The basic goal of this study is to make a significant contribution to the understanding of the cellular mechanism of insulin action and the regulation of cellular metabolism in general. A comprehension of the mechanism of insulin action is crucial both from a basic science point of view and from a practical standpoint to understand the pathophysiology and to potentially design new methods of treatment of diabetes mellitus. The glucose transport protein will be the focus of the investigation. Specifically, the role of Ca2+- and phospholipid-sensitive protein kinase (protein kinase C, also known as Ca, PL-PK) in insulin mediated phosphorylation of the glucose transporter will be investigated. Recent evidence indicates that the glucose transport protein is phosphorylated by both insulin and protein kinase C. It is my hypothesis that phosphorylation of the glucose transport protein regulates glucose transport and the mechanism of insulin-induced phosphorylation of the glucose transporter may be mediated via protein kinase C. The experimental approach includes the following specific aims: i) characterization of phosphorylation of the glucose transport protein in isolated adipocyte plasma membranes and microsomes, ii) characterization of changes in phosphorylation of the glucose transporter in plasma membranes and microsomes from adipocytes pre-treated with insulin or activators (e.g., phospholipase C, diacylglycerol, the phorbol ester tetradecanoylphorbol acetate) and inhibitors (e.g., gossypol, quercitin, polymyxin B) of protein kinase C and iii) investigation of the effects of the above agents on phosphorylation of the glucose transport protein in intact adipocytes. In all experiments, changes in phosphorylation of the glucose transport protein will be correlated with changes in glucose transport activity and 3H cytochalasin B binding to the glucose transporter. These studies should add considerably to our knowledge of the molecular mechanism by which insulin stimulates glucose transport.
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