flies carrying mutations in dilp genes show changes to aging, metabolism, sleep, and immunity, among other phenotypes. Further, it will be interesting to determine whether the sex-specific regulation of sun is observed in any other contexts, and whether it will influence sex differences in phenotypes associated with altered IIS activity, such as life span (Millington, 2021).
While these findings on sun and dilp2 provide mechanistic insight into the molecular basis for the larger body size of females reared on a protein-rich diet, a key finding from this study was the identification of sex determination gene tra as the factor that confers plasticity to females. Normally, nutrient-dependent body size plasticity is higher in females than in males in a protein-rich context. In females lacking a functional Tra protein, however, this increased nutrient-dependent body size plasticity was abolished. In males, which normally lack a functional Tra protein, ectopic Tra expression conferred increased nutrient-dependent body size plasticity. While a previous study showed that on the 2Y diet Tra promotes Dilp2 secretion , the current study extends this finding in two ways: by identifying sun as one link between Tra, Dilp2, and changes to IIS activity; and by showing that Tra regulates sun mRNA via conserved transcriptional coactivator Srl. While previous studies discovered Srl as the factor that promotes sun mRNA levels in response to dietary protein in a mixed-sex larval population, the current findings reveal a previously unrecognized sex-specific role for Srl in regulating transcription. Because loss of Tra reduces Srl transcriptional activity, this new link between Tra and Srl suggests an additional way in which Tra may impact gene expression beyond its canonical downstream targets dsx and fru. While this builds on recent studies that reveal a number of additional Tra-regulated genes, it will be important to determine whether these additional Tra-regulated genes including sun represent direct targets of Tra/Srl. Future studies will also be needed to elucidate how Tra impacts Srl transcriptional activity in a context-dependent manner. However, uncovering a connection between a sex determination gene and a key regulator of genes involved in mitochondrial function suggests an additional mechanism that may contribute to sex differences in phenotypes affected by mitochondrial function (e.g., lifespan). In addition, it will be critical to explore how the presence of Tra allows an individual to couple dietary protein with body size. Because the tra locus is regulated both by alternative splicing and transcription, and Tra protein is regulated by phosphorylation, this study highlights the importance of additional studies on the regulation of the tra genomic locus and Tra protein throughout development to gain mechanistic insight into its effects on nutrient-dependent body size plasticity (Millington, 2021).
While the main outcome of this work was to reveal the molecular mechanisms that regulate the sex difference in nutrient-dependent body size plasticity, this study also provides some insight into how genes that contribute to nutrient-dependent body size plasticity affect female fecundity and male fertility. The findings align well with previous studies demonstrating that increased nutrient availability during development and a larger female body size confers increased ovariole number and fertility, as females lacking either dilp2 or fat body-derived sun were unable to augment egg production in a protein-rich context. Given that previous studies demonstrate IIS activity influences germline stem cells in the ovary in adult flies, there is a clear reproductive benefit that arises from the tight coupling between nutrient availability, IIS activity, and body size in females. In males, however, the relationship between fertility and body size remains less clear. While larger males are more reproductively successful both in the wild and in laboratory conditions. Given that this study revealed no significant increase in the number of progeny produced by larger males, the fertility benefits that accompany a larger body size in males may be context-dependent. For example, a larger body size increases the ability of males to outcompete smaller males. Thus, in crowded situations, a bigger body may provide significant fertility gains. However, in conditions where nutrients are limiting, an imbalance in the allocation of energy from food to growth rather than to reproduction may decrease fertility. Future studies will need to resolve the relationship between body size and fertility in males, as this will suggest the ultimate reason(s) for the sex difference in nutrient-dependent body size plasticity (Millington, 2021).
Female sub-fertility, a prominent complication due to Type 1 diabetes (T1D), is generally attributed to disturbances in menstrual cycles and/or ovarian defects/disorders. T1D women, however, are high in oxidative stress, although the impact of the same on their reproduction and associated events remains unknown. Therefore, the repercussions of elevated oxidative stress on the sperm fate (storage/utilization) in the reproductive tract milieu of T1D females and their fertility using the Drosophila T1D model (Df[dilp1-5]), which lacks insulin-like peptides and displays reduced female fertility. Df[dilp1-5] females were mated to normal males and thereafter sperm storage and/or utilization were examined in conjunction with oxidative stress parameters in mated Df[dilp1-5] females at different time points. Also, the impact of antioxidant (Amla or Vitamin C) supplementation on the above oxidative stress parameters in Df[dilp1-5] females and the consequences on their sperm and fertility levels were examined. Df[dilp1-5] females showed elevated oxidative stress parameters and a few of their reproductive tract proteins are oxidatively modified. Also, these females stored significantly fewer sperm and also did not utilize sperm as efficiently as their controls. Surprisingly, amelioration of the oxidative stress in Df[dilp1-5] females' milieu through antioxidant (Amla or vitamin C) supplementation enhanced sperm storage and improved fertility. Hyperglycemia coupled with elevated oxidative stress within the female reproductive tract environment affects the sperm fate, thereby reducing female fertility in T1D. In addition, these findings suggest that antioxidant supplementation may substantially aid in the mitigation of sub-fertility in T1D females (Gupta, 2023)
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).
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).
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).
Evolutionarily conserved insulin/insulin-like growth factor (IGF) signaling (IIS) correlates nutrient levels to metabolism and growth, thereby playing crucial roles in development and adult fitness. In the fruit fly Drosophila, ImpL2, an ortholog of IGFBP7, binds to and inhibits the function of Drosophila insulin-like peptides. In this study, a temperature-sensitive mutation was isolated in the insulin receptor (InR) gene as a spontaneous revertant in ImpL2 null mutants. The p.Y902C missense mutation is located at the functionally conserved amino acid residue of the first fibronectin type III domain of InR. The hypomorphic InR mutant animals showed a temperature-dependent reduction in IIS and body size. The mutant animals also exhibited metabolic defects, such as increased triglyceride and carbohydrate levels. Metabolomic analysis further revealed that defects in InR caused dysregulation of amino acid and ribonucleotide metabolism. It was also observed that InR mutant females produced tiny irregular-shaped embryos with reduced fecundity. In summary, this novel allele of InR is a valuable tool for the Drosophila genetic model of insulin resistance and type 2 diabetes (Banzai, 2023).
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).
Progressive lipid loss of adipose tissue is a major feature of cancer-associated cachexia. In addition to systemic immune/inflammatory effects in response to tumor progression, tumor-secreted cachectic ligands also play essential roles in tumor-induced lipid loss. However, the mechanisms of tumor-adipose tissue interaction in lipid homeostasis are not fully understood. The yki-gut tumors were induced in fruit flies. Lipid metabolic assays were performed to investigate the lipolysis level of different types of insulin-like growth factor binding protein-3 (IGFBP-3) treated cells. Immunoblotting was used to display phenotypes of tumor cells and adipocytes. Quantitative polymerase chain reaction (qPCR) analysis was carried out to examine the gene expression levels such as Acc1, Acly, and Fasn et al . This study revealed that tumor-derived IGFBP-3 was an important ligand directly causing lipid loss in matured adipocytes. IGFBP-3, which is highly expressed in cachectic tumor cells, antagonized insulin/IGF-like signaling (IIS) and impaired the balance between lipolysis and lipogenesis in 3T3-L1 adipocytes. Conditioned medium from cachectic tumor cells, such as Capan-1 and C26 cells, contained excessive IGFBP-3 that potently induced lipolysis in adipocyted. Notably, neutralization of IGFBP-3 by neutralizing antibody in the conditioned medium of cachectic tumor cells significantly alleviated the lipolytic effect and restored lipid storage in adipocytes. Furthermore, cachectic tumor cells were resistant to IGFBP-3 inhibition of IIS, ensuring their escape from IGFBP-3-associated growth suppression. Finally, cachectic tumor-derived ImpL2, the IGFBP-3 homolog, also impaired lipid homeostasis of host cells in an established cancer-cachexia model in Drosophila. Most importantly, IGFBP-3 was highly expressed in cancer tissues in pancreatic and colorectal cancer patients, especially higher in the sera of cachectic cancer patients than non-cachexia cancer patients. This study demonstrates that tumor-derived IGFBP-3 plays a critical role in cachexia-associated lipid loss and could be a biomarker for diagnosis of cachexia in cancer patients (Wang, 2023).
Although microbiome-host interactions are usual at steady state, gut microbiota dysbiosis can unbalance the physiological and behavioral parameters of the host, mostly via yet not understood mechanisms. Using the Drosophila model, this study investigated the consequences of a gut chronic dysbiosis on the host physiology. The results show that adult flies chronically infected with the non-pathogenic Erwinia carotorova caotovora bacteria displayed organ degeneration resembling wasting-like phenotypes reminiscent of Metabolic Syndrome associated pathologies. Genetic manipulations demonstrate that a local reduction of insulin signaling consecutive to a peptidoglycan-dependent NF-κB (Relish) activation in the excretory system of the flies is responsible for several of the observed phenotypes. This work establishes a functional crosstalk between bacteria-derived peptidoglycan and the immune NF-κB cascade that contributes to the onset of metabolic disorders by reducing insulin signal transduction. Giving the high degree of evolutionary conservation of the mechanisms and pathways involved, this study is likely to provide a helpful model to elucidate the contribution of altered intestinal microbiota in triggering human chronic kidney diseases (Zugasti, 2020).
Food and reproduction are the fundamental needs for all animals. However, the neural mechanisms that orchestrate nutrient intake and sexual behaviors are not well understood. This study found that sugar feeding immediately suppresses sexual drive of male Drosophila, a regulation mediated by insulin that acts on insulin receptors on the courtship-promoting P1 neurons. The same pathway was co-opted by anaphrodisiac pheromones to suppress sexual hyperactivity to suboptimal mates. Activated by repulsive pheromones, male-specific PPK23 neurons on the leg tarsus release crustacean cardioactive peptide (CCAP) that acts on CCAP receptor on the insulin-producing cells in the brain to trigger insulin release, which then inhibits P1 neurons. These results reveal how male flies avoid promiscuity by balancing the weight between aphrodisiac and anaphrodisiac inputs from multiple peripheral sensory pathways and nutritional states. Such a regulation enables male animals to make an appropriate mating decision under fluctuating feeding conditions (Zhang, 2022).
This work identified a neuropeptidergic neural circuit underlying mating decision, and a direct link is revealed between the 'metabolic center' and the 'sex center' in the Drosophila brain. First, sugar feeding largely suppressed male sexual drive toward a virgin female, and this metabolic state-dependent neural control relied on ILP2 and ILP5. A suppression on P1 neurons was induced in vitro with the activation of IPCs, which was triggered by loading sugar. This conclusion was further validated by the fact that the decrease of P1 neuron activity was weaker when InR was knock down specifically on P1 neurons. It was also demonstrated that leg tarsal PPK23 M cells release CCAP once contacting aversive pheromone and this inhibitory signal activates IPCs in the brain via CCAP-R to release ILP2/5. IPC-derived ILP2/5 furthermore suppresses the activity of courtship-promoting P1 neurons to ultimately shut down male's sexual drive toward inappropriate mating targets. Together with inhibition on P1 by the tandem connections of PPK23 M cells and mAL neurons, male flies maintain high selectivity against inappropriate mates (Zhang, 2022).
As the central hub in control of male courtship, P1 neurons in the brain integrate both the external and internal information. The former includes sensory inputs from a potential mating target. While visual and olfactory cues are reported to trigger a male's propensity to court, contact pheromones can be either attractive or repulsive and gate the perception for other sensory pathways. The internal state, on the other hand, represents the male's readiness to mate, e.g., his mating history and nutritional state. P1 neurons constantly monitor the internal state of a male to evaluate the readiness to court or mate. For example, dopaminergic neurons control the mating drive of a male, and neuropeptide Drosulfakinin (DSK) and NPF impose an inhibitory tone on P1 neurons by encoding either mating experiences or nutritional state. In addition, sleep regulates mating via the functional interaction between circadian neurons and P1. Insulin signaling reportedly plays a well-established role in homeostatic regulation. This study revealed an unprecedented role of insulin in modulating male sexual behavior. On the basis of the fact that insulin level changes under numerous physiological conditions, such as feeding, temperature change, and sleep, the current findings raised the question whether, under such conditions, insulin regulates sexual activity via InR on P1. The present data support the existence of feeding/mating interaction via the insulin signaling (Zhang, 2022).
Further investigation is needed to reveal the biological significance of courtship inhibition immediately after a sugar meal. In the studies of human and mouse, sugar intake reduces the level of testosterone, an effect likely mediated with insulin. This adversity of sugar intake on libido may be important for the animals' fitness. There is an immediate boost of the blood sugar level after a sugar meal. Insulin is then secreted to help move the sugar from the blood into the cells. During this process, the sexual activity may be momentarily inhibited to foster sugar uptake. It still needs further investigation how decreased sexual behaviors contribute to energy storage. Another interesting question is whether other nutrition-related hormones such as Adipokinetic hormone (Akh) and Unpaired 2 (Upd2) also regulate sexual activity in flies. It is already reported that DSK and NPF are both required to tune a male's sexual drive. Recently, it was reported that protein intake caused postprandial sleepiness that may be critical for protein metabolism, suggesting that animals' nutritional homeostasis is critical in maintaining the balance of their feeding, reproduction, and other behaviors (Zhang, 2022).
Another intriguing question that warrants further investigation is whether insulin released upon contacting aversive pheromones would cause certain metabolic consequences to the males. It was reported that exposing male flies to female pheromone but preventing them from mating reduced males' life span. As deterring courtship is relatively fast, it would be interesting to look at the long-term effects after a male is exposed to aversive pheromones (Zhang, 2022).
Insulin was implicated to regulate female sexual receptivity. The female flies with mutations in their insulin-like protein genes exhibit a higher sexual activity, a similar defect as seen in male flies in current study. However, the neural circuit controlling mating in male and female shows profound sexual dimorphism. It thus raises an interesting question: How does insulin regulate the sexual drive in females? A virgin female's receptivity is controlled by double-sex-positive neurons in the brain. Among them, clusters of neurons of pCd and pC1 play a determinant role in female's sex behaviors. It is still an open question whether these neurons express InR and, if so, under what circumstance are they inhibited by insulin release. Insulin is essential for vitellogenesis in female flies, suggesting that insulin signaling may play differential roles at different reproductive stages (Zhang, 2022).
Cell proliferation is dependent on growth factors insulin and IGF1. This study sought to identify interactors of IRS1, the most proximal mediator of insulin/IGF1 signaling, that regulate cell proliferation. Using proximity-dependent biotin identification (BioID), 40 proteins were detected displaying proximal interactions with IRS1, including DCAF7 and its interacting partners DYRK1A and DYRK1B. In HepG2 cells, DCAF7 knockdown attenuated cell proliferation by inducing cell cycle arrest at G2. DCAF7 expression was required for insulin-stimulated AKT phosphorylation, and its absence promoted nuclear localization of the transcription factor FOXO1. DCAF7 knockdown induced expression of FOXO1-target genes implicated in G2 cell cycle inhibition, correlating with G2 cell cycle arrest. In Drosophila melanogaster, wing-specific knockdown of DCAF7/wap (wings apart) caused smaller wing size and lower wing cell number; the latter recovered upon double knockdown of wap and dfoxo. It is proposed that DCAF7 regulates cell proliferation and cell cycle via IRS1-FOXO1 signaling, of relevance to whole organism growth (Frendo-Cumbo, 2022).
Crawling insects, when starved, tend to have fewer head wavings and travel in straighter tracks in search of food. This study used the Drosophila melanogaster larva to investigate whether this flexibility in the insect's navigation strategy arises during early olfactory processing and, if so, how. A critical role is demonstrated for Keystone-LN, an inhibitory local neuron in the antennal lobe, in implementing head-sweep behavior. Keystone-LN responds to odor stimuli, and its inhibitory output is required for a larva to successfully navigate attractive and aversive odor gradients. Insulin signaling in Keystone-LN likely mediates the starvation-dependent changes in head-sweep magnitude, shaping the larva's odor-guided movement. These findings demonstrate how flexibility in an insect's navigation strategy can arise from context-dependent modulation of inhibitory neurons in an early sensory processing center. They raise new questions about modulating a circuit's inhibitory output to implement changes in a goal-directed movement (Odell, 2022).
Animals control their developmental schedule in accordance with internal states and external environments. In Drosophila larvae, it is well established that nutrient status is sensed by different internal organs, which in turn regulate production of insulin-like peptides and thereby control growth. In contrast, the impact of the chemosensory system on larval development remains largely unclear. A genetic screen was performed to identify gustatory receptor (Gr) neurons regulating growth and development; Gr28a-expressing neurons were found to be required for proper progression of larval growth. Gr28a is expressed in a subset of peripheral internal sensory neurons, which directly extend their axons to insulin-producing cells (IPCs) in the central nervous system. Silencing of Gr28a-expressing neurons blocked insulin-like peptide release from IPCs and suppressed larval growth during the mid-larval period. These results indicate that Gr28a-expressing neurons promote larval development by directly regulating growth-promoting endocrine signaling in a stage-specific manner (Ohhara, 2022).
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)
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).
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).
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).
Adult progenitor cells in the trachea of Drosophila larvae are activated and migrate out of niches when metamorphosis induces tracheal remodeling. In response to metabolic deficiency in decaying tracheal branches, signaling by the insulin pathway controls the progenitor cells by regulating Yorkie (Yki)-dependent proliferation and migration. Yki, a transcription coactivator that is regulated by Hippo signaling, promotes transcriptional activation of cell cycle regulators and components of the extracellular matrix in tracheal progenitor cells. These findings reveal that regulation of Yki signaling by the insulin pathway governs proliferation and migration of tracheal progenitor cells, thereby identifying the regulatory mechanism by which metabolic depression drives progenitor cell activation and cell division that underlies tracheal remodeling (Li, 2022).
Increasing reports suggest insulin signalling pathway as a putative drug target against polyglutamine [poly(Q)] disorders, such as Huntington's disease (HD), Spinocerebellar ataxias (SCA) 1, 2, 3 etc. However, studies on drug-based stimulation of insulin signalling cascade to mitigate poly(Q) pathogenesis are lacking. This study adopted an evidence-based approach to examine if some established insulin stimulating drug can be utilized to restrict poly(Q) aetiology in Drosophila disease models. For the first time, this study reports that glipizide, an FDA approved anti-diabetic drug upregulates insulin signalling in poly(Q) expressing tissues and restricts formation of inclusion bodies and neurodegeneration. Moreover, it reinstates the chromatin architecture by improving histone acetylation, which is otherwise abrogated due to poly(Q) toxicity. In view of the functional conservation of insulin signalling pathway in Drosophila and humans, this finding strongly suggests that glipizide can be repurposed as an effective treatment strategy against the neurodegenerative poly(Q) disorders. Also, with appropriate validation studies in mammalian disease models, glipizide could be subsequently considered for the clinical trials in human patients (Tandon, 2023).
Insulin-like peptides (Ilps) play crucial roles in nearly all life stages of insects. Ilp8 is involved in developmental stability, stress resistance and female fecundity in several insect species, but the underlying mechanisms are not fully understood. This study reports the functional characterization of Ilp8s in three fly species, including Bactrocera dorsalis, Drosophila mercatorum and Drosophila melanogaster. Phylogenetic analyses were performed to identify and characterize insect Ilp8s. The amino acid sequences of fly Ilp8s were aligned and the three-dimensional structures of fly Ilp8s were constructed and compared. The tissue specific expression pattern of fly Ilp8s were examined by qRT-PCR. In Bactrocera dorsalis and Dro../sophila mercatorum, dsRNAs were injected into virgin females to inhibit the expression of Ilp8 and the impacts on female fecundity were examined. In Drosophila melanogaster, the female fecundity of Ilp8 loss-of-function mutant was compared with wild type control flies. The mutant fruit fly strain was also used for sexual behavioral analysis and transcriptomic analysis. Orthologs of Ilp8s are found in major groups of insects except for the lepidopterans and coleopterans, and Ilp8s are found to be well separated from other Ilps in three fly species. The key motif and the predicted three-dimensional structure of fly Ilp8s are well conserved. Ilp8 is specifically expressed in the ovary and are essential for female fecundity in three fly species. Behavior analysis demonstrates that Ilp8 mutation impairs female sexual attractiveness in fruit fly, which results in decreased mating success and is likely the cause of fecundity reduction. Further transcriptomic analysis indicates that Ilp8 might influence metabolism, immune activity, oocyte development as well as hormone homeostasis to collectively regulate female fecundity in the fruit fly. These findings support a universal role of insect Ilp8 in female fecundity, and also provide novel clues for understanding the modes of action of Ilp8 (Li, 2023).
Although painful stimuli elicit defensive responses including escape behavior for survival, starved animals often prioritize feeding over escape even in a noxious environment. This behavioral priority is typically mediated by suppression of noxious inputs through descending control in the brain, yet underlying molecular and cellular mechanisms are incompletely understood. This study identified a cluster of GABAergic neurons in Drosophila larval brain, designated as SEZ-localized Descending GABAergic neurons (SDGs), that project descending axons onto the axon terminals of the peripheral nociceptive neurons and prevent presynaptic activity through GABA(B) receptors. Remarkably, glucose feeding to starved larvae causes sustained activation of SDGs through glucose-sensing neurons and subsequent insulin signaling in SDGs, which attenuates nociception and thereby suppresses escape behavior in response to multiple noxious stimuli. These findings illustrate a neural mechanism by which sugar sensing neurons in the brain engages descending GABAergic neurons in nociceptive gating to achieve hierarchical interaction between feeding and escape behavior (Nakamizo-Dojo, 2023).
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).
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). 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).
Understanding how different cell types acquire their motile behaviour is central to many normal and pathological processes. Drosophila border cells represent a powerful model to address this question and to specifically decipher the mechanisms controlling collective cell migration. This study has identified the Drosophila Insulin/Insulin-like growth factor Signalling (IIS) pathway as a key regulator controlling actin dynamics in border cells, independently of its function in growth control. Loss of IIS activity blocks the formation of actin-rich long cellular extensions that are important for the delamination and the migration of the invasive cluster. IIS specifically activates the expression of the actin regulator chickadee, the Drosophila homolog of Profilin, essential for promoting the formation of actin extensions and migration through the egg chamber. In this process, the transcription factor dFoxO acts as a repressor of chickadee expression. Altogether, these results show that local activation of IIS controls collective cell migration through regulation of actin homeostasis and protrusion dynamics (Ghiglione, 2018).
This study identified the Insulin/IGF-Signalling (IIS) pathway as a key regulator of border cell migration during Drosophila oogenesis. Activation of dInR at the onset of migration promotes actin dynamics in the outer border cells, the subpopulation of cells known to drive migration. In this process, the canonical IIS pathway is shown to act through the inhibition of the transcription factor FoxO, which leads to the de- repression of chic/profilin. High levels of Profilin in turn facilitate actin polymerization and the formation of dynamic protrusions and of specific, actin long cellular extensions which are required for delamination and proper migration of the invasive cell cluster (Ghiglione, 2018).
The conserved IIS pathway couples nutritional cues with cellular metabolism, which in turn is essential for coordinating development with growth conditions. The systemic action of the IIS pathway thus makes it difficult to discriminate between chronic versus more acute or specific roles in particular cellular processes and during morphogenesis. In this context, border cells provide a powerful model to specifically address the role of the IIS pathway on cellular motility. During Drosophila oogenesis, the IIS pathway acts both in the germline and somatic cells to adjust egg chamber maturation rates to protein availability. This study used the FLP/FRT system that to show that chronic downregulation of IIS in border cells impairs their migration, a process that can be associated with metabolic defects. Interestingly, acute manipulation of IIS in border cells using the Gal4/Gal80ts system, shows that IIS downregulation can also block cluster migration specifically, a phenotype that can be rescued partly by restoring Profilin expression. These data argue for an active control of cell migration by IIS, independently of cellular fitness. This view is consistent with previous work showing that in ex vivo experiments, Insulin-containing culture medium is necessary to support egg chamber development and border cell migration (Ghiglione, 2018).
Border cells migrate towards the oocyte to make the micropyle, an opening that allows oocyte fertilization through the chorion. In this process, border cell migration needs to be synchronized with oocyte growth. It is proposed that the dual role of IIS for both egg chamber growth and border cell migration could help coordinating migratory events with organ maturation, thereby ensuring robust morphogenesis important for fertility (Ghiglione, 2018).
Actin dynamics is essential to a multitude of cellular and morphogenetic processes, therefore understanding the diverse modes of actin regulation is of prime interest. Members of the IIS pathway have been linked to actin regulation in a number of normal and pathological processes. For example, IIS plays an important role in neuronal guidance or wound healing. Additionally, PI3K has been shown to couple glycolytic flux with actin dynamics, while AKT participates to epithelial-to-mesenchymal transition required to drive mesoderm formation during gastrulation. Accumulating evidence also indicates that PI3K/AKT controls the migratory phenotype of metastatic cells. In breast cancer cells, AKT enhances cell migration and invasion through increased filopodia formation, which can be blocked with a specific AKT inhibitor. These observations suggest a model in which AKT activation potentially influences cell motility through direct modulation of actin, which is supported by studies showing that actin preferentially binds to phosphorylated AKT at pseudopodia sites. Despite these evidences, the view is fragmented and data are lacking to demonstrate a clear role of the full canonical pathway in cytoskeleton plasticity. In particular, the requirement of IIS transcriptional regulation in this process remained elusive. This report reveals that canonical IIS acts through inhibition of the transcription factor FoxO to control a major actin regulator, Profilin. These data provide a molecular mechanism as to how FoxO can control actin remodeling, which may be generalized to other processes where actin dynamics is particularly important. For example, during wound healing in Drosophila larvae, formation of an acto-myosin cable has been shown to depend on PI3K activation and redistribution of the transcription factor FoxO (Ghiglione, 2018).
In conclusion, these findings establish the canonical IIS pathway as a gene regulatory network important for collective cell migration. The data also provide a novel mechanism by which actin homeostasis and organization is regulated transcriptionally in a dynamic migratory process. In this mechanism, the formation of actin-rich protrusions is constitutively and negatively controlled by the transcription factor FoxO, whose inhibition by IIS signalling can generate peak levels of actin polymerization required for delamination and migration. It will be interesting to establish whether the control of Profilin expression through IIS signalling represents a general mechanism controlling actin remodeling in cell and tissue morphogenesis (Ghiglione, 2018).
The core functions of the insulin/insulin-like signaling and target of rapamycin (IIS/TOR) pathway are nutrient sensing, energy homeostasis, growth, and regulation of stress responses. This pathway is also known to interact directly and indirectly with the sex determination regulatory hierarchy. The IIS/TOR pathway plays a role in directing sexually dimorphic traits, including dimorphism of growth, metabolism, stress and behavior. To understand the degree to which the environmentally responsive insulin signaling pathway contributes to sexual dimorphism of gene expression, the effect of perturbation of the pathway on gene expression was examined in male and female Drosophila heads. The data reveal a large effect of insulin signaling on gene expression, with greater than 50% of genes examined changing expression. Males and females have a shared gene expression response to knock-down of InR function, with significant enrichment for pathways involved in metabolism. Perturbation of insulin signaling has a greater impact on gene expression in males, with more genes changing expression and with gene expression differences of larger magnitude. Primarily as a consequence of the response in males, this study found that reduced insulin signaling results in a striking increase in sex-differential expression. This includes sex-differences in expression of immune, defense and stress response genes, genes involved in modulating reproductive behavior, genes linking insulin signaling and ageing, and in the insulin signaling pathway itself. These results demonstrate that perturbation of insulin signaling results in thousands of genes displaying sex differences in expression that are not differentially expressed in control conditions. Thus, insulin signaling may play a role in variability of somatic, sex-differential expression. The finding that perturbation of the IIS/TOR pathway results in an altered landscape of sex-differential expression suggests a role of insulin signaling in the physiological underpinnings of trade-offs, sexual conflict and sex differences in expression variability (Graze. 2018).
Males and females typically pursue divergent reproductive strategies and accordingly require different dietary compositions to maximise their fitness. This study moves from identifying sex-specific optimal diets to understanding the molecular mechanisms that underlie male and female responses to dietary variation in Drosophila melanogaster. Male and female gene expression was examined on male-optimal (carbohydrate-rich) and female-optimal (protein-rich) diets. The sexes share a large core of metabolic genes that are concordantly regulated in response to dietary composition. However, smaller sets of genes were observed with divergent and opposing regulation, most notably in reproductive genes which are over-expressed on each sex's optimal diet. These results suggest that nutrient sensing output emanating from a shared metabolic machinery are reversed in males and females, leading to opposing diet-dependent regulation of reproduction in males and females. Further analysis and experiments suggest that this reverse regulation occurs within the IIS/TOR network (Camus, 2019).
Metabolic diseases such as type 2 diabetes are associated with increased cancer incidence. This study shows that hyperinsulinemia promotes epithelial tumorigenesis by abrogating cell competition. In Drosophila eye imaginal epithelium, oncogenic scribble (scrib) mutant cells are eliminated by cell competition when surrounded by wild-type cells. Through a genetic screen, this study found that flies heterozygous for the insulin receptor substrate chico allow scrib cells to evade cell competition and develop into tumors. Intriguingly, chico is required in the brain's insulin-producing cells (IPCs) to execute cell competition remotely. Mechanistically, chico downregulation in IPCs causes hyperinsulinemia by upregulating a Drosophila insulin Dilp2, which activates insulin-mTOR signaling and thus boosts protein synthesis in scrib cells. A diet-induced increase in insulin levels also triggers scrib tumorigenesis, and pharmacological repression of protein synthesis prevents hyperinsulinemia-induced scrib overgrowth. These findings provide an in vivo mechanistic link between metabolic disease and cancer risk via systemic regulation of cell competition (Sanaki, 2020).
Metabolic diseases such as type 2 diabetes and obesity are often accompanied by hyperinsulinemia, which is characterized by high levels of circulating insulin. In epidemiology, hyperinsulinemia has been implicated in increased cancer incidence. For instance, the risk of liver, pancreas, endometrium, kidney, and bladder cancers increases 1.5- to 2-fold in people with hyperinsulinemia. Although previous studies in Drosophila and rodents unveiled some aspects of the mechanism by which hyperinsulinemia promotes tumor growth and malignancy, the underlying mechanisms are still largely unknown (Sanaki, 2020).
Most cancers originate from epithelial cells that frequently lose apicobasal polarity during tumor progression. In Drosophila imaginal epithelium, loss-of-function mutations in evolutionarily conserved apicobasal polarity genes, such as scrib or discs large (dlg), disrupt epithelial integrity and result in tumorous overgrowth. Intriguingly, such oncogenic polarity-deficient cells do not overproliferate but are eliminated from the tissue when surrounded by wild-type cells, a phenomenon called tumor-suppressive cell competition. Previous work found multiple mechanisms that drive this cell elimination via cell-cell interaction between scrib and wild-type cells, which include Sas-PTP10D ligand-receptor interaction, Slit-Robo2-Ena/VASP-mediated scrib cell extrusion, and engulfment of scrib cells by wild-type cells. Through a genetic screen in Drosophila, this study found an unexpected new regulatory mechanism whereby hyperinsulinemia systemically abrogates tumor-suppressive cell competition and thus causes tumorigenesis in the epithelium. These data could provide a mechanistic explanation for the epidemiological evidence that links hyperinsulinemia and cancer incidence, thus contributing to a better understanding of cancer biology in vivo (Sanaki, 2020).
This study found that hyperinsulinemia in flies systemically suppresses cell competition in the eye epithelium, leading to tumorous overproliferation of polarity-deficient cells that are normally eliminated when surrounded by wild-type cells. It has been reported that high-sugar diet promotes tumor growth and metastasis of fly tumors with elevated Ras and Src signaling, providing a model of how abnormal physiology promotes tumor progression. In addition, studies in mice have shown that high-fat diet-induced obesity suppresses extrusion of oncogenic RasV12-expressing cells from mice intestine and that endogenous hyperinsulinemia contributes to pancreatic ductal adenocarcinoma. Thus, abnormal physiology, especially hyperinsulinemia, has a promotive effect on tumor development and progression, yet the mechanism by which hyperinsulinemia controls the initial step of tumorigenesis has been unclear. The current observations indicate that chico heterozygous mutant or IPCs-specific chico-knockdown larvae can be used as a Drosophila model of hyperinsulinemia. Consistently, although chico homozygous mutant flies drastically decrease their body weight, chico heterozygous mutant flies show increased body weight, implying a phenotypic outcome of hyperinsulinemia (Sanaki, 2020).
The findings that hyperinsulinemia systemically abrogates tumor-suppressive cell competition by boosting InR-TOR-mediated protein synthesis in pre-malignant cells may provide an in vivo mechanistic link between metabolic diseases and cancer risk. Previous work has shown that Sas-PTP10D signaling in scrib cells promotes their elimination by repressing epidermal growth factor receptor (EGFR) signaling. Defects in Sas-PTP10D signaling attenuates scrib cell elimination via cooperation between EGFR-Ras and TNF-JNK signaling, which leads to activation of the Hippo pathway effector Yorkie (Yki). On the other hand, this study found that hyperinsulinemia attenuates scrib cell elimination by fueling insulin-mTor signaling. Given that these two signaling pathways are independent, there would be no direct cross talk between Sas-PTP10D signaling and hyperinsulinemia-driven tumorigenesis. Rather, it is possible that both Sas-PTP10D inactivation (Yki activation) and insulin signaling activation (Tor activation) lead to the same biological outcome, namely, elevation of protein synthesis, which could explain how insulin signaling overrides Sas-PTP10D signaling (Sanaki, 2020).
Notably, differential levels of protein synthesis between cells has long been implicated in regulating classical Minute cell competition, which is a competitive elimination of cells with a heterozygous mutation for a ribosomal protein gene. In addition, recent work has found that losers of cell competition triggered by different mutations such as Minute, Myc, Mahjong, and Hel25E commonly show lower protein synthesis levels than that neighboring winners do (Nagata, 2019). Moreover, insulin-TOR signaling has been shown to control cell competition during mouse embryonic development. These observations suggest that differential levels of insulin-TOR signaling and protein synthesis between cells are the key for cell competition. Supporting this notion, scrib-induced cell competition can be compromised either by introducing Minute mutation in wild-type winners or by overexpressing Myc in scrib losers. These data show that scrib cells are insensitive to environmental insulin and thus are lower in insulin-TOR signaling and protein synthesis levels compared with that of the neighbors, and hyperinsulinemia reverses this balance and causes scrib tumorigenesis. Given that a drug treatment targeting cellular metabolism could prevent hyperinsulinemia-driven tumorigenesis, cancer risk risen by metabolic diseases may become controllable in the future (Sanaki, 2020).
Formation of short term memory is energetically costly and by the reason of restricted availability of food or fluctuations in energy expanses, efficient metabolic homeostasis modulating different needs like survival, growth, reproduction, or investment in longer lasting memories is crucial. Whilst equipped with cellular and molecular pre-requisites for formation of a protein synthesis dependent long-term memory (LTM), its existence in the larval stage of Drosophila remains elusive. Considering it from the viewpoint that larval brain structures are completely rebuilt during metamorphosis, and that this process depends completely on accumulated energy stores formed during the larval stage, investing in LTM represents an unnecessary expenditure. However, as an alternative, Drosophila larvae are equipped with the capacity to form a protein synthesis independent so-called larval anaesthesia resistant memory (lARM), which is consolidated in terms of being insensitive to cold-shock treatments. Motivated by the fact that LTM formation causes an increase in energy uptake in Drosophila adults, this study tested the question of whether an energy surplus can induce the formation of LTM in the larval stage. Surprisingly, increasing the metabolic state by feeding Drosophila larvae the disaccharide sucrose directly before aversive olfactory conditioning led to the formation of a protein synthesis dependent longer lasting memory. Moreover, formation of this memory component is accompanied by the suppression of lARM. It was ascertained that insulin receptors (InRs) expressed in the mushroom body Kenyon cells suppresses the formation of lARM and induces the formation of a protein synthesis dependent longer lasting memory in Drosophila larvae. Given the numerical simplicity of the larval nervous system this work offers a unique prospect to study the impact of insulin signaling on the formation of protein synthesis dependent memories on a molecular level (Eschment, 2020).
Insecticide resistance is an ongoing challenge in agriculture and disease vector control. This study demonstrates a novel strategy to attenuate resistance. This study used genomics tools to target fundamental energy-associated pathways and identified a potential "Achilles' heel" for resistance, a resistance-associated protein that, upon inhibition, results in a substantial loss in the resistance phenotype. Specifically, the gene expression profiles and structural variations of the insulin/insulin-like growth factor signaling (IIS) pathway genes were compared in DDT-susceptible (91-C) and -resistant (91-R) Drosophila melanogaster (Drosophila) strains. A total of eight and seven IIS transcripts were up- and down-regulated, respectively, in 91-R compared to 91-C. A total of 114 nonsynonymous mutations were observed between 91-C and 91-R, of which 51.8% were fixed. Among the differentially expressed transcripts, phosphoenolpyruvate carboxykinase (PEPCK), down-regulated in 91-R, encoded the greatest number of amino acid changes, prompting the performance of PEPCK inhibitor-pesticide exposure bioassays. The inhibitor of PEPCK, hydrazine sulfate, resulted in a 161- to 218-fold decrease in the DDT resistance phenotype (91-R) and more than a 4- to 5-fold increase in susceptibility in 91-C. A second target protein, Glycogen synthase kinase 3β (GSK3β-PO), had one amino acid difference between 91-C and 91-R, and the corresponding transcript was also down-regulated in 91-R. A GSK3β-PO inhibitor, lithium chloride, likewise reduced the resistance but to a lesser extent than did hydrazine sulfate for PEPCK. This study has demonstrated the potential role of IIS genes in DDT resistance and the potential discovery of an "Achilles' heel" against pesticide resistance in this pathway (Zhang, 2021a).
Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, this study found that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. 253 genes were identified whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, it was found that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. These results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies (Nakamura, 2020).
Insulin signaling in Drosophila has a significant role in regulating growth, metabolism, fecundity, stress response, and longevity. The molecular mechanism by which insulin signaling regulates these vital processes is dependent on the nutrient status and oxygen availability of the organism. In a genetic screen to identify novel genes that regulate Drosophila insulin signalling, Lumens interrupted (lint), a gene that has previously been shown to act in tracheal development, was discovered. The knockdown of lint gene expression using a Dilp2Gal4 driver which expresses in the neuronal insulin-producing cells (IPCs), led to defects in systemic insulin signaling, metabolic status and growth. However, this analysis of lint knockdown phenotypes revealed that downregulation of lint in the trachea and not IPCs was responsible for the growth phenotypes, as the Gal4 driver is also expressed in the tracheal system. We found various tracheal terminal branch defects, including reduction in the length as well as number of branches in the lint knockdown background. This study reveals that substantial effects of lint downregulation arose because of tracheal defects, which induced tissue hypoxia, altered systemic insulin/TOR signaling, and resulted in effects on developmental growth regulation (Pathak, 2021).
Reduced activity of the insulin/IGF signalling network increases health during ageing in multiple species. Diverse and tissue-specific mechanisms drive the health improvement. Tissue-specific transcriptional and proteomic profiling were performed of long-lived Drosophila dilp2-3,5 mutants, and tissue-specific regulation was identified of &gy;3600 transcripts and >3700 proteins. Most expression changes were regulated post-transcriptionally in the fat body, and only in mutants infected with the endosymbiotic bacteria, Wolbachia pipientis, which increases their lifespan. Bioinformatic analysis identified reduced co-translational ER targeting of secreted and membrane-associated proteins and increased DNA damage/repair response proteins. Accordingly, age-related DNA damage and genome instability were lower in fat body of the mutant, and overexpression of a minichromosome maintenance protein subunit extended lifespan. Proteins involved in carbohydrate metabolism showed altered expression in the mutant intestine, and gut-specific overexpression of a lysosomal mannosidase increased autophagy, gut homeostasis, and lifespan. These processes are candidates for combatting ageing-related decline in other organisms (Tain, 2021).
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).
The insulin-IGF-1 signaling (IIS) pathway is conserved throughout multicellular organisms and regulates many traits, including aging, reproduction, feeding, metabolism, stress resistance, and growth. This study presents evidence of a survival-sustaining role for IIS in a subset of gut cells in Drosophila melanogaster, namely the intestinal stem cells (ISCs) and progenitor cells. Using RNAi to knockdown the insulin receptor, inhibition of IIS in ISCs was found to statistically shortened the lifespan of experimental flies compared with non-knockdown controls, and also shortened their survival under starvation or malnutrition conditions. These flies also showed decreased reproduction and feeding, and had lower amounts of glycogen and glucose in the body. In addition, increased expression was observed for the Drosophila transcripts for the insulin-like peptides dilp2, dilp5, and dilp6. This may reflect increased insulin signaling in peripheral tissues supported by up-regulation of the target of the brain insulin gene (tobi). In contrast, activation of IIS (via knockdown of the insulin pathway inhibitor PTEN) in intestinal stem and progenitor cells decreased fly resistance to malnutrition, potentially by affecting adipokinetic hormone signaling. Finally, Pten knockdown to enhance IIS also activated JAK-STAT signaling in gut tissue by up-regulation of upd2, upd3, and soc36 genes, as well as genes encoding the EGF receptor ligands spitz and vein. These results clearly demonstrate that manipulating insulin levels may be used to modulate various fly traits, which are important determinants of organismal survival (Strilbytska, 2020).
Insulin/insulin-like growth factor signaling (IIS) is a conserved mechanism to regulate animal physiology in response to nutrition. IIS activity controls gene expression, but only a subset of transcriptional regulators (TRs) targeted by the IIS pathway is currently known. This study reports the results of an unbiased screen for Drosophila TRs phosphorylated in an IIS-dependent manner. To conduct the screen, a library was built of 857 V5/Strep-tagged TRs under the control of Copper-inducible metallothionein promoter (pMt). The insulin-induced phosphorylation changes were detected by using Phos-tag SDS-PAGE and Western blotting. Eight proteins were found to display increased phosphorylation after acute insulin treatment. In each case, the insulin-induced phosphorylation was abrogated by mTORC1 inhibitor rapamycin. The hits included two components of the NURF complex (NURF38 and NURF55). bHLHZip transcription factor Max, as well as the Drosophila ortholog of human proliferation-associated 2G4 (dPA2G4). Subsequent experiments revealed that the expression of the dPA2G4 gene was promoted by the mTOR pathway, likely through transcription factor Myc. Furthermore, NURF38 was found to be necessary for growth in larvae, consistent with the role of IIS/mTOR pathway in growth control (Liu, 2020).
The insulin/IGF-signaling pathway is central in control of nutrient-dependent growth during development, and in adult physiology and longevity. Eight insulin-like peptides (DILP1-8) have been identified in Drosophila, and several of these are known to regulate growth, metabolism, reproduction, stress responses, and lifespan. However, the functional role of DILP1 is far from understood. Previous work has shown that dilp1/DILP1 is transiently expressed mainly during the pupal stage and the first days of adult life. The role of dilp1 in the pupa, as well as in the first week of adult life, was studied, and some comparisons were made to dilp6 that displays a similar pupal expression profile, but is expressed in fat body rather than brain neurosecretory cells. Mutation of dilp1 diminishes organismal weight during pupal development, whereas overexpression increases it, similar to dilp6 manipulations. No growth effects of dilp1 or dilp6 manipulations were detected during larval development. It was next show that dilp1 and dilp6 increase metabolic rate in the late pupa and promote lipids as the primary source of catabolic energy. Effects of dilp1 manipulations can also be seen in the adult fly. In newly eclosed female flies, survival during starvation is strongly diminished in dilp1 mutants, but not in dilp2 and dilp1/dilp2 mutants, whereas in older flies, only the double mutants display reduced starvation resistance. Starvation resistance is not affected in male dilp1 mutant flies, suggesting a sex dimorphism in dilp1 function. Overexpression of dilp1 also decreases survival during starvation in female flies and increases egg laying and decreases egg to pupal viability. In conclusion, dilp1 and dilp6 overexpression promotes metabolism and growth of adult tissues during the pupal stage, likely by utilization of stored lipids. Some of the effects of the dilp1 manipulations may carry over from the pupa to affect physiology in young adults, but the data also suggest that dilp1 signaling is important in metabolism and stress resistance in the adult stage (Liao, 2020).
The insulin/IGF signaling (IIS) pathway plays a central role in nutrient-dependent growth control during development, as well as in adult physiology and aging. More specifically, in mammals, insulin, IGFs, and relaxins act on different types of receptors to regulate metabolism, growth, and reproduction. This class of peptide hormones has been well conserved over evolution and therefore the genetically tractable fly Drosophila is an attractive model system for investigating IIS mechanisms. Eight insulin-like peptides (DILP1-8), each encoded on a separate gene, have been identified in Drosophila. The genes encoding these DILPs display differential temporal and tissue-specific expression profiles, suggesting that they have different functions. Specifically, DILP1, 2, 3, and 5 are mainly expressed in median neurosecretory cells located in the dorsal midline of the brain, designated insulin-producing cells (IPCs). The IPC-derived DILPs can be released into the open circulation from axon terminations in the corpora cardiaca, the anterior aorta, the foregut, and the crop. Genetic ablation of the IPCs reduces growth and alters metabolism, and results in increased resistance to several forms of stress and prolongs lifespan (Liao, 2020).
The functions of the individual DILPs produced by the IPCs may vary depending on the stage of the Drosophila life cycle. Already, the temporal expression patterns hint that DILP1-3 and 5 play different roles during development. Thus, whereas DILP2 and 5 are relatively highly expressed during larval and adult stages, DILP1 and 6 are almost exclusively expressed during pupal stages under normal conditions (Liao, 2020).
DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1 is different from the other insulin-like peptides tested (Liao, 2020).
DILP1 is unique among the IPC-produced peptides since it can be detected primarily during the pupal stage (a non-feeding stage) and the first few days of adult life when residual larval/pupal fat body is present. Furthermore, in female flies kept in adult reproductive diapause, where feeding is strongly reduced, dilp1/DILP1 expression is also high (Liu, 2016). The temporal expression profile of
dilp1/DILP1 resembles that of dilp6/DILP6 although the latter peptide is primarily produced by the fat body, not IPCs. Since DILP6 was shown to regulate growth of adult tissues during pupal development, it was asked whether also DILP1 plays a role in growth control. It is known that overexpression of several of the DILPs is sufficient to increase body growth through an increase in cell size and cell number, and especially DILP2 produces a substantial increase in body weight. In contrast, not all single dilp mutants display a decreased body mass. The dilp1, dilp2, and dilp6 single mutants display slightly decreased body weight, whereas the dilp3, dilp4, dilp5, and dilp7 single mutants display normal body weight. However, a triple mutation of dilp2, 3, and 5 causes a drastically reduced body weight, and a dilp1-4,5 mutation results in a further reduction. Note that several of the above studies do not show bona fide effects on cell or organismal growth (e.g., volume or cell numbers/sizes); they only provide body mass data (Liao, 2020).
There is a distinction between how DILPs act in growth regulation. DILPs other than DILP1 and DILP6 promote growth primarily during the larval stages (both feeding and wandering stages) when their expression is high. This nutrient-dependent growth is relatively well-understood and is critical for production of the steroid hormone ecdysone and thereby developmental timing and induction of developmental transitions such as larval molts and pupariation. The growth in the pupal stage, which primarily affects imaginal discs and therefore adult tissues, is far less studied. This study investigated the role of dilp1/DILP1 in growth regulation in Drosophila in comparison to dilp6/DILP6. For this, both bona fide size of body and/or wings were determined and wet weights were provided, and thus it was possible to distinguish between growth and increase of body mass. Mutation of dilp1 diminishes body weight (but not body size), whereas ectopic dilp1 expression promotes organismal growth by increasing both weight and size during the pupal stage, similar to dilp6. Thus, we cannot unequivocally show a role of dilp1 in organismal growth, but it does regulate body mass, suggesting that dilp1 affects metabolism and energy stores. Determination of metabolic rate (MR) and respiratory quotient (RQ) as well as triacylglyceride (TAG) levels during late pupal development provides evidence that dilp1 and dilp6 increase the MR and that the associated increased metabolic cost is fueled by increased lipid catabolism (Liao, 2020).
Since dilp1/DILP1 levels are high the first week of adult life, the role of dilp1 mutation and overexpression on early adult physiology was determined, including metabolism stress resistance and fecundity. Interestingly, the newly eclosed dilp1 mutant flies are less resistant to starvation than controls and dilp2 mutants. Thus, dilp1 acts differently from other dilps for which it has been shown that reduced signaling increases survival during starvation. Also, early egg laying and female fecundity are affected by dilp1 overexpression, and in general, dilp1 manipulations produce more prominent effects in female flies (Liao, 2020).
Taken together, these data suggest that ectopic expression of dilp1/DILP1 promotes growth of adult tissues during the pupal stage, and that this process mainly utilizes stored lipids to fuel the increased MR. The DILP1 signaling also affects the metabolism in the young adult fly, and sex dimorphic effects of altered signaling on stress responses and fecundity were seen (Liao, 2020).
This study shows that dilp1 gain of function stimulates adult tissue growth and increases metabolic rate (MR) during the pupal stage, and also affects adult physiology, especially during the first days of adult life. These stages correspond to the time when dilp1 is normally expressed. The gain of function experiments in this study suggest that the developmental role of ectopic dilp1 could be similar to that of dilp6, namely, to stimulate growth of adult tissues during pupal development. It was furthermore shown that in the adult fly, dilp1 is upregulated during starvation and genetic gain and loss of function of dilp1 signaling diminishes the flies' survival under starvation conditions in a sex-specific manner. These novel findings, combined with previous data that demonstrated high levels of dilp1 during adult reproductive diapause and the role of dilp1 as a pro-longevity factor during aging, suggest a wide-ranging importance of this signaling system. Not only does dilp1 expression correlate with stages of non-feeding (or reduced feeding), these stages are also associated with lack of reproductive activity and encompass the pupa, newly eclosed flies, and diapausing flies. Under normal conditions, the transient expression of dilp1/DILP1 during the first few days of adult life may be associated with a metabolic transition (remodeling from larval to adult fat body) and the process of sexual maturation (gonad growth and differentiation). The data also suggest that dilp1 affects physiology more prominently in young female flies than in males, which might be associated with ovary maturation (Liao, 2020).
It is also interesting to note that the diminished starvation resistance in dilp1 and dilp1/dilp2 mutants is opposite to the phenotype seen after IPC ablation, mutation of dilp1-4, or diminishing IIS by other genetic interventions. Thus, in recently eclosed flies, dilp1 appears to promote starvation resistance rather than diminishing it. Furthermore, the decreased survival during starvation in female dilp1 mutants is the opposite of that shown in dilp6 mutants, indicating that dilp1 action is different from the other insulin-like peptides tested (Liao, 2020).
In Drosophila, the final body size is determined mainly by nutrient-dependent hormonal action during the larval feeding stage. However, some regulation of adult body size can also occur after the cessation of the larval feeding stage, and this process is mediated by dilp6 acting on adult tissue growth in the pupa in an ecdysone-dependent manner. This is likely a mechanism to ensure growth of adult tissues if the larva is exposed to shortage of nutrition during its feeding stage. The current findings suggest that dilp1 can function as another regulator of growth during the pupal stage. Overexpression of dilp1 promotes organismal growth in the pupa, probably at the cost of stored nutrients derived from the larval feeding stage. This is supported by respiratory quotient (RQ) data that clearly show a shift from mixed-energy substrate metabolism in control flies toward almost pure lipid catabolism at the end of pupal development in the dilp1 overexpression flies (also seen for dilp6 gain of function in these experiments). Furthermore, triacylglycerol (TAG) (but not carbohydrate) levels in dilp1 overexpression pupae were clearly decreased, which likely reflects the shift in catabolic energy substrate also seen in the RQ using respirometry. It should be noted that insects predominantly use lipids as fuel during metamorphosis and dilp1 overexpression increases lipid catabolism. This study hence suggests that dilp1 can parallel dilp6 in balancing adult tissue growth and storage of nutrient resources during pupal development. This is interesting since dilp6 is an IGF-like peptide that is produced in the nutrient sensing fat body, whereas the source of the insulin-like dilp1 is the brain IPCs (Liao, 2020).
In contrast to the dilp1 gain of function, the experiments with dilp1 mutant flies did not show a clear effect on adult body growth, only a decrease in weight. Is this a result of compensation by other DILPs? Previous work showed that young adult dilp1 mutant flies display increased dilp6 and vice versa, suggesting feedback between these two peptide hormones in adults. During the pupal stage, this feedback appears less prominent in dilp1 mutants and no effects were detected on (dilp2, dilp3), or dilp6 levels. Furthermore, overexpression of dilp1 in fat body or IPCs has no effect on pupal levels of dilp2 and dilp6. Thus, at present, there is no evidence for compensatory changes in other dilps/DILPs in pupae with dilp1 manipulations. However, under normal conditions (in wild-type pupae), dilp6 levels are far higher than those of dilp1, which could buffer the effects of changes in dilp1 signaling (Liao, 2020).
DILPs and IIS are involved in modulating responses to starvation, desiccation, and oxidative stress in Drosophila. Flies with ablated IPCs or genetically diminished IIS display increased resistance to several forms of stress, including starvation . Conversely, overexpression of dilp2 increases mortality in Drosophila. This study found that young dilp1 mutant flies displayed diminished starvation resistance. In both recently eclosed and 3-day-old flies, mutation of dilp1 decreased survival during starvation (but not in 6- to 7-day-old flies) (Liao, 2020).
Action of dilp1 in the adult fly is also linked to reproductive diapause in females, where feeding is strongly reduced, and both peptide and transcript are upregulated. Related to this, dilp1 mRNA was found to upregulated during starvation in 12-day-old flies. Furthermore, it was shown that expression of dilp1 (dilp1 rescue) increases lifespan in dilp1/dilp2 double mutants, suggesting that loss of dilp2 induces dilp1 as a factor that promotes longevity. Thus, dilp1 activity is beneficial also during adult life, even though its expression under normal conditions is very low. This pro-longevity effect of dilp1 is in contrast to dilp2, 3, and 5 and the mechanisms behind this effect are of great interest to unveil (Liao, 2020).
A previous study showed that in wild-type (Canton S) Drosophila, DILP1 expression in young adults is sex-dimorphic with higher levels in females. In line with this, starvation resistance in young flies is diminished only in female dilp1 mutant and dilp1 overexpression flies. Thus, taken together, previous work showed that dilp1 displays a sex-specific expression and this study shows female-specific function in young adult Drosophila. It is tempting to speculate that the more prominent role of dilp1 in female flies is linked to metabolism associated with reproductive physiology and early ovary maturation, which is also reflected in the dilp1 upregulation during reproductive diapause. In fact, this study shows that egg-laying increased after dilp1 overexpression, and an earlier study demonstrated a decreased egg laying in dilp1 mutant flies. Part of the sex dimorphic effects on body weight of young adults after dilp1 manipulations might be a result of a differential role of dilp1 in water homeostasis (Liao, 2020).
This study shows that IPC-derived dilp1 displays several similarities to the fat body-produced dilp6, including temporal expression pattern, growth promotion, effects on adult stress resistance and lifespan. Additionally, dilp1 may play a role in regulation of nutrient utilization and metabolism during the first few days of adult life, especially in females. At this time, larval fat body is still present and utilized as energy fuel/nutrient store and this source also contributes to egg development. Curiously, there is a change in the action of DILP1 between the pupal and adult stages from being able to stimulate growth (agonist of dInR, like DILP6) in pupae, to acting in a manner opposite to DILP2, DILP6, and other DILPs in adults in regulation of lifespan and stress responses. Only one dInR is known so far (excluding the G protein-coupled receptors for the relaxin-like DILP7 and DILP8). Thus, the mechanisms behind this apparent switch in function of DILP1 signaling remain an open question. One possibility is that DILP1 acts via different signaling pathways downstream the dInR in pupae and adults. An obvious difference between these two stages is the presence of larval-derived fat body in the pupa and during the first few days of adults and its replacement by functional adult fat body in later stages. Perhaps dInR-mediated action differs in these types of fat body when activated by DILP1. Another possibility is stage-specific expression of insulin/IGF-binding proteins such as SDR, ALS, and Imp-L2 that could affect the activity of DILP1 in particular (Liao, 2020).
In the future, it would be interesting to investigate whether DILP1 acts differently on larval/pupal and adult fat body, or act on different downstream signaling in the two stages of the life cycle. Also, the possibility that dilp1 and dilp6 interact to regulate growth and metabolism in Drosophila is worth pursuing (Liao, 2020).
Endogenous clocks enable organisms to adapt cellular processes, physiology, and behavior to daily variation in environmental conditions. Metabolic processes in cyanobacteria to humans are under the influence of the circadian clock, and dysregulation of the circadian clock causes metabolic disorders. In mouse and Drosophila, the circadian clock influences translation of factors involved in ribosome biogenesis and synchronizes protein synthesis. Notably, nutrition signals are mediated by the insulin receptor/target of rapamycin (InR/TOR) pathways to regulate cellular metabolism and growth. However, the role of the circadian clock in Drosophila brain development and the potential impact of clock impairment on neural circuit formation and function is less understood. This study demonstrates that changes in light stimuli or disruption of the molecular circadian clock cause a defect in neural stem cell growth and proliferation. Moreover, this study shows that disturbed cell growth and proliferation are accompanied by reduced nucleolar size indicative of impaired ribosomal biogenesis. Further, this study defines that light and clock independently affect the InR/TOR growth regulatory pathway due to the effect on regulators of protein biosynthesis. Altogether, these data suggest that alterations in InR/TOR signaling induced by changes in light conditions or disruption of the molecular clock have an impact on growth and proliferation properties of neural stem cells in the developing Drosophila brain (Dapergola, 2021).
In Drosophila raised in nutrient-rich conditions, female body size is approximately 30% larger than male body size due to an increased rate of growth and differential weight loss during the larval period. While the mechanisms that control this sex difference in body size remain incompletely understood, recent studies suggest that the insulin/insulin-like growth factor signaling pathway (IIS) plays a role in the sex-specific regulation of processes that influence body size during development. In larvae, IIS activity differs between the sexes, and there is evidence of sex-specific regulation of IIS ligands. Yet, knowledge is lacking of how changes to IIS activity impact body size in each sex, as the majority of studies on IIS and body size use single- or mixed-sex groups of larvae and/or adult flies. The goal of the current study was to clarify the body size requirement for IIS activity in each sex. To achieve this goal, established genetic approaches were used to enhance, or inhibit, IIS activity, and pupal size was quantified in males and females. Overall, genotypes that inhibited IIS activity caused a female-biased decrease in body size, whereas genotypes that augmented IIS activity caused a male-specific increase in body size. These data extend the current understanding of body size regulation by showing that most changes to IIS pathway activity have sex-biased effects, and highlights the importance of analyzing body size data according to sex (Millington, 2021).
Sexual-size dimorphism (SSD) is replete among animals, but while the selective pressures that drive the evolution of SSD have been well studied, the developmental mechanisms upon which these pressures act are poorly understood. SSD in D. melanogaster reflects elevated levels of nutritional plasticity in females versus males, such that SSD increases with dietary intake and body size, a phenomenon called sex-specific plasticity (SSP). Additional data indicate that while body size in both sexes responds to variation in protein level, only female body size is sensitive to variation in carbohydrate level. This study explored whether these difference in sensitivity at the morphological level are reflected by differences in how the insulin/IGF-signaling (IIS) and TOR-signaling pathways respond to changes in carbohydrates and proteins in females versus males, using a nutritional geometry approach.The IIS-regulated transcripts of 4E-BP and InR most strongly correlated with body size in females and males, respectively, but neither responded to carbohydrate level and so could not explain the sex-specific response to body size to dietary carbohydrate. Transcripts regulated by TOR-signaling did, however, respond to dietary carbohydrate in a sex-specific manner. In females, expression of dILP5 positively correlated with body size, while expression of dILP2,3 and 8, was elevated on diets with a low concentration of both carbohydrate and protein. In contrast, lower levels of dILP2 and 5 protein were observed in the brains of females fed on low concentration diets. No effect of diet on dILP expression in males was detectec. Although females and males show sex-specific transcriptional responses to changes in protein and carbohydrate, the patterns of expression do not support a simple model of the regulation of body-size SSP by either insulin- or TOR-signaling. The data also indicate a complex relationship between carbohydrate and protein level, dILP expression and dILP peptide levels in the brain. In general, diet quality and sex both affect the transcriptional response to changes in diet quantity, and so should be considered in future studies that explore the effect of nutrition on body size (McDonald, 2021).
This study shows that the dietary lipid cholesterol, which is required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the nutrient sensor TOR, which is regulated by the Niemann-Pick-type-C 1 (Npc1) cholesterol transporter, in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to an accelerated attainment of a nutritional checkpoint that normally ensures that animals do not initiate maturation prematurely. These findings, therefore, show that a Npc1-TOR signaling system couples the sensing of the lipid cholesterol with cellular and systemic growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the connection between body fat (obesity) and early puberty (Texada, 2022).
Animal growth and development depend upon nutrient availability. Therefore, specialized cells and tissues have arisen that sense nutritional inputs and adjust growth and developmental programs via systemic hormonal pathways. In most eumetazoans, these include the conserved insulin-like peptide and steroid-hormone signaling systems. These become dysfunctional when nutrient levels exceed their physiologically normal range. Overloading of the insulin system leads to obesity, metabolic syndrome, insulin resistance, and other pathophysiologies, and overnutrition also leads to precocious puberty associated with childhood obesity (Texada, 2022).
The early life of many animals is a nonreproductive stage of rapid growth, terminated at some nutritional threshold that signals readiness to become a reproductively fit adult. In animals as diverse as humans and insects, this transition is driven by steroid hormones - gonadal steroids including testosterone and estrogen trigger mammalian puberty, and insect metamorphosis is initiated by ecdysone, produced in the prothoracic gland (PG). Similar neuroendocrine cascades regulate insect and mammalian steroidogenesis, including the orthologous neuropeptides Allatostatin A/Kisspeptin and Corazonin/gonadotropin-releasing hormone (GnRH) as well as analogous steroid-feedback circuits. These axes are clearly linked to the metabolic state of the animal, including attainment of a certain critical size. However, the mechanisms of body-size estimation and the effects of nutritional status are not completely understood. Recent work in Drosophila suggests that progression to adulthood is gated by a checkpoint system monitoring tissue growth and nutritional status. When animals reach a 'critical weight' (CW), they become committed to completing their development and maturation, irrespective of further nutritional inputs, whereas animals starved before this checkpoint is satisfied halt their progression to adulthood. This suggests that the CW checkpoint assesses the animal's nutritional state, but the specific nutrients required, and the mechanisms by which their levels are sensed, are incompletely defined (Texada, 2022).
In Drosophila, nutritional input drives growth and maturation through the insulin pathway. Nutrient intake, of amino acids in particular, is sensed via the fat body (analogous to mammalian adipose tissue) and the glia of the blood-brain barrier (BBB). These tissues release factors that regulate the expression and release of Drosophila insulin-like peptides (ILPs) from the insulin-producing cells (IPCs) within the brain, which share functional and developmental homologies with mammalian pancreatic beta cells. These ILPs promote systemic growth through the conserved insulin-receptor/PI3K/Akt pathway. Insulin also promotes PG ecdysone production, linking nutrition directly to developmental progression (Texada, 2022).
Human puberty-triggering CW appears to be linked to body-fat stores, which may explain the link between childhood obesity and early puberty. Despite this, the mechanism by which adiposity affects puberty initiation is unclear. Furthermore, the role of cholesterol has not been considered, even though adipose tissue is a major cholesterol storage depot, especially in obesity. Sterols such as cholesterol have membrane-structural functions but also play important signaling roles, and sterols are required as substrates for steroid-hormone production. Insects, including Drosophila, have lost the ability to synthesize sterols de novo and thus must acquire them through feeding. Mammals are cholesterol prototrophs, but most intracellular cholesterol still comes from low-density-lipoprotein (LDL)-mediated cellular uptake of dietary cholesterol. In both taxa, consumed sterols are transported in the circulatory system bound within lipoprotein particles (LPPs such as mammalian LDL/HDL), and target tissues take them up through a variety of mechanisms including receptor-mediated endocytosis. LPP-bound sterols are extracted in the lysosome and inserted into the lysosomal membrane by membrane-integral transport proteins. The primary such protein, Niemann-Pick-type-C 1 (Npc1), underlies the Niemann-Pick type C lysosomal storage disorder; without Npc1 function, cholesterol accumulates in endosomal-lysosomal compartments, leading to increased intracellular cholesterol signaling. Thus, Npc1 seems to be part of a mechanism by which cells regulate cholesterol signaling (Texada, 2022).
This study set out to determine whether, and the routes by which, cholesterol might regulate Drosophila larval growth. The findings show that dietary cholesterol dose-responsively promotes growth and accelerates development by increasing insulin signaling. Cholesterol sensing is mediated by the target of rapamycin (TOR) pathway in the cells of the fat body and the glia of the BBB, which remotely induce the expression and release of ILPs from the IPCs. Enhancing cholesterol signaling in the PG also promotes TOR activity, drives endoreduplication, and leads to premature attainment of the CW checkpoint. Thus, dietary cholesterol accelerates growth through insulin signaling and leads to early maturation through effects on steroidogenesis, effects which are mediated by promoting TOR activity in sensing tissues (Texada, 2022).
Nutrition is one of the most important influences on developmental growth and maturation. Malnutrition or disease can impair growth and delay puberty, whereas obese children enter puberty early. Similarly, Drosophila larvae exposed to poor nutrition, tissue damage, or inflammation delay their development, whereas rich conditions promote rapid growth and maturation. These environmental factors are coupled to the appropriate gating of steroid production via internal checkpoints, one of which is a nutrition-dependent CW required to initiate the maturation process. This suggests that signals reflecting nutritional state and body-fat storage play a key role in activating the neuroendocrine pathways that trigger puberty. Although studies suggest that the adipokine leptin may be involved, the mechanisms linking body fat to puberty are poorly defined, and the potential involvement of lifestyles associated with excessive accumulation of cholesterol, one of the most important lipids, has not been considered. In humans, white adipose tissue is the main site of cholesterol storage and can contain over half the body's total cholesterol in obesity. The results show that dietary cholesterol intake promotes systemic body growth through insulin-dependent pathways and that animals raised on high dietary cholesterol initiate maturation earlier. Cholesterol is sensed through an Npc1-regulated TOR-mediated mechanism in the fat body and the glial cells of the BBB, which relay information to the IPCs within the brain to promote insulin expression and release, thus coupling growth and maturation with cholesterol status (Texada, 2022).
Insect CW likely evolved as a mechanism ensuring that maturation will not occur unless the animal has accumulated adequate nutrient stores to survive the nonfeeding metamorphosis period and has completed sufficient growth to produce an adult of proper size and thus of maximal fitness. Likewise, the link between body fat and maturation in humans probably ensures adequate stores of fat before maturation onset to support pregnancy and reproductive success. In Drosophila, insulin signaling plays a critical role in coordinating steroidogenesis with nutritional conditions. Insulin acts upon the PG and induces a small ecdysone peak early in L3 that is correlated with CW attainment. In combination with nutrition-related signaling mediated via insulin, nutrient availability is also assessed directly in the PG and is coupled to irreversible endoreduplication that permits ecdysone production at CW. the current findings show that accumulation of cholesterol in the PG, induced by the loss of Npc1a, drives a remarkable TOR-dependent increase in endoreduplication and leads to inappropriate attainment of CW. Taken together, These findings indicate that cholesterol sensed by the BBB glia and the fat body promotes growth through insulin signaling and that cholesterol sensed by the PG accelerates maturation through ecdysone signaling (Texada, 2022).
Loss of Npc1 function in humans leads to the Niemann-Pick lysosomal storage disorder, marked by intracellular cholesterol accumulation. Although neurodegeneration is the hallmark of NPC disease, including in a Drosophila model, alterations in glial, adipose, hepatic, and endocrine systems are also components of NPC syndrome. In humans, Npc1 itself is strongly expressed in glia and in adipose tissues, especially in obese individuals, and variants in Npc1 are associated with obesity, type-2 diabetes, and hepatic lipid dysfunction. These findings link glial and adipose-tissue cholesterol sensing through Npc1 to systemic growth and metabolic control through effects on insulin signaling. It was also found that intracellular cholesterol accumulation driven by Npc1 loss leads to hyperactivation of TOR that drives increases in DNA replication and cell growth. TOR activity is frequently upregulated in cancer, and the results therefore provide mechanistic insight for understanding the emerging link between cholesterol and a range of cancers (Texada, 2022).
As the coupling of nutrition with growth and maturation is ancient and highly conserved, this work provides a foundation for understanding how cholesterol is coupled to developmental growth and maturation initiation in humans. These findings link a high concentration of this particular lipid in adipose tissues to the neuroendocrine initiation of maturation, which may explain the critical link between obesity (body fat) and early puberty (Texada, 2022).
Insulin signaling promotes anabolic metabolism to regulate cell growth through multi-omic interactions. To obtain a comprehensive view of the cellular responses to insulin, a trans-omic network of insulin action was constructed in Drosophila cells that involves the integration of multi-omic data sets. In this network, 14 transcription factors, including Myc, coordinately upregulate the gene expression of anabolic processes such as nucleotide synthesis, transcription, and translation, consistent with decreases in metabolites such as nucleotide triphosphates and proteinogenic amino acids required for transcription and translation. Next, as cell growth is required for cell proliferation and insulin can stimulate proliferation in a context-dependent manner, the trans-omic network was integrated with results from a CRISPR functional screen for cell proliferation. This analysis validates the role of a Myc-mediated subnetwork that coordinates the activation of genes involved in anabolic processes required for cell growth (Terakawa, 2022).
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).
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 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).
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).
In mammals, blood glucose levels likely play a role in appetite regulation yet the mechanisms underlying this phenomenon remain opaque. Mechanisms can often be explored from Drosophila genetic approaches. To determine if circulating sugars might be involved in Drosophila feeding behaviors, hemolymph glucose and trehalose, and food ingestion were scored in larvae subjected to various diets, genetic mutations, or RNAi. Larvae with glucose elevations, hyperglycemia, were found to have an aversion to feeding; however, trehalose levels do not track with feeding behavior. It was further discovered that insulins and SLC5A11 may participate in glucose-regulated feeding. To see if food aversion might be an appropriate screening method for hyperglycemia candidates, a food aversion screen was developed to score larvae with abnormal feeding for glucose. It was found that many feeding defective larvae have glucose elevations. These findings highlight intriguing roles for glucose in fly biology as a potential cue and regulator of appetite (Ugrankar, 2018).
Developing organisms use fine-tuning mechanisms to adjust body growth to ever-changing nutritional conditions. In Drosophila, the secretory activity of insulin-producing cells (IPCs) is central to couple systemic growth with amino acids availability. This study identified a subpopulation of inhibitory neurons contacting the IPCs (IPC-connecting neurons or ICNs) that play a key role in this coupling. ICNs respond to growth-blocking peptides (GBPs), a family of fat-body-derived signals produced upon availability of dietary amino acids. GBPs are atypical ligands for the fly EGF receptor (EGFR). Upon activation of EGFR by adipose GBPs, ICN-mediated inhibition of IPC function is relieved, allowing insulin secretion. Tnis study reveals an unexpected role for EGF-like metabolic hormones and EGFR signaling as critical modulators of neural activity, coupling insulin secretion to the nutritional status (Meschi, 2019).
Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. This study found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, it will be used to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic (Keliinui, 2022).
CD133, also called Prominin-1, is a biomarker for mammalian stem cells. It is involved in cell growth, development, and tumor biology. However, the function of CD133 at the organismal level has not been investigated. This study found that prominin-like (promL) loss-of-function mutant flies show an extended lifespan and metabolic defects such as increased circulating carbohydrates, lipid storage, and starvation resistance. The mRNA expression levels of Drosophila insulin-like peptides (Dilps) were reduced in loss-of-function promL mutants. Furthermore, the level of phosphorylated AKT, a downstream component of insulin signaling, was lower in promL loss-of-function mutants than in the w- control flies. Importantly, the PromL protein is predominantly expressed in the pars intercerebralis region with insulin producing cells (IPCs) of the adult brain. When promL was inhibited in IPCs, these flies showed an extended lifespan, metabolic defects, and reduced insulin signaling. These results indicate that the promL gene regulates longevity and glucose metabolism by controlling insulin signaling in Drosophila (Ryu, 2018).
Adipose tissue is essential for metabolic homeostasis, balancing lipid storage and mobilisation based on nutritional status. This is coordinated by insulin, which triggers kinase signalling cascades to modulate numerous metabolic proteins, leading to increased glucose uptake and anabolic processes like lipogenesis. Given recent evidence that glucose is dispensable for adipocyte respiration, this study sought to test whether glucose is necessary for insulin-stimulated anabolism. Examining lipogenesis in cultured adipocytes, glucose was essential for insulin to stimulate the synthesis of fatty acids and glyceride-glycerol. Importantly, glucose was dispensable for lipogenesis in the absence of insulin, suggesting distinct carbon sources are used with or without insulin. Metabolic tracing studies revealed glucose was required for insulin to stimulate pathways providing carbon substrate, NADPH, and glycerol 3'-phosphate for lipid synthesis and storage. Glucose also displaced leucine as a lipogenic substrate and was necessary to suppress fatty acid oxidation. Together, glucose provided substrates and metabolic control for insulin to promote lipogenesis in adipocytes. This contrasted with the suppression of lipolysis by insulin signalling, which occurred independently of glucose. Given previous observations that signal transduction acts primarily before glucose uptake in adipocytes, these data are consistent with a model whereby insulin initially utilises protein phosphorylation to stimulate lipid anabolism, which is sustained by subsequent glucose metabolism. Consequently, lipid abundance was sensitive to glucose availability, both during adipogenesis and in Drosophila flies in vivo. Together, these data highlight the importance of glucose metabolism to support insulin action, providing a complementary regulatory mechanism to signal transduction to stimulate adipose anabolism (Krycer, 2020).
The volumes of sugar solutions ingested and amounts of different carbohydrates eaten were measured in fruit fly lines with mutated genes for Drosophila insulin-like peptides (DILPs). The wild type w(1118) flies consumed 20-40 μg of fructose or glucose per day regardless of carbohydrate concentration. This relatively constant amount of consumed carbohydrate was regulated due to satiety-driven decreases in the ingested volume of sugar solution, a so-called "compensatory feeding" strategy. This decrease was not observed for flies fed sucrose solutions. The dilp3 mutant and quadruple mutant dilp1-4 showed no "compensatory feeding" when fed glucose but these two mutants consumed larger amounts of sucrose than the wild type from solutions with carbohydrate concentrations equal to or higher than 4%. Flies with mutations of dilp2, dilp3, dilp4, dilp5, and dilp6 genes consumed larger amounts of carbohydrate from 4-10% sucrose solutions as compared to the wild type. Mutations of DILPs affected appetite mainly for sucrose and glucose, but the least for fructose. The presented data confirm the hypothesis that DILPs are involved in the regulation of fly appetite in response to type and concentration of carbohydrate (Semaniuk, 2021).
Fasting in mammals promotes increases in circulating glucagon and decreases in circulating insulin that stimulate catabolic programs and facilitate a transition from glucose to lipid burning. The second messenger cAMP mediates effects of glucagon on fasting metabolism, in part by promoting the phosphorylation of CREB and the dephosphorylation of the cAMP-regulated transcriptional coactivators (CRTCs) in hepatocytes. In Drosophila, fasting also triggers activation of the single Crtc homolog in neurons, via the PKA-mediated phosphorylation and inhibition of salt-inducible kinases. Crtc mutant flies are more sensitive to starvation and oxidative stress, although the underlying mechanism remains unclear. This study used RNA sequencing to identify Crtc target genes that are up-regulated in response to starvation. Crtc was found to stimulate a subset of fasting-inducible genes that have conserved CREB binding sites. In keeping with its role in the starvation response, Crtc was found to induce the expression of genes that inhibit insulin secretion (Lst) and insulin signaling (Impl2). In parallel, Crtc also promoted the expression of genes involved in one-carbon (1-C) metabolism. Within the 1-C pathway, Crtc stimulated the expression of enzymes that encode modulators of S-adenosyl-methionine metabolism (Gnmt and Sardh) and purine synthesis (ade2 and AdSl) Collectively, these results point to an important role for the CREB/CRTC pathway in promoting energy balance in the context of nutrient stress (Wang, 2021).
The nervous and endocrine systems coordinately monitor and regulate nutrient availability to maintain energy homeostasis. Sensory detection of food regulates internal nutrient availability in a manner that anticipates food intake, but sensory pathways that promote anticipatory physiological changes remain unclear. This study identified serotonergic (5-HT) neurons as critical mediators that transform gustatory detection by sensory neurons into the activation of insulin-producing cells and enteric neurons in Drosophila. One class of 5-HT neurons responds to gustatory detection of sugars, excites insulin-producing cells, and limits consumption, suggesting that they anticipate increased nutrient levels and prevent overconsumption. A second class of 5-HT neurons responds to gustatory detection of bitter compounds and activates enteric neurons to promote gastric motility, likely to stimulate digestion and increase circulating nutrients upon food rejection. These studies demonstrate that 5-HT neurons relay acute gustatory detection to divergent pathways for longer-term stabilization of circulating nutrients (Yao, 2022).
This study identified two classes of 5-HT neurons that play distinct roles in gustatory processing and function independently to promote nutrient homeostasis. Sugar-SELs, located in the lateral subesophageal ganglion (SEL) respond to sugar gustatory detection, promote insulin-producing cell (IPC) activity, and reduce feeding drive, suggesting that they prevent overconsumption in nutrient rich environments. Bitter-SELs respond to bitter taste detection and promote crop contractions, likely to utilize stored food upon food rejection. Thus, 5-HT neurons coordinate endocrine and digestive function in anticipation of altered food intake (Yao, 2022).
5-HT profoundly modulates appetite and feeding across animal species. In humans and rodents, the global effect of brain 5-HT signaling is suppression of food intake; however, the involvement of multiple brain regions (including the hypothalamus, solitary tract nucleus, and parabrachial nuclei) and multiple 5-HT receptors (e.g., 5-HT1B, 5-HT2C, 5-HT6) underscores the complex nature of 5-HT modulation of appetite and feeding. Studies in invertebrates have likewise demonstrated multiple, sometimes opposite, roles for 5-HT neurons in regulating feeding. For instance, in Drosophila adults, activating all 5-HT neurons suppresses food intake whereas activating a smaller yet diverse subset promotes food intake, suggesting heterogeneity in 5-HT feeding regulation. With the exception of a few cases where specific 5-HT neurons that influence feeding have been identified—for example, 5-HT neurons that promote pharyngeal pumping to enhance ingestion in C. elegans and Drosophila larvae—the diversity of 5-HT neurons that contributes to feeding regulation and nutrient homeostasis remains largely uncharacterized (Yao, 2022).
This study identified multiple classes of 5-HT neurons that are activated by gustatory detection and signal to the endocrine and digestive systems to influence nutrient availability. These studies reveal 5-HT neurons that have different projection patterns, relay sugar and bitter gustatory information to different downstream targets, and regulate internal nutrient availability by distinct mechanisms. These studies provide insight into the multifaceted roles of 5-HT neurons in gustatory processing, feeding regulation, and nutrient homeostasis, highlighting the importance of understanding the myriad functions of 5-HT at the neural circuit level (Yao, 2022).
Food-derived sensory cues are used as anticipatory signals to regulate endocrine function and feeding drive. Work in mammals has demonstrated that there are two phases of insulin release in response to food consumption, a pre-absorptive phase in response to sensory detection of food and a post-absorptive phase in response to elevated blood glucose levels. The pre-absorptive phase (cephalic phase) is triggered by sensory detection prior to food consumption and nutrient absorption. The neural circuit that underlies pre-absorptive insulin release, however, is not fully understood (Yao, 2022).
The current findings suggest that insulin release in anticipation of food consumption is a process shared in flies and mammals, perhaps indicating an effective strategy to promote rapid nutrient storage during feeding. Using in vivo calcium imaging, this study found that fly IPCs are rapidly excited by sugar gustatory detection independent of consumption. The findings that sugar-SELs respond to sugar taste detection and activate IPCs suggest a neural circuit mechanism for pre-absorptive insulin release independent of ingestion. Moreover, as gustatory sensory neurons detect both nutritive and non-nutritive sugars and are inputs to sugar-SELs and IPCs, it is anticipated that sugar-SELs, IPCs, and pre-absorptive insulin release are also activated by non-nutritive sugars, but this requires further investigation. The finding that fly IPCs are activated by sugar taste detection contrasts with previous whole-brain imaging studies, likely indicating signal detection limits. In addition to identifying sugar taste responses in IPCs, this study also identified the sugar-SELs as a specific neural pathway mediating the preparatory insulin response (Yao, 2022).
Knocking down 5-HT2A in IPCs reduces but does not abolish gustatory-induced IPC activity and does not impact consumption. One caveat of this approach is that RNAi reduces gene expression and may not produce complete loss-of-function phenotypes. The IPCs are activated by many nutritional state signals, including multiple pathways that report circulating sugars and signals from the intestine and fat body. The current work shows that external nutrients in the form of sugar taste detection also activate this important hub and identifies sugar-SELs as a defined pathway conveying the sugar taste signal. As IPCs release multiple peptides, the activation of IPCs by sugar-SELs may coordinate widespread changes in metabolism and behavior. Thus, this study has identified a specific class of 5-HT neurons that participates in the preparatory insulin response and the reduction of feeding drive in response to sugar gustatory detection, shedding light on the neural circuit mechanisms that anticipate sugar consumption (Yao, 2022).
A surprising finding from this study is that bitter-SELs, which respond to bitter gustatory detection, promote contractions of the crop food storage organ. While there is evidence that intestinal bitter detection modulates gastrointestinal physiology, the regulation of gastrointestinal function by bitter gustatory detection is less examined. Activation of bitter gustatory neurons, bitter-SELs, and 5-HT7 neurons all promote crop contractions, although rates differ, possibly based on optogenetic activation strength or propagation of activity to the crop (Yao, 2022).
Why do bitter compounds promote crop contractions? It was reasoned that because bitter compounds are feeding deterrents, frequent encounters with bitter compounds may prevent food intake, leading to depletion of internal nutrients. Under such conditions, bitter-SELs may promote crop motility to utilize food reserves in anticipation of limited food intake. It is therefore proposed that bitter gustatory compounds may have an unappreciated role in predicting food scarcity and stimulating digestion as a preparatory response (Yao, 2022).
Previous studies in Drosophila have demonstrated that bitter taste detection elicits inhibition of proboscis extension and suppression of consumption. In addition, detection of bitter compounds drives avoidance behavior and increased locomotion, likely to promote departure to new areas. The current findings suggest an additional role for bitter taste detection in promoting mobilization of food stores to increase circulating nutrients. These actions are aligned in mitigating the impact of a potentially toxic food source by limiting consumption, promoting relocation, and maintaining internal nutrient levels. Whereas bitter taste detection rapidly leads to consumption inhibition and increased locomotion, whether bitter taste detection activates crop contractions acutely or only under specific conditions of food deprivation is not resolved. In addition, although increased crop contractions were observed upon activation of bitter-SELs, it remains to be examined whether this impacts internal circulating nutrients. Bitter-SELs may also influence additional aspects of feeding behavior not tested in this study, including modulating consumption under different physiological states or when different feeding assays or different bitter compounds are used (Yao, 2022).
In addition to the hypocerebral ganglion (HCG), bitter-SELs project to diverse targets, suggesting that they may carry out other functions besides enteric modulation. For example, bitter-SELs broadly arborize on the dorsal surface of the VNC. Thus, bitter-SELs may also set the 5-HT tone in the VNC or secrete 5-HT into the hemolymph to modulate target tissues in a paracrine or endocrine fashion (Yao, 2022).
This study found that 5-HT neurons are critical nodes in the circuits that transform gustatory detection into changes in endocrine and digestive function. Although the timescale of activation of sugar-SELs and bitter-SELs and the dynamics of 5-HT release requires further investigation, 5-HT receptors are metabotropic receptors ideally suited for transforming transient neural signals into more sustained cellular responses. In this regard, neuromodulatory circuits are prime candidates for eliciting preparatory responses that require the transformation of neural signals across time scales. This work thus sheds light on neural circuit mechanisms that translate external sensory cues into preparatory physiological responses and suggests that neuromodulators such as 5-HT may contribute to anticipatory mechanisms in other animals (Yao, 2022).
Studying the microbial symbionts of eukaryotic hosts has revealed a range of interactions that benefit host biology. Most eukaryotes are also infected by parasites that adversely affect host biology for their own benefit. However, it is largely unclear whether the ability of parasites to develop in hosts also depends on host-associated symbionts, e.g., the gut microbiota. The effects were studied of parasitic wasp Leptopilina boulardi (Lb) and its host Drosophila melanogaster. Results showed that Lb successfully develops in conventional hosts (CN) with a gut microbiota but fails to develop in axenic hosts (AX) without a gut microbiota. Developing Lb larvae consume fat body cells that store lipids. It was also determined that much larger amounts of lipid accumulate in fat body cells of parasitized CN hosts than parasitized AX hosts. CN hosts parasitized by Lb exhibited large increases in the abundance of the bacterium Acetobacter pomorum in the gut, but did not affect the abundance of Lactobacillus fructivorans which is another common member of the host gut microbiota. However, AX hosts inoculated with A. pomorum and/or L. fructivorans did not rescue development of Lb. In contrast, AX larvae inoculated with A. pomorum plus other identified gut community members including a Bacillus sp. substantially rescued Lb development. Rescue was further associated with increased lipid accumulation in host fat body cells. Insulin-like peptides increased in brain neurosecretory cells of parasitized CN larvae. Lipid accumulation in the fat body of CN hosts was further associated with reduced Bmm lipase activity mediated by insulin/insulin-like growth factor signaling (IIS). Altogether, these results identify a previously unknown role for the gut microbiota in defining host permissiveness for a parasite. These findings also identify a new paradigm for parasite manipulation of host metabolism that depends on insulin signaling and the gut microbiota (Zhao, 2022).
Cellular Insulin signaling (IS) shows a remarkable high molecular and functional conservation. Insulin-producing cells respond directly to nutritional cues in circulation and receive modulatory input from connected neuronal networks. Neuronal control integrates a wide range of variables including dietary change or environmental temperature. Although it is shown that neuronal input is sufficient to regulate Insulin-producing cells, the physiological relevance of this network remains elusive. In Drosophila melanogaster, Insulin-like peptide7-producing neurons are wired with Insulin-producing cells. The former cells regulate the latter to facilitate larval development at high temperatures, and to regulate systemic Insulin signaling in adults feeding on calorie-rich food lacking dietary yeast. These results demonstrate a role for neuronal innervation of Insulin-producing cells important for fruit flies to survive unfavorable environmental conditions (Prince, 2021).
This study has analyzed the role of dIlp7-producing neurons in different thermal treatments. D7Ns are active on yeast diets, but show no activity in animals kept on yeast-free corn food (CF). Activated D7Ns are required to respond to heat stress. In addition, dIlp7 produced by D7Ns regulates dIlp2/dIlp3-induced Insulin signaling (IS) on CF, and yeast products are able to supplement efficiently for the loss of this neuropeptide (Prince, 2021).
The generative cycle of Drosophila is divided into feeding and non-feeding stages. Due to the absence of food intake during embryonic and pupal development these stages highly rely on internal energy stores. In contrast, larvae and adults need to absorb food to survive and develop. The insulin signaling cascade is one metabolic circuit to regulate the absorption and internal turnover of macronutrients. In addition, the cascade is essential to provide thermal resistance for ectothermic insects. All feeding stages of Drosophila express four neuronal Insulin-like peptides, namely dIlp 2, 3, 5, and 7 . Larvae with functionally compromised Insulin-producing cells (IPCs) kept on yeast diets are heat sensitive, slow in development and small in size (Prince, 2021).
Dietary yeast increase intracellular Ca2+ levels of IPCs, elevate systemic IS and support survival at high temperatures. This study found that IPCs with high Ca2+ are not sufficient to rescue larval survival at high temperatures on yeast-free CF. Therefore, it was speculated that yeast products likely activate additional neurons involved in heat stress responses. It was shown that animals kept on yeast increase Ca2+ in D7Ns (Linneweber, 2014). D7Ns connect to IPCs and are able to stimulate the latter. This study shows that, on CF, D7Ns are low on Ca2+ with respect to yeast-fed animals and that induced Ca2+ levels in D7Ns improve larval heat resistance on CF. In addition, larvae with inactivated D7Ns kept on yeast show poor survival at high temperatures. Thus, D7Ns are one integral part of the heat response and it is speculated that these neurons directly communicate with IPCs. D7Ns secrete a multitude of neuropeptides including dIlp7. DIlp7 mutants kept on yeast food (YF) are slightly heat sensitive, and due to such relative high survival rates, is is deemed unlikely that dIlp7 is one main cue crucial to withstand thermal treatments (Prince, 2021).
D7Ns are inactive on CF and attempts were made to identify dIlp candidates responsible for IS on yeast-free diets. Interestingly, dIlp2, dIlp3, and dIlp7 were identified as essential for larval development. Moreover, genetic interactions revealed that δdIlp2,3 double mutants are unable to survive on CF. In stark contrast, δdilp2,7 and δdilp2-3,7 animals rescued the lethality shown by single mutants. These findings indicate a new metabolic link between dIlp7 and dIlp2 essential for larval development in yeast-free environments. However, wild larvae grow in microbe-rich environments, such as rotting fruits, and have likely access to dietary yeast. Adult flies sometimes feed on yeast-poor diets or avoid yeast in response to cold. Therefore, adults kept on CF were sampled. Adult δdilp7 flies show reduced IS levels and higher lethality rates with respect to genetic controls. Moreover, the combined absence of dIlp2 and dIlp7 pronounced the observed adult lethality on CF. Thus, larval and adult dIlp7 signaling is likely very different (Prince, 2021).
It was reported that dIlp7 is expressed in the subesophageal ganglion region of the brain and suggested that D7Ns regulate the feeding behavior (Cognigni, 2011). Therefore, reduced feeding of dIlp7 mutants could explain the lower IS levels on CF. This study has shown that, on CF, δdilp2, and δdilp7 mutants ingest food faster, have a longer retention time of the ingested material and are able to absorb macronutrients. Therefore, the idea that these flies are starving on CF is not favored. It is more likely that dIlp7 is required to stimulate IPCs to maintain basic dIlp levels in circulation. To test for this possibility, wthe predicted target receptor of dIlp7, the G-protein-coupled rector Lgr3 was knocked down. The loss of Lgr3 results in low IS levels on CF. In contrast, on YF, all tested genotypes show IS comparable to controls. Taken together, it is concluded that neuronal dIlp7/Lgr3 signaling controls IPCs in adults kept on yeast-free diets. As such dIlp7 secures a basic amount of systemic IS and therefore, likely contributes to thermal resistance of adult flies. However, required adult tracking on CF at low temperatures appeared impractical to confirm this idea (Prince, 2021).
Neuronal innervation of IPCs is established in many animals and modulates metabolic signals. The current findings indicate that food products can overwrite such neuronal stimulation. In Drosophila, a dual role for D7Ns was found: (1) these neurons facilitate the heat response of larvae feeding on yeast and (2) they form a metabolic circuit that enables adult flies to thrive on yeast-free diets if required. In mice and humans, pancreatic islets are directly innervated; however, the role of this neuronal stimulation in response to dietary cues is not well understood. This study has identified the importance of D7Ns and their product, dIlp7, in regulating IS in response to dietary quality. These findings provide new insights into the neuronal stimulation of IPCs within a given ecological context and provide a model to study neuronal innervation of insulin producing cells (Prince, 2021).
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).
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 number of experimental results demonstrated the impact of long-term IIS manipulations on the nervous system. For example, systemic injections of IGF-1 mimicked some of the effects of exercise in the brain, and genetically reduced IGF-1 signaling in the whole organism reduced inflammation and neuronal loss in a mouse Alzheimer disease model. Likewise, chronic IIS manipulations only in the nervous system can have consequences on the whole organism: attenuated IR substrate/IR substrate 2 signaling in aging brains promotes healthy metabolism and extends the lifespan in mice, and neuron-specific reduction of IIS increases longevity in Drosophila. At the synaptic level, basal IGF-1 activity has recently been shown to regulate ongoing neuronal activity in hippocampal circuits. While infusion of IGF-1 does not appear to have short-term influence on Cx43 levels in various regions of the rat brain, no study so far has examined the effect of chronic IIS manipulations in the aging nervous system on GJs (Augustin, 2017).
This work demonstrated a role for IIS in regulating the trafficking of gap junctional proteins that is conserved over the large evolutionary distance between Drosophila and humans, and between different cell types. Elevated IIS induces the targeting of GJ proteins to lysosomes and degradation, thereby decreasing their cell surface assembly (Augustin, 2017).
Specifically, reduced insulin signaling throughout adulthood leads to Rab4/11-mediated increase in the synaptic targeting of Shak-B-encoded gap junctional components in the Drosophila escape response circuit, resulting in the maintenance of the 'youthful' functional output even in old flies. Previous studies demonstrated a positive effect of reduced insulin signaling on neuronal circuit function. For example, visual acuity is improved in mice with reduced insulin signaling in the visual cortex. In the nematode C. elegans, mutations of the IR gene resulted in improved chemical transmission at the neuromuscular synapse, and delayed decline in the synaptic function with age. The current findings have revealed a novel restorative and adaptive cellular mechanism by which lowered IIS can maintain electrical transmission in a neuronal circuit during aging, and that could potentially be harnessed to prevent decline in neuronal function. A recent report demonstrated a negative effect of neuron-specific IIS reduction on age-specific walking behavior in Drosophila, suggesting that the effect of insulin signaling depends on the type of neuron(s) mediating a specific behavior. For example, physiological roles of different (chemical) neuronal circuits can be preferentially mediated by either evoked or spontaneous transmission. Interestingly, blockade of insulin signaling has opposing effects on these 2 types of transmission, possibly explaining some of the seemingly contradictory experimental data about the role of IIS in the nervous system. Together, these findings indicate that studies of insulin signaling in the nervous system should be circuit- and synapse type-specific, taking into consideration the physiological properties of the neuronal system under study, and precluding simplified generalizations about the effectiveness of specific IIS manipulations across the nervous system (Augustin, 2017).
The increasing age of global populations highlights the urgent need to understand the biological underpinnings of ageing. To this end, inhibition of the insulin/insulin-like signalling (IIS) pathway can extend healthy lifespan in diverse animal species, but with trade-offs including delayed development. It is possible that distinct cell types underlie effects on development and ageing; cell-type-specific strategies could therefore potentially avoid negative trade-offs when targeting diseases of ageing, including prevalent neurodegenerative diseases. The highly conserved diversity of neuronal and non-neuronal (glial) cell types in the Drosophila nervous system makes it an attractive system to address this possibility. This study has thus investigated whether IIS in distinct glial cell populations differentially modulates development and lifespan in Drosophila. Glia-specific IIS inhibition, using several genetic means, delays development while extending healthy lifespan. The effects on lifespan can be recapitulated by adult-onset IIS inhibition, whereas developmental IIS inhibition is dispensable for modulation of lifespan. Notably, the effects observed on both lifespan and development act through the PI3K branch of the IIS pathway and are dependent on the transcription factor FOXO. Finally, IIS inhibition in several glial subtypes can delay development without extending lifespan, whereas the same manipulations in astrocyte-like glia alone are sufficient to extend lifespan without altering developmental timing. These findings reveal a role for distinct glial subpopulations in the organism-wide modulation of development and lifespan, with IIS in astrocyte-like glia contributing to lifespan modulation but not to developmental timing. These results enable a more complete picture of the cell-type-specific effects of the IIS network, a pathway whose evolutionary conservation in humans make it tractable for therapeutic interventions. These findings therefore underscore the necessity for cell-type-specific strategies to optimise interventions for the diseases of ageing (Woodling, 2020).
The Drosophila genome encodes eight insulin/IGF-like peptide (dilp) paralogs, including tandem-encoded dilp1 and dilp2. This study finds that dilp1 is highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Dilp1 and dilp2 single and double mutants were used to describe interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1-dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 - dilp2 double mutants. dilp1 and dilp2 interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. Repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro-longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. The data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro-longevity insulin-like peptide (Post, 2018).
Based on mutational analyses of the insulin receptor (daf-2, InR) and its associated adaptor proteins and signaling elements, numerous studies in C. elegans and Drosophila established that decreased insulin/IGF signaling (IIS) extends lifespan. Studies on how reduced IIS in Drosophila systemically slows aging also reveal systems of feedback where repressed IIS in peripheral tissue decreases DILP2 production in brain insulin-producing cells (IPC), which may then reinforce a stable state of longevity assurance. This study finds that expression of dilp1 is required for loss of dilp2 to extend longevity. This novel observation contrasts with conventional interpretations where reduced insulin ligand is required to slow aging: Elevated dilp1 is associated with longevity in dilp2 mutants, and transgene expression of dilp1 increases longevity (Post, 2018).
dilp1 and dilp2 are encoded in tandem, likely having arisen from a duplication event. Perhaps as a result, some aspects of dilp1 and dilp2 are regulated in common: Both are expressed in IPCs, are regulated by sNPF, and have strongly correlated responses to dietary composition. Nonetheless, the paralogs are differentially expressed throughout development. While dilp2 is expressed in larvae, dilp1 expression is elevated in the pupal stage when dilp2 expression is minimal. In reproductive adults, dilp1 expression decreases substantially after eclosion and dilp2 expression increases (Post, 2018).
Furthermore, DILP1 production is associated with adult reproductive diapause. IIS regulates adult reproductive diapause in Drosophila, a somatic state that prolongs survival during inclement seasons. DILP1 may stimulate these diapause pro-longevity pathways, while expression in nondiapause adults is sufficient to extend survival even in optimal environments (Post, 2018).
The current data suggest a hypothesis whereby dilp1 extends longevity in part through induction of adipokinetic hormone (AKH), which is also increased during reproductive diapause and acts as a functional homolog of mammalian glucagon. Critically, AKH secretion has been shown to increase Drosophila lifespan and to induce triacylglycerides and free fatty acid catabolism. Here, it is noted that dilp1 mutants were more sensitive to starvation than wild-type and dilp2 mutants, as might occur if DILP1 and AKH help mobilize nutrients during fasting and diapause. Mammalian insulin and glucagon inversely regulate glucose storage and glycogen breakdown, while insulin decreases glucagon mRNA expression. It is propose that DILP2 in Drosophila indirectly regulates AKH by repressing dilp1 expression, while DILP1 otherwise induces AKH (Post, 2018).
A further connection between dilp1 and diapause involves juvenile hormone (JH). In many insects, adult reproductive diapause and its accompanied longevity are maintained by the absence of JH. Furthermore, ablation of JH-producing cells in adult Drosophila is sufficient to extend lifespan, and JH is greatly reduced in long-lived Drosophila insulin receptor mutants. In each case, exogenous treatment of long-lived flies with a JH analog (methoprene) restores survival to the level of wild-type or nondiapause controls. JH is a terpenoid hormone that interacts with a transcriptional complex consisting of Met (methoprene tolerant), Taimen, and Kruppel homolog 1 (Kr-h1). As well, JH induces expression of kr-h1 mRNA, and this serves as a reliable proxy for functionally active JH. This study finds that dilp2 mutants have reduced kr-h1 mRNA, while the titer of this message is similar to that of wild-type in dilp1 - dilp2 double mutants. DILP1 may normally repress JH activity, as would occur in diapause when DILP1 is highly expressed. Such JH repression may contribute to longevity assurance during diapause as well as in dilp2 mutant flies maintained in laboratory conditions (Post, 2018).
Does DILP1 act as an insulin receptor agonist or inhibitor? Inhibitory DILP1 could directly interact with the insulin receptor to suppress IIS, potentially even in the presence of other insulin peptides. Such action could induce programs for longevity assurance that are associated with activated FOXO. Alternatively, DILP1 may act as a typical insulin receptor agonist that induces autophosphorylation and represses FOXO. In this case, to extend lifespan, DILP1 should stimulate cellular responses distinct from those produced by other insulin peptides such as DILP2 or DILP5. Through a third potential mechanism, DILP1 may interact with binding proteins such as IMPL2 or dALS to indirectly inhibit IIS output. These distinctions may be resolvednin a future study using synthetic DILP1 applied to cells in culture (Post, 2018).
A precedent exists from C. elegans where some insulin-like peptides are thought to function as antagonists. In genetic analyses, ins-23 and ins-18 stimulate larval diapause and longevity, while ins-1 promotes Dauer formation during development and longevity in adulthood. Moreover, C. elegans ins-6 acts through DAF-2 to suppress ins-7 expression in neuronal circuits to affect olfactory learning, where ins-7 expression inhibits DAF-2 signaling. These studies propose that additional amino acid residues of specific insulin peptides contribute to their distinct functions, and notably, the B-chain of DILP1 has an extended N-terminus relative to other DILP sequences (Post, 2018).
While dFOXO and DAF-16 are intimately associated with how reduced IIS regulates aging in Drosophila and C. elegans, in the current work, the behavior of FOXO does not correspond with how longevity is controlled epistatically by dilp1 and dilp2. Mutation of dilp2 did not impact FOXO activity, as measured by expression of target genes InR and 4eBP, and interactions with dilp1 did not modify this result. Some precedence suggests only a limited role for dfoxo as the mediator of reduced IIS in aging, as dfoxo only partially rescues longevity benefits of chico mutants, revealing that IIS extends lifespan through some FOXO-independent pathways. On the other hand, dilp1 expression from a transgene in the dilp1-2 double mutant background did induce FOXO targets. Differences among these results might arise if whole animal analysis of dFOXO targets obscures its role when IIS regulates aging through actions in specific tissues. In this vein, this study found that dilp2 controls thorax ERK signaling but not AKT, suggesting that dilp2 mutants may activate muscle-specific ERK/MAPK anti-aging programs (Post, 2018).
Dilp1 and dilp2 redundantly regulate glycogen levels and blood sugar, while these dilp loci interact synergistically to modulate dilp5 expression and starvation sensitivity. In contrast, dilp1 and dilp2 interact in a classic epistatic fashion to modulate longevity and AKH. Such distinct types of genetic interactions may reflect unique ways DILP1 and DILP2 stimulate different outcomes from their common tyrosine kinase insulin-like receptor, along with outcomes based on cell-specific responses. Understanding how and what is stimulated by DILP1 in the absence of dilp2 will likely reveal critical outputs that specify longevity assurance (Post, 2018).
Circular RNAs (circRNAs) are abundant and accumulate with age in neurons of diverse species. However, only few circRNAs have been functionally characterized, and their role during aging has not been addressed. This study uses transcriptome profiling during aging and find that accumulation of circRNAs is slowed down in long-lived insulin mutant flies. Next, the in vivo function was tested of a circRNA generated by the sulfateless gene (circSfl), which is consistently upregulated, particularly in the brain and muscle, of diverse long-lived insulin mutants. Strikingly, lifespan extension of insulin mutants is dependent on circSfl, and overexpression of circSfl alone is sufficient to extend the lifespan. Moreover, circSfl is translated into a protein that shares the N terminus and potentially some functions with the full-length Sfl protein encoded by the host gene. This study demonstrates that insulin signaling affects global circRNA accumulation and reveals an important role of circSfl during aging in vivo (Weigelt, 2020).
Circular RNAs (circRNAs) were originally identified more than 30 years ago, but for a long time they were thought to be by-products of the mRNA splicing process without a specific function; hence, they were not investigated further. Recently, circRNAs have been discovered in fungi, protists, and plants; C. elegans; Drosophila; mice; and humans. The majority of circRNA are generated by backsplicing of exons of protein-coding genes ('host genes'), and reverse complementary regions in the introns flanking circRNA-producing exons are crucial for circularization. Despite the high abundance and expression of certain circRNAs, only a few circRNAs have been functionally characterized; for instance, human CDR1as, which acts as an effective microRNA sponge. More recently, two independent reports have shown that a subset of circRNAs might be translated. circRNAs are enriched in neuronal tissues such as Drosophila heads and the mammalian brain. Furthermore, circRNAs have been shown to accumulate with age in C. elegans, in Drosophila heads and photoreceptor neurons, and in the mouse cortex and hippocampus but not in mouse heart tissue. However, a function of circRNAs in the aging process has not yet been revealed (Weigelt, 2020).
The nutrient-sensing insulin/insulin-like growth factor signaling (IIS) pathway is a key regulator of aging, metabolism, reproduction, and growth and is evolutionarily conserved from worms and flies to mice and humans. Downregulation of IIS pathway activity pharmacologically or by genetic modification extends the lifespan in C. elegans, Drosophila, and mice. In Drosophila, simultaneous knockout of three of the seven insulin-like peptides (dilp2-3,5) results in a robust lifespan extension of 30%-50% and ameliorates the age-related decline in sleep quality, suggesting that the healthspan is also extended. Proteome analysis of long-lived insulin mutants (genetic ablation of insulin-producing cells) revealed that the response to reduced insulin signaling and lifespan extension are highly tissue specific (Weigelt, 2020).
This study has characterized the functional link between circRNAs and insulin-mediated lifespan extension and aging. Tissue-specific, genome-wide, next-generation sequencing was used of wild-type and dilp2-3,5 mutant flies and hundreds of differentially expressed circRNAs were identified, including the circRNA encoded by the sulfateless (sfl) gene (hereafter referred to as circSfl). circSfl was highly upregulated in all tissues of several long-lived insulin mutants, and overexpression of circSfl alone was sufficient to extend the lifespan. Finally, evidence is provided that circSfl is translated into a small protein that may share some function with the protein encoded by the linear sfl transcripts. Importantly, overexpression of just the circSfl open reading frame (ORF) from a linear transcript was sufficient to extend longevity, implicating the protein encoded by circSfl in lifespan regulation. This study demonstrated that circRNAs are actively involved in the aging process and can influence the lifespan (Weigelt, 2020).
One of the most striking discoveries about circRNAs is the observation that they accumulate with age in neuronal tissues of diverse species. Several hypotheses regarding why circRNAs accumulate with age have been proposed. First, circRNAs are more stable compared with linear RNA molecules. Second, it has been suggested that circRNAs accumulate with age specifically in neuronal tissue because neurons are mostly post-mitotic, and, therefore, the more stable circRNAs are not degraded by proliferation or cell death. However, if this theory holds true, then circRNAs should accumulate in most tissues of the fruit fly because Drosophila is a mainly post-mitotic organism. Third, alternative splicing is increased and more error prone with age, potentially resulting in more backsplicing of circRNAs. This study showed that circRNA accumulation with age is slowed down in long-lived insulin mutants. This might point toward the third theory of why circRNAs accumulate with age and is supported by the finding that the splicing factor SFA-1 is required for dietary restriction-induced longevity in nematodes, highlighting the importance of splicing for lifespan extension upon deregulated nutrient sensing. These findings demonstrated that accumulation of circRNAs with age is malleable, suggesting that accumulation of circRNAs might be a potential aging biomarker (Weigelt, 2020).
This study identified several circRNAs that were differentially regulated in response to reduced insulin signaling in dilp 2-3,5 mutants, including circSfl. circSfl was also upregulated in two other insulin mutant flies that have an extended lifespan, and the upregulation is dependent on the dFoxo transcription factor, an essential mediator of longevity downstream of IIS. Notably, the magnitude of upregulation of circSfl in these mutants was correlated with the magnitude of the lifespan extension, with strong, up to 7-fold upregulation in the very long-lived dilp 2-3,5 mutants and only mildly upregulated in MNC-ablated flies and dFoxo overexpression flies (1.5- to 2-fold), which show a more mild lifespan extension. Similarly, the linear transcript Sfl RB was only upregulated in dilp2-3,5 mutant flies and not in the two other insulin mutants, suggesting that longevity and expression of the linear isoform can be uncoupled. Interestingly, neither circSfl nor the linear Sfl isoforms are upregulated upon rapamycin treatment or dietary restriction or in mth1 mutant flies, suggesting that upregulation of circSfl is not a general hallmark of lifespan-extending interventions in flies but specific to IIS-mediated longevity (Weigelt, 2020).
To overexpress circRNAs in vivo, different UAS constructs were tested. As expected, overexpression of the circRNA exon without its flanking introns did not lead to increased circRNA expression because flanking introns are required for biogenesis of circRNAs. In contrast, introducing reverse complementary matching flanking introns strongly increased biogenesis of circRNAs in vivo. These results are in line with previous studies that expressed circRNAs by engineering reverse complementary introns in zebrafish and in vitro. However, the first study that overexpressed a circRNA (circMbl) in vivo in Drosophila used a minigene construct including the circMbl exon and around 100 bp of the natural flanking introns but no inverted repeats. CircMbl overexpression led to a 4-fold increase in the circMbl expression level, much less than the strong overexpression achieved by engineered flanking introns. In summary, the mutants demonstrate that circRNAs can be efficiently overexpressed in Drosophila using engineered, reverse complementary matches in flanking introns that increase circRNA biogenesis in vivo (Weigelt, 2020).
Furthermore, sflΔex2 mutant flies were generated that lacked the Sfl RA-specific exon 2, and it was demonstrated that circSfl biogenesis is dependent on Sfl RA. Combination of sflΔex2 mutants with dilp 2-3,5 mutants revealed that the lifespan extension of dilp 2-3,5 mutants is partly dependent on the presence of this exon. The biogenesis of circRNAs is poorly understood, but several RNA binding proteins have been shown recently to inhibit or promote circularization, including Muscleblind, Quaking, and Adar1. It is tempting to speculate that an RNA binding protein might bind to the Sfl RA-specific exon and promote biogenesis of circSfl, which is abolished in sflΔex2 mutant flies. Because sflΔex2 mutants affect biogenesis of circSfl and expression of the linear Sfl RA isoform, it is currently not possible to formally exclude a role of the linear splice variant in insulin-mediated longevity. However, several lines of evidence suggest circSfl as the causal factor in this context. First, although Sfl RA expression was lost in sflΔex2 mutants, overall expression of linear Sfl was not affected. This is consistent with the finding that Sfl protein levels were not changed in dilp 2-3,5 mutant flies despite differential alternative splicing of the RA and RB isoforms. Thus, modifying exon 2 expression levels does not seem to affect Sfl protein levels, which can affect the lifespan. In addition, overexpression of circSfl and a linear transcript encoding the circSfl protein was sufficient to extend the lifespan, directly linking circSfl expression with longevity regulation. Given that most circRNAs are embedded in a host gene, generation of specific circRNA mutants that do not affect the host gene has been very challenging in the field. Because siRNA-mediated knockdown was not efficient in the case of circSfl, in new strategies should be tested in the future (e.g., by modification or deletion of the flanking introns that affect circRNA biogenesis), which can then be used to verify the hypothesis (Weigelt, 2020).
This study presented several lines of evidence showing that circSfl might be translated into a protein that is identical to the N terminus arising from linear Sfl transcripts. Sequence homology analysis showed that the in-frame stop codon after the circRNA-specific backsplice junction is conserved within Drosophila species that are separated by 10-20 million years of evolution, suggesting that the protein encoded by circSfl might also be conserved between these species. However, the identical stop codon could not be detected in more distantly related insect species, like honeybees or mosquitoes, which could indicate that the circlSfl protein is specific to Drosophila species or that other stop codons more downstream are used in other insects. Pamudurti (2017) previously identified 37 potentially translated circRNAs in Drosophila using ribosome footprinting on wild-type Drosophila heads; however, they failed to detect circSfl. Similarly, in the polysome profiling experiment, only very few circSfl reads were detected in wild-type fat bodies. In contrast, in dilp 2-3,5 mutant fat bodies, circSfl was one of the most abundant circRNAs, consistent with the insulin-dependent increase in circSfl transcript and protein levels. Furthermore, this study has shown that the protein encoded by circSfl and the protein arising from the linear Sfl transcripts can positively affect the lifespan of flies. This finding might indicate that both proteins affect the lifespan through overlapping mechanisms or by interacting with each other. Because the protein encoded by circSfl lacks the catalytic domain, it is unlikely that it acts as an active enzyme. Thus, circSfl may interact with proteins similar to the Sfl full-length protein in the cytoplasm or the membrane. For example, one could imagine that circSfl might interact with a repressor of the Sfl full-length protein, promoting the activity of Sfl and extending the lifespan. Alternatively, the truncated circSfl protein could also act as a dominant-negative protein because Sfl overexpression has also been suggested to cause a loss-of-function phenotype. Noteworthy is that overexpression of circSfl and Sfl caused tissue-specific effects on longevity, which could indicate that they work via different mechanisms or that different expression levels in different tissues are needed for the beneficial effects of the two proteins on lifespan. Interestingly, overexpression of circSfl and Sfl only extended the female but not the male lifespan despite upregulation of circSfl in dilp2-3,5 mutant males. This might reflect the gender bias in insulin-mediated longevity, in which females often show stronger effects than males (Austad and Fischer, 2016) (Weigelt, 2020).
The sfl gene in Drosophila encodes an Ndst and catalyzes synthesis of heparan sulfate (a glycosaminoglycan) by sulfation of the N and 6-O position of GlcNAc. Heparan sulfate is essential for wingless and fibroblast growth factor (FGF) receptor signaling, and full knockout of sfl is embryonic lethal. Sfl has been suggested to be localized to the Golgi apparatus and may be involved in the unfolded protein response. Furthermore, knockdown of Sfl increased the autophagy machinery and ubiquitinated proteins and reduced the climbing ability of flies, suggesting that Sfl is required for protein homeostasis and health. Similarly, this study has demonstrated that neuron-specific knockdown of Sfl is detrimental for the lifespan but that neuron-specific overexpression of Sfl extends the lifespan. Furthermore, this suty demonstrated, by genetic epistasis experiments, that Dally might contribute to the Sfl-mediated lifespan extension. Previous studies have demonstrated that overexpression of Sfl increases heparan sulfate levels and disrupts normal Wingless (Wg) and Decapentaplegic (Dpp) signaling, with the latter being controlled by Dally. However, similar to Sfl, the role of Dally has been characterized extensively during development but has not yet been implicated in aging (Weigelt, 2020).
In summary, this study demonstrated that neuronal circRNA accumulation with age is malleable and reduced in long-lived insulin mutants. Furthermore, this study established an efficient method to overexpress circRNAs in vivo by using reverse complementary introns. Interestingly, this study showed that a single circRNA (circSfl) can extend the lifespan in Drosophila. It is proposed that circSfl is translated into a protein that shares the same N terminus with the full-length protein arising from linear transcripts and potentially similar functions. This study will help to further elucidate the molecular mechanisms underlying longevity and provides unique insights into the in vivo function of circRNAs (Weigelt, 2020).
A limitation of this study is that it is currently unclear whether the circRNA-derived peptide and the full-length Sfl protein affect the lifespan by the same or by independent mechanisms. Lifespan extension by the full-length Sfl protein is dependent on its direct downstream target, the Dally protein. Thus, to address whether lifespan extension upon circSfl overexpression works in a similar way and also requires Dally, epistasis experiments were performed by co-overexpression of the circSfl protein and dally RNAi and the lifespan of these flies was measured. These experiments had to be terminated because of the current coronavirus crisis. In the future, it will be very interesting to further elucidate the mechanism of lifespan extension by circSfl and Sfl using genetic epistasis experiments (Weigelt, 2020).
Mutations of the Drosophila melanogaster insulin/IGF signaling system slow aging, while also affecting growth and reproduction. To understand this pleiotropy, an allelic series of single codon substitutions was produced in the Drosophila insulin receptor, InR. Substitutions were generated using homologous recombination, and each was related to emerging models of receptor tyrosine kinase structure and function. Three mutations when combined as trans-heterozygotes extended lifespan while retarding growth and fecundity. These genotypes reduced insulin-stimulated Akt phosphorylation, suggesting they impede kinase catalytic domain function. Among these genotypes, longevity was negatively correlated with egg production, consistent with life-history trade-off theory. In contrast, one mutation (InR353) was located in the kinase insert domain, a poorly characterized element found in all receptor tyrosine kinases. Remarkably, wild-type heterozygotes with InR353 robustly extended lifespan without affecting growth or reproduction and retained capacity to fully phosphorylate Akt. The Drosophila insulin receptor kinase insert domain contains a previously unrecognized SH2 binding motif. It is proposed the kinase insert domain interacts with SH2-associated adapter proteins to affect aging through mechanisms that retain insulin sensitivity and are independent of reproduction (Yamamoto, 2021).
Ohhara, Y., Hoshino, G., Imahori, K., Matsuyuki, T. and Yamakawa-Kobayashi, K. (2021). The Nutrient-Responsive Molecular Chaperone Hsp90 Supports Growth and Development in Drosophila. Front Physiol 12: 690564. PubMed ID: 34239451
Animals can sense internal nutrients, such as amino acids/proteins, and are able to modify their developmental programs in accordance with their nutrient status. In the fruit fly, Drosophila melanogaster, amino acid/protein is sensed by the fat body, an insect adipose tissue, through a nutrient sensor, target of rapamycin (TOR) complex 1 (TORC1). TORC1 promotes the secretion of various peptide hormones from the fat body in an amino acid/protein-dependent manner. Fat-body-derived peptide hormones stimulate the release of insulin-like peptides, which are essential growth-promoting anabolic hormones, from neuroendocrine cells called insulin-producing cells (IPCs). Although the importance of TORC1 and the fat body-IPC axis has been elucidated, the mechanism by which TORC1 regulates the expression of insulinotropic signal peptides remains unclear. This study shows that an evolutionarily conserved molecular chaperone, heat shock protein 90 (Hsp90), promotes the expression of insulinotropic signal peptides. Fat-body-selective Hsp90 knockdown caused the transcriptional downregulation of insulinotropic signal peptides. IPC activity and systemic growth were also impaired in fat-body-selective Hsp90 knockdown animals. Furthermore, Hsp90 expression depended on protein/amino acid availability and TORC1 signaling. These results strongly suggest that Hsp90 serves as a nutrient-responsive gene that upregulates the fat body-IPC axis and systemic growth. It is proposed that Hsp90 is induced in a nutrient-dependent manner to support anabolic metabolism during the juvenile growth period (Ohhara, 2021).
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).
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).
Fruit flies have eight identified Drosophila insulin-like peptides (DILPs) that are involved in the regulation of carbohydrate concentrations in hemolymph as well as in accumulation of storage metabolites. This study investigated diet-dependent roles of DILPs encoded by the genes dilp1-5, and dilp7 in the regulation of insect appetite, food choice, accumulation of triglycerides, glycogen, glucose, and trehalose in fruit fly bodies and carbohydrates in hemolymph. The wild type and the mutant lines demonstrate compensatory feeding for carbohydrates. However, mutants on dilp2, dilp3, dilp5, and dilp7 showed higher consumption of proteins on high yeast diets. High nutrient diets led to a moderate increase in concentration of glucose in hemolymph of the wild type flies. Mutations on dilp genes changed this pattern. The dilp2 mutation led to a drop in glycogen levels independently of diet. Lack of dilp3 led to dramatic increase in circulating trehalose and glycogen levels, especially at low protein consumption. Lack of dilp5 led to decreased levels of glycogen and triglycerides for all diets, whereas knockout on dilp7 caused increase in glycogen levels and simultaneous decrease in triglyceride levels at low protein consumption. Fruit fly appetite was influenced by dilp3 and dilp7 genes. These data contribute to the understanding of Drosophila as a model for further studies of metabolic diseases (Semaniuk, 2018).
In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. This study shows that the growth and body size response to moderate hypoxia (10% O(2)) in Drosophila melanogaster is systemically regulated via the steroid hormone ecdysone. Hypoxia increases level of circulating ecdysone and inhibition of ecdysone synthesis ameliorates the negative effect of low oxygen on growth. This study also shows that the effect of ecdysone on growth under hypoxia is through suppression of the insulin/IGF-signaling pathway, via increased expression of the insulin-binding protein Imp-L2. These data indicate that growth suppression in hypoxic Drosophila larvae is accomplished by a systemic endocrine mechanism that overlaps with the mechanism that slows growth at low nutrition. This suggests the existence of growth-regulatory mechanisms that respond to general environmental perturbation rather than individual environmental factors (Kapali, 2022).
Regulation of circadian behavior and physiology by the Drosophila brain clock requires communication from central clock neurons to downstream output regions, but the mechanism by which clock cells regulate downstream targets is not known. This study shows that the pars intercerebralis (PI), previously identified as a target of the morning cells in the clock network, also receives input from evening cells. It was determined that morning and evening clock neurons have time-of-day-dependent connectivity to the PI, which is regulated by specific peptides as well as by fast neurotransmitters. Interestingly, PI cells that secrete the peptide DH44, and control rest:activity rhythms, are inhibited by clock inputs while insulin-producing cells (IPCs) are activated, indicating that the same clock cells can use different mechanisms to drive cycling in output neurons. Inputs of morning cells to IPCs are relevant for the circadian rhythm of feeding, reinforcing the role of the PI as a circadian relay that controls multiple behavioral outputs. These findings provide mechanisms by which clock neurons signal to nonclock cells to drive rhythms of behavior (Barber, 2021).
Many physiological and behavioral processes across organisms exhibit daily rhythms controlled by an internal circadian system. The temporal organization conferred by circadian clocks allows anticipation of environmental changes and the coordination of biochemical and physiological processes within and across cells and tissues. Work in Drosophila and other organisms has elucidated the molecular basis of circadian pacemaking in the brain, but a molecular understanding of how time-of-day information is relayed from brain clock circuitry to downstream output regions that regulate circadian physiology and behavior is still lacking (Barber, 2021).
The Drosophila clock network consists of ~150 neurons that express the core clock transcription-translation feedback loop genes (1). Of these, the ventrolateral neurons (LNvs) and dorsal lateral (LNd) clock neurons are important pacemakers for driving circadian rhythms in constant darkness, and coordinating the activity of other neurons in the clock network. Indeed, robust rhythms are an emergent property of the clock network as a whole. The rhythmic pattern of locomotor activity in light:dark (LD) cycles, which consists of morning and evening peaks that anticipate dawn and dusk, respectively, is also attributed to specific clock cells. The LNvs act through the neuropeptide pigment-dispersing factor (PDF) to drive the morning bout of locomotor activity while LNd clock neurons are termed evening neurons due to their role in regulating evening anticipatory activity. LNd neurons are a molecularly heterogeneous population with subpopulations expressing different signaling molecules including the classical transmitter acetylcholine and the neuropeptides Ion transport peptide (ITP), NPF, and sNPF DN1 dorsal neurons, which receive input from LNvs, also promote morning activity and exhibit highest activity in the predawn and morning hours. DN1 neurons express glutamate and the neuropeptide DH31, both of which have previously described roles in regulating circadian rhythms. The diverse array of signaling molecules expressed by neurons in the clock network offers many possibilities for signaling to clock output brain regions that lack their own clocks but are important for the generation of behavioral rhythm (Barber, 2021).
A major clock output region in Drosophila is the pars intercerebralis (PI), a protohypothalamic region implicated in regulating circadian locomotor rhythms and peripheral cycling. Like the hypothalamus, the PI controls aspects of sleep and feeding and comprises a population of neurosecretory cells with heterogeneous peptide expression. PI neurons that express the neuropeptide diuretic hormone 44 (DH44), the fly ortholog of corticotropin-releasing factor, modulate rest:activity rhythms. On the other hand, PI neurons producing Drosophila insulin-like peptides are implicated in circadian gene expression in the fat body, but have not been linked to behavioral rhythms (Barber, 2021).
As PI cells do not express the molecular clock machinery, time-of-day information must be relayed directly or indirectly from clock neurons to the PI. Previous work showed that the DN1 clock neurons project to two groups of PI neurons, the diuretic hormone 44 positive (DH44+) cells and the insulin-producing cells (IPCs). However, while hyperactivation of DH44+ neurons or RNAi knockdown of DH44 peptide significantly dampens rest:activity rhythms in Drosophila, silencing of DN1 neurons has a considerably weaker effect. Thus, there must be additional upstream inputs to PI neurons that maintain rest:activity rhythms in the absence of DN1 signals. It was hypothesized that robust circadian control of the PI would require inputs from both morning- and evening-active clock neurons, including perhaps long-distance signals directly from LNvs. This study sought to determine if evening cells project to the PI and also investigated clock-PI signaling at different times of day. It was demonstrated that both DH44+ neurons and IPCs receive time-of-day-dependent inputs from both CRYPTOCHROME-negative LNds and DN1s. Surprisingly, clock neurons inhibit DH44+ neurons while activating IPCs, indicating that the same clock cells use multiple mechanisms simultaneously to drive cycling in output neurons. It was also shown that morning clock cells and PI-secreted insulin-like peptides are required for rhythms of feeding (Barber, 2021).
The Drosophila PI was previously described as a protohypothalamic region that serves as a circadian output hub; while inputs from DN1 clock neurons were shown, relevance of other clock neurons or the signaling molecules involved in transducing time-of-day cues from the clock to the PI were not described. This study demonstrates that time-of-day information arrives at the PI from multiple clock neuron populations through cholinergic and glutamatergic signaling. This study further identified an output role for the IPCs of the PI in regulating circadian feeding, which depends on inputs from morning-active, but not evening-active, clock neurons (Barber, 2021).
The response of PI neurons to clock neuron stimulation is time-of-day dependent. DH44+ neurons are inhibited-i.e., they exhibit a reduction in intracellular calcium-by both DN1 and LNd neuron stimulation in the morning, with little to no effect of stimulation of clock neurons in the evening. Given that LNds drive the evening peak of locomotor activity, it is surprising that their stimulation only inhibits DH44+ neurons in the morning; however, inhibition in the morning is consistent with the normal pattern of activity of DH44+ neurons. DH44+ neurons are evening active in both LD and DD conditions, and while they regulate both the morning and evening peaks of locomotor activity in LD, their contribution to the evening peak is larger. It is likely that DH44+ neurons, which are implicated in several processes, receive nonclock inputs that drive locomotor activity, and they require silencing by the clock to inhibit locomotion outside the morning and evening activity peaks. Thus, silencing by DN1s may serve to delineate the morning peak of activity; on the other hand, early day silencing of DH44+ neurons by LNds may be one mechanism by which LNds drive evening activity (Barber, 2021).
The response of IPCs to DN1 stimulation is time-of-day dependent. The response to LNd stimulation also shows an apparent difference between morning and evening, but, in fact, some cells respond at both times. The IPC population is larger than the DH44+ population and is known to be heterogeneous in terms of basal activity and signaling molecule expression. The functional relevance of LNd signaling to IPCs is unclear, but could be linked to the role of these cells in promoting arousal. However, the DN1 inputs likely contribute to the circadian rhythm of feeding. As noted above, this is consistent with the higher firing of IPCs in the morning and the morning peak of the feeding rhythm in w1118 flies, as well as with the finding that DILPs are required for rhythmic feeding. In further support of this idea, this study reports that feeding rhythms depend upon the activity of morning-active LNvs, which signal through DN1s, but not evening-active LNds. These finding of loss of feeding rhythms on yeast/sucrose diet with genetic ablation of two insulin-like peptides differs from a previous study that observed maintenance of rhythmic feeding on sucrose-only food upon adult silencing of IPCs. It is speculated that developmental changes caused by genetic ablation of insulin(s) result in disorganized feeding patterns and/or that rhythmic protein, but not sugar, feeding is regulated by brain insulins (Barber, 2021).
The finding that nAChR and mGluR signaling contribute to inhibition in one case (input from DN1 and LNds on to DH44 neurons) and activation in another (input from DN1 and LNds on to IPCs) raises the question of what determines the nature of the effect. The most straightforward explanation would be a difference in the receptor subtypes expressed in the two cell groups. Alternatively, other signaling components specific to each cell type could account for the differential response. Regardless, it is clear that the actions of known neurotransmitters can be regulated by the clock in different ways to effect rhythmic output. As noted above, acetylcholine is secreted by the LNds and likely also DN1s, but to date, the only known source of glutamate in the clock network are the DN1s (16, 20). This suggests that glutamatergic input from the DN1s can modulate the effect of LNds on PI neurons (Barber, 2021).
Despite the effect of clock cell-secreted neuropeptides on PI cells and the role of fast neurotransmitters in signaling from clock cells to the PI, knockdown of neuropeptide/neurotransmitter receptors in the PI has little effect on behavioral rhythms. There are many possible explanations for lack of a phenotype-low mRNA levels remaining after knockdown are sufficient for function; receptor subtypes act redundantly in the regulation of rhythms; knockdown in the PI alone is insufficient to yield a phenotype; or these molecules affect physiological rhythms other than those of rest:activity. While DH31 and NPF have been implicated in the control of rest:activity rhythms, loss of acetylcholine and glutamate have not been linked to behavioral rhythms. Although knockdown of receptors for these molecules in the PI did not yield a phenotype, reduced evening activity was seen with DH44-driven knockdown of PDFR. This is in contrast to the effect of loss-of-function pdf mutants in which evening activity remains high, but is shifted ~1 h early. It is possible that DH44+ cell-mediated reduced evening activity is compensated in pdfr mutants. PDF is secreted by LNvs, which are not known to synapse onto the PI, but do project to the dorsal brain, suggesting diffusion of PDF across a small region. Limited diffusion of PDF is supported by a previous study showing that overexpression of PDF in cells that project to the dorsal brain produces behavioral phenotypes (Barber, 2021).
These findings elucidate some of the complexity underlying circadian control in neural circuits. Through the use of neuropeptides and fast neurotransmitters coupled with time-of-day-specific actions on downstream neurons, clock cells are able to drive multiple outputs at varying timescales. Behavioral outputs require that rhythmic patterns of activity be propagated through many parts of the brain, and this is likely the case. Previous work has shown that hugin+ neurons, which are downstream of the PI, show rhythmic peptide release, indicating that cycling is propagated to second order output neurons. In addition, clock neurons are linked to rhythmic neural activity in locomotor centers of the fly brain. While a rhythm in spiking activity may be sufficient to transmit some outputs, it is known that other variations in electrical activity (e.g., temporal coding from DN1 neurons) may also contribute to circadian behaviors. As in mammals, different output rhythms also map to different output circuits in flies. Thus, Drosophila take advantage not only of diverse neural output mechanisms to regulate aspects of circadian behavior, but also use distinct neuropeptidergic output cells to regulate locomotor and feeding rhythms. DH44+ neurons are important for locomotor, but not feeding, rhythms, while the opposite is true for IPCs. At the same time, proximity of output neurons controlling locomotor activity with those that control feeding likely allows for integration of multiple signals to coordinate behavior, thus contributing to organismal fitness (Barber, 2021).
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).
Growth and maturation are coordinated processes in all animals. Integration of internal cues, such as signalling pathways, with external cues such as nutritional status is paramount for an orderly progression of development in function of growth. In Drosophila, this involves insulin and steroid signalling, but the underlying mechanisms and their coordination are incompletely understood. This study shows that bioactive 20-hydroxyecdysone production by the enzyme Shade in the fat body is a nutrient-dependent process. Under fed conditions, Shade plays a role in growth control. The trachea and the insulin-producing cells in the brain are identified as direct targets through which 20-hydroxyecdysone regulates insulin-signaling. The identification of the trachea-dependent regulation of insulin-signaling exposes an important variable that may have been overlooked in other studies focusing on insulin-signaling in Drosophila. These findings provide a potentially conserved, novel mechanism by which nutrition can modulate steroid hormone bioactivation, reveal an important caveat of a commonly used transgenic tool to study IPC function and yield further insights as to how steroid and insulin signalling are coordinated during development to regulate growth and developmental timing (Buhler, 2018).
Metabolic conditions affect the developmental tempo of animals. Developmental gene regulatory networks (GRNs) must therefore synchronize their dynamics with a variable timescale. This study found that layered repression of genes couples GRN output with variable metabolism. When repressors of transcription or mRNA and protein stability are lost, fewer errors in Drosophila development occur when metabolism is lowered. This study demonstrates the universality of this phenomenon by eliminating the entire microRNA family of repressors. Development to maturity can be largely rescued when metabolism is reduced. Using a mathematical model that replicates GRN dynamics, lowering metabolism was found to suppress the emergence of developmental errors by curtailing the influence of auxiliary repressors on GRN output. This study experimentally shows that gene expression dynamics are less affected by loss of repressors when metabolism is reduced. Thus, layered repression provides robustness through error suppression and may provide an evolutionary route to a shorter reproductive cycle (Cassidy, 2019).
Developmental patterns arise from directed dynamics of cell-cell signaling and gene regulation. The sensory organs of Drosophila are a classic system with which to study these phenomena. A broad collection of gene mutations has specific effects on the formation of various sensory organs. The affected genes encode transcription factors, microRNAs, signaling factors, and other gene regulators. This study used such mutations to readdress the relationship between reduced metabolism and phenotype suppression. This was done by scoring Drosophila sensory mutant phenotypes under conditions of reduced energy metabolism (Cassidy, 2019).
To reduce metabolism, animals were generated that had genetic ablation of their insulin-producing cells (IPCs) in the brain. IPC ablation limits synthesis and release of insulin-like peptides (ILPs) and reduces the amount of glucose cells consume. It also reduces the abundance of the mitochondrial ATP synthase enzyme complex in cells. Moreover, inhibition of ILP production reduces the whole-body metabolic rate of Drosophila, as measured by calorimetry. IPC ablation results in 70% slower development and small but normally proportioned adults. Therefore, reduced ILP production by IPC ablation broadly decreases cellular energy metabolism and slows development (Cassidy, 2019).
Yan is a transcription factor that maintains cells of the developing compound eye in a progenitor-like state. The protein is transiently expressed in cells and is cleared from differentiating photoreceptor (R) cells by multiple repressors acting on its transcription, mRNA stability, and protein stability. The microRNA miR-7 represses post-transcriptional expression of Yan in the developing eye. When the mir-7 gene was specifically ablated in the compound eye of an otherwise wild-type animal, it resulted in small malformed adult eyes caused by errors in R cell differentiation. This phenotype was highly penetrant in genetically mosaic animals. However, when energy metabolism was slowed by IPC ablation, loss of mir-7 was much less important for the formation of correctly patterned eyes. Mutations affecting post-translational modification of Yan were examined. The epidermal growth factor (EGF) and Sevenless (Sev) receptor tyrosine kinases activate MAP kinase in the progenitors of R7 photoreceptors. MAP kinase phosphorylates Yan protein, leading to its ubiquitin-mediated degradation. This clearance of Yan protein enables differentiation of R7 cells, and when sev is mutated, cells completely fail to differentiate as R7 photoreceptors. However, slowing metabolism allowed a small but significant number of sev mutant cells to become R7 photoreceptors. Importantly, because the sev mutant makes no protein products, rescue of the mutant phenotype was not simply due to more functional Sev protein molecules being present in slowly metabolizing cells (Cassidy, 2019).
The transcription factor Hairy directly represses transcription of the achaete and scute genes during selection of cells for bristle fates. Mutation of hairy causes some individuals to develop ectopic large bristles. However, this effect of hairy mutation was strongly suppressed when energy metabolism was slowed. A similar effect was seen on a cis-regulatory module (CRM) that represses transcription of the wingless (wg) gene. The Sternopleural (Sp-1) mutation is present in a CRM located on the 3' flank of wg, causing wg misexpression and development of ectopic bristles. However, the ectopic bristle phenotype of the wgSp-1 mutant was almost totally reversed under conditions of slowed energy metabolism (Cassidy, 2019).
In conclusion, IPC ablation suppressed developmental phenotypes caused by mutations in genes that repress other genes at the transcriptional, post-transcriptional, and post-translational levels (Cassidy, 2019).
This study has shown that multi-layered weak repression within GRNs plays an unexpected function in synchronizing gene expression dynamics with the variable pace of the developmental program. Multiple repressors are required for accelerated development when metabolism is high, and they become functionally redundant when metabolism is low. Multiple repressors therefore allow reliable development across a broader range of metabolic conditions than tolerated otherwise (Cassidy, 2019).
This model explains long-standing observations linking nutrient limitation to suppression of mutant phenotypes. Presumably, such mutations cripple regulatory genes acting on developmental GRNs. This model might also offer an explanation for why animals that undergo above-normal growth exhibit compromised development. Wild-type GRNs might function across a limited range of metabolism, with functionality breaking down when metabolism exceeds that range (Cassidy, 2019).
Another mechanism to explain phenotype suppression relies on a steady-state and not dynamic perspective of gene expression. Genome-wide gene expression patterns could conceivably change with organismal growth rate. This is the case for chemostat-grown yeast cells, where the expression of 27% of all genes correlates with growth rate. Most genes associated with stress response are overexpressed when cells grow at a slow rate. Such stress-responsive expression could modulate global processes such as protein folding and turnover, among others, and attenuate phenotypes when metabolism is slowed. Indeed, molecular chaperones have been found to affect the penetrance of diverse gene mutations in C. elegans and Drosophila. However, this steady-state model does not explain why gene expression dynamics are conditionally dependent on the availability of repressors. This study found that repression of Yan and Sens expression by microRNAs becomes more redundant when metabolic rates are slowed. Nevertheless, phenotype suppression might be due to a combination of mechanisms, including steady-state stress response and gene expression dynamics (Cassidy, 2019).
Varied analyses carried out in this study suggest that the relationship between metabolism and gene expression dynamics is widespread. The entire family of 466 microRNAs in Drosophila melanogaster was found to become much less essential for development when energy metabolism is slowed. The extensive literature on microRNA function in Drosophila implicates them in practically all facets of the fruit fly's life. Various explanations have been provided for why this family of weak repressors has flourished in the animal kingdom, chief among them the idea that they act as buffers for gene expression. This study now posits that microRNAs also provide a robust means for developmental processes to accommodate fluctuations in metabolism (Cassidy, 2019).
Raising animals at lower temperatures can suppress the phenotypes of mutations that are not classical ts alleles. Indeed, loss of sens repression by miR-9a has less impact on bristle development when temperature is lowered. Because metabolic rate varies with temperature, it is possible that temperature-dependent phenotype suppression may also be attributed to a relaxed requirement for coupling gene expression dynamics to a metabolism-dependent timescale. This notion was explored using a modeling framework, and the results are inconclusive. It is anticipated that error suppression will be weak when temperature-modulated expression dynamics are the sole cause. This is because temperature should affect the rates of both anabolic and catabolic processes involved in gene expression. In contrast, limiting ATP availability or protein translation reduces the rates of anabolic reactions but not all catabolic reactions. This asymmetric effect on different steps in gene expression is a major reason why gene repression becomes less important when ATP availability or protein translation is limited (Cassidy, 2019).
Metabolic conditions drive variation of the intrinsic developmental tempo of each species. This study has shown that layered weak repression within GRNs enables these fluctuations to occur without causing developmental errors. Metabolic conditions change in both space and time. Perhaps the selective advantage of a reliable developmental outcome amidst variable environmental conditions is a driving force in the evolution of gene regulatory networks (Cassidy, 2019).
Energy-sensing neural circuits decide to expend or conserve resources based, in part, on the tonic, steady-state, energy-store information they receive. Tonic signals, in the form of adipose tissue-derived adipokines, set the baseline level of activity in the energy-sensing neurons, thereby providing context for interpretation of additional inputs. However, the mechanism by which tonic adipokine information establishes steady-state neuronal function has heretofore been unclear. This study shows that under conditions of nutrient surplus, Upd2, a Drosophila leptin ortholog, regulates actin-based synapse reorganization to reduce bouton number in an inhibitory circuit, thus establishing a neural tone that is permissive for insulin release. Unexpectedly, this study found that insulin feeds back on these same inhibitory neurons to conversely increase bouton number, resulting in maintenance of negative tone. These results point to a mechanism by which two surplus-sensing hormonal systems, Upd2/leptin and insulin, converge on a neuronal circuit with opposing outcomes to establish energy-store-dependent neuron activity (Brent, 2020).
Animal feeding is controlled by external sensory cues and internal metabolic states. Does it also depend on enteric neurons that sense mechanical cues to signal fullness of the digestive tract? This study identified a group of piezo-expressing neurons innervating the Drosophila crop (the fly equivalent of the stomach) that monitor crop volume to avoid food overconsumption. These neurons reside in the pars intercerebralis (PI), a neuro-secretory center in the brain involved in homeostatic control, and express insulin-like peptides with well-established roles in regulating food intake and metabolism. Piezo knockdown in these neurons of wild-type flies phenocopies the food overconsumption phenotype of piezo-null mutant flies. Conversely, expression of either fly Piezo or mammalian Piezo1 in these neurons of piezo-null mutants suppresses the overconsumption phenotype. Importantly, Piezo(+) neurons at the PI are activated directly by crop distension, thus conveying a rapid satiety signal along the "brain-gut axis" to control feeding (Wang, 2020).
In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. This study found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels were found of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, this study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding understanding of the comprehensive regulatory mechanisms of final body size in animals (Yuan, 2020).
This study has discovered that in the insect fat body, 20E activates AMPK in two ways: By up-regulating the mRNA levels of all three AMPK subunits and by inducing energy stress to activate AMPK (Yuan, 2020).
The transcription levels of all three AMPK subunits, the protein level of AMPKα, and the phosphorylation level of AMPKα were all elevated in the Bombyx fat body at ∼4M and in the Drosophila fat body during pupariation, showing developmental profiles that were consistent with those of 20E signaling. Both the gain-of-function and loss-of-function experiments further demonstrate that AMPK is transcriptionally activated by 20E signaling. According to preliminary data, 20E-EcR-USP does not directly induce the expression of the AMPK-PP2A subunit genes, and further studies should be performed to investigate the detailed mechanisms whereby the 20E-triggered transcriptional cascade is involved in this transcriptional activation (Yuan, 2020).
20E is well known to act through the insect larval central nervous system (CNS) to induce wandering behavior and escape from food. Moreover, 20E slowly reduces insect feeding behavior and, thus, food consumption. Nevertheless, both the induction of wandering behavior and the reduction of feeding behavior can cause energy stress, such as sugar starvation, which ultimately increases the cellular AMP/ATP ratio, leading to the activation of AMPK. According to the Bombyx fat body results at ~4M, such a poor nutrition status promoted AMPK activity and inhibited IIS. Altogether, 20E slowly induces a sugar starvation-like condition to activate AMPK in the fat body by modulating CNS-controlled feeding behavior and wandering behavior in insects (Yuan, 2020).
IIS is an anabolic pathway, while AMPK accounts for catabolism, thus it naturally exists a mutual inhibition between IIS and AMPK. AMPK and PP2A might affect each other, and the AMPK-PP2A axis has been documented in mammalian cells. This study confirmed that the AMPK-PP2A axis exists in the Drosophila fat body, linking the antagonism of IIS by 20E (Yuan, 2020).
In addition to the dephosphorylation of AKT by PP2A, PP2A also dephosphorylates S6K, playing a key role in the attenuation of IIS and its downstream TORC1 activity. These studies determined that PP2A not only dephosphorylates AKT and inhibits TORC1 activity but also dephosphorylates InR and inactivates PI3K, showing that PP2A inhibits IIS starting from the dephosphorylation of InR. It is hypothesized that PP2A might dephosphorylate InR, PI3K, and AKT and, thus, inhibit IIS in an integrative manner (Yuan, 2020).
Finally, this study demonstrated that 20E activates the AMPK-PP2A axis to antagonize IIS in the insect fat body. After blocking either AMPK or PP2A, 20E no longer antagonizes IIS in the fat body. In summary, the AMPK-PP2A axis in the insect fat body is activated by 20E to antagonize IIS (Yuan, 2020).
Considering the similar regulatory functions in the antagonism of IIS by 20E, the possible relationship between miR-8/Ush and AMPK-PP2A was examined. Via bioinformatics prediction, it was found that miR-8 does not target AMPK or PP2A transcripts. Meanwhile, preliminary data showed that overexpression of AMPKCA or PP2ACA did not affect Ush expression in the fat body. Thus, it is supposed that AMPK-PP2A should function in parallel with miR-8 in the antagonism of IIS by 20E. It is concluded that the AMPK-PP2A axis is a crucial, but not a unique, pathway linking 20E to IIS (Yuan, 2020).
Previous studies and the current results together indicate that, similar to the inhibition of IIS in the larval fat body, activation of the fat body AMPK-PP2A axis reduces food consumption and thus restricts growth rate and body size in Drosophila. In other words, the AMPK-PP2A axis and IIS in the fat body play opposite developmental roles in regulating the larval growth rate and body size, and one crucial reason should be the modulation of feeding behavior and thus food consumption (Yuan, 2020).
The insect fat body, which is analogous to the mammalian liver, functions as an energy reservoir and nutrient sensor to regulate developmental timing. Fat body-derived amino acid signals, which involve Slimfast (the amino acid transporter) and TORC1 signaling, reactivate quiescent neuroblasts and finally control larval growth by regulating the synthesis and release of insulin/IGF. In addition to amino acid-dependent signals, certain other growth-promoting factors, such as CCHamide-2 and Unpaired 2, secreted from the fat body also affect the brain to remotely control insulin/IGF secretion in Drosophila. Moreover, IIS acts as the center of energy and nutrition response and positively regulates the larval growth rate partially by inhibiting autophagy in the Drosophila fat body. In contrast, 20E negatively regulates the larval growth rate by impeding IIS in the Drosophila fat body. Interestingly, preliminary results suggest that the AMPK-PP2A axis had little effect on fat body autophagy during normal feeding conditions and that TORC1 in the fat body plays little role in regulating the larval growth rate (Yuan, 2020).
It is likely that activation of the AMPK-PP2A axis and the inhibition of IIS in the fat body might affect the nutritional and endocrinal functions of this tissue. These changes in the fat body should cause the reduction of food consumption, resulting in the restriction of growth rate and body size. Investigating the detailed molecular mechanisms of how food consumption and its related feeding behavior and wandering behavior are regulated by hormonal and nutritional signals in the fat body might open a new window for understanding the regulatory mechanisms of final body size in insects. In future, it is worthwhile to examine whether the CNS. as well as neuropeptides and neurotransmitters, are involved in this regulation. Taking these data together, a model is proposed in which 20E antagonizes IIS by activating the AMPK-PP2A axis in the fat body to restrict the larval growth rate in insects. This study expands understanding of the comprehensive regulatory mechanisms underlying final body size determination in animals (Yuan, 2020).
The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, this study shows that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status (Ingaramo, 2020).
Insulin plays a major role in connecting nutrient availability to energy homeostasis by regulating metabolic pathways. Defects in insulin signalling is the primary cause for diabetes, obesity and various metabolic disorders. Nutritional status during growth and developmental stages play a crucial role in determining adult size, fecundity and ageing. However, the association between developmental nutrition and adult metabolic disorders has not been fully explored. This study addresses the effects of nutrient status during the larval growth phase on adult metabolism in Drosophila. Restricted food supply in larvae led to higher fat reserves and starvation resistance in mature adult flies, which is attributed to low insulin signalling. A lesser amount of stored fat was mobilised during early adult stages and during acute starvation, which accounts for the metabolic effects. Furthermore, larval diet influenced the expression of fat mobilisation genes brummer and lipid storage droplet-2 in adult flies, which led to the metabolic phenotypes reported in this study. Thus, the restricted nutrient environment in developing larvae led to adaptive changes that entrain the adult flies for scarce food availability (Rehman, 2021).
The insulin-producing cells (IPCs), a group of 14 neurons in the Drosophila brain, regulate numerous processes, including energy homeostasis, lifespan, stress response, fecundity, and various behaviors, such as foraging and sleep. Despite their importance, little is known about the development and the factors that regulate morphological and functional differentiation of IPCs. This study describes the use of a new transgenic reporter to characterize the role of the Drosophila L1-CAM homolog Neuroglian (Nrg), and the transmembrane Semaphorin-1a (Sema-1a) and its receptor Plexin A (PlexA) in the differentiation of the insulin-producing neurons. Loss of Nrg results in defasciculation and abnormal neurite branching, including ectopic neurites in the IPC neurons. Cell-type specific RNAi knockdown experiments reveal that Nrg, Sema-1a and PlexA are required in IPCs and glia to control normal morphological differentiation of IPCs albeit with a stronger contribution of Nrg and Sema-1a in glia and of PlexA in the IPCs. These observations provide new insights into the development of the IPC neurons and identify a novel role for Sema-1a in glia. In addition, this study shows that Nrg, Sema-1a and PlexA in glia and IPCs not only regulate morphological but also functional differentiation of the IPCs and that the functional deficits are likely independent of the morphological phenotypes. The requirements of nrg, Sema-1a, and PlexA in IPC development and the expression of their vertebrate counterparts in the hypothalamic-pituitary axis, suggest that these functions may be evolutionarily conserved in the establishment of vertebrate endocrine systems (Clements, 2021).
The brain plays a key role in energy homeostasis, detecting nutrients, metabolites and circulating hormones from peripheral organs and integrating this information to control food intake and energy expenditure. This study shows that a group of neurons in the Drosophila larval brain expresses the adiponectin receptor (AdipoR) and controls systemic growth and metabolism through insulin signaling. glucose-regulated protein 78 (Grp78) was identified as a circulating antagonist of AdipoR function produced by fat cells in response to dietary sugar. This study further showed that central AdipoR signaling inhibits peripheral Juvenile Hormone (JH) response, promoting insulin signaling. In conclusion, this study identified a neuroendocrine axis whereby AdipoR-positive neurons control systemic insulin response (Arquier, 2021).
Epigenetic regulation relies on the activity of enzymes that use sentinel metabolites as cofactors to modify DNA or histone proteins. Thus, fluctuations in cellular metabolite levels have been reported to affect chromatin modifications. However, whether epigenetic modifiers also affect the levels of these metabolites and thereby impinge on downstream metabolic pathways remains largely unknown. This study tested this notion by investigating the function of N-alpha-acetyltransferase 40 (NAA40), the enzyme responsible for N-terminal acetylation of histones H2A and H4, which has been previously implicated with metabolic-associated conditions such as age-dependent hepatic steatosis and calorie-restriction-mediated longevity. Using metabolomic and lipidomic approaches, this study found that depletion of NAA40 in murine hepatocytes leads to significant increase in intracellular acetyl-CoA levels, which associates with enhanced lipid synthesis demonstrated by upregulation in de novo lipogenesis genes as well as increased levels of diglycerides and triglycerides. Consistently, the increase in these lipid species coincide with the accumulation of cytoplasmic lipid droplets and impaired insulin signalling indicated by decreased glucose uptake. However, the effect of NAA40 on lipid droplet formation is independent of insulin. In addition, the induction in lipid synthesis is replicated in vivo in the Drosophila melanogaster larval fat body. Finally, supporting these results, this study found a strong association of NAA40 expression with insulin sensitivity in obese patients. Overall, these findings demonstrate that NAA40 affects the levels of cellular acetyl-CoA, thereby impacting lipid synthesis and insulin signalling. This study reveals a novel path through which histone-modifying enzymes influence cellular metabolism with potential implications in metabolic disorders.
Insulin/insulin-like growth factor (IGF) signaling (IIS) controls many aspects of development and physiology. In Drosophila, a conserved family of insulin-like peptides called Dilps is produced by brain neurosecretory cells, and it regulates organismal growth and developmental timing. To accomplish these systemic functions, the Dilps are secreted into the general circulation, and they signal to peripheral tissues in an endocrine fashion. This study describes the local uptake and storage of Dilps in the corpora cardiaca (CC), an endocrine organ composed of alpha cell homologs known to produce the glucagon-like adipokinetic hormone (AKH). Dilp uptake by the CC relies on the expression of an IGF-binding protein called ImpL2. Following their uptake, immunogold staining demonstrates that Dilps are co-packaged with AKH in dense-core vesicles for secretion. In response to nutrient shortage, this specific Dilp reservoir is released and activates IIS in a paracrine manner in the prothoracic gland. This stimulates the production of the steroid hormone ecdysone and initiates entry into pupal development. This study has therefore uncovered a sparing mechanism whereby insulin stores in CC serve to locally activate IIS and the production of ecdysone in the PG, accelerating developmental progression in adverse food conditions (Ghosh, 2022).
The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. This study shows that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, hyperglycemia was found to produce chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3K(RNAi) flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3K(RNAi) larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, these results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, these findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications (Mascolo, 2022).
Insulin regulation is a hallmark of health, and impaired insulin signaling promotes metabolic diseases like diabetes mellitus. However, current assays for measuring insulin signaling in all animals remain semi-quantitative and lack the sensitivity, tissue-specificity or temporal resolution needed to quantify in vivo physiological signaling dynamics. Insulin signal transduction is remarkably conserved across metazoans, including insulin-dependent phosphorylation and regulation of Akt/Protein kinase B. This study generated transgenic fruit flies permitting tissue-specific expression of an immunoepitope-labelled Akt (AktHF). Enzyme-linked immunosorption assays (ELISA) were developed to quantify picomolar levels of phosphorylated (pAktHF) and total AktHF in single flies, revealing dynamic tissue-specific physiological regulation of pAktHF in response to fasting and re-feeding, exogenous insulin, or targeted genetic suppression of established insulin signaling regulators. Genetic screening revealed Pp1-87B as an unrecognized regulator of Akt and insulin signaling. Tools and concepts of this study provide opportunities to discover tissue-specific regulators of in vivo insulin signaling responses (Tsao, 2023).
Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. This study shows that Eiger, the Drosophila TNF-alpha, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-alpha converting enzyme (TACE) family protein CG7908 is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, this study has identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-alpha that mediates adaptive response to nutrient deprivation (Agrawal, 2016).
This study shows that the fly TNF Eiger functions as a metabolic hormone produced by the fat body in response to chronic protein deprivation. Elevated circulating levels of human TNF-α and TNFR are also observed in malnutrition conditions and in catabolic states associated with cachexia induced by sepsis and cancer. Moreover, this study presents evidence that the molecular mechanism leading to decreased insulin expression by TNF-α is conserved in mammalian β cells. Therefore, activation of TNF signaling could be an evolutionary conserved response to undernutrient imbalance, recently highjacked as an adaptive response to continuous nutritional surplus. Drosophila Eiger has so far mostly been implicated in local, autonomous responses, activating JNK signaling in the cells or tissues that express it. In recent studies, however, it has been postulated that Egr can diffuse outside of its expression domain in a paracrine manner. The current data now demonstrate that Egr circulates in the hemolymph and acts remotely, allowing crossorgan communication. This raises questions relative to the mode of transport of Eiger in the hemolymph and its specificity of action on remote target tissues. Indeed, although overexpression of Egr in fat cells leads to a strong increase in Egr levels in the hemolymph, flies show no obvious defects, suggesting that secreted Egr has limited access to peripheral tissues while in the hemolymph. Previous studies have shown that human and mouse TNF-α efficiently cross the blood-brain barrier (BBB) after i.v. injection and are detected in the cerebrospinal fluid in a process requiring the presence of TNF receptors in glial cells. This study shows that hemolymph Eiger can penetrate the brain and access to the insulin-producing cells. It will be important to evaluate in future experiments the mechanisms by which Egr travels in the hemolymph and across the larval BBB (Agrawal, 2016).
This study shows that an important aspect of Egr secretion relies on its shedding from the membrane by the convertase enzyme TACE. Drosophila TACE was recently shown to be required for Egr function in a tumor model using activated ras (Chabu, 2014). This study shows that transcription of TACE, but not egr, is induced in fat cells under low-protein diet (LPD) and that adipose TACE activity is critical for metabolic adaptation to low protein. Moreover, a genetic link was identified between TACE expression and TORC1, the main amino acid sensor in fat cells. Interestingly, REPTOR and REPTOR-BP, two transcription factors that are responsible for most of the transcription response to TORC1 inhibition, are not required for TACE expression, suggesting that an alternative mechanism is required for TACE induction in response to TORC1 inhibition following exposure to LPD. Vertebrate TACE/ADAM17 acts on a small number of cytokines and growth factors including TNF-α and several membrane receptors (Menghini, 2013). Mice deficient in TACE function are lean and resistant to high-fat-diet-induced obesity and diabetes type 2, a range of phenotypes that could be linked to TNF-α shedding defects. In mammals, TACE activity is controlled through balanced expression of the Tissue Inhibitor of Metalloprotease 3 (TIMP3). Although a TIMP3 homolog exists in Drosophila, there is no indication that it participates in modulating Drosophila TACE activity in addition to TACE transcriptional activation observed in LPD (Agrawal, 2016).
Circulating Eiger produced by adipose cells remotely acts on the brain neurosecretory cells that produce insulin (IPCs), leading to general body growth inhibition. This correlates with specific expression of the TNF receptor Grindelwald in the IPCs. Indeed, knocking down Grnd in these neurons mimics the effect of knocking down Egr in the fat body. No effect is observed upon knocking down the other fly TNFR Wengen in IPCs, indicating that Grnd mediates Egr metabolic action and that specific targeting of TNF signaling to the IPCs is the consequence of localized Grnd expression. As a consequence of Grnd activation, JNK signaling is elevated in the IPCs of animals raised on a LPD. TNF signaling is not required in the IPCs for the retention of Dilp2 observed upon acute protein starvation. By contrast, activation of JNK in larval IPCs leads to reduced expression of Dilp2 and Dilp5, two major circulating Dilps. Strikingly, this is reminiscent of the role described for JNK in vertebrate pancreatic β cells. Indeed, JNK inhibitors increase insulin expression in Langerhans islets from obese mice, suggesting that JNK represses expression of the insulin gene. These results are also in line with the present finding that TNF-α inhibits INS1 and INS2 gene transcription from Min6 cells and mouse islets (Agrawal, 2016).
In conclusion, this work unravels an anciently conserved mechanism by which TNF signaling mediates direct response to low nutrient through a fat-brain crossorgan communication leading to the modulation of growth. Other signals contributing to reduction of insulin signaling in condition of nutrient shortage have recently been identified in Drosophila. Systemic Hedgehog is produced by gut cells in response to low nutrients and targets both fat cells and ecdysone-producing cells to adapt larval growth to nutrient shortage. A hormone called Limostatin produced by the corpora cardiaca in low-glucose condition acts directly on adult insulin-producing cells to block insulin secretion. These findings together with the present work indicate that a variety of signals allow the integration of different physiological contexts for the proper control of insulin production (Agrawal, 2016).
Eukaryotic Tribbles proteins are pseudoenzymes that regulate multiple aspects of intracellular signalling. Both Drosophila melanogaster and mammalian members of this family of pseudokinases act as negative regulators of insulin signalling. Mammalian tribbles pseudokinase (TRIB) genes have also been linked to insulin resistance and type 2 diabetes mellitus. Type 2 diabetes mellitus is associated with increased body weight, sleep problems and increased long-term mortality. This study investigated how manipulating the expression of Tribbles impacts body weight, sleep and mortality. Overexpression of Drosophila tribbles (trbl) in the fly fat body reduces both body weight and lifespan in adult flies without affecting food intake. Furthermore, it decreases the levels of Drosophila insulin-like peptide 2 (DILP2; ILP2) and increases night-time sleep. The three genes encoding TRIBs of mammals, TRIB1, TRIB2 and TRIB3, show both common and unique features. As the three human TRIB genes share features with Drosophila trbl, this study further explored the links between TRIB genetic variants and both body weight and sleep in the human population. Associations were identified between the polymorphisms and expression levels of the pseudokinases and markers of body weight and sleep duration. It is concluded that Tribbles pseudokinases are involved in the control of body weight, lifespan and sleep (Popovic, 2023).
The insulin signalling pathway is evolutionarily conserved throughout metazoans, playing key roles in development, growth, and metabolism. Misregulation of this pathway is associated with a multitude of disease states including diabetes, cancer, and neurodegeneration. The human insulin receptor gene (INSR) is widely expressed throughout development and was previously described as a 'housekeeping' gene. Yet, there is abundant evidence that this gene is expressed in a cell-type specific manner, with dynamic regulation in response to environmental signals. The Drosophila insulin-like receptor gene (InR) is homologous to the human INSR gene and was previously shown to be regulated by multiple transcriptional elements located primarily within the introns of the gene. These elements were roughly defined in ~1.5 kbp segments, but an understanding of the potential detailed mechanisms of their regulation is lacking. The substructure of these cis-regulatory elements was characterized in Drosophila S2 cells, focusing on regulation through the ecdysone receptor (EcR) and the dFOXO transcription factor. By identifying specific locations of activators and repressors within 300 bp subelements, this study showed that some previously identified enhancers consist of relatively compact clusters of activators, while others have a distributed architecture not amenable to further reduction. In addition, these assays uncovered a long-range repressive action of unliganded EcR. The complex transcriptional circuitry likely endows InR with a highly flexible and tissue-specific response to tune insulin signalling. Further studies will provide insights to demonstrate the impact of natural variation in this gene's regulation, applicable to human genetic studies (Thompson, 2023).
Of the nine genes of the American cockroach, Periplaneta americana, coding for peptides related to insulin and insulin-like growth factor, seven show significant expression in the central nervous system as demonstrated by the polymerase chain reaction on reverse transcribed RNA. In situ hybridisation shows that five of those are expressed by cells in the pars intercerebralis. Antisera raised to the predicted peptides show that these cells are neuroendocrine in nature and project to the corpora cardiaca. Interestingly, there are at least three cell types that each express different genes. This contrasts with Drosophila where a single cell type expresses a number of genes expressing several such peptides. Whereas in Drosophila the neuroendocrine cells producing insulin-like peptides also express sulfakinins, the arthropod orthologs of gastrin and cholecystokinin, in Periplaneta the sulfakinins are produced by different cells. Other neuropeptides known to be produced by the pars intercerebralis in Periplaneta and other insect species, such as the CRF-like diuretic hormone, neuroparsin, leucokinin or myosuppressin, neither colocalize with an insulin-related peptide. The separate cellular localization of these peptides and the existence of multiple insulin receptors in this species implies a more complex regulation by insulin and IGF-related peptides in cockroaches than in the fruit fly (Veenstra, 2023).
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