07-1517 Sigma-AldrichAnti-phospho-IRS-2 (Ser388) Antibody

Human ApoE4 accelerates memory decline in ageing and in Alzheimer's disease. Although intranasal insulin can improve cognition, this has little effect in ApoE4 subjects. To understand this ApoE genotype-dependent effect, we examined brain insulin signaling in huApoE3 and huApoE4 targeted replacement (TR) mice. At 32 weeks, lower insulin receptor substrate 1 (IRS1) at S636/639 and Akt phosphorylation at T308 were detected in fasting huApoE4 TR mice as compared to fasting huApoE3 TR mice. These changes in fasting huApoE4 TR mice were linked to lower brain glucose content and have no effect on plasma glucose level. However, at 72 weeks of age, these early changes were accompanied by reduction in IRS2 expression, IRS1 phosphorylation at Y608, Akt phosphorylation at S473, and MAPK (p38 and p44/42) activation in the fasting huApoE4 TR mice. The lower brain glucose was significantly associated with higher brain insulin in the aged huApoE4 TR mice. These results show that ApoE4 reduces brain insulin signaling and glucose level leading to higher insulin content.

Insulin receptor substrates (IRSs) are tyrosine-phosphorylated following stimulation with insulin, insulin-like growth factors (IGFs), and interleukins. A key question is whether different IRSs play different roles to mediate insulin's metabolic and growth-promoting effects. In a novel system of insulin receptor-deficient hepatocytes, insulin fails to (i) stimulate glucose phosphorylation, (ii) enhance glycogen synthesis, (iii) suppress glucose production, and (iv) promote mitogenesis. However, insulin's ability to induce IRS-1 and gab-1 phosphorylation and binding to phosphatidylinositol (PI) 3-kinase is unaffected, by virtue of the compensatory actions of IGF-1 receptors. In contrast, phosphorylation of IRS-2 and generation of IRS-2/PI 3-kinase complexes are markedly reduced. Thus, absence of insulin receptors selectively reduces IRS-2, but not IRS-1 phosphorylation, and the impairment of IRS-2 activation is associated with lack of insulin effects. To address whether phosphorylation of additional IRSs is also affected, we analyzed phosphotyrosine-containing proteins in PI 3-kinase immunoprecipitates from insulin-treated cells. However, these experiments indicate that IRS-1 and IRS-2 are the main PI 3-kinase-bound proteins in hepatocytes. These data identify IRS-2 as the main effector of both the metabolic and growth-promoting actions of insulin through PI 3-kinase in hepatocytes, and IRS-1 as the main substrate mediating the mitogenic actions of IGF-1 receptors.