The long-term objective of this proposal continues to be the delineation of the mechanism by which acetyl-CoA carboxylase (ACC), a major rate-limiting enzyme for fatty acid biosynthesis, is regulated by hormones, especially insulin, with a particular focus on the regulation of its phosphorylation state in intact cells and in vitro by several protein kinases and phosphatases. The regulation of this enzyme by insulin has proven to be an excellent """"""""reporter"""""""" system, which may yield more general insight into the mechanism of insulin action. In extension of these studies, three major specific aims are proposed. First, we will explore the mechanism by which insulin and other ACC stimulators promote the dephosphorylation of ACC coincident with its activation in rat hepatoma cells. In these studies, we plan to determine site- specific alterations in ACC phosphorylation after stimulation, to purify a major ACC regulatory kinase and to characterize the regulation of ACC kinases/phosphatases in hepatoma cells. Second, we plan to detail the mechanism underlying differences in ACC specific activity in varying clones of Reuber hepatoma cells and to select and subclone other lines with alterations in ACC activity or content. Differences in ACC phosphorylation and kinase/phosphatase activity will be compared; cells will be selected for subcloning based on Nile Red 0 fluorescence and cell sorting and after cell killing with an ACC inhibitor, TOFA. Third, we plan to purify and characterize an insulinomimetic oligosaccharide autocrine factor derived from hepatoma cells in order to determine its structure, its potential loci of action in intact cells and in vitro and whether the factor is generated in response to insulin and other stimulators, the latter to address the important question of its role in insulin-stimulated signal transduction in hepatoma and other cells. The results of these investigations will further our understanding of the regulation of fatty acid synthesis and may provide important insight into the mechanism of insulin action. This would supply information needed to understand abnormalities in cellular function in diabetes mellitus with respect to fatty acid and lipoprotein metabolism.
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