The Drosophila insulin receptor InR is similar to its mammalian counterpart in deduced amino acid sequence, subunit structure, and ligand-stimulated protein tyrosine kinase activity. The function of this receptor in D. melanogaster is not yet known. However, a role in development is suggested by the observations that levels of insulin-stimulated kinase activity and expression of InR mRNA are maximal during Drosophila midembryogenesis. In this study, a 2.9-kilobase (kb) cDNA clone corresponding to both the InR tyrosine kinase domain and some of the 3' untranslated sequence was used to determine the tissue distribution of InR mRNA during development. Two principal mRNAs of 11 and 8.6 kb hybridize with the InR cDNA in Northern (RNA) blot analysis. The abundance of the 8.6-kb mRNA increases transiently in early embryos, whereas the 11-kb species is most abundant during midembryogenesis. A similar pattern of expression has been determined by Northern analysis, using an InR genomic clone. In situ hybridization revealed InR transcripts in the ovaries of adult flies, in which the transcripts appear to be synthesized by nurse cells for eventual storage as maternal RNA in the mature oocyte. Throughout embryogenesis, InR transcripts are ubiquitously expressed, although after midembryogenesis, higher levels are detected in the developing nervous system. Nervous system expression remains elevated throughout the larval stages and persists in the adult, in which the cortex of the brain and ganglion cells are among the most prominently labeled tissues. In larvae, the imaginal disc cells exhibit comparatively high levels of InR mRNA expression. The broad distribution of InR mRNA in embryos and imaginal discs is compatible with a role for InR in anabolic processes required for cell growth. The apparently elevated expression of InR mRNA in nervous tissue during mid- and late-embryogenesis coincides with a period of active neurite outgrowth and suggests that dIRH may be involved in this process (Garofalo, 1988).
A monoclonal antibody (Mab E1C) has been generated that recognizes the differentiated nervous system in Drosophila embryos. At the cellular blastoderm stage, Mab E1C behaves as a general ectodermal marker but, in subsequent stages, it also labels the mesoderm. As neurogenesis takes place, staining increases within the neuromeres and is almost exclusively restricted to the nervous tissue by the time neuronal differentiation is completed. In third instar larvae, Mab E1C stains the central nervous system as well as the imaginal discs, which display a staining pattern related to their degree of neuronal differentiation. No labelling can be detected in adult brains or ovaries. Western blots are consistent with this developmental profile and allow the characterization of a major glycoprotein of 135 X 10(3) Mr (135K) that cosediments with a membrane fraction prepared from embryos. Additional glycoproteins (100K and 80K) are extracted from embryo homogenates by immunoaffinity procedures. In larvae, the 100K polypeptide is not detected. The properties of the 135K and 100K components are highly reminiscent of the molecular pattern of the Drosophila insulin receptor homologue. It has been shown that a Mab directed against the human insulin receptor stains the same cells as Mab E1C in imaginal discs and in the CNS. Moreover, this Mab cross-reacts with the 135K and 100K components of the embryonic antigen E1C (Piovant, 1988).
Insulin and insulin-like growth factor (IGF) receptors are members of the tyrosine kinase family of receptors, and are thought to play an important role in the development and differentiation of neurons. The presence of an insulin-like peptide and an insulin receptor (InR) at the body wall neuromuscular junction of developing Drosophila larvae is reported. InR-like immunoreactivity is found in all body wall muscles at the motor nerve branching regions, where it surrounds synaptic boutons. The identity of this immunoreactivity as an InR was confirmed by two additional schemes: in vivo binding of labeled insulin and immunolocalization of phosphotyrosine. Both methods produce staining patterns markedly similar to InR-like immunoreactivity. The presence of an InR in whole larvae was also shown by receptor binding assays. This receptor is more specific for insulin (> 25-fold) than for IGF II, and does not appear to bind IGF I. Among the 30 muscle fibers per hemisegment, insulin-like immunoreactivity is found only on one fiber, and is localized to a subset of morphologically distinct synaptic boutons. Staining in the CNS is limited to several cell bodies in the brain lobes and in a segmental pattern throughout most of the abdominal ganglia, as well as in varicosities along the neuropil areas of the ventral ganglion and brain lobes. Insulin-like peptide and InR are first detected by early larval development, well after neuromuscular transmission begins (Gorczyca, 1993).
The isolation of the Drosophila insulin receptor gene and the recent analysis of loss of function mutants have clearly implicated insulin signaling in embryonic nervous system development. The presence of insulin in the embryo is studied and cellular processes affected by insulin in embryonic neural cells are characterized. A fraction of the cells (7.5%) in the 15-18 h Drosophila embryo contain insulin immunoreactivity. In the embryonic-derived cell line Schneider 1, human insulin is capable of stimulating proliferation and neural differentiation. Thus, the action of insulin on the developing Drosophila nervous system appears to be as pleiotropic as in vertebrates (Pimentel, 1996).
Adult flies contain a specific, high-affinity insulin-binding protein. Insulin-dependent protein tyrosine kinase activity has now been identified in Drosophila. Activity first appears at 6-12 h of embryogenesis, increases during the 12-18-h period and falls to low levels in the adult. 125I-insulin has been cross-linked specifically and with high affinity to a protein (Mr = 135,000) throughout embryogenesis and in the adult. However, during the 6-12- and 12-18-h periods of embryogenesis when insulin-dependent protein tyrosine kinase activity is expressed, another protein (Mr = 100,000) becomes cross-linked to 125I-insulin. Crosslinking to both proteins is competitively inhibited by the addition of 100 nM insulin. It is concluded that the insulin-binding and insulin-dependent protein tyrosine kinase activities of the mammalian insulin receptor are conserved in Drosophila. However, the insulin-dependent protein tyrosine kinase activity of the receptor is detected only during specific times in embryogenesis (Petruzzelli, 1985).
The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the alpha and beta subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding alpha subunits of 110 or 120 kilodaltons (kDa), a 95-kDa beta subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The beta subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the alpha subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved (Fernandez-Almonacid, 1987).
The cloning and primary structure of the Drosophila Insulin receptor gene (InR), functional expression of the predicted polypeptide, and the isolation of mutations in the InR locus are reported. These data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The InR proreceptor [M(r) 280 kDa] is processed proteolytically to generate an insulin-binding alpha subunit [M(r) 120 kDa] and a beta subunit [M(r) 170 kDa] with protein tyrosine kinase domain. The InR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension that contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C-terminus) from the InR gene. These subunits assemble into mature InR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of InR have different signaling potentials. Finally, loss of function mutations in the InR gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are all affected by InR mutations (Fernandez, 1995).
The Insulin-like receptor (InR) gene is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand-activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the InR gene and identified six independent mutations that led to a loss of expression or function of the receptor protein. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for Inr in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size (Chen, 1996).
Genetic analyses suggest that the TSC genes act in a parallel pathway that converges on the insulin pathway downstream from Akt. The most convincing evidence for a functional link between the TSC genes and insulin signaling comes from the observation that heterozygosity of TSC1 or TSC2/gigas is sufficient to rescue the lethality of loss-of-function InR mutants. This argues that the TSC genes are intimately linked to insulin signaling, rather than functioning in a totally independent cell-growth pathway. These results suggest that the TSC tumor suppressor genes are novel negative regulators of insulin signaling, and modulating the activities of the TSC genes might provide a potential way to correct insulin signaling defects in certain diseases such as diabetes and obesity (Gao, 2001).
Previous studies have shown that loss of inr or Akt leads to decreased cell size. To investigate the relationship between inr, Akt, and the TSC genes, TSC1;Akt and TSC1;inr double-mutant clones were studied. Cells homozygous for a strong allele of inr, or a null allele of Akt are smaller, and are rarely recovered in adult eye clones because of cell competition during development. However, TSC1;inr or TSC1;Akt1 double-mutant cells showed a similar cell size increase as that observed in TSC1- cells. Furthermore, the competitive disadvantage of inr and Akt mutant cells is also rescued in the TSC1;inr or TSC1;Akt1 double-mutant clones, resulting in larger clones that contained more cells. This result suggests that TSC1 acts genetically downstream from Akt. This observation is compatible with either TSC1 acting molecularly downstream from Akt in the linear InR-PI3K-Akt pathway, or TSC1 acting in a parallel pathway that converges on the insulin pathway downstream from Akt (Gao, 2001).
To distinguish between these two possibilities, cells were generated that were doubly mutant for null alleles of PTEN and TSC1. PTEN is a negative regulator of the InR-PI3K-Akt pathway, and loss of PTEN results in increased Akt activity and cellular growth. It was reasoned that if TSC1 acts downstream from Akt within the InR-PI3K-Akt pathway, it might be expected that PTEN;TSC1 double-mutant cells would show a similar cell-size phenotype to either single mutant. However, if TSC1 acts parallel to the InR-PI3K-Akt the pathway, it might be expected that PTEN;TSC1 double-mutant cells would show additive effects on cell size as compared with each single mutant. PTEN;TSC1 double-mutant photoreceptors are 2.9 times the size of wild-type cells, as compared with 1.9 for PTEN - and 1.8 for TSC1 -. This result strongly suggests that the TSC genes function in a parallel pathway that converges on the insulin pathway at a point downstream from Akt (Gao, 2001).
In the course of these studies, a striking genetic interaction between the TSC genes and inr mutations was observed. Flies homozygous for a strong loss-of-function inr allele, are larval lethal. However, homozygous inr mutant flies that are heterozygous for TSC1 can survive to adults, suggesting that a mere 50% reduction in the dosage of the TSC1 gene can rescue the developmental arrest of an inr mutant (Gao, 2001).
Similarly, heterozygosity of TSC2 is sufficient to rescue the lethality of another inr mutant. Flies carrying the allelic combination inr353/inrl(3)05545 are 100% lethal. However, approximately 39% of inr353/inrl(3)05545 flies that are heterozygous for TSC2 can survive to adults. Taken together, these results provide convincing in vivo evidence that the TSC genes are negative regulators of insulin signaling in development (Gao, 2001).
The Drosophila gene insulin-like receptor (InR) is homologous to mammalian insulin receptors as well as to C. elegans daf-2, a signal transducer regulating worm dauer formation and adult longevity. A heteroallelic, hypomorphic genotype of mutant InR is described that yields dwarf females with up to an 85% extension of adult longevity and dwarf males with reduced late age-specific mortality. Treatment of the long-lived InR dwarfs with a juvenile hormone analog restores life expectancy toward that of wild-type controls. It is concluded that juvenile hormone deficiency, which results from InR signal pathway mutation, is sufficient to extend life-span, and that in flies, insulin-like ligands nonautonomously mediate aging through retardation of growth or activation of specific endocrine tissue (Tatar, 2001).
Molecular similarity between fly InR and worm daf-2 suggests that mutants of InR in flies should affect adult life-span, as do mutants of daf-2 in worms. InR and daf-2 are members of the insulin receptor family with homology to mammalian insulin and insulin-like growth factor-1 (IGF-1) receptors. Worms carrying temperature-sensitive mutations in daf-2 form dauers at high temperatures, but at lower temperatures develop directly into adults with extended longevity and resistance to exogenous stress. Genotypes homozygous for mutant InR have been reported to be lethal, but several heteroallelic combinations of InR alleles produce viable, dwarf adults that are slow to develop: InREC34/InRE19 and InRGC25/InRE19, and InRE19/InRp5545. In addition, InRE19/InRE19 is viable and dwarf, once crossed into a new isogenic background. Dwarf females eclose with extremely immature ovaries, and the egg chambers of young adults remain previtellogenic (Tatar, 2001).
Measurement of INR kinase activity indicates that the InRp5545 and InRE19 alleles both confer loss of INR function. Basal activity of heterozygotes +/InRE19 and +/InRp5545 was 45% of that of the wild type. Insulin stimulation increases kinase activity of INR from +/+ and +/InRE19 flies by 60%, but only by 26% from +/InRp5545 flies. Basal kinase activity of INR from InRE19/InRE19 and from InRp5545/InRE19 flies is 14% and 11% of that of the wild type, respectively; neither type is stimulated by insulin. InRp5545 is a P-element insertion in exon-1; the molecular lesion of InRE19 has yet to be identified, but it does not appear to occur in the known coding region of the gene (Tatar, 2001).
Life tables of InR mutant adults were compared to concurrent cohorts of a wild-type coisogenic strain. Dwarfs of InREC34/InRE19 and InRGC25/InRE19 are short-lived. Dwarf InRE19/InRE19 and nondwarf +/InRp5545 have moderately reduced survival; nondwarf +/InRE19 individuals are normal. In contrast, females of InRp5545/InRE19 are 85% longer lived than wild-type controls and overall present reduced age-specific mortality. The life-span of female D. melanogaster is also extended by mutation of the insulin receptor substrate homolog chico. Survivorship among male InR genotypes follows the pattern observed for females. Relative to the wild type, InRp5545/InRE19 males exhibit high mortality as early adults, but because of reduced mortality at late ages, dwarf life expectancy at 10 days is 43% greater than that of controls. It is likely that not all InR alleles increase longevity because the gene is highly pleiotropic, with some alleles producing developmental defects that carry over to the adult stage, which counterbalance positive effects of the allele upon aging (Tatar, 2001).
The fact that InR mutants are nonvitellogenic suggests a plausible mechanism for the extended longevity of InRp5545/InRE19 flies. Drosophila overwinter as adults in a reproductive diapause where egg development is arrested at previtellogenic stages. In many insects, including Drosophila, reproductive diapause is proximally controlled through down-regulation of juvenile hormone (JH) synthesis by the corpora allata (CA). Ovaries of InR dwarf females morphologically resemble ovaries of diapause wild-type flies, and exogenous application of the JH analog methoprene to dwarf females initiates vitellogenesis. Females of InRE19/InRE19 respond to a single treatment of methoprene in a dose-dependent manner, but females of InRp5545/InRE19 require continuous exposure to hormone to induce any vitellogenesis. Direct assay of adult JH synthesis verified that CA activity is reduced in InR dwarfs to about 23% of the wild-type level. Because reduced JH synthesis is seen in InRE19/InRE19 flies, which exhibit normal life-span, as well as in long-lived InRp5545/InRE19 flies, the simple lack of JH may not be enough to extend longevity (Tatar, 2001).
Loss of corpora allata JH accounts for dwarf infertility. Mutation of InR may increase longevity because infertility reduces allocation of metabolic resources to reproduction and frees resources for somatic maintenance or because reduced JH in mutant flies induces specific physiological mechanisms of somatic persistence normally expressed during adult reproductive diapause. Adult D. melanogaster in reproductive diapause age at negligible rates and are stress resistant; these traits are reversed by treatment with methoprene. Extended survival is characteristic of adult reproductive diapause in acridid grasshoppers and in the monarch butterfly, and surgical ablation of the corpora allata to eliminate adult JH synthesis induces both diapause and increased longevity. Consistent with the notion that reduced JH synthesis can directly extend life-span, InR dwarf flies show somatic physiological changes: (1) triglycerides are elevated fourfold (F = 32.2, P < 0.001), as observed in diapause D. triauraria and in dwarf D. melanogaster mutant for chico, and (2) Cu/Zn-superoxide dismutase concentration is increased twofold, as is characteristic of long-lived mutants of C. elegans. Measured in young adults, no difference in mass-specific metabolic rate is detected. It is suggested that infertility need not be the direct cause of slowed aging in InR mutants; JH may simply control both fertility and life-span (Tatar, 2001).
To test directly whether JH modulates survival in InRp5545/InRE19 female dwarfs, a test was made of whether treatment with methoprene restores wild-type longevity to these mutants, even if it does not fully restore fertility. In concurrent trials of dwarf and wild-type flies, survival of methoprene-treated InRp5545/InRE19 females is reduced toward the level observed in coisogenic controls. This rescue is physiological rather than toxicological because, in wild-type controls, methoprene produces no significant change relative to ethanol-treated flies (Tatar, 2001).
The InR pathway may alter endocrine function in two ways. Adult CA is derived from neurosecretory tissue of the larval ring gland. Adult dwarf CA may be immature upon metamorphosis as a result of cell autonomous effects of InR upon the development of neuroendocrine cells. A second way InR may alter endocrine function is that JH secretion by CA may be impaired by reduced neuropeptide transmission in the adult brain, due to a reduction of INR function in brain areas where it is normally expressed (Tatar, 2001).
In C. elegans, the insulin/IGF-1 pathway influences dauer formation, fertility, and aging in part through nonautonomous, secondary signaling; sterility is not required for extended longevity in C. elegans because some long-lived daf-2 are fully fertile. For Drosophila, InR affects neurosecretory tissue specialized for secretion of juvenile hormone. Therefore, mutations in the insulin signaling pathway in flies autonomously affect cell proliferation, growth, and body size, but nonautonomously affect diapause, reproduction, and life-span through effects upon specific neuroendocrine cells. Deficiency in a juvenoid-like hormone signal in worms and in flies may extend longevity because its absence leads to the inappropriate expression of parallel physiological programs normally reserved for dauer or diapause (Tatar, 2001).
This invertebrate model may have parallels with mammalian aging. Ames and Snell mice are mutant for the genes Prop-1 or Pit-1, respectively, and are defective for pituitary development. Consequently, they are deficient in growth hormone, prolactin, and thyroid-stimulating hormone, leading to hypothyroidism and presumably reduced synthesis of thyroxin, a retinoid hormone with potential functional similarity to JH. These mice are phenotypically dwarf, mildly obese, and long-lived. A remarkably similar phenotype is observed in mice lacking insulin receptor function in the central nervous system or those lacking the chico homolog, IRS-2, in all tissues: increased fat mass and infertility with accompanying neuroendocrine deficiency. Although effects on life-span in these mice remain to be determined, the concordance of phenotypes suggests that insulin signaling may be central to a common mechanism that exists across taxa for the neuroendocrine regulation of metabolism and the reproductive state, and their associated consequences upon aging (Tatar, 2001).
The Drosophila gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode C. elegans, insulin/IGF signaling regulates adult longevity. Mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span is not a result of impaired oogenesis in chico females, nor is it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved (Clancy, 2001).
In Drosophila, the insulin/IGF receptor INR, the insulin receptor substrate Chico, the phosphatidylinositol 3-kinase (PI3K) Dp110/p60, and the PI3K target protein kinase B (PKB, also known as DAkt1) form a signaling pathway that regulates growth and size. The effects on aging of hypomorphic mutations in Inr (equivalent to daf-2) and PKB, and null mutations in chico and the catalytic (Dp110, equivalent to age-1) and adapter (p60) PI3K subunits were examined. All mutants were tested as heterozygotes. chico1 and PKB3 homozygotes and InrGC25/InrE19 transheterozygotes, which form viable dwarf adults, were also examined. The remaining mutations were homozygous lethal (Clancy, 2001).
Most mutants tested had normal or significantly decreased life-span. For example, PKB3 homozygotes and InrGC25/InrE19 flies are short-lived. By contrast, chico1 extends life-span. Homozygous chico1 females exhibit an increase of median and maximum life-span of up to 48% and 41%, respectively. chico1 heterozygotes also exhibit increases in median life-span of up to 36% and 13% in females and males, respectively. Homozygous males, however, are slightly short-lived (Clancy, 2001).
Of the mutations tested, only chico1 increases life-span. This may be because the effect of reduced IIS on life-span depends on the degree to which signaling is reduced. Unlike the other null mutations in IIS genes tested, chico1 is not homozygous lethal, presumably because the INR receptor can signal to PI3K directly, as well as indirectly via Chico. Thus, chico1 mutants may be long-lived because of the relatively mild reduction in pathway activity that they bring about. Notably, severe IIS mutations in C. elegans can cause premature mortality in some adults, although the maximum life-span of populations is invariably increased. This is probably why InrGC25/InrE19 flies are short-lived: Demographic analysis indicates that a reduction in the age-specific mortality rate acceleration occurs, whose effect on survival is masked by an elevated rate of age-independent mortality. Furthermore, a different heteroallelic Drosophila Inr mutant to that tested here exhibits an 85% increase in female life-span. By contrast, in short-lived PKB3 populations, no reduction in mortality rate acceleration is seen. This raises the possibility that a second pathway downstream of chico might regulate aging in Drosophila. Interestingly, Chico contains potential binding sites for the Drk/Grb2 docking protein, consistent with signaling via Ras/mitogen-activated protein kinase (Clancy, 2001). 30 March 2008PDK1 regulates growth through Akt and S6K in Drosophila
The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, dPDK1 (FlyBase name: Protein kinase 61C) controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K. Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).
The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK11/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP3 promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP3, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP3-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).
These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).
Insulin/IGF signaling during development controls growth and size, possibly by coordinating the activities of the Ras and PI 3-kinase signaling pathways. In vertebrates, the IR and IGFR act through IRS1-IRS4 proteins, which are multifunctional adaptors that link insulin and IGF signaling to the Ras/MAPK and phosphoinositide 3'-kinase (PI 3-kinase) signaling pathways. The pleckstrin homology domain (PH) and phosphotyrosine binding domain (PTB) of the IRS proteins are believed to mediate binding to phosphoinositol phosphates and the juxtamembrane NPXY motif of IR/IGFR, respectively. Grb2 (Drosophila homolog Drk) is an adaptor protein containing SH2 and SH3 domains. It has been suggested that Grb2 may, via its binding to IRS, link insulin/IGF to the Ras/MAPK pathway and thereby control proliferation. The Drosophila homolog of the SH2 domain containing p85 PI 3-kinase adaptor subunit, p60, binds Chico/IRS and thereby recruits the p110 catalytic subunit of PI 3-kinase [which converts phosphoinositol(4,5)P2 (PtdIns(4,5)P2) into phosphoinositol(3,4,5)P3 (PtdIns(3,4,5)P3)] to the plasma membrane. The p110 PI 3-kinase belongs to the class I PI 3-kinases implicated in the metabolic effects of insulin. The classical effectors that mediate the biological outcomes of insulin and IGF downstream of IRS have been divided into two functional branches: the Ras/MAPK proliferation pathway, and the PI 3-kinase metabolic, growth and survival pathway (Oldham, 2002).
To analyze the role of the different domains of Chico/IRS under physiological conditions, a panel of effector site mutants was created in a genomic rescue construct for chico that disrupts the PH or the PTB domains or the putative binding sites of Drk/Grb2 and p60. The constructs include the cis-regulatory sequences that permit expression of chico in its normal spatial and temporal pattern. The wild-type chico construct fully restores the defects of chico homozygous null mutants. In this manner, the effector site mutants were assayed for the ability to rescue the three different phenotypes associated with complete loss of Chico function: body size reduction, female sterility and lipid alterations. The Drk/Grb2 consensus binding site mutant is able to fully rescue the reduced weight to the same extent as the wild-type rescue construct. Therefore, the presence of a functional Drk binding site in Chico and thus the link to the activation of the Ras/MAPK kinase pathway is not required for its wild-type function. In contrast, the PH and PTB domain mutants and the double p60 PI 3-kinase binding site mutant were unable to rescue the reduced body weight. The latter result is surprising because InR contains additional functional PI 3-kinase binding sites in its C-terminal tail, an extension shared only with the C. elegans InR homolog, Daf-2, and not the mammalian IR or IGFR. This suggests that the presence of additional p60 binding sites in the InR C-terminal tail is not sufficient in vivo to mediate wild-type levels of growth and proliferation in the absence of the Chico p60 PI 3-kinase binding sites and that the InR C-terminal tail may contribute only low levels of PI 3-kinase signaling. Although the PTB domain mutant fails to restore normal body weight, it rescues the female sterility associated with the loss of Chico function. With the exception of the full rescue of the lipid accumulation observed in Drk/Grb2 mutant, all the other effectors only partially restore the change in lipid accumulation (Oldham, 2002).
To test whether increasing PtdInsP3 levels in an InR or PI 3-kinase p110 mutant background is sufficient to restore growth, the function of a negative regulator of the insulin pathway was eliminated. The 3'-phosphoinositol-specific lipid phosphatase, PTEN acts as a negative regulator of the PI 3-kinase pathway by converting PtdInsP3 generated by PI 3-kinase into PtdInsP2. Used were a null (Pten2L117) and a hypomorphic (Pten2L100) allele of Pten, identified in a screen for genes involved in growth control. As shown by HPLC analysis of the phospholipids in extracts of Pten mutant larvae, the loss of PTEN function results in a 2-fold increase in PtdInsP3 levels. This is consistent with the increase in PtdInsP3 seen in Pten-deleted murine fibroblasts. One prominent biological effect of these increased PtdInsP3 levels in Drosophila is a substantial increase in size in both larvae and pupae. To test whether loss of PTEN function, and consequently increased PtdInsP3 levels, is sufficient to restore growth or viability in InR null mutants, InR and Pten double mutants were generated by creating mosaic animals using the eyeless-Flipase (eyFlp) tissue-specific recombination system. In such animals, the head consists of homozygous mutant tissue, whereas the rest of the body is heterozygous for the same mutation. While loss of PTEN function (Pten2L117) in the head results in a fly with a disproportionately larger head (with more and larger cells), loss of InR function (InR327) results in flies with smaller heads (pinhead) compared to the wild type. Heads doubly mutant for Pten2L117and InR327, however, are almost the size of heads singly mutant for Pten2L117. Also, two different lethal heteroallelic InR combinations (InR304/InR327 or InR304/InR25), which arrest at the second larval instar stage, develop to the pupal stage (15%-17% of 33% expected) and even to pharate adults in the presence of reduced PTEN levels (Pten2L117/Pten2L100). These results demonstrate that complete loss of PTEN function can largely substitute for InR-mediated growth and proliferation in the absence of InR function and that the Ras/MAPK pathway plays little or no role in the InR mediated control of cell growth. This notion is further supported by the observation that complete loss of InR function in the compound eye does not result in a loss of photoreceptors, a hallmark of loss of Ras pathway function (Oldham, 2002).
The rescue of lethal, null InR mutant combinations to near viability by reducing PTEN activity strengthens the argument that a PtdInsP3-dependent signaling pathway is the primary effector for InR-derived growth and proliferation. In support of this observation, PI 3-kinase and Akt have been isolated as retroviral oncogenes, suggesting that activation of PI 3-kinase and Akt is sufficient to mediate growth, proliferation, and oncogenesis in vertebrate systems. In Drosophila and mammals, overexpression of PI 3-kinase causes increased growth; but this is not sufficient for proliferation as is the removal of Pten. From this premise, it has been proposed that PI 3-kinase and PTEN regulate similar yet distinct pathways. Alternatively, it is possible that they do function uniquely in the same pathway and that the difference may be due to altered location and function because of overexpression, or to differential feedback of PI 3-kinase versus PTEN. For example, since PI 3-kinase has been shown to act as a serine/threonine protein kinase on IRS, this may have a negative feedback effect on the insulin pathway that might not be evident in Pten loss-of-function mutations. Nevertheless, PI 3-kinase is absolutely critical in controlling size because using an allelic series of PI 3-kinase mutants in combination with the ey-Flp sytem resulted in a range of different head sizes. Furthermore, expressing an activated and dominant-negative form of PI 3-kinase in Drosophila imaginal discs or the heart of the mouse also leads to a corresponding increase or decrease in cell and organ size. Thus, the PI 3-kinase/PTEN cycle can be considered a dedicated growth rheostat, and the InR pathway is an evolutionary conserved module for regulating the range of growth and size (Oldham, 2002).
Loss of PTEN function results in a metabolically similar phenotype as loss of murine PTP1B (Ptpn1), an IR-specific tyrosine phosphatase, in that hyperactivation of the IR pathway causes resistance to high-fat-diet-induced obesity because of increased basal metabolism. These metabolic lipid effects have likely been conserved during evolution because the increased lipid levels in chico mutants are reminiscent of the enhanced lipid content in Irs2 deleted and NIRKO mice (Oldham, 2002).
Collectively, these data firmly establish Drosophila as a valid model organism for the study of metabolic diseases like diabetes and obesity as well as for the study of growth disorders like cancer. Pten mutant flies are larger in size due to increased cell size and number, but have a corresponding decrease in energy stores, a situation completely opposite that of mutations in positive components of the insulin signaling pathway like InR, chico, PI 3-kinase, and dAkt. These large viable Pten mutants show that a reduction of PTEN function is sufficient for increased organism size. This fact suggests that the four-fold size difference between viable InR and Pten mutants can simply be controlled by the amount of PtdInsP3 and this phenomenon may possibly be extended to vertebrate size regulation. Thus, in Drosophila, the InR/PI 3-kinase/PTEN pathway combines both metabolism and growth control into one pathway that later diverged into two separate, yet interacting systems in mammals (Oldham, 2002).
Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).
Stem cells reside in specialized niches that provide signals required for their maintenance and division. Tissue-extrinsic signals can also modify stem cell activity, although this is poorly understood. This study reports that neural-derived Drosophila insulin-like peptides (DILPs) directly regulate germline stem cell division rate, demonstrating that signals mediating the ovarian response to nutritional input can modify stem cell activity in a niche-independent manner. A crucial direct role is demonstrated for DILPs in controlling germline cyst growth and vitellogenesis (LaFever, 2005).
Germline and somatic stem cells support oogenesis throughout adult life in Drosophila. Germline stem cells (GSCs) reside within a specialized niche where they are exposed to a unique combination of signals required for stem cell function. However, GSCs are also controlled by tissue-extrinsic signals, such as Drosophila insulin-like peptides (DILPs), which mediate the ovarian response to nutrition. On a protein-rich diet, germline and somatic stem cells have high division rates, and their progeny exhibit high division and development rates. On a protein-poor diet or under reduced insulin signaling, rates of division and development are reduced, and progression through vitellogenesis is blocked. It remains unclear, however, how DILPs control the response of GSCs in coordination with their differentiating progeny and with follicle cells (LaFever, 2005).
In adult females, DILPs are produced in two clusters of medial neurosecretory cells in the brain, and stage 10 follicle cells express dilp5 mRNA. Ablation of brain DILP-producing cells results in reduced egg production rates and a partial block in vitellogenesis. To examine the role of the brain DILP-producing cells in previtellogenic stages, they were ablated and follicle cell proliferation rates were measured. Females missing brain DILP-producing cells (ablated) have a severely impaired ability to up-regulate follicle cell proliferation in response to a protein-rich diet. The rate of germline development is reduced in coordination with follicle cell divisions because no abnormalities are observed in previtellogenic egg chambers. Ablation of DILP-producing cells reduces the size of eclosed adults and delays development. Ablated females in which these developmental defects are rescued by an hs-dilp2 transgene expressed during larval stages show a reduced follicle cell proliferation rate, comparable to that of nonrescued, ablated females. Thus, the impaired response to a protein-rich diet is not a secondary consequence of the developmental defects. Moreover, the 2.3-fold delay caused by ablation of brain DILP-producing cells is very similar to that caused by blocking reception of DILP signals by the germ line. This indicates that the brain is the major source of DILPs that determine the rate of egg chamber development with little, if any, contribution from dilp5-expressing follicle cells (LaFever, 2005).
To examine whether DILPs control the rate of germline development directly or indirectly, germline cysts unable to respond to DILPs were created by inducing Drosophila insulin receptor (dinr) mutant clones using the flipase (FLP)/FLP-recognition target (FRT) technique. Germline cysts homozygous for dinr339, a genetic null allele, had normal morphology and correct cell number; however, 83% of these cysts were developmentally delayed, showing markedly decreased size relative to neighboring wild-type egg chambers. Further quantification of these data showed a 2.4-fold delay in the development of dinr339 cysts. Similar results were obtained for germline cysts homozygous for dinrE19 and dinr353, which are viable hypomorphic alleles; 78% and 64% of dinrE19 and dinr353 cysts, respectively, were developmentally delayed. These results reveal that dinr function is required cell autonomously for a normal rate of germline cyst development. Thus, the rate of cyst development is regulated by a DILP signal that is received directly (LaFever, 2005).
Progression through vitellogenesis requires DILP signaling; however, it has been unclear whether this role is direct. Reduced juvenile hormone levels are present in homozygous viable dinr mutants, and the block in yolk uptake in these mutants can be partially bypassed by treatment with methoprene, a juvenile hormone analog, suggesting an indirect role for DILPs in promoting vitellogenesis. To specifically address whether direct activation of germline cysts by DILPs is required for vitellogenesis, mosaic ovarioles were examined in which the entire germ line was homozygous dinr mutant for the ability of their egg chambers to undergo vitellogenesis. All egg chambers containing dinr339 or dinrE19 homozygous mutant cysts failed to progress through vitellogenesis and degenerated. In the case of dinr353, the allele with the higher level of dinr activity, only one out of six egg chambers containing homozygous mutant cysts failed to undergo vitellogenesis. These results suggest that the level of insulin signaling within the germ line controls vitellogenesis, revealing a direct role for DILPs in this process. Moreover, complete loss of dinr function in the germ line results in a complete block in vitellogenesis, whereas this block is partial upon ablation of brain DILP-producing cells. Thus, DILP5 expressed in stage 10 follicle cells likely signals in combination with brain DILPs to regulate vitellogenesis (LaFever, 2005).
It was next asked whether DILPs control GSC division rate directly by binding to receptors on their surface (a cell-autonomous requirement for dinr in GSCs) or indirectly by regulating signals produced by niche cells (a non-cell-autonomous requirement). dinr mosaic ovarioles containing one wild-type and one mutant GSC were examined and the number of wild-type versus mutant cystoblasts and cysts present in their germaria was counted. Because each cystoblast or cyst corresponds to one GSC division, the ratio of mutant to wild-type cystoblasts and cysts is a measure of their relative division rates. For dinr339 homozygous mutant GSCs, a relative division rate of 0.31 was found, whereas, for wild-type GSCs, it was 0.90. Similarly, the relative division rates of dinr353 and dinrE19 GSCs were 0.55 and 0.65, respectively. Thus, dinr homozygous mutant GSCs divide more slowly than wild-type GSCs, and GSC division rate appears sensitive to the level of dinr activity. These results demonstrate that GSCs directly receive the DILP signal to regulate their division rate without mediation by the stem cell niche (LaFever, 2005).
Germline and somatic cells respond to nutritional status in a coordinated manner; however, it is unclear whether somatic cells receive the DILP signal directly (a cell-autonomous role of dinr in follicle cell proliferation) as does the germ line, or indirectly through secondary signals (a non-cell-autonomous role). The percentages of dinr mutant and control follicle cells were measured in mosaic ovarioles carrying one wild-type and one dinr mutant somatic stem cell. If follicle cells receive the DILP signal directly, the reduced level of insulin signaling in dinr mutant follicle cells should result in lower rates of proliferation (i.e., fewer mutant than control follicle cells should be observed), whereas if they receive the signal indirectly, the proliferation rates should be similar. In dinrE19 mosaic ovarioles, 51% of follicle cells were wild-type and 49% were mutant, indicating similar proliferation rates. dinr mutant follicle cells appeared to enter the endoreplicative cycle normally, but pycnotic (degenerating) nuclei and cell death were observed within dinrE19 and dinr339 mutant follicle cell clones starting at stage 8. These results reveal that although a reduction in dinr activity delays germline cyst development cell autonomously, it does not cause a cell-autonomous reduction in follicle cell proliferation rate. Furthermore, in ovarioles carrying a fully dinr mutant germ line, excess follicle cells were not observed, showing that proliferation of surrounding wild-type follicle cells remains coordinated with germline growth. These results suggest that follicle cells respond indirectly to increased DILP levels through a secondary signal from the germ line. Similar degrees of coordination between germ line and soma have been observed in the presence of developmentally delayed dMyc mutant germline clones (LaFever, 2005).
These data demonstrate that tissue-extrinsic DILP signals can directly modify GSC proliferative activity, acting in parallel to signals from their niche. Evidence is provided that, in addition to its previously reported indirect roles in Drosophila and mammals through secondary hormonal signals, insulin signaling plays a crucial direct role during Drosophila oogenesis in regulating not only GSC division rate but also germline cyst development rate and progression through vitellogenesis. Insulin may, therefore, have important direct roles in mammalian oogenesis. Finally, the data suggest that the coordinated response of germline and somatic cells to nutrition involves communication between these tissues. These results have broad significance, in light of the long-known effects of nutrition on human fertility and of the high degree of conservation of insulin signaling functions (LaFever, 2005).
Alcedo, J. and Kenyon, C. (2004). Regulation of C. elegans longevity by specific gustatory and olfactory neurons. Neuron 41: 45-55. 14715134
Araki, E., et al. (1994). Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372: 186-190. 7526222
Bateman, J. M. and McNeill, H. (2004). Temporal control of differentiation by the Insulin receptor/Tor pathway in Drosophila. Cell 119: 87-96. 15454083
Baugh, L. R. and Sternberg, P. W. (2006). DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest. Curr. Biol. 16(8): 780-5. 16631585
Britton, J. S. and Edgar, B. A. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125(11): 2149-58. 9570778
Britton, J. S., et al. (2002). Drosophila's insulin/pi3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2: 239-249. 11832249
Brogiolo, W., et al. (2001). An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Current Biology 11: 213-221. 11250149
Butler, A. A. and Roith, D. L. (2001). Control of growth by the somatropic axis: Growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63: 141-164. 11181952
Chen, C., Jack, J. and Garofalo, R. S. (1996). The Drosophila insulin receptor is required for normal growth. Endocrinology 137(3): 846-856. 8603594
Clancy, D. J., et al. (2001). Extension of life-span by loss of Chico, a Drosophila Insulin receptor substrate protein. Science 292: 104-6. 11292874
Danielsen, A. G., et al. (1995). Activation of protein kinase Ca inhibits signaling by members of the insulin receptor family. J. Biol. Chem. 270(37): 21600-21605. 7545165
Dennis, P., et al. (2001). Mammalian TOR: a homeostatic ATP sensor. Science 294: 1102-1105. 11691993
Fernandez, R., et al. (1995). The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential. EMBO J. 14(14): 3373-3384. 7628438
Fernandez-Almonacid, R. and Rosen, O. M. (1987). Structure and ligand specificity of the Drosophila melanogaster insulin receptor. Molec. Cell. Biol. 7: 2718-2727. 3118188
Fernandez, A. M., et al. (2001). Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes. Genes Dev. 15: 1926-1934. 11485987
Gao, X. and Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 15: 1383-1392. 11390358
Garofalo, R. S. and Rosen, O. M. (1988). Tissue localization of Drosophila melanogaster insulin receptor transcripts during development. Molec. Cell. Biol. 8: 1638-1647. 2454394
Gorczyca, M., Augart, C. and Budnik, V. (1993). Insulin-like receptor and insulin-like peptide are localized at neuromuscular junctions in Drosophila. J. Neurosci. 13(9): 3692-3704. 8366341
Hetru, C., et al. (1991). Isolation and structural characterization of an insulin-related molecule, a predominant neuropeptide from Locusta migratoria Eur. J. Biochem. 201: 495-499
Hopper, N. A. (2006). The adaptor protein soc-1/Gab1 modifies growth factor receptor output in C. elegans. Genetics 173(1): 163-75. 16547100
Iiboshi, Y., et al. (1999). Amino acid-dependent control of p70(s6k). Involvement of tRNA aminoacylation in the regulation. J. Biol. Chem. 274: 1092-1099. 9873056
Ikeya, T., et al. (2002). Nutrient-dependent expression of Insulin-like Peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12: 1293-1300. 12176357
Kawakami, A., et al. (1989). Structure and organization of four clustered genes that encode bombyxin, an insulin-related brain secretory peptide of the silkmoth Bombyx mori Proc. Natl. Acad. Sci. 86: 6843-6847. 2674935
Kim, H., Rogers, M. J., Richmond, J. E. and McIntire, S. L. (2004). SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans. Nature 430: 891-896. Medline abstract: 15318222
Kleijn, M. and Proud, C. G. (2000). Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells. Biochem. J. 347: 399-406. 10749669
Lee, R. Y., Hench, J. and Ruvkun, G. (2001). Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr. Biol. 11(24):1950-7. 11747821
Lee, S. S., Kennedy, S., Tolonen, A. C. and Ruvkun, G. (2003). DAF-16 target genes that control C. elegans life-span and metabolism. Science 300(5619): 644-7. 12690206
LaFever. L. and Drummond-Barbosa, D. (2005). Direct control of germline stem cell division and cyst growth by neural insulin in Drosophila. Science 309: 1071-1073. Medline abstract: 16099985
Leibiger, B., et al. (2001). Selective insulin signaling through a and b insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic cells. Molec. Cell 7: 559-570. 11463381
Lin, K., Dorman, J.B., Rodan, A., and Kenyon, C. (1997). daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278: 1319-1322. 9360933
Lin, K., Hsin, H., Libina, N. and Kenyon, C. (2001). Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat. Genet. 28(2): 139-45. 11381260
Marin-Hincapie, M. and Garofalo, R. S. (1995). Drosophila insulin receptor: Lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation. Endocrinology 136(6): 2357-2366. 7750456
Marin-Hincapie, M. and Garofalo, R. S. (1999). The carboxyl terminal extension of the Drosophila insulin receptor homologue binds IRS-1 and influences cell survival. J. Biol. Chem. 274(35): 24987-24994. 10455177
Marr, M. T., D'Alessio, J. A., Puig, O. and Tjian, R. (2007). IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev. 21(2): 175-83. Medline abstract: 17234883
Masumura, M., Ishizaki, H., Nagata, K., Kataoka, H., Suzuki, A. and Mizoguchi, A. (1997). Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 118: 349-357. 9440228
Masumura, M., et al. (2000). Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori. Gen. Comp. Endocrinol. 118: 393-399. 10843790
Montanaro, F. and Carbonetto, S. (2003). Targeting dystroglycan in the brain. Neuron 37: 193-196. Medline abstract: 12546815
Murakami, S. and Johnson, T. E. (2001). The OLD-1 positive regulator of longevity and stress resistance is under DAF-16 regulation in Caenorhabditis elegans. Curr. Biol. 11(19): 1517-23. 11591319
Nishida, Y., et al. (1986). Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor. Biochem. biophys. Res. Commun. 141: 474-481. 3099787
Ogg, S., Paradis, S., Gottlieb, S., Patterson, G. I., Lee, L., Tissenbaum, H. A. and Ruvkun, G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389: 994-999. 9353126
Oldham, S., et al. (2000). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14: 2689-2694. 11069885
Oldham, S., et al. (2002). The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP3 levels. Development 129: 4103-4109. 12163412
Ookuma, S., Fukuda, M. and Nishida, E. (2003). Identification of a DAF-16 transcriptional target gene, scl-1, that regulates longevity and stress resistance in Caenorhabditis elegans. Curr. Biol. 13(5): 427-31. 1262019
Piovant, M., and Lena, P. (1988). Membrane glycoproteins immunologically related to the human insulin receptor are associated with presumptive neuronal territories and developing neurones in Drosophila melanogaster. Development. 103(1): 145-56. 3143540
Petruzzelli, L., et al. (1985). Acquisition of insulin-dependent protein tyrosine kinase activity during Drosophila embryogenesis. J. Biol. Chem. 260(30): 16072-5. 3934169
Petruzzelli, L., et al. (1986). Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibody. Proc. Natl. Acad. Sci. 83(13): 4710-4. 3014506
Piekny, A. J., Wissmann, A. and Mains, P. E. (2000). Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase. Genetics 156(4): 1671-89. 11102366
Pimentel, B., et al. (1996). Insulin acts as an embryonic growth factor for Drosophila neural cells. Biochem. biophys. Res. Commun. 226(3): 855-861. 8831701
Poltilove, R. M. K., et al. (2000). Characterization of Drosophila insulin receptor substrate. J. Biol. Chem. 275(30): 23346-23354. 10801879
Puig, O., Marr, M. T., Ruhf, R. L. and Tjian, R. (2003). Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway. Genes and Development 17: 2006-2020. 12893776
Puig, O. and Tjian, R. (2005). Transcriptional feedback control of insulin receptor by dFOXO/FOXO1. Genes Dev. 19(20): 2435-46. 16230533
Qu, Q. and Smith, F. I. (2004). Alpha-dystroglycan interactions affect cerebellar granule neuron migration. J. Neurosci. Res. 76: 771-782. Medline abstract: 15160389
Raught, B., Gingras, A. C. and Sonenberg, N. (2001). The target of rapamycin (TOR) proteins. Proc. Natl. Acad. Sci. 98: 7037-7044. 11416184
Richard-Parpaillon, L., et al. (2002). The IGF pathway regulates head formation by inhibiting Wnt signaling in Xenopus. Dev. Biol. 244: 407-417. 11944947
Rintelen, F., Stocker, H., Thomas, G. and Hafen, E. (2001). PDK1 regulates growth through Akt and S6K in Drosophila. Proc. Natl. Acad. Sci. 98: 15020-15025. 11752451
Ruan, Y., et al. (1995). The Drosophila insulin receptor contains a novel carboxyl-terminal extension likely to play an important role in signal transduction. J. Biol. Chem. 270(9): 4236-4243. 7876183
Rulifson, E. J., Kim, S. K. and Nusse, R. (2002). Ablation of insulin-producing neurons in flies: Growth and diabetic phenotypes. Science 296: 1118-1120. 12004130
Samuelson, A. V., Carr, C. E. and Ruvkun, G. (2007). Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants. Genes Dev. 21(22): 2976-94. PubMed citation: 18006689
Schmelzle, T. and Hall, M. N. (2000). TOR, a central controller of cell growth. Cell 103: 253-262. 11057898
Shcherbata, H. R., et al. (2007). Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy. EMBO J. 26(2): 481-93. Medline abstract: 17215867
Smit, A. B., et al. (1988). Growth-controlling molluscan neurons produce the precursor of an insulin-related peptide. Nature 331: 535-538
Tamemoto, H., et al. (1994). Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372(6502): 182-6. 7969452
Tatar, M., et al. (2001). A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292: 107-10. 11292875
Tiessen, J., Underwood, L. and Ketelslegers, J. (1999) Regulation of insulin growth factor-1 in starvation and injury. Nutr. Rev. 57: 167-176. 10439629
Tissenbaum, H. A. and Guarente, L. (2001). Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410(6825): 227-30. 11242085
Wessells, R. J., Fitzgerald, E., Cypser, J. R., Tatar, M. and Bodmer, R. (2004). Insulin regulation of heart function in aging fruit flies. Nat. Genet. 36(12): 1275-81. 15565107
Withers, D. J., et al. (1998). Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391: 900-904. 9495343
Wu, Q., Zhang, Y., Xu, J. and Shen, P. (2005). Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila. Proc. Natl. Acad. Sci. 102(37): 13289-94. 16150727
Xu, G. G. and Rothenberg, P. L. (2001). Insulin receptor signaling in the beta-cell influences insulin gene expression and insulin content: evidence for autocrine beta-cell regulation. Diabetes 47: 1243-1252. 9703324
Yamaguchi, T., Fernandez, R. and Roth, R.A. (1995). Comparison of the signaling abilities of the Drosophila and human insulin receptors in mammalian cells. Biochemistry 34(15): 4962-4968. 7711018
Yeh, T. C., et al. (1996). Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase. J. Biol. Chem. 271(6): 2921-2928. 8621681
Yenush, L., et al. (1996). The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. Molec. Cell. Biol. 16(5): 2509-2517. 8628319
Yu, H. and Larsen, P. L. (2001). DAF-16-dependent and independent expression targets of DAF-2 insulin receptor-like pathway in Caenorhabditis elegans include FKBPs. J. Mol. Biol. 314(5): 1017-28. 11743719
Zhang, H., et al. (2000). Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14: 2712-2724. 11069888
date revised: 30 March 2008
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.