Drosophila Insulin-like Peptides Research Articles

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

Members of the insulin peptide family have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. Drosophila insulin-like peptides (Dilps) promote tissue growth through the single insulin-like receptor (InR). Despite the important role of Dilps in nutrient-dependent growth control, the molecular mechanism that regulates the activity of circulating Dilps is not well understood. Here, we report the function of a novel secreted decoy of InR (SDR) as a negative regulator of insulin signaling. SDR is predominantly expressed in glia 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). We further demonstrate that SDR is constantly secreted into the hemolymph independent of nutritional status and is essential for adjusting insulin signaling under adverse food conditions. We propose that Drosophila uses a secreted decoy to fine-tune systemic growth against fluctuations of circulating insulin levels.

Drosophila Cytokine Unpaired 2 Regulates Physiological Homeostasis by Remotely Controlling Insulin Secretion

In Drosophila, the fat body (FB), a functional analog of the vertebrate adipose tissue, is the nutrient sensor that conveys the nutrient status to the insulin-producing cells (IPCs) in the fly brain to release Drosophila insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. We identify Unpaired 2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the fed state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, we find that human Leptin can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin.

ADrosophilamodel for fetal alcohol syndrome disorders: role for the insulin pathway

SUMMARYPrenatal exposure to ethanol in humans results in a wide range of developmental abnormalities, including growth deficiency, developmental delay, reduced brain size, permanent neurobehavioral abnormalities and fetal death. Here we describe the use of Drosophila melanogaster as a model for exploring the effects of ethanol exposure on development and behavior. We show that developmental ethanol exposure causes reduced viability, developmental delay and reduced adult body size. We find that flies reared on ethanol-containing food have smaller brains and imaginal discs, which is due to reduced cell division rather than increased apoptosis. Additionally, we show that, as in mammals, flies reared on ethanol have altered responses to ethanol vapor exposure as adults, including increased locomotor activation, resistance to the sedating effects of the drug and reduced tolerance development upon repeated ethanol exposure. We have found that the developmental and behavioral defects are largely due to the effects of ethanol on insulin signaling; specifically, a reduction in Drosophila insulin-like peptide (Dilp) and insulin receptor expression. Transgenic expression of Dilp proteins in the larval brain suppressed both the developmental and behavioral abnormalities displayed by ethanol-reared adult flies. Our results thus establish Drosophila as a useful model system to uncover the complex etiology of fetal alcohol syndrome.

Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.

Nutrition-Responsive Glia Control Exit of Neural Stem Cells from Quiescence

SummaryThe systemic regulation of stem cells ensures that they meet the needs of the organism during growth and in response to injury. A key point of regulation is the decision between quiescence and proliferation. During development, Drosophila neural stem cells (neuroblasts) transit through a period of quiescence separating distinct embryonic and postembryonic phases of proliferation. It is known that neuroblasts exit quiescence via a hitherto unknown pathway in response to a nutrition-dependent signal from the fat body. We have identified a population of glial cells that produce insulin/IGF-like peptides in response to nutrition, and we show that the insulin/IGF receptor pathway is necessary for neuroblasts to exit quiescence. The forced expression of insulin/IGF-like peptides in glia, or activation of PI3K/Akt signaling in neuroblasts, can drive neuroblast growth and proliferation in the absence of dietary protein and thus uncouple neuroblasts from systemic control.

Blocking O-Linked GlcNAc Cycling in Drosophila Insulin-producing Cells Perturbs Glucose-Insulin Homeostasis

A dynamic cycle of O-linked GlcNAc (O-GlcNAc) addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively, in a process that serves as the final step in a nutrient-driven "hexosamine-signaling pathway." Evidence points to a role for O-GlcNAc cycling in diabetes and insulin resistance. We have used Drosophila melanogaster to determine whether O-GlcNAc metabolism plays a role in modulating Drosophila insulin-like peptide (dilp) production and insulin signaling. We employed transgenesis to either overexpress or knock down Drosophila Ogt(sxc) and Oga in insulin-producing cells (IPCs) or fat bodies using the GAL4-UAS system. Knockdown of Ogt decreased Dilp2, Dilp3, and Dilp5 production, with reduced body size and decreased phosphorylation of Akt in vivo. In contrast, knockdown of Oga increased Dilp2, Dilp3, and Dilp5 production, increased body size, and enhanced phosphorylation of Akt in vivo. However, knockdown of either Ogt(sxc) or Oga in the IPCs increased the hemolymph carbohydrate concentration. Furthermore, phosphorylation of Akt stimulated by extraneous insulin in an ex vivo cultured fat body of third instar larvae was diminished in strains subjected to IPC knockdown of Ogt or Oga. Knockdown of O-GlcNAc cycling enzymes in the fat body dramatically reduced neutral lipid stores. These results demonstrate that altered O-GlcNAc cycling in Drosophila IPCs modulates insulin production and influences the insulin responsiveness of peripheral tissues. The observed phenotypes in O-GlcNAc cycling mimic pancreatic β-cell dysfunction and glucose toxicity related to sustained hyperglycemia in mammals.

Stem Cell Dynamics in Response to Nutrient Availability

When nutrient availability becomes limited, animals must actively adjust their metabolism to allocate limited resources and maintain tissue homeostasis. However, it is poorly understood how tissues maintained by adult stem cells respond to chronic changes in metabolism. To begin to address this question, we fed flies a diet lacking protein (protein starvation) and assayed both germline and intestinal stem cells. Our results revealed a decrease in stem cell proliferation and a reduction in stem cell number; however, a small pool of active stem cells remained. Upon refeeding, stem cell number increased dramatically, indicating that the remaining stem cells are competent to respond quickly to changes in nutritional status. Stem cell maintenance is critically dependent upon intrinsic and extrinsic factors that act to regulate stem cell behavior. Activation of the insulin/IGF signaling pathway in stem cells and adjacent support cells in the germline was sufficient to suppress stem cell loss during starvation. Therefore, our data indicate that stem cells can directly sense changes in the systemic environment to coordinate their behavior with the nutritional status of the animal, providing a paradigm for maintaining tissue homeostasis under metabolic stress.

Molecular evolution and functional characterization of Drosophila insulin-like peptides.

Multicellular animals match costly activities, such as growth and reproduction, to the environment through nutrient-sensing pathways. The insulin/IGF signaling (IIS) pathway plays key roles in growth, metabolism, stress resistance, reproduction, and longevity in diverse organisms including mammals. Invertebrate genomes often contain multiple genes encoding insulin-like ligands, including seven Drosophila insulin-like peptides (DILPs). We investigated the evolution, diversification, redundancy, and functions of the DILPs, combining evolutionary analysis, based on the completed genome sequences of 12 Drosophila species, and functional analysis, based on newly-generated knock-out mutations for all 7 dilp genes in D. melanogaster. Diversification of the 7 DILPs preceded diversification of Drosophila species, with stable gene diversification and family membership, suggesting stabilising selection for gene function. Gene knock-outs demonstrated both synergy and compensation of expression between different DILPs, notably with DILP3 required for normal expression of DILPs 2 and 5 in brain neurosecretory cells and expression of DILP6 in the fat body compensating for loss of brain DILPs. Loss of DILP2 increased lifespan and loss of DILP6 reduced growth, while loss of DILP7 did not affect fertility, contrary to its proposed role as a Drosophila relaxin. Importantly, loss of DILPs produced in the brain greatly extended lifespan but only in the presence of the endosymbiontic bacterium Wolbachia, demonstrating a specific interaction between IIS and Wolbachia in lifespan regulation. Furthermore, loss of brain DILPs blocked the responses of lifespan and fecundity to dietary restriction (DR) and the DR response of these mutants suggests that IIS extends lifespan through mechanisms that both overlap with those of DR and through additional mechanisms that are independent of those at work in DR. Evolutionary conservation has thus been accompanied by synergy, redundancy, and functional differentiation between DILPs, and these features may themselves be of evolutionary advantage.

Dominant-negative Dmp53 extends life span through the dTOR pathway in D. melanogaster

Expression of dominant-negative (DN) versions of the Drosophila ortholog of the tumor suppressor p53 extends fly life span in a Calorie Restriction (CR) dependent manner. DN-Dmp53 expression furthermore leads to reduction of Drosophila insulin-like peptide (dILP) 2 mRNA levels and a decrease in insulin/insulin-like growth factor-signaling activity (IIS) in the fly fat body. It is unclear by which mechanisms DN-Dmp53 extends longevity, and whether modulation of insulin-signaling activity plays a pivotal role in life span regulation by Dmp53. Here we show that life span extension due to DN-Dmp53 expression is likely due to reduction of Dmp53 activity and that decreased Dmp53 activity does not extend life span when dILP2 is concomitantly over expressed. Furthermore, extended longevity due to DN-Dmp53 expression does not further extend the life span of flies over expressing the IIS associated transcription factor dFoxO, indicating that DN-Dmp53-dependent life span extension may be related to IIS. However, reduction of dFoxO levels does not decrease DN-Dmp53-dependent longevity extension. Interestingly, when DN-Dmp53 is expressed in flies lacking the translation initiation controlling factor Thor/4E-BP, the downstream target of dTOR signaling, no increase in life span is observed. Taken together, these data suggest that Dmp53 may affect life span by differentially engaging the IIS and dTor pathways.

A Drosophila Insulin-like Peptide Promotes Growth during Nonfeeding States

SummaryIn metazoans, tissue growth relies on the availability of nutrients—stored internally or obtained from the environment—and the resulting activation of insulin/IGF signaling (IIS). In Drosophila, growth is mediated by seven Drosophila insulin-like peptides (Dilps), acting through a canonical IIS pathway. During the larval period, animals feed and Dilps produced by the brain couple nutrient uptake with systemic growth. We show here that, during metamorphosis, when feeding stops, a specific DILP (Dilp6) is produced by the fat body and relays the growth signal. Expression of DILP6 during pupal development is controlled by the steroid hormone ecdysone. Remarkably, DILP6 expression is also induced upon starvation, and both its developmental and environmental expression require the Drosophila FoxO transcription factor. This study reveals a specific class of ILPs induced upon metabolic stress that promotes growth in conditions of nutritional deprivation or following developmentally induced cessation of feeding.

Deletion of Drosophila insulin-like peptides causes growth defects and metabolic abnormalities

Insulin/Insulin-like growth factor signaling regulates homeostasis and growth in mammals, and is implicated in diseases from diabetes to cancer. In Drosophila melanogaster, as in other invertebrates, multiple Insulin-Like Peptides (DILPs) are encoded by a family of related genes. To assess DILPs' physiological roles, we generated small deficiencies that uncover single or multiple dilps, generating genetic loss-of-function mutations. Deletion of dilps1-5 generated homozygotes that are small, severely growth-delayed, and poorly viable and fertile. These animals display reduced metabolic activity, decreased triglyceride levels and prematurely activate autophagy, indicative of "starvation in the midst of plenty," a hallmark of Type I diabetes. Furthermore, circulating sugar levels are elevated in Df [dilp1-5] homozygotes during eating and fasting. In contrast, Df[dilp6] or Df[dilp7] animals showed no major metabolic defects. We discuss physiological differences between mammals and insects that may explain the unexpected survival of lean, 'diabetic' flies.

Expression of human uncoupling protein-3 in Drosophila insulin-producing cells increases insulin-like peptide (DILP) levels and shortens lifespan

Uncoupling proteins (UCPs) can dissipate mitochondrial protonmotive force by increasing the proton conductance of the inner membrane and through this effect could decrease ROS production, ameliorate oxidative stress and extend lifespan. We investigated whether ubiquitous, pan-neuronal or neurosecretory cell-specific expression of human UCP3 (hUCP3) in adult Drosophila melanogaster affected lifespan. Low, ubiquitous expression of hUCP3 at levels found in rodent skeletal muscle mitochondria did not affect proton conductance in mitochondria isolated from whole flies, but high pan-neuronal expression of hUCP3 increased the proton conductance of mitochondria isolated from fly heads. Expression of hUCP3 at moderate levels in adult neurons led to a marginal lifespan-extension in males. However, high expression of hUCP3 in neuronal tissue shortened lifespan. The life-shortening effect was replicated when hUCP3 was expressed specifically in median neurosecretory cells (mNSC), which express three of the Drosophila insulin-like peptides (DILPs). Expression of hUCP3 in the mNSC did not alter expression of dilp2, dilp3 or dilp5 mRNA, but led to increased amounts of DILP2 in fly heads. These data suggest that lowering mitochondrial coupling by high expression of hUCP3 alters mNSC function in a way that appears to increase DILP-levels in fly heads and lead to a concomitant decrease in lifespan.

Opposing Effects of Dietary Protein and Sugar Regulate a Transcriptional Target of Drosophila Insulin-like Peptide Signaling

Specific neurosecretory cells of the Drosophila brain express insulin-like peptides (dilps), which regulate growth, glucose homeostasis, and aging. Through microarray analysis of flies in which the insulin-producing cells (IPCs) were ablated, we identified a target gene, target of brain insulin (tobi), that encodes an evolutionarily conserved alpha-glucosidase. Flies with lowered tobi levels are viable, whereas tobi overexpression causes severe growth defects and a decrease in body glycogen. Interestingly, tobi expression is increased by dietary protein and decreased by dietary sugar. This pattern is reminiscent of mammalian glucagon secretion, which is increased by protein intake and decreased by sugar intake, suggesting that tobi is regulated by a glucagon analog. tobi expression is also eliminated upon ablation of neuroendocrine cells that produce adipokinetic hormone (AKH), an analog of glucagon. tobi is thus a target of the insulin- and glucagon-like signaling system that responds oppositely to dietary protein and sugar.

Dynamics of the action of dFOXO on adult mortality in Drosophila

The insulin/insulin growth factor (IGF)-like signaling (IIS) pathway has a conserved role in regulating lifespan in Caenorhabditis elegans, Drosophila and mice. Extension of lifespan by reduced IIS has been shown in C. elegans to require the key IIS target, forkhead box class O (FOXO) transcription factor, DAF-16. dFOXO, the Drosophila DAF-16 orthologue, is also an IIS target, and its overexpression in adult fat body increases lifespan. In C. elegans, IIS acts exclusively during adulthood to determine adult survival. We show here, using an inducible overexpression system, that in Drosophila continuous dFOXO overexpression in adult fat body reduces mortality rate throughout adulthood. We switched the IIS status of the flies at different adult ages and examined the effects of these switches on dFOXO expression and mortality rates. dFOXO protein levels were switched up or down by the inducible expression system at all ages examined. If IIS status is reversed early in adulthood, similar to the effects of another intervention that reduces adult mortality in Drosophila, dietary restriction (DR), there is a complete switch of subsequent mortality rate to that of flies chronically exposed to the new IIS regime. At this age, IIS thus acts acutely to determine risk of death. Mortality rates continued to respond to a switch in IIS status up to 4 weeks of adult age, but not thereafter. However, unlike DR, as IIS status was altered at progressively later ages, mortality rates showed incomplete switching and responded with progressively smaller changes. These findings indicate that alteration of expression levels of dFOXO may have declining effects on IIS status with age, that there could be some process that prevents or lessens the physiological response to a switch in IIS status or that, unlike DR, this pathway regulates aging-related damage. The decreased mortality and increased lifespan of dFOXO overexpressing flies was uncoupled from any effect on female fecundity and from expression levels of Drosophila insulin-like peptides in the brain.

Specification and development of the pars intercerebralis and pars lateralis, neuroendocrine command centers in the Drosophila brain

The central neuroendocrine system in the Drosophila brain includes two centers, the pars intercerebralis (PI) and pars lateralis (PL). The PI and PL contain neurosecretory cells (NSCs) which project their axons to the ring gland, a complex of peripheral endocrine glands flanking the aorta. We present here a developmental and genetic study of the PI and PL. The PI and PL are derived from adjacent neurectodermal placodes in the dorso-medial head. The placodes invaginate during late embryogenesis and become attached to the brain primordium. The PI placode and its derivatives express the homeobox gene Dchx1 and can be followed until the late pupal stage. NSCs labeled by the expression of Drosophila insulin-like peptide (Dilp), FMRF, and myomodulin form part of the Dchx1 expressing PI domain. NSCs of the PL can be followed throughout development by their expression of the adhesion molecule FasII. Decapentaplegic (Dpp), secreted along the dorsal midline of the early embryo, inhibits the formation of the PI and PL placodes; loss of the signal results in an unpaired, enlarged placodeal ectoderm. The other early activated signaling pathway, EGFR, is positively required for the maintenance of the PI placode. Of the dorso-medially expressed head gap genes, only tailless (tll) is required for the specification of the PI. Absence of the corpora cardiaca, the endocrine gland innervated by neurosecretory cells of the PI and PL, does not affect the formation of the PI/PL, indicating that inductive stimuli from their target tissue are not essential for early PI/PL development.

JNK Extends Life Span and Limits Growth by Antagonizing Cellular and Organism-Wide Responses to Insulin Signaling

Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling (IIS) pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by IIS, extends life span in conditions of low IIS activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. Here we show that JNK requires Foxo to extend life span in Drosophila. JNK antagonizes IIS, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict IIS activity systemically by repressing IIS ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity.

Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands.

The insulin/insulin-like growth factor-like signaling pathway, present in all multicellular organisms, regulates diverse functions including growth, development, fecundity, metabolic homeostasis, and lifespan. In flies, ligands of the insulin/insulin-like growth factor-like signaling pathway, the Drosophila insulin-like peptides, regulate growth and hemolymph carbohydrate homeostasis during development and are expressed in a stage- and tissue-specific manner. Here, we show that ablation of Drosophila insulin-like peptide-producing median neurosecretory cells in the brain leads to increased fasting glucose levels in the hemolymph of adults similar to that found in diabetic mammals. They also exhibit increased storage of lipid and carbohydrate, reduced fecundity, and reduced tolerance of heat and cold. However, the ablated flies show an extension of median and maximal lifespan and increased resistance to oxidative stress and starvation.

Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body.

In Drosophila melanogaster, ageing is slowed when insulin-like signalling is reduced: life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (chico) are mutated, or when insulin-producing cells are ablated. But we have yet to resolve when insulin affects ageing, or whether insulin signals regulate ageing directly or indirectly through secondary hormones. Caenorhabditis elegans lifespan is also extended when insulin signalling is inhibited in certain tissues, or when repressed in adult worms, and this requires the forkhead transcription factor (FOXO) encoded by daf-16 (ref. 6). The D. melanogaster insulin-like receptor mediates phosphorylation of dFOXO, the equivalent of nematode daf-16 and mammalian FOXO3a. We demonstrate here that dFOXO regulates D. melanogaster ageing when activated in the adult pericerebral fat body. We further show that this limited activation of dFOXO reduces expression of the Drosophila insulin-like peptide dilp-2 synthesized in neurons, and represses endogenous insulin-dependent signalling in peripheral fat body. These findings suggest that autonomous and non-autonomous roles of insulin signalling combine to control ageing.

α-Endosulfine, a potential regulator of insulin secretion, is required for adult tissue growth control in Drosophila

α-Endosulfine is a small protein that has been proposed to regulate ion channel activity and insulin secretion, but in vivo studies have been lacking. We have previously established the Drosophila ovary as a model system in which to study adult tissue growth regulation, and demonstrated a role of the insulin pathway in the proliferative response of ovarian cells to nutritional changes. Here, we find that the Drosophila α-endosulfine ( dendos) gene, whose protein is expressed in germline and somatic cells of the ovary, as well as in the brain and certain regions of the intestine, is also required for this response. This requirement is non-cell autonomous, which is consistent with a role of dendos in secretion of Drosophila insulin-like peptides (DILPs), required for the proliferative response to nutritional changes. Our results show that dendos is also required for a distinct process in oogenesis, namely, the osmotic regulation of stage 14 oocytes, and that this requirement is cell autonomous, consistent with the role in ion channel regulation suggested by studies of the mammalian homologues.

Nutrient-Dependent Expression of Insulin-like Peptides from Neuroendocrine Cells in the CNS Contributes to Growth Regulation in Drosophila

Background: The insulin/IGF-1 signaling pathway controls cellular and organismal growth in many multicellular organisms. In Drosophila, genetic defects in components of the insulin signaling pathway produce small flies that are delayed in development and possess fewer and smaller cells as well as female sterility, reminiscent of the phenotypes of starved flies.Results: Here we establish a causal link between nutrient availability and insulin-dependent growth. We show that in addition to the Drosophila insulin-like peptide 2 (dilp2) gene, overexpression of dilp1 and dilp3-7 is sufficient to promote growth. Three of the dilp genes are expressed in seven median neurosecretory cells (m-NSCs) in the brain. These m-NSCs possess axon terminals in the larval endocrine gland and on the aorta, from which DILPs may be released into the circulatory system. Although expressed in the same cells, the expression of the three genes is controlled by unrelated cis-regulatory elements. The expression of two of the three genes is regulated by nutrient availability. Genetic ablation of these neurosecretory cells mimics the phenotype of starved or insulin signaling mutant flies.Conclusions: These results point to a conserved role of the neuroendocrine axis in growth control in multicellular organisms.