REGULATION OF CHOLESTEROL METABOLISM

描述(申请人提供)：胆固醇平衡是通过胆固醇摄取、合成、运输、降解和分泌的协调调节来维持的。胆固醇动态平衡的丧失会导致疾病，如动脉粥样硬化和胆固醇结石。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磺化氧化甾醇，5-胆固酮-3b，25-二醇-3-硫酸盐，它随着胆固醇输送到原代肝细胞线粒体的增加而显著增加。这种氧化甾醇在这些细胞的线粒体和细胞核中发现了高浓度，并似乎能够激活编码胆固醇7a-羟基酶(CYP7A1)的基因和胆固醇转运蛋白(ABCA1，ABCG5，ABCG8)。我们的初步数据表明，胆固醇可以首先在线粒体的25位羟化，然后在3位羟基处进行硫酸盐化。然后，这种水溶性产物被运出线粒体，进入细胞核。这种硫化的25-羟基胆固醇似乎激活了核受体(S)，并上调胆汁酸的合成和胆固醇的分泌。我们假设这种硫化胆固醇衍生物是一种新的胆固醇代谢途径的中间产物。这项应用的总体目标是表征这一新途径的中间产物，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动态平衡中可能发挥的作用。其具体目的是：1.鉴定和确定新型硫酸盐化氧化甾醇(S)在细胞核、线粒体和培养液中的化学结构；化学合成这种新型的核硫酸化氧化甾醇；2.阐明这种新型的核硫酸化氧化甾醇在原代肝细胞中的代谢途径；3.利用化学合成的核氧化甾醇来探讨这种新型的硫酸盐化氧化甾醇在维持胆固醇动态平衡中的作用。这项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类代谢提供基础信息，这可能为治疗高胆固醇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.