Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Oral vanadate administration has been demonstrated to normalize blood glucose levels in ob/ob and db/db mice and streptozotocin (STZ) diabetic rats. The exact mechanism of this vanadate effect is uncertain, since there are no consistent effects on the insulin receptor tyrosine kinase activity or phosphotyrosine phosphatase activity. We have therefore studied the postreceptor actions of vanadate, focusing our attention on the steady-state levels of mRNA of enzymes involved in carbohydrate metabolism. When compared with their lean (ob/+) controls, the livers of ob/ob mice exhibited an approximately 90% reduction in the levels of phosphoenolpyruvate carboxykinase (PEPCK) mRNA and twofold to fivefold higher levels of the mRNAs for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the "liver beta-cell" glucose transporter (GLUT2), and the proto-oncogene c-myc. Administration of sodium vanadate (0.25 mg/mL) in the drinking water of ob/ob mice over a 45-day period resulted in a near normalization of blood glucose and increased PEPCK mRNA levels more than ninefold. Starvation of the ob/ob mice for 24 to 48 hours also increased PEPCK mRNA levels by fourfold to 15-fold. Vanadate treatment did not alter mRNA levels of any other proteins studied and had no effect on PEPCK mRNA in ob/+ mice. However, 1 to 100 mumol/L vanadate produced a concentration-dependent increase in PEPCK mRNA levels in an H35 hepatoma cell line, an effect opposite to the suppression of PEPCK mRNA produced by insulin. In summary, hyperglycemia in the ob/ob mouse is characterized by decreased expression of PEPCK and increased expression of GAPDH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin receptor substrate 1 (IRS-1) is the primary cytosolic substrate of the insulin and insulin-like growth factor-I (IGF-I) receptors. Following tyrosine phosphorylation IRS-1 binds to and activates specific proteins containing SH2 domains. Using biochemical and immunocytochemical techniques, we have mapped the distribution of IRS-1 in the CNS of the adult rat and compared it with that of insulin and IGF-I receptors and phosphatidylinositol 3-kinase (PI-3 kinase), a signaling molecule functionally related to IRS-1. Immunoprecipitation and Western blotting experiments demonstrate the presence of substantial amounts of IRS-1, insulin receptor, and PI-3 kinase in the brain. IRS-1 immunoreactivity is widely distributed in neurons from several areas of the brain and spinal cord. The cerebral cortex, the hippocampus, many hypothalamic and thalamic nuclei, the basal ganglia, the cerebellar cortex, the brainstem nuclei, and the lamina X of the spinal cord are particularly rich of immunopositive nerve cells. In these areas most of the neurons immunoreactive for IRS-1 are also stained by either anti-insulin receptor or anti-IGF-I receptor antibodies as well as PI-3 kinase antiserum. IRS-1 immunostaining was very weak or totally absent in neurons of the olfactory bulb, the supraoptic and paraventricular nuclei, the mesencephalic trigeminal nucleus, and the granule cell layer of the cerebellum, despite the fact that these areas were immunolabeled with antibodies against insulin or IGF-I receptors and/or PI-3 kinase. These results show that neurons in the adult rat CNS are endowed with some of the components of the early signaling pathway for growth factors of the insulin/IGF-I family, although IRS-1 has a distribution distinct from that of the two receptors.

IRS-1 (insulin receptor substrate-1) is a major substrate of the insulin receptor. Rat and human IRS-1 cDNAs, and human and mouse IRS-1 genes have been cloned so far. They show high homology in nucleic acids and amino acids levels, which indicate the high conservation of IRS-1 across the species. Interestingly, the entire coding region is contained in the 1st exon in the IRS-1 gene. The promoter of the mouse IRS-1 gene lacks TATA and CAAT boxes but contains 9 potential Spl binding sites, indicating that IRS-1 is a "housekeeping" gene. By deletion analysis, two positively and two negatively regulating fragments are identified in the promoter. In cultured adipocytes, insulin and dexamethasone down regulate IRS-1 expression by different mechanisms. Insulin down regulates at the post-translational level by shortening the protein half life, and dexamethasone down regulates at the post-transcriptional level mainly by shortening the mRNA half life.

The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.

Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PT 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 microM) with > 95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp70 S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin- and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp90 S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp70 S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp90 S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.

The role of insulin receptor tyrosine kinase activity in stimulation of intracellular enzymes linked to insulin action [phosphatidylinositol 3-kinase (PtdIns 3-kinase), microtubule-associated protein (MAP) kinase, and S6 kinases] was studied in Chinese hamster ovary cells which overexpress wild type human insulin receptors, receptors with reduced kinase activity due to substitution of Phe for Tyr1146 (single-Phe), Tyr1150,1151 (double-Phe), and Tyr1146,1150,1151 (triple-Phe), or kinase-inactive receptors with a substitution of Ala for Lys1018 in the ATP binding site (A1018). We have previously shown that receptor autophosphorylation and kinase activity of these mutants were reduced by approximately 50, 65, 85, and 100%, respectively. Glycogen and DNA synthesis parallel the level of receptor autophosphorylation and kinase activity; however, receptor serine and threonine phosphorylation was independent of receptor tyrosine kinase activity and receptor internalization was completely dependent on maximal receptor kinase activity. Overexpression of the wild type insulin receptor increased both maximal insulin receptor substrate-1-associated and total insulin-stimulated PtdIns 3-kinase activity, as well as S6 and MAP kinase activities 2.0- to 3.6-fold. In addition there was a leftward shift of the dose-response curves for PtdIns 3-kinase and S6 kinases by approximately 10-fold. Expression of the single- and double-Phe mutant receptors also enhanced maximal PtdIns 3-kinase activity, but had no effect on insulin sensitivity, whereas expression of either the triple-Phe or kinase-inactive receptors did not enhance insulin stimulation or increase insulin sensitivity as compared to the control cells. When comparing the mutant and wild type receptors, differences in insulin sensitivity were least for insulin-stimulated MAP kinase and greatest for S6 kinase; with the latter there was greater than a 1000-fold difference in insulin sensitivity when cells that overexpress wild type vs. kinase-inactive insulin receptors were compared. Thus, the level of insulin receptor tyrosine autophosphorylation and kinase activity regulate both maximal activation and insulin sensitivity of these intracellular kinases in the insulin action pathway which may lead to glycogen and/or DNA synthesis. The differential sensitivity of these enzymes to changes in receptor activation suggests that they may be differently coupled to the receptor kinase.

Insulin rapidly stimulates tyrosine kinase activity of its receptor resulting in phosphorylation of its cytosolic substrate insulin receptor substrate 1 (IRS-1), which in turn associates with and activates the enzyme phosphatidylinositol 3-kinase (PI 3-kinase). In the present study we have examined these three initial steps in insulin action during the differentiation of 3T3-F442A adipocytes and after treatment with dexamethasone or insulin. The differentiation of 3T3-F442A cells was characterized by a 13-fold increase in insulin receptor protein, a 9-fold increase in IRS-1, and a 10- and 4.5-fold increase in their insulin-stimulated phosphorylation, respectively. The mRNA expression of these two proteins showed a similar 8-fold increase during differentiation. In addition there was a 3.5-fold increase in PI 3-kinase protein [85 kilodalton (kDa) subunit] and a 16-fold increase in IRS-1-associated PI 3-kinase activity between day 0 and day 8 of differentiation. Dexamethasone (1 microM) treatment of differentiated cells induced a further 48% (P 0.05) increase in insulin receptor level, but the autophosphorylation of the receptor was decreased by 31 +/- 1% (P 0.02). At the same time there was a decrease by 56 +/- 4% (P 0.005) in IRS-1 protein and by 31 +/- 1% (P 0.001) in IRS-1 phosphorylation. The expression of insulin receptor mRNA was unchanged, but the expression of IRS-1 mRNA was decreased by approximately 75% after dexamethasone. By contrast, dexamethasone induced a 69% increase in the level of PI 3-kinase as determined by immunoblotting. The combined effect of decreased IRS-1 phosphorylation and increased PI 3-kinase protein was a minimal change (15% decrease) in the association/activation between IRS-1 and PI 3-kinase. Chronic treatment with 100 nM insulin induced a time- and dose-dependent decrease in insulin receptor and IRS-1 protein levels reaching a nadir of 34 +/- 5% (P 0.005) and 39 +/- 5% (P 0.01) of control levels after 24 h, respectively. There was an even more marked decrease in the phosphorylation level of these proteins. Chronic insulin treatment also produced a 30% decrease in PI 3-kinase protein levels and a approximately 50% decrease in the association/activation between IRS-1/PI 3-kinase. The expression of insulin receptor and IRS-1 mRNA was unchanged during chronic insulin treatment.(ABSTRACT TRUNCATED AT 400 WORDS)