Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster

An insulin-degrading enzyme (IDE) from the cytoplasm of Drosophila Kc cells has been purified and characterized. The purified enzyme is a monomer with an s value of 7.2 S, an apparent Km for porcine insulin of 3 microM, and a specific activity of 3.3 nmol of porcine insulin degraded/(min.mg). N-Terminal sequence analysis of the gel-purified enzyme gave a single, serine-rich sequence. The Drosophila IDE shares a number of properties in common with its mammalian counterpart. The enzyme could be specifically affinity-labeled with [125I]insulin, has a molecular weight of 110K, and has a pI of 5.3. Although Drosophila Kc cells grow at room temperature, the optimal enzyme activity assay conditions parallel those of the mammalian IDE: 37 degrees C and a pH range of 7-8. The Drosophila IDE activity, like the mammalian enzymes, is inhibited by bacitracin and sulfhydryl-specific reagents. Similarly, the Drosophila IDE activity is insensitive to glutathione as well as protease inhibitors such as aprotinin and leupeptin. Insulin-like growth factor II, equine insulin, and porcine insulin compete for degradation of [125I]insulin at comparable concentrations (approximately 10(-6) M), whereas insulin-like growth factor I and the individual A and B chains of insulin are less effective. The high degree of evolutionary conservation between the Drosophila and mammalian IDE suggests an important role for this enzyme in the metabolism of insulin and also provides further evidence for the existence of a complete insulin-like system in invertebrate organisms such as Drosophila.     

Comparison of the enzymatic and biochemical properties of human insulin-degrading enzyme and Escherichia coli protease III.

The enzymatic and biochemical properties of human insulin-degrading enzyme and Escherichia coli protease III have been compared. Both enzymes were found to degrade insulin in such a way that its receptor binding activity was rapidly lost but its precipitability in trichloracetic acid was only slightly decreased. Both enzymes were also found to be inhibited by chelating agents. The bacterial enzyme, which could be purified in large amounts, was found to contain 0.6 mol of zinc per mol of enzyme but no detectable manganese. The mammalian enzyme but not the bacterial one was inhibited by a sulfhydryl alkylating agent. The two enzymes also differed in substrate specificity. The mammalian enzyme degraded insulin much better than insulin-like growth factor II, whereas the bacterial enzyme degraded them equally. The mammalian enzyme could be labeled by cross-linking to insulin = bombyxin II much greater than insulin-like growth factor I and II much greater than relaxin, while the bacterial enzyme was labeled by insulin-like growth factor II greater than insulin = insulin-like growth factor I much greater than relaxin much greater than bombyxin. Finally, sucrose gradient centrifugation and cross-linking studies both in vitro and in vivo indicated that active human enzyme partially existed as a homo- or heterodimer, whereas the bacterial enzyme was active as a monomer.     

Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites

Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with 125I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. Sequencing of the B chain fragments demonstrated that the Drosophila enzyme cleaves the B chain of insulin at four sites between residues B10 and B11, B14 and B15, B16 and B17, and B25 and B26. These cleavage sites correspond to four of the seven cleavage sites generated by the mammalian insulin protease. These results demonstrate that all the insulin cleavage sites generated by the Drosophila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function.     

Nutrient control of neural stem cells

The physiological status of an organism is able to influence stem cell behaviour to ensure that stem cells meet the needs of the organism during growth, and in response to injury and environmental changes. In particular, the brain is sensitive to metabolic fluctuations. Here we discuss how nutritional status is able to regulate systemic and local insulin/IGF signalling so as to control aspects of neural stem behaviour. Recent results have begun to reveal how systemic signals are relayed to neural stem cells through local interactions with a glial niche. Although much still remains to be discovered, emerging parallels between the regulation of Drosophila and mammalian stem cells suggest a conserved mechanism for how the brain responds to changes in nutritional state.     

An insulin epidermal growth factor-binding protein from Drosophila has insulin-degrading activity

We have recently described the purification and characterization of an insulin-degrading enzyme (IDE) from Drosophila melanogaster that can cleave porcine insulin, is highly conserved through evolution and is developmentally regulated. We now report that the IDE is, in fact, an insulin EGF-binding protein (dp100) that we had isolated previously from Drosophila using an antihuman EGF receptor antiserum. This conclusion is based upon the following evidence. (a) dp100, identified by its ability to cross-link to labeled insulin, EGF, and transforming growth factor-alpha (TGF-alpha), and to be immunoprecipitated by anti-EGF receptor antisera, copurifies with the IDE activity. Thus, the purified IDE can be affinity labeled with either 125I-insulin, 125I-EGF, or 125I-TGF-alpha, and this labeling is specifically inhibited with unlabeled insulin, EGF, and the insulin B chain. (b) The antiserum to the human EGF receptor, which recognizes dp100, is able to specifically immunoprecipitate the insulin-degrading activity. (c) The purified IDE preparation contains a single protein of 110 kD which is recognized by both the anti-EGF receptor antiserum and anti-Drosophila IDE antiserum. (d) Polyclonal antiserum to the purified IDE, which specifically recognized only the 110-kD band in Drosophila Kc cells, immunoprecipitates dp100 cross-linked to 125I-TGF-alpha and dp100 cross-linked to 125I-insulin from the purified IDE preparation. (e) EGF, which competes with insulin for binding to dp100, also inhibits the degradation of insulin by the purified IDE. These results raise the possibility that a functional interaction between the insulin and EGF growth factor families can occur which is mediated by the insulin-degrading enzyme.     

Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. We found that dCbl was highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increased sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl resulted in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulated the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolished the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increased insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells.     

Characterization of a Drosophila protein that binds both epidermal growth factor and insulin-related growth factors

The identification of a novel protein from Drosophila melanogaster that binds both mammalian epidermal growth factor (EGF) and insulin has been reported (Thompson, K. L., S. J. Decker, and M. R. Rosner, 1985, Proc. Natl. Acad. Sci. USA., 82:8443-8447). This 100-kD protein (designated dp100) is also recognized by an antiserum against the human EGF receptor. To further characterize the properties of this protein, we have determined the binding spectrum, glycosylation state, and cellular distribution of dp100. Our results indicate that dp100 binds to other insulin-like and EGF-like growth factors with dissociation constants ranging from 10(-6) to 10(-9) M, and these ligands compete with each other for binding to dp100. All other ligands tested, including platelet-derived growth factor, transforming growth factor-beta, nerve growth factor, and glucagon, either did not bind or bound with a Kd greater than 10(-6) M. Unlike the Drosophila insulin receptor, dp100 does not bind to wheat germ agglutinin and is present in a cytoplasmic as well as a membrane-bound form that cannot be differentiated by two-dimensional PAGE. Further, dp100 is the sole transforming growth factor-alpha-binding protein detected by affinity labeling in Drosophila Kc cells. These results indicate that dp100 shares properties in common with, but distinct from, the Drosophila homologues of the insulin and EGF receptors.     

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.     

Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface

Glypicans are heparan sulfate proteoglycans that are attached to the cell surface by a GPI (glycosylphosphatidylinositol)anchor. Glypicans regulate the activity of Wnts, Hedgehogs,bone morphogenetic proteins and fibroblast growth factors. In the particular case of Wnts, it has been proposed that GPI-anchored glypicans stimulate Wnt signalling by facilitating and/or stabilizing the interaction between Wnts and their cell surface receptors. On the other hand, when glypicans are secreted to the extracellular environment, they can act as competitive inhibitors of Wnt. Genetic screens in Drosophila have recently identified a novel inhibitor of Wnt signalling named Notum. The Wnt inhibiting activity of Notum was associated with its ability to release Dlp [Dally (Division abnormally delayed)-like protein; a Drosophila glypican] from the cell surface by cleaving the GPI anchor. Because these studies showed that the other Drosophila glypican Dally was not released from the cell surface by Notum,it remains unclear whether this enzyme is able to cleave glypicans from mammalian cells. Furthermore, it is also not known whether Notum cleaves GPI-anchored proteins that are not members of the glypican family. Here, we show that mammalian Notum can cleave several mammalian glypicans. Moreover, we demonstrate that Notum is able to release GPI-anchored proteins other than glypicans. Another important finding of the present study is that,unlike GPI-phospholipase D, the other mammalian enzyme that cleaves GPI-anchored proteins, Notum is active in the extracellular environment. Finally, by using a cellular system in which GPC3 (glypican-3) stimulates Wnt signalling, we show that Notum can act as a negative regulator of this growth factor.     

Mutation of the gene encoding the ubiquitin activating enzyme ubal causes tissue overgrowth in Drosophila

Protein ubiquitination has been shown to regulate a wide variety of cellular process including cell cycle progression, protein trafficking and apoptosis. Most regulation of ubiquitination occurs at the level of E2 or E3 enzymes and their interactions with specific substrates. In a screen for mutations that cause tissue overgrowth, we recovered multiple mutations in the Drosophila Uba1 gene that encodes the E1 enzyme that is required for the first step of most, if not all, ubiquitination reactions. Previous studies with yeast and mammalian cells have shown that disrupting E1 function results in a cell-cycle arrest. Here we show that in the developing Drosophila eye, clones of cells that are homozygous for partial loss of function alleles of Uba1 show defects in apoptosis. Moreover, clones homozygous for stronger or complete loss of function alleles of Uba1, that are predicted to have a global defect on ubiquitination, survive poorly but are able to stimulate the overgrowth of adjacent wild-type tissue. Experiments with mammalian cells show that reducing the level of RNA of the mammalian Uba1 ortholog, UBE1, also results in increased expression of specific growth factor genes. Our studies show that a reduction in E1 activity can promote tissue growth in a multicellular organism and raise the possibility that changes in E1 activity may occur during normal development or in cancer.     

Identification of a fibroblast growth factor-binding protein in Drosophila melanogaster.

As assessed by competitive binding and protein-crosslinking experiments, Drosophila melanogaster cells possess basic fibroblast growth factor (bFGF)-specific binding proteins that are similar to FGF receptors on vertebrate cells in molecular weight and binding affinity; these D. melanogaster cells, however, have no detectable binding proteins for acidic fibroblast growth factor (aFGF). Consistent with the presence of bFGF-specific binding proteins, D. melanogaster cells degrade bFGF but not aFGF. These results indicate the conservation of heparin-binding growth factors and receptors between vertebrates and D. melanogaster.     

Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation via an intracellular pathway.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved enzyme that has been implicated in cellular insulin degradation, but its site of action and importance in regulating insulin degradation have not been clearly established. We addressed this question by examining the effects of overexpressing IDE on insulin degradation in COS cells, using both human IDE (hIDE) and its Drosophila homolog (dIDE). The dIDE, which was recently cloned in our laboratory, has 46% amino acid identity with hIDE, degrades insulin with comparable efficiency, and is readily expressed in mammalian cells. Transient expression of dIDE or hIDE in COS monkey kidney cells led to a 5- to 7-fold increase in the rate of degradation of extracellular insulin, indicating that IDE can regulate cellular insulin degradation. Insulin-degrading activity in the medium was very low and could not account for the difference between transfected and control cells. To further localize the site of IDE action, the fate of insulin after receptor binding was examined. The dIDE-transfected cells displayed increased degradation of prebound insulin compared to control cells. This increase in degradation was observed even when excess unlabeled insulin was added to block reuptake or extracellular degradation. These results indicate that IDE acts at least in part within the cell. The lysosomotropic agents chloroquine and NH4Cl did not affect the increase in insulin degradation produced by transfection with dIDE, indicating that the lysosomal and IDE-mediated pathways of insulin degradation are independent. The results demonstrate that IDE can regulate the degradation of insulin by intact cells via an intracellular pathway.     

The Drosophila VEGF receptor homolog is expressed in hemocytes.

Several signalling pathways have been defined by studies of genes originally characterised in Drosophila. However, some mammalian signalling systems have so far escaped discovery in the fly. Here, we describe the identification and characterisation of fly homologs for the mammalian vascular endothelial growth factor/platelet derived growth factor (VEGF/PDGF) and the VEGF receptor. The Drosophila factor (DmVEGF-1) gene has two splice variants and is expressed during all stages, the signal distribution during embryogenesis being ubiquitous. The receptor (DmVEGFR) gene has several splice variants; the variations affecting only the extracellular domain. The most prominent form is expressed in cells of the embryonic haematopoietic cell lineage, starting in the mesodermal area of the head around stage 10 of embryogenesis. Expression persists in hemocytes as embryonic development proceeds and the cells migrate posteriorly. In a fly strain carrying a deletion uncovering the DmVEGFR gene, hemocytes are still present, but their migration is hampered and the hemocytes remain mainly in the anterior end close to their origin. These data suggest that the VEGF/PDGF signalling system may regulate the migration of the Drosophila embryonic haemocyte precursor cells.     

Identification of insulin receptors on the mucosal surface of colon epithelial cells

Brush-border membranes were isolated from the mucosal surface of rabbit proximal colon epithelial cells by a procedure involving Ca2+ precipitation. Ouabain-insensitive K+-phosphatase, a marker enzyme for the colon brush-border membrane, was enriched 17-fold by this technique, while no enrichment was observed in the activity of ouabain-sensitive K+-phosphatase, a marker for the basal-lateral membrane. Insulin binding studies revealed a dose-dependent inhibition of 125I-insulin binding with porcine insulin and approximately 4 X 10(-9) M insulin was required to produce 50% inhibition of 125I-insulin binding, while desoctapeptide insulin, insulin-like growth factor I, and A chain of insulin had less effect on 125I-insulin binding. This is the first demonstration of the existence of high-affinity insulin binding sites on the brush-border membrane of mammalian colon epithelial cells. Subsequent studies with the cross-linking agent disuccinimidyl suberate confirmed the presence of insulin binding sites in these membranes and autoradiography of polyacrylamide gels revealed that the binding subunit of the colon epithelial cell brush-border insulin receptor is similar in size to that observed in hepatic tissue. Interestingly, the insulin binding capacity/mg of protein of this preparation is high, suggesting that large numbers of insulin receptors are present in vivo on the mucosal surface of colon epithelial cells. The potential physiological role of these previously unrecognized insulin receptors is discussed.     

p42/mitogen-activated protein kinase as a converging target for different growth factor signaling pathways: use of pertussis toxin as a discrimination factor.

Mitogen-activated protein (MAP) kinase is a 42-kDa serine/threonine-specific protein kinase that requires phosphorylation on both tyrosine and threonine residues for activity. This enzyme is rapidly and transiently activated in quiescent cells after addition of various agonists, including insulin, epidermal growth factor, platelet-derived growth factor, and phorbol esters. We show here that addition of the growth factors thrombin or basic fibroblast growth factor to CCL39 fibroblasts rapidly induces tyrosine phosphorylation of the p42 MAP kinase protein and concomitantly stimulates MAP kinase enzymatic activity. To elucidate the signaling pathways utilized in this activation, we took advantage of the sensitivity of CCL39 cells to the toxin of bordetella pertussis, which ADP-ribosylates two Gi proteins in this cell system. We show that pretreatment of cells with the toxin inhibited thrombin stimulation of MAP kinase by greater than 75% but had no detectable effect on the stimulation induced by basic fibroblast growth factor. We also demonstrate that these two growth factors that synergize for mitogenicity are able to cooperate in activation of MAP kinase and that this synergism is partially sensitive to pertussis toxin. Finally, we describe a 44-kDa protein, the tyrosine phosphorylation of which appears to be coregulated with p42 MAP kinase. We conclude that p42 MAP kinase (and the pp44 protein) are at or are downstream from a point of convergence of two different receptor-induced signaling pathways and might well play a key role in integrating those signals.     

Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides

In metazoans, factors of the insulin family control growth, metabolism, longevity, and fertility in response to environmental cues. In Drosophila, a family of seven insulin-like peptides, called Dilps, activate a common insulin receptor. Some Dilp peptides carry both metabolic and growth functions, raising the possibility that various binding partners specify their functions. Here we identify dALS, the fly ortholog of the vertebrate insulin-like growth factor (IGF)-binding protein acid-labile subunit (ALS), as a Dilp partner that forms a circulating trimeric complex with one molecule of Dilp and one molecule of Imp-L2, an IgG-family molecule distantly related to mammalian IGF-binding proteins (IGFBPs). We further show that dALS antagonizes Dilp function to control animal growth as well as carbohydrate and fat metabolism. These results lead us to propose an evolutionary perspective in which ALS function appeared prior to the separation between metabolic and growth effects that are associated with vertebrate insulin and IGFs.     

A Conserved Role for L1 as a Transmembrane Link Between Neuronal Adhesion and Membrane Cytoskeleton Assembly

The L1-family of cell adhesion molecules is involved in many important aspects of nervous system development. Mutations in the human L1-CAM gene cause a complicated array of neurological phenotypes; however, the molecular basis of these effects cannot be explained by a simple loss of adhesive function. Human L1-CAM and its Drosophila homolog neuroglian are rather divergent in sequence, with the highest degree of amino acid sequence conservation between segments of their cytoplasmic domains. In an attempt to elucidate the fundamental functions shared between these distantly related members of the L1-family, we demonstrate here that the extracellular domains of mammalian L1-CAMs and Drosophila neuroglian are both able to induce the aggregation of transfected Drosophila S2 cells in vitro. To a limited degree they even interact with each other in cell adhesion and neurite outgrowth assays. The cytoplasmic domains of human L1-CAM and neuroglian are both able to interact with the Drosophila homolog of the cytoskeletal linker protein ankyrin. Moreover the recruitment of ankyrin to cell-cell contacts is completely dependent on L1-mediated cell adhesion. These findings support a model of L1 function in which the phenotypes of human L1-CAM mutations result from a disruption of the link between the extracellular environment and the neuronal cytoskeleton.     

The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis.

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.     

Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha

The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from beta cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3'-phosphoinositide-dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse beta cell substantially affects compartment size and function. There was a significant increase in both beta-cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.