EVIDENCE THAT IRS-2 PHOSPHORYLATION IS REQUIRED FOR INSULIN ACTION INHEPATOCYTES

Insulin receptor substrates (IRSs) are tyrosine-phosphorylated followi ng stimulation with insulin, insulinlike growth factors (IGFs), and in terleukins. A key questionis whether different IRSs play different rol es to mediate insulin's metabolic and growth-promoting effects. In a n ovel system of insulin receptor-deficient hepatocytes, insulin fails t o (i) stimulate glucose phosphorylation, (ii) enhance glycogen synthes is, (iii) suppress glucose production, and (iv) promote mitogenesis, H owever, insulin's ability to induce IRS-1 and gab-1 phosphorylation an d binding to phosphatidylinositol (PI) 3-kinase is unaffected, by virt ue of the compensatory actions of IGF-1 receptors, In contrast, phosph orylation of IRS-2 and generation of IRS-2/PI 3-kinase complexes are m arkedly reduced. Thus, absence of insulin receptors selectively reduce s IRS-2, but not IRS-1 phosphorylation, and the impairment of IRS-S ac tivation is associated with lack of insulin effects. To address whethe r phosphorylation of additional IRSs is also affected, we analyzed pho sphotyrosine-containing proteins in PI 3-kinase immunoprecipitates fro m insulin-treated cells, However, these experiments indicate that IRS- 1 and IRS-2 are the main PI 3-kinase bound proteins in hepatocytes, Th ese data identify IRS-2 as the main effector of both the metabolic and growth-promoting actions of insulin through PI 3-kinase in hepatocyte s, and IRS-1 as the main substrate mediating the mitogenic actions of IGF-1 receptors.