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to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance.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.

The activation of p21ras by receptor tyrosine kinases involves the translocation of the growth factor receptor bound protein 2-mammalian son of sevenless protein (Grb2-SOS) complex to the plasma membrane where p21ras is localized. Insulin receptors induce p21ras-GTP formation by two possible mechanisms: tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent association with Grb2, or Shc phosphorylation and its subsequent association with Grb2. We investigated the contribution of the major tyrosine autophosphorylation sites Tyr1158, Tyr1162, and Tyr1163 of the insulin receptor to IRS1.Grb2 and Shc.Grb2 association and the formation of p21ras-GTP. Chinese hamster ovary-derived cell lines were used overexpressing mutant insulin receptors in which the major tyrosine autophosphorylation sites were stepwise replaced by phenylalanines. In cell lines expressing wild type or mutant Y1158F,Y1162,Y1163 (FYY) receptors, insulin stimulated tyrosine phosphorylation of IRS1 and Shc and the formation of IRS1.Grb2 and Shc.Grb2 protein complexes, together with an increase in p21ras-GTP. Cell lines expressing mutant Y1158,Y1162F,Y1163F (YFF) receptors showed insulin-induced tyrosine phosphorylation of Shc, Shc.Grb2 complex formation, and p21ras-GTP formation, whereas tyrosine phosphorylation of IRS1 was strongly decreased and formation of IRS1.Grb2 complexes was undetectable. The activity of FYY and YFF receptors to mediate p21ras-GTP formation correlated with their activity to induce Shc phosphorylation and Shc.Grb2 association. The mutant insulin receptors Y1158F,Y1162F,Y1163 and Y1158F,Y1162F,Y1163F were inactive in inducing any of these responses. We conclude that phosphorylation of Tyr1158 and Tyr1162 of the insulin receptor is linked to distinct post-receptor processes and that YFF receptors generate p21ras-GTP via the Shc.Grb2 pathway rather than one involving IRS1.Grb2 interaction.

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 principal substrate for the insulin and insulin-like growth factor-1 (IGF-1) receptors is the cytoplasmic protein insulin-receptor substrate-1 (IRS-1/pp185). After tyrosine phosphorylation at several sites, IRS-1 binds to and activates phosphatidylinositol-3'-OH kinase (PI(3)K) and several other proteins containing SH2 (Src-homology 2) domains. To elucidate the role of IRS-1 in insulin/IGF-1 action, we created IRS-1-deficient mice by targeted gene mutation. These mice had no IRS-1 and showed no evidence of IRS-1 phosphorylation or IRS-1-associated PI(3)K activity. They also had a 50 per cent reduction in intrauterine growth, impaired glucose tolerance, and a decrease in insulin/IGF-1-stimulated glucose uptake in vivo and in vitro. The residual insulin/IGF-1 action correlated with the appearance of a new tyrosine-phosphorylated protein (IRS-2) which binds to PI(3)K, but is slightly larger than and immunologically distinct from IRS-1. Our results provide evidence for IRS-1-dependent and IRS-1-independent pathways of insulin/IGF-1 signalling and for the existence of an alternative substrate of these receptor kinases.

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.

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.

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)