PI3K has been shown to play a role in the regulation of cell growth and function by a variety of growth factors, oncogenes, and other factors that act via tyrosine kinases. It has also been implicated in the mechanism of insulin action. Thus, three years ago, the applicant showed that insulin stimulates PI3K to produce the reaction products PIP2 and PIP3 in CHO cells transfected with normal human insulin receptors. During the preceding grant period, this work was extended to cells with mutant insulin receptors and to mammalian tissues. Of particular note were studies in isolated rat adipocytes, in which it was found that insulin stimulates the appearance of PI3K activity in both plasma and low density membranes, where it is associated with IRS-1, a recently identified substrate of the insulin receptor tyrosine kinase. The applicant has also found that PI3K in unstimulated adipocytes is located principally in the cytosol and that its activation by insulin is inhibited by norepinephrine. Based on these findings, studies are proposed with the following aims: 1) to determine whether the stimulatory effect of insulin on P13K in the two membranes occurs in parallel or in sequence, and how it is correlated to the tyrosyl phosphorylation of IRS-1; 2) to characterize the membrane vesicles in which PI3K activity is expressed in response to insulin with respect to the presence of proteins such as the glucose transporter and insulin receptor; 3) to define the time course of synthesis and metabolism of PIP3 and PIP2 in vivo. It will be determined whether these lipids are produced in parallel or in sequence, and at what subcellular sites they are generated and metabolized; and 4) to explore the physiological role of P13K in the adipocyte, and the mechanism by which its activation by insulin is antagonized by norepinephrine.
