Mechanisms of insulin signaling and the role of caveolae

Abstract: Insulin regulates metabolic as well as mitogenic processes in target cells, involving a large number of mediators of signal transduction. In its role as a growth factor, insulin stimulates cell growth, in a process we demonstrate requires the participation of the Raf-1 kinase.Caveolae are invaginations of the plasma membrane, involved in signal transduction and intracellular transport of cholesterol. Caveolae are enriched in cholesterol, sphingolipids and the constituent protein caveolin. Herein we report that the insulin receptor is located in caveolae of plasma membrane from adipocytes. By confocal and electron microscopy we show co-localization of caveolin and the insulin receptor. Additionally, the insulin receptor independently of insulin stimulation is enriched in caveolae isolated by cell fractionation.Cholesterol depletion has been shown to flatten caveolae and affect processes which occur in these domains. We show that depletion of cholesterol in adipocytes destroys caveolae and inhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1 ), without affecting insulin receptor ligand binding or its autophosphorylation. Cholesterol-depleted adipocytes showed a decreased insulin-stimulated glucose uptake and phosphorylation of A TP citrate-lyase. Cholesterol depletion did not affect insulin's effect on the MAPK kinases ERK 1/2. She, which has been described to mediate an alternative pathway to that mediated by IRS-1 for insulin mitogenic regulation, was not involved in the regulation of the MAP kinases by insulin in adipocytes. We conclude that some other mediators which are not dependent on caveolae integrity must exist for regulation of this pathway.The effects of cholesterol depletion on caveolae and insulin signaling prompted us to study caveolae in models of insulin resistance. We show that adipocytes from the obese and insulin resistant Zucker fa/fa rats have reduced amounts of cholesterol in caveolae compared with their lean littermates. Adipocytes of Zucker fa/fa rats have been shown to express high levels of TNF-α. We demonstrate that TNF-α treatment lowers the amount of cholesterol in caveolae in adipocytes from normal rats.The results presented in this thesis demonstrate that insulin signaling originates in caveolae invaginations of the plasma membrane where the insulin receptor is located. Caveolae are required for certain metabolic effects of insulin but not for activation of the MAP kinase pathway, a scenario similar to what is found in cases of insulin resistance and type 2 diabetes. Moreover, alteration of the amount of cholesterol or caveolae leads to insulin resistance, suggesting that caveolae play a central role in insulin resistance and diabetes.