REGULATION OF CHOLESTEROL METABOLISM

描述（由申请人提供）：胆固醇稳态是通过胆固醇摄取、合成、运输、降解和分泌的协调调节来维持的。胆固醇体内平衡的丧失会导致疾病，如动脉粥样硬化和胆固醇胆结石。动脉粥样硬化及其临床后遗症是美国和工业化国家过早死亡和残疾的主要原因。我们最近发现并表征了一种新的核硫酸化氧甾醇，5-胆固醇-3b， 25-二醇3-硫酸盐，随着原代肝细胞中胆固醇向线粒体输送的增加，其含量急剧增加。在这些细胞的线粒体和细胞核中发现了高浓度的这种氧甾醇，并且似乎能够激活编码胆固醇7a-羟化酶（CYP7A1）和胆固醇转运蛋白（ABCA1, ABCG5, ABCG8）的基因。我们的初步数据表明，胆固醇可以在线粒体中首先在25位羟基化，然后在3-羟基位置硫酸化。水溶性产物随后被运输出线粒体并进入细胞核。这种硫酸化的25-OH胆固醇似乎激活核受体并上调胆汁酸合成和胆固醇分泌。我们假设这种硫酸盐胆固醇衍生物是一种新的胆固醇代谢途径的中间产物。本应用程序的总体目标是表征该新途径中的中间体，确定该途径中关键酶的细胞位置，并探索该途径在细胞胆固醇稳态中可能发挥的作用。具体目标是：1。表征和确定核、线粒体和培养基中新型硫酸氧化甾醇的化学结构；用化学方法合成这种核硫酸化的氧甾醇；2.阐明这种新型核硫酸氧甾醇在原代肝细胞中的代谢途径；和3。利用化学合成的核氧化甾醇，探讨这种新型硫酸氧化甾醇在维持胆固醇稳态中的作用。本研究的成功完成将为细胞内氧甾醇调节细胞内胆固醇和脂质代谢提供基础信息，可能为治疗高胆固醇血症和高脂血症提供新的治疗途径。

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.