REGULATION OF HEPATIC CHOLESTERYL ESTER METABOLISM

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Hepatic free cholesterol is secreted directly into the bile, is used to form bile acids, is required for lipoprotein production, and regulates the synthesis of cholesterol and receptors for LDL uptake. Cholesteryl ester formation, by acyl CoA: cholesterol acyl transferase (ACAT) and break down by cholesterol ester hydrolase (CEH) is one mechanism to control the cellular content of free cholesterol. Recent studies suggest that covalent phosphorylation regulates the activities of ACAT and the rate-limiting enzymes for cholesterol and bile acid synthesis in the liver and CEH activity in peripheral tissues. The long term objective of these studies is to understand the role of rapid regulation of these enzymes in the overall economy of liver cholesterol. This project is specifically directed toward studies on the effects of phosphorylation on ACAT and CEH. One specific aim is to obtain direct evidence that phosphorylation activates ACAT. These studies involve purifying ACAT, incorporating 32P into ACAT with protein kinases, and demonstrating that cyclic changes in its phosphate content, produced by alternative treatment with phosphatase and protein kinase, correlate with ACAT activity. A second aim is to determine whether hepatic CEH is also controlled by phosphorylation. In this regard, studies similar to those described for ACAT will be carried out with CEH. A third aim is to identify factors that affect cholesteryl ester synthesis and hydrolysis in freshly isolated hepatocytes and to correlate such changes with the activities and phosphorylation states of ACAT and CEH. Initial studies will utilize dibutyryl cAMP. Other agents will include hormones that stimulate protein kinase activity, such as glucagon, vasopressin, and catecholamines, 25-hydroxycholesterol, and mevalonate. The final aim is to examine the effects of in vivo manipulations, such as cholesterol and cholestyramine feeding and mevalonate administration, on the activities and phosphorylation states of ACAT and CEH. These studies have implications for our understanding of atherosclerosis. The balance between cholesterol and its esters may influence cholesterol excretion and hepatic synthesis and uptake of lipoproteins, thereby affecting plasma lipoprotein concentrations. Better knowledge of the regulation of ACAT and CEH in the liver may provide insights into their control in peripheral tissues where cholesteryl ester hydrolysis is required for cholesterol removal by high density lipoproteins.