Genetic analysis of insulin signaling in Drosophila

Transcription

1 156 Review Genetic analysis of insulin signaling in Drosophila Robert S. Garofalo Studies in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans have revealed that components of the insulin signaling pathway have been highly conserved during evolution. Genetic analysis in Drosophila suggests that structural conservation also extends to the functional level. Flies carrying mutations that reduce insulin signaling have a growth deficiency phenotype similar to that seen in mice with disruptions of genes encoding insulin-like growth factors (IGFs) or the IGF-I receptor. Recent studies in flies have demonstrated a role for the insulin signaling pathway in the regulation of metabolism, reproduction and lifespan via modulation of central neuroendocrine pathways. Similarly, mice with loss of brain insulin receptors or insulin receptor substrate 2 deficiency exhibit neuroendocrine defects and female infertility. These parallels suggest that the insulin system has multiple conserved roles, acting directly to modulate growth and indirectly, via the neuroendocrine system, to modulate peripheral physiology in response to changes in nutrient availability. Published online: 22 March 2002 Robert S. Garofalo Dept Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, MS , Groton, CT 06340, USA. robert_s_garofalo@ groton.pfizer.com The presence of an insulin-like hormone in insects was proposed in the 1970s [1 3]. Since then, molecular genetic analysis in the fruit fly Drosophila melanogaster has demonstrated the presence of an extremely well conserved insulin signaling system (Fig. 1). Genetic studies have helped us to understand relationships among some of the proteins already known from mammalian systems, and have also allowed the identification of some novel players in the insulin signaling cascade and in insulin-regulated growth control. A role for insulin signaling in the control of lifespan and reproduction, in addition to metabolism, has also been recently revealed by genetic analysis in Drosophila [4,5] and Caenorhabditis elegans (reviewed in [6]). Here, I summarize what has been learnt about the insulin signaling pathway from recent molecular genetic studies in Drosophila, and the implications for our understanding of insulin action in mammals. The Drosophila insulin receptor Determination of the complete structure of InR (see Glossary) [7,8], confirmed the results of biochemical studies suggesting that the Drosophila receptor, like its mammalian counterparts, comprised two α and two β subunits, with a cytoplasmic tyrosine kinase in its β subunit that was activated upon insulin binding [9]. The degree of sequence conservation between the fly and human receptors in some domains, especially the kinase domain, is remarkable, given the evolutionary distance between these organisms. The Drosophila receptor also binds mammalian insulin with reasonably high affinity (K d 15 nm [10]), consistent with structural conservation of the insulin-binding site. One distinction between the Drosophila and human insulin receptors is the presence of a C-terminal extension of ~400 amino acids in the Drosophila protein [7,8]. A role for this domain in signal transduction is suggested by the presence of several potential tyrosine phosphorylation sites and three NPXYXXM motifs, which might be involved in binding SH2 or PTB domain-containing proteins after tyrosine phosphorylation. The C-terminal domain is phosphorylated and can bind IRS-1 [11,12] and PI3K [13] when the Drosophila receptor is expressed in mammalian cells, but the function of the extension in flies has yet to be explored. Evidence suggests that most INRs in embryos are processed proteolytically to remove the C-terminal extension [14]. This processing might be developmentally regulated or tissue specific, suggesting that INR might employ two modes of signal transduction. One, utilized by the full-length receptor, might involve direct binding of SH2 or PTB domain-containing proteins to the receptor after autophosphorylation. The second mechanism, utilized by the truncated β subunit, might resemble that used by the human insulin receptor, in which phosphorylation of an adaptor protein CHICO [15], is the first post-receptor step in signal transduction. Drosophila with null mutations in chico are viable, indicating that INR can signal in the absence of an IRS protein. This suggests that the C-terminal extension is functional, although not to a normal level, given the growth deficiency phenotype of chico mutants [15]. Insulin-like peptides in Drosophila The ability of INR to bind mammalian insulin suggests that insulin-like ligands should be present in Drosophila. Accordingly, a new family of genes was recently identified that encodes seven Drosophila insulin-like peptides [16] (dilp1 7). The deduced amino acid sequences predict a signal peptide, a B chain, a C peptide and an A chain. The presence of consensus proteolytic processing sites suggests that the mature peptides are composed of A and B chains and lack the C peptide, as in mammalian insulin. The functions of the seven DILP proteins are unknown, but a genetic interaction with InR has been demonstrated for dilp2, which is the most closely related to /02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S (01)

2 Review 157 dtor Drosophila DILP1 7 INR CHICO Dp110, p60 PI3K Dstpk61 Dakt DPTEN Caenorhabditis elegans ins-1 37 DAF-2? Age-1 PI3K pdk-1 AKT-1, AKT-2 DAF-18 PTEN Mammals Insulin, IGF-I, IGF-II Insulin receptor, IGF-IR, IRR IRS1 4 PI3K PDK-1 Akt/PKB PTEN mtor neurons, again consistent with a neuroendocrine function. Overexpression of the human gene encoding insulin and ins-1 in worms (which encodes the peptide most closely related to human insulin) enhances the daf-2 mutant phenotype, rather than rescuing it [19], suggesting that DAF-2 signaling is further reduced in the presence of elevated ligand. The authors suggest that INS-1 and insulin are acting as DAF-2 antagonists [19]. However, insulin resistance in mammals is observed when insulin levels are raised by as little as 50% [20], suggesting that the two- to 20-fold overexpression of ins-1 might induce resistance or DAF-2 downregulation. Biochemical studies of DAF-2 activation will be required to define clearly the functional relationship between DAF-2 and its ligands. Notably, recent studies have shown that the mammalian insulin peptide hormone family is larger than previously thought, comprising at least eight members: insulin, IGF-I and IGF-II, relaxin and the novel putative insulin-like peptides, insulin-like 3 6 (reviewed in [21]). ds6k FKHR GSK-3/Shaggy DAF-16 FKHR FKHR S6K GSK-3 TRENDS in Endocrinology & Metabolism Fig. 1. Evolutionary conservation of insulin signaling pathways across species. Insulin-like peptides are present in Drosophila and Caenorhabditis elegans in addition to mammals, and activate homologous receptors. An IRS homolog is present in Drosophila (chico) but has yet to be demonstrated in C. elegans. All three pathways signal through PI3K to Akt/PKB homologs, and are negatively regulated at this step by homologs of the lipid phosphatase PTEN. In mammals, Akt/PKB inhibits the kinase GSK-3, and the transcriptional activity of the FKHRs. DAF-16 is the C. elegans FKHR homolog, and was identified as a negative regulator of insulin signaling by genetic analysis. Both GSK-3 and FKHR homologs are present in Drosophila, but their relationship to the insulin signaling pathway has not yet been described (broken line). mtor and dtor are upstream activators of S6K, most probably in response to nutrient levels, and act in concert with signals from the PI3K pathway to upregulate translation. The extensive conservation of the insulin signaling pathway across these divergent species suggests that genetic analysis in the more experimentally tractable model organisms will continue to provide novel insights relevant to insulin actions in mammals. Broken lines indicate interactions that are predicted based on functional conservation, but have not yet been demonstrated. Abbreviations: see Glossary. mammalian insulin [16]. The different dilp genes have distinct expression patterns, suggesting distinct functions [16]. dilp2, in particular, exhibits broad expression in embryos and throughout imaginal discs [16], similar to that seen for InR [17]. Expression of dilp mrna was also observed in a discrete set of cells in the larval brain that might correspond to neurosecretory cells [16]. Similarly, an antibody raised against a DILP peptide labeled medial neurosecretory cells in the Drosophila brain [18]. The axons of these neurosecretory neurons extend to the larval ring gland complex, which is involved in the synthesis and release of hormones, such as JH, suggesting that DILPs via INR could regulate the release of other hormones. Notably, flies carrying InR mutations have an 80% decrease in the level of JH [4], consistent with such a role. Analysis of the Caenorhabditis elegans genome suggests the presence of as many as 37 ins genes, although only two are predicted to code for cleaved C peptides [19]. They are expressed primarily in Insulin signaling and growth control The most obvious role of insulin signaling uncovered by genetic analysis in Drosophila is in the regulation of growth and body size. The ability to generate visible aberrant phenotypes by perturbation of cellular growth has allowed studies of growth regulation to progress rapidly. However, strong InR mutations are recessive embryonic lethal [8,22], indicating an essential function for InR during normal development beyond growth control. This is consistent with the position of InR as the first step in the signaling pathway and suggests that InR has multiple outputs. Likewise, the phenotype of mice lacking insulin receptors is more severely altered than that of mice lacking IRS proteins, suggesting that in both mammals and in flies, multiple substrates are required to mediate insulin action [23]. Nonetheless, mutations in InR and all genes examined thus far encoding proteins downstream of InR in the IRS-1 PI3K pathway alter cellular and organismal growth (Table 1; Fig. 2). Hypomorphic InR alleles and some heteroallelic combinations produce viable adults with striking growth deficiency [16,22]. Body size is reduced by ~50% because of decreased cell numbers and size [16,22]. The growth deficiency phenotype of chico mutants resembles that of InR mutations [15] (Table 1), suggesting that most of the growth regulatory functions of INR require CHICO. However, heterozygosity for a hypomorphic InR allele in a null chico background causes a further reduction in wing and eye size, but one that results from a reduction in cell number alone [15]. This suggests either that the cells have reached a minimum size or that the effect of INR on cell size is entirely mediated through CHICO, but that a CHICOindependent pathway emanating from INR can influence cell number.

3 158 Review Table 1. Growth phenotypes of mutations in the insulin receptor signaling pathway in Drosophila a,b Gene Lethality of mutant alleles Body size Cell size Cell number Cell cycle distribution Developmental delay c (days) Apoptosis Other Refs InR Recessive embryonic Adult E by E E Not reported 10 Not reported Female sterile; [4,16,22] lethal; some 45 60% Dtriglycerides; heteroallelic Dlifespan combinations viable chico Homozygous null Adult E by E E = to wild type 2 3 No change Female sterile; [5,15] mutations viable 55 65% Dtriglycerides; Dlifespan Dp110, Larval/pupal lethal Larvae E E E p60: DG1; No adults D by dominantnegative Small fat bodies [24,28] p60 Dp110 OE: DG2 eclose Dp110 Dakt Larval lethal; embryonic Adult E (Dakt1 E = = to wild type No adults D [27 29] lethal in absence of larval rescue) eclose maternal mrna ds6k 25% eclose as viable Adult E by 46% E = = to wild type 3 5 Not reported Female sterile [34] adults dtor Larval lethal Larvae E E E DG1 No adults No growth after [35,36] eclose 2nd instar DPTEN Late embryonic/early DLOF, DLOF, Not reported No adults DGOF [28,30] larval lethal E GOF EGOF eclose a Abbreviations: Dp110, p60, catalytic and adaptor subunits of phosphatidylinositol 3-kinase; GOF, gain-of-function mutation; LOF, loss-of-function mutation; OE, overexpression; p60, dominant-negative mutant of p60; and see Glossary. b E, decreased; D, increased; =, no difference. c Delay in days to adult eclosion, relative to wild type. Mutations in the Dp110 gene produce small larvae that are incapable of growth beyond the size reached in the early third instar [24]. The larvae remain viable for up to 20 days (normal third instar lasts two days), but do not increase in size. Mutations in the p60 adaptor protein confer a similar but less severely altered phenotype [24]. Larvae comprise two principal types of tissue: larval cells that do not undergo mitosis but increase in size owing to endoreplication, and actively mitotic imaginal disc tissue epithelial sheets that proliferate in the larvae and differentiate during pupation into the adult organs of the fly. Dp110- and p60-mutant larvae lack most imaginal disc tissue, suggesting that PI3K function is required for disc cell proliferation. Furthermore, the inability of the larvae to grow suggests that the increase in larval cell size also depends on PI3K function. Clonal analysis of cells homozygous for Dp110 or p60 mutations confirmed that PI3K function is required for both cell growth and proliferation to proceed normally [24]. Notably, increasing PI3K activity by ectopic overexpression of Dp110 increased cell size but not cell number [24], suggesting that PI3K activity, although necessary for normal proliferation, is not by itself sufficient to induce it. This is in contrast to overexpression of InR, which increases both the number and size of cells in the Drosophila eye [16], again suggesting that PI3K-dependent and -independent outputs from INR are involved in growth regulation. The phosphoinositide products of PI3K regulate a series of kinases, including PDK-1 and PKB/Akt (reviewed in [25]). A PDK-1 homolog has been identified in Drosophila [26], but the effects of mutations in this gene have not yet been described. However, loss-of-function mutations in Dakt are larval lethals [27]. Complete loss of embryonic Dakt function by elimination of maternally encoded Dakt mrnas leads to extensive apoptosis of embryonic cells and death [27]. Synthesis of high levels of a dominant-negative Dp110 PI3K in embryos confers a phenotype similar to complete loss of Dakt [28], suggesting that the PI3K Dakt pathway regulates embryonic cell survival. Interestingly, although loss of PI3K or Dakt activity can induce apoptosis, overexpression of Dakt does not alter the normal rate of apoptosis in eye discs [29]. Thus, Dakt activity is required for cell survival, but by itself is not sufficient to override the normal controls on programmed cell death. Mutations in Dakt reduce cell size [28,29] and conversely, overexpression of Dakt increases cell size [29]. However, unlike mutations in the upstream genes InR and Dp110, Dakt mutations do not affect cell number [28,29], suggesting that Dakt might be a point at which growth and proliferation signals diverge (Fig. 2). PKB/Akt activity is regulated by PI3K via synthesis of 3-phosphoinositides, and by the lipid phosphatase PTEN, via degradation of 3-phosphoinositides [28]. Accordingly, overexpression of the Drosophila homolog DPTEN confers a phenotype similar to that of dominant-negative Dp110: a decrease in cell number and size [28,30]. Dakt overexpression provides significant rescue of the growth deficiency phenotype induced either by dominant-negative Dp110 or overexpression of DPTEN, indicating that

4 Review 159 chico DILP1 DILP7 INR Dp110, p60, PI3K? Dakt? DPTEN ds6k Cell growth Ras MAPK dtor Cell proliferation Tsc1/Tsc2 TRENDS in Endocrinology & Metabolism Fig. 2. The insulin signaling pathway in growth control of Drosophila. Activation of INR by DILPs 1 7 stimulates both cell growth and proliferation. Proliferation induced by INR involves both PI3K-dependent and -independent pathways, with the PI3K-independent pathway acting via Ras MAPK. Components downstream of PI3K (Dakt and ds6k) stimulate cell growth, but not proliferation. ds6k is a major target of dtor, but dtor appears to exert effects on both growth and proliferation, suggesting that it has other targets. Tsc1 and Tsc2 suppress both cell growth and proliferation, acting at ds6k to suppress growth, and at an unknown site to suppress proliferation. Broken lines indicate interactions that are predicted based on functional conservation, but have not yet been demonstrated. Abbreviations: see Glossary. Dakt is downstream of both [28]. Notably, Dp110 overexpression was not sufficient to increase cell number [24], whereas a loss-of-function mutation in DPTEN was [30]. Because both should lead to an increase in 3-phosphoinositides, this suggests either that DPTEN has other targets, or that the increase in 3-phosphoinositides induced by DPTEN inactivation is greater or more persistent than that induced by increased Dp110 activity in the presence of functional DPTEN. Consistent with the role of Dakt in growth regulation in flies, mice with disruption of the Akt1 gene are 15 20% smaller than are their wild-type littermates [31]. TOR is a conserved protein of the phosphatidylinositol kinase-related kinase family that has been implicated in growth control via its regulation of translation initiation in response to nutrient levels (reviewed in [32]). A major downstream target of TOR is S6K, which phosphorylates ribosomal protein S6, leading to upregulation of translation of a subset of mrnas that contain an oligopyrimidine tract at their transcriptional start site (reviewed in [33]). The activity of S6K is regulated by insulin and other growth factors, but the mechanism and the role of TOR in this process is not completely understood. Genetic analysis of the Drosophila homologs dtor and ds6k demonstrates a role for these proteins in cellular growth control [34 36]. Flies homozygous for ds6k mutations are viable but are dwarfs, solely because of decreased cell size [34]. Strong dtor mutations confer a more severely altered phenotype: larvae are dramatically reduced in size with little or no imaginal disc tissue and die during the second larval instar [35,36]. Thus, dtor affects both cell proliferation and cell growth. Consistent with this, clonal analysis of dtor mutant imaginal disc cells reveals that they are smaller and have a reduced rate of cell proliferation [36]. The phenotype of dtor mutant larvae resembles that induced by amino acid deprivation, consistent with a role for dtor in regulating growth in response to nutrient levels ([36] and references therein). The loss of ds6k kinase activity in dtor mutants [35], in addition to the ability of activated ds6k to rescue the lethal phenotype of some dtor mutant alleles [36], indicates that ds6k is a major effector of dtor. However, the adult dtor mutants rescued by activated ds6k overexpression are still reduced in size [36], suggesting that ds6k does not mediate all the functions of dtor and that other signals are required to achieve full body size. Notably, ds6k activity appears to be normal in dwarf chico mutants [35], suggesting that normal cell size requires ds6k in addition to signals emanating from the PI3K pathway (such as Akt kinase activity). Likewise, activation of the PI3K pathway appears to require dtor function for growth, as indicated by the requirement of dtor for the overgrowth phenotype induced by loss-offunction mutations in DPTEN [35,36]. Together, these data suggest that normal growth requires signals both from growth factors, via PI3K Dakt, and from nutrients, via dtor. Whether these converge on ds6k, or whether Dakt and ds6k act cooperatively in parallel to promote growth, should be clarified by further genetic analysis. Two novel genes involved in insulin-mediated growth control are the Drosophila homologs of TSC1 and TSC2, human tumor suppressor genes responsible for the genetic disorder TSC [37 39]. TSC is characterized by benign tumors called hamartomas, consisting of differentiated but disorganized and sometimes giant cells. TSC1 and TSC2 act together as a complex in both flies and mammals, and TSC2 has GAP activity for the small GTPases, rap1 and rab5 [40,41]. The Drosophila Tsc1- and Tsc2-mutant phenotypes resemble those conferred by alterations in the activity of the lipid phosphatase, DPTEN: decreased cell size with overexpression of Tsc1 and Tsc2 and increased cell size and number with loss-of-function mutations in either gene. Tsc1 and Tsc2 overexpression blocked the overgrowth induced by overexpression of InR, suggesting that Tsc1 and Tsc2 negatively regulate insulin signaling to growth and proliferative targets [38,39]. The large cell phenotype characteristic of Tsc1 mutations is not suppressed by mutations in InR or Dakt, or by overexpression of DPTEN, but is suppressed by mutations in ds6k [39]. These data suggest that Tsc1 and Tsc2 affect growth at a point

5 160 Review Non-autonomous, nutrient sensing Neuroendocrine system Juvenile hormone (flies) Leptin (mammals) Lifespan Metabolism Insulin Reproduction Cell autonomous Cell growth and proliferation Body size Acute nutrient storage Glycogen, lipid and protein synthesis Fig. 3. Cell autonomous and non-autonomous actions of insulin. The actions of insulin can be divided into those that are cell autonomous (direct effects on cell growth and proliferation and acute nutrient storage) and those that are non-autonomous (modulation of neuroendocrine pathways in response to changing nutrient levels, which in turn coordinate regulation of metabolism, reproduction and lifespan). The data summarized in this review indicate that both types of insulin action have been conserved during evolution. The high degree of structural and functional conservation across such divergent species suggests that the insulin signaling pathway arose very early as a means of adjusting growth, physiology and energy storage to meet environmental demands. downstream of Dakt but upstream of ds6k [39]. Whether Tsc1 and Tsc2 are part of a linear pathway from INR to ds6k or reside in a parallel pathway that converges with the insulin pathway remains to be determined. Nonetheless, the function of the TSC proteins as growth regulators is conserved in flies and mammals, and their interaction with the insulin signaling pathway observed in flies is probably relevant to their mechanism of action in mammals. All of the mutations described above affect cell growth in a cell-autonomous fashion (Fig. 3), indicating that the function of INR and downstream gene products is required in the affected cells, rather than regulating growth via a secondary, possibly secreted, signal. In addition, the growth effects are seen in the absence of changes in cell differentiation or patterning, suggesting that insulin signaling, at least in the postembryonic stages, does not regulate these processes. Strong InR mutations do disrupt embryonic nervous system development [8], as might be expected from the high level of expression of InR in the developing nervous system [17]. It is probable that INR plays a role in neuroblast and glial cell proliferation, because large populations of neurons and glia are missing in InR mutants [8]. However, it has not been determined whether InR mutations affect differentiation per se, or inhibit neurite outgrowth, processes in which insulin or IGFs are thought to play a role in mammalian systems [42]. The growth-deficiency phenotype of InR pathway mutations in Drosophila is reminiscent of that seen in mice with disruptions in genes of the IGF pathway (reviewed in [43]) and in mice lacking IRS-1 [44,45], indicating that the growth regulatory function of the insulin family of hormones evolved early and has been maintained. Insulin signaling and metabolism, reproduction and lifespan Recent data also implicate INR signaling in metabolic regulation in flies. The first genetic evidence that insulin signaling impacted on metabolism in lower organisms came from daf-2 mutations in C. elegans. Worms with decreased DAF-2 function have greater lipid stores than their wild-type counterparts [46]. Similarly, dwarf flies with mutations in chico [15] and InR [4,16] exhibit up to a fivefold increase in stored triglyceride. Because insulin receptor activation stimulates lipogenesis in mammalian adipocytes, the increased lipid stores in worms or flies with reduced insulin signaling seems counterintuitive. These data suggest either that the function of insulin signaling in these organisms with regard to energy storage differs from that in mammals, or conversely, that the decrease in InR function impacts on energy storage via another mechanism. The phenotype conferred by hypomorphic InR or chico mutations also includes female sterility [15,22] and, as recently shown, extended lifespan [4,5]. Interestingly, flies exhibit female sterility, increased triglyceride stores and prolonged lifespan during diapause, a physiological adaptation to harsh environmental conditions, such as low nutrient availability or low temperature (reviewed in [47]). Increased stress resistance is also manifested during diapause [47] and this, in conjunction with heightened energy stores, enhances survival and hence the probability of reproduction. Diapause is functionally analogous to the dauer stage of C. elegans, and strong daf-2 mutations induce a constitutive dauer [6]. Entry into diapause in flies is initiated by a decrease in the level of JH [47]. The similarity between diapause and the InR mutant phenotype suggested that reduced INR function might be impacting on JH levels, and indeed, JH was found to be reduced by 80% as a result of InR mutations [4]. In addition, application of an exogenous JH analog to long-lived InR dwarfs rescues the female sterility and restores lifespan back to the wild-type duration, indicating that decreased JH is sufficient to account for the effect of InR mutations on both lifespan and reproductive capacity [4]. Thus, INR signaling appears to act centrally to control release of a neuroendocrine hormone that, in turn, regulates peripheral functions, such as reproduction, somatic stress resistance and energy storage. These data suggest that mutations leading to decreased INR function mimic signaling in the context of low nutrient availability and that central INR serves as a key upstream regulator of the neuroendocrine system. The level of INR activity could reflect environmental conditions (such as nutrient levels),

6 Review 161 with a decrease in activity initiating a neuroendocrine-mediated physiological program designed to enhance survival. Consistent with this notion, the proliferative response of ovarian follicle cells to increased nutrients requires a functional INR signaling pathway [48]. Because effects of InR mutations on reproduction and lifespan are mediated via a neuroendocrine signal, they differ from those on growth and body size in that they are not cell autonomous (Fig. 3). Does this role of INR have a parallel in mammalian systems? The role of insulin in the regulation of food intake and energy expenditure via actions in the brain has been studied for over 20 years (reviewed in [49]). Insulin from the periphery acts in the hypothalamus to inhibit food intake and increase energy expenditure. Conversely, lower insulin levels in response to decreased food intake or energy stores act centrally on neuroendocrine pathways to suppress energy expenditure and stimulate eating. Thus, the increased triglyceride storage in InR mutant flies probably reflects a well-conserved role for insulin signaling in the central regulation of whole body energy stores in response to nutrient availability. Consistent with this is the phenotype of mice lacking IRS-2 [50], or lacking insulin receptors only in their central nervous system [51] (NIRKO mice), which have increased body fat and food intake and female infertility owing to reduced pituitary gonadotropin release. It will be of interest to determine whether the lifespan of NIRKO mice is extended. Therefore, although the importance of centrally acting insulin in regulation of food intake has been acknowledged for some time, the InR phenotype in Drosophila suggests that the role of insulin in central modulation of neuroendocrine pathways is part of a conserved system for coordination of metabolism, reproduction and lifespan, which arose to allow adaptation to changing environments (Fig. 3). Increased lifespan in response to caloric restriction in mammals ([6] and references therein) could reflect the functional persistence of such a system. What has not been approachable in the Drosophila studies is the function of the insulin pathway under conditions of nutrient excess, as is encountered in much of the developed world, where diabetes is increasing at an alarming rate. If the analogy between insulin function in worms, Drosophila and mammals is extended, one might propose that central insulin resistance induced by elevated plasma insulin and/or triglycerides in mammals is interpreted by the hypothalamus as decreased insulin signaling owing to low nutrient availability. The hypothalamus and pituitary then perform their evolutionarily conserved role and adjust peripheral physiology to adapt to this perceived deficit: increasing energy storage and inhibiting reproduction. However, this response, which serves the organism well in situations of low nutrient availability, could exacerbate weight gain in a situation of nutrient excess, and contribute to the reproductive complications of insulin resistance (e.g. polycystic ovary syndrome). Another area yet to be examined in Drosophila is the role of INR in acute regulation of enzymes of glucose storage and utilization. This function of insulin is also probably conserved. The induction of hyperglycemia in blowflies by surgical removal of the medial neurosecretory cells [3], which contain a DILP in Drosophila [18], is consistent with a function for INR signaling in acute metabolic regulation. Key enzymes, such as Drosophila glycogen phosphorylase [52], are closely related to the mammalian enzymes. The enzyme GSK-3 plays an important role in the regulation of glucose storage in mammalian cells (reviewed in [53]), but a role for its Drosophila homolog Shaggy Zeste-white3 in metabolism has yet to be established. The design of genetic screens to look at alteration of metabolic endpoints needs to be undertaken to identify all of the gene products that interact with InR and downstream proteins to regulate energy metabolism. Such an analysis might uncover novel components involved in insulin action, some of which might be targets for therapeutic intervention in diabetes. Conclusions Molecular and genetic analysis of the insulin signaling pathway in Drosophila has revealed Glossary Age-1: Caenorhabditis elegans homolog of PI3K CHICO: Drosophila homolog of IRS daf-2: gene encoding Caenorhabditis elegans insulin receptor homolog Dakt: gene encoding Drosophila homolog of PKB/Akt dilp1 7: Drosophila insulin-like peptide genes 1 7 Dp110: gene encoding Drosophila homolog of the catalytic subunit of PI3K Dstpk61: Drosophila homolog of PDK-1 FKHR: forkhead-related transcription factor GAP: GTPase-activating protein. GSK-3: glycogen synthase kinase-3 IGF: insulin-like growth factor InR: insulin receptor gene in Drosophila ins-1 37: Caenorhabditis elegans insulin-like peptide genes 1 37 IRR: insulin receptor-related receptor IRS: insulin receptor substrate JH: juvenile hormone MAPK: mitogen-activated protein kinase p60: Drosophila homolog of the p85 adaptor subunit of PI3K PDK-1: phosphatidylinositol-dependent kinase-1 PI3K: phosphatidylinositol 3-kinase PKB: protein kinase B, also known as Akt PTB domain: phosphotyrosine-binding domain PTEN: phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase S6K: p70 ribosomal S6 kinase Shaggy: Drosophila homolog of GSK-3 SH2 domain: src homology domain 2 TOR: target of rapamycin (dtor, Drosophila TOR; mtor, mammalian TOR) TSC: tuberous sclerosis complex

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