Human type 2 diabetes is characterized by defects in both insulin action and insulin secretion. It has been difficult to identify a single molecular abnormality underlying these features. Insulin-receptor substrates (IRS proteins) may be involved in type 2 diabetes: they mediate pleiotropic signals initiated by receptors for insulin and other cytokines. Disruption of IRS-1 in mice retards growth, but diabetes does not develop because insulin secretion increases to compensate for the mild resistance to insulin. Here we show that disruption of IRS-2 impairs both peripheral insulin signalling and pancreatic beta-cell function. IRS-2-deficient mice show progressive deterioration of glucose homeostasis because of insulin resistance in the liver and skeletal muscle and a lack of beta-cell compensation for this insulin resistance. Our results indicate that dysfunction of IRS-2 may contribute to the pathophysiology of human type 2 diabetes.

Intracellular insulin signaling involves a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (IRS) and the various isoforms of SH2 domain signaling molecules that can interact with these substrates. In this study, we have evaluated the roles of IRS-1 and IRS-2 in signaling to the phosphatidylinositol (PI) 3-kinase pathway in the ob/ob mouse, a model of the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. We find that the levels of expression of both IRS-1 and IRS-2 are decreased approximately 50% in muscle, whereas in liver the decrease is significantly greater for IRS-2 (72%) than for IRS-1 (29%). This results in differential decreases in IRS-1 and IRS-2 phosphorylation, docking of the p85alpha regulatory subunit of PI 3-kinase, and activation of this enzyme in these two insulin target tissues. In ob/ob liver there is also a change in expression of the alternatively spliced isoforms of the regulatory subunits for PI 3-kinase that was detected at the protein and mRNA level. This resulted in a 45% decrease in the p85alpha form of PI 3-kinase, a ninefold increase in the AS53/p55alpha, and a twofold increase in p50alpha isoforms. Thus, there are multiple alterations in the early steps of insulin signaling in the ob/ob mouse, with differential regulation of IRS-1 and IRS-2, various PI 3-kinase regulatory isoforms, and a lack of compensation for the decrease in insulin signaling by any of the known alternative pathways at these levels.

Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55 PIK , a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH 2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identity to those in p85. Phosphatidylinositol 3-kinase (PI-3 kinase) is a common signaling element which plays a role in the regulation of a broad array of biological responses by activated receptors for hormones, growth factors, cytokines, and antigens (6,13,15,42,46,48,61). It is composed of a 110-kDa catalytic subunit (p110) associated with an 85-kDa regulatory subunit (p85) that contains one Src homology 3 (SH3) domain, homology to the breakpoint cluster region (bcr) gene, two proline-rich motifs, and two SH2 domains (11). Interestingly, p110␣ displays dual catalytic specificity, as it phosphorylates the D-3 position of phosphatidylinositol and its phosphorylated derivatives and serine residues in p85 and insulin receptor substrate 1 (IRS-1) (12, 32). Mammalian p110 is homologous to VPS34, a Saccharomyces cerevisiae PI-3 kinase which is involved in vacuolar protein sorting (49); however, the molecular role of PI-3 kinase in mammalian cells is unclear (22).The p85 regulatory subunit has a broad potential to couple the PI-3 kinase to multiple signaling elements by employing its SH3 domain, proline-rich motifs, bcr homology region, or SH2 domains (24). Most activated receptors with tyrosine kinase activity engage the SH2 domains in p85 through phosphorylated YXXM motifs in the receptors themselves or a closely associated subunit (52). The platelet-derived growth factor receptor, one of the best-characterized systems, associates directly with the SH2 domains in p85 at a phosphorylated YMDM motif in the kinase insert region (61). Inhibition of PI-3 kinase catalytic activity with wortmannin or disruption of p85 function by site-directed mutagenesis blocks several growth factor-stimulated processes, including mitogenesis and antiapoptosis (61, 67), differentiation (27), receptor trafficking (23), chemotaxis (31, 45), membrane ruffling (29, 64), and insulin-stimulated glucose transport (7,16,44) and Xenopus oocyte maturation (8, 9). In addition, PI-3 kinase appears to be required for the stimulation of p70S6k by platelet-derived growth factor and insulin and probably other growth factors (7,10,40). On the basis of these results, PI-3 kinase plays a central role in cellular signaling.Insulin regulates PI-3 kinase by tyrosine phosphorylation of IRS-1 and IRS-2, multipotential docking proteins which contain multiple potential tyrosine phosphorylation sites, including several YXXM motifs (41, 58). In addition to insulin and insulin-like growth factor 1 (IGF-1), ...