The long-term objective of this proposal is to increase our knowledge of the cellular mechanisms of insulin action. When insulin binds to its receptor at the cell surface signals are transmitted to the enzymes regulated by insulin through signal transduction cascades. However, the molecular details of these pathways remain incompletely understood. Defects in these signalling cascades lead to peripheral insulin resistance and the development of type II diabetes mellitus, a common and devastating disease in this country. In almost all patients these defects lie downstream of the insulin receptor itself. A more complete understanding of insulin-stimulated signalling pathways will lead to a greater understanding of the underlying causes of insulin resistance and diabetes, and potentially to identification of better targets for the treatment of this disease. The focus of this proposal is the characterization of a unique insulin-stimulated signalling pathway involving tyrosine phosphorylation of caveolin. Caveolin is a structural component of specialized cell surface domains termed caveolae. This phosphorylation is both insulin-specific and cell type-dependent, occurring only in cells that are highly insulin responsive. These properties suggest that this pathway may play a key role in the cellular effects of insulin. The 3T3-L1 cell culture model of adipocytes was chosen for its convenience and extraordinary insulin-responsiveness, particularly in the regulation of glycogen metabolism. To obtain large amounts of material for protein isolation the cultured primary adipocyte system will also be used.
The specific aims of this proposal are to: 1. Determine the signalling pathway leading to caveolin phosphorylation through the heterologous expression of wild type and mutant forms of candidate upstream signalling molecules. 2. Identify proteins that bind phosphorylated caveolin using biochemical and molecular approaches. 3. Determine the effect of environmental factors on caveolin phosphorylation through the manipulation of experimental conditions. 4. Correlate caveolin phosphorylation with known cellular effects of insulin using heterologous expression of signalling molecules to modulate caveolin phosphorylation.
