REGULATION OF CHOLESTEROL METABOLISM

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DESCRIPTION (provided by applicant): Cholesterol homeostasis is maintained through the coordinate regulation of cholesterol uptake, synthesis, transport, degradation, and secretion. Loss of cholesterol homeostasis causes diseases, such as atherosclerosis and cholesterol gallstones. Atherosclerosis and its clinical sequelae are a major cause of premature death and disability in the United States and in the industrialized would. We recently discovered and characterized a novel nuclear sulfated oxysterol, 5-cholesten-3b, 25-diol 3- sulphate, which increased dramatically with increased cholesterol delivery to mitochondria in primary hepatocytes. This oxysterol was found in high concentrations in the mitochondria and nuclei of these cells, and appears capable of activating the genes encoding cholesterol 7a-hydroxylase (CYP7A1), and cholesterol transporters (ABCA1, ABCG5, ABCG8). Our preliminary data indicate that cholesterol can be hydroxylated at 25-position first, and then sulfated at 3-hydroxy position in mitochondria. The water-soluble product is then transported out of the mitochondria and enters to the nucleus. This sulfated 25-OH cholesterol seems to activate nuclear receptor(s) and upregulates bile acid synthesis and cholesterol secretion. We hypothesize that this sulfated cholesterol derivative is an intermediate in a novel cholesterol metabolism pathway. The overall objective of this application is to characterize the intermediates in this novel pathway, determine the cellular location of the key enzymes in the pathway, and explore the role this pathway may play in cellular cholesterol homeostasis. the specific aims are: 1. To characterize and determine the chemical structures of the novel sulfated oxysterol(s) in nuclei, mitochondria, and culture media; to chemically synthesize this nuclear sulfated oxysterol; 2.To elucidate the metabolic pathway of this novel nuclear sulfated oxysterol in primary hepatocytes; and 3. To explore the role that this novel sulfated oxysterol plays in the maintenance of cholesterol homeostasis using the chemically synthesized nuclear oxysterol. The successful completion of this study will provide fundamental information regarding intracellular oxysterols regulate intracellular cholesterol and lipids metabolism, which may represent new therapeutic approaches to treatment of hypercholesterolemia and hyperlipidemia.