Lipid Transport, Bile Acid Synthesis, and Cholesterol Homeostasis

建议书摘要/摘要 肝脏脂代谢调节紊乱存在于多种重要疾病中。特别是， 非酒精性脂肪性肝病(NAFLD)是一种肝脏脂质稳态的紊乱，其中胆固醇和 甘油三酯在肝细胞中积累。部分NAFLD患者进展为肝细胞坏死， 炎症和进行性纤维化。非酒精性脂肪肝现在被认为是美国肝硬变的主要原因。 非酒精性脂肪性肝病的肝脏脂肪堆积似乎部分是通过诱导未折叠蛋白产生损伤。 内质网的反应(UPR)；类似于在内质网发现的事件的进展 动脉粥样硬化的发展。与动脉粥样硬化一样，减少细胞内脂肪堆积的治疗 可能通过抑制UPR而减轻NAFLD的肝损伤。 最近，我们已经确认线粒体胆固醇的传递和氧化是 调节肝脂代谢。线粒体胆固醇传递蛋白的表达增加， 在肝细胞中，StARD1被发现下调胆固醇生物合成途径，而上调胆固醇生物合成途径 胆固醇降解和分泌的途径。StARD1过度表达的后果包括 显著降低细胞内中性脂(胆固醇和甘油三酯)，增加关键核水平 在脂质稳态中起重要作用的受体，以及HMG CoA还原酶乙酰辅酶A的表达减少 羧基酶和脂肪酸合成酶(胆固醇和脂肪生物合成中的速率决定酶 酸)。我们现在已经证明，肝脏StARD1的过表达通过 线粒体CYP27A1启动的通路以及由此产生的氧固醇产物是调节分子 能够调节由此引起的脂类代谢的变化。细胞色素P27A1是一种普遍存在的线粒体 酶，我们的初步数据表明，StARD1/CyP27A1途径可能调节脂质稳态 在许多肝外组织中也是如此。在由CYP27A1基因缺失引起的遗传性疾病CTX中， 缺乏这些调节的氧固醇与各种组织中脂质的积累有关(包括 肝脏)、加速的动脉粥样硬化和神经损伤。 这一更新应用的目的是进一步阐明StARD1/CyP27A1途径在乳癌中的作用。 胆固醇氧化在调节肝脏脂质动态平衡中的作用我们假设StARD1作为一个 细胞内胆固醇可利用性传感器。当胆固醇过多时，线粒体胆固醇 交付量增加，导致CYP27A1衍生的氧化甾醇产量增加。由此产生的氧类固醇 然后通过与核受体结合来调节脂质代谢。我们进一步假设，对此的刺激 肝脏途径可能是治疗非酒精性脂肪性肝病的一种有用的策略。 本文提出了研究这一假说的四个具体目标。特定目标1将使用选择性StARD1 在完整和敲除模型中过表达以确定StARD1/CYP27A1途径是否源于氧固醇 负责激活关键的核受体，控制参与基因的表达 调节胆固醇、脂肪酸和胆汁酸的动态平衡；以及随后决定如何表达 编码途径中的一部分与各自的核受体激活相关。在具体目标2中，我们建议 StARD1/CyP27A1途径激活核受体的机制研究 衍生的氧化甾醇参与脂类的动态平衡。具体目标3将确定StarD1/CyP27A1的作用 降低肝细胞中未折叠蛋白反应(UPR)的胆固醇代谢途径。 特定目标4将首次测试StARD1表达增加的药理潜力，以防止 或者在代表人类疾病的动物模型中逆转肝脏脂肪堆积的紊乱。

Proposal Summary/Abstract Disordered regulation of hepatic lipid metabolism is found in a variety of important disorders. In particular, nonalcoholic fatty liver disease (NAFLD) is a disorder of hepatic lipid homeostasis in which both cholesterol and triglycerides accumulate in hepatocytes. A fraction of patients with NAFLD progress to liver cell necrosis, inflammation, and progressive fibrosis. NAFLD is a now recognized as a leading cause of cirrhosis in the U.S. Hepatic lipid accumulation in NAFLD appears to produce injury in part by inducing the unfolded protein response (UPR) in the endoplasmic reticulum; a progression of similar events to those found with the development of atherosclerosis. As with atherogenesis, treatments that reduce intracellular lipid accumulation may attenuate liver injury in NAFLD by repressing the UPR. Recently, we have identified mitochondrial cholesterol delivery and oxidation as crucial steps in the regulation of hepatic lipid metabolism. Increased expression of the mitochondrial cholesterol delivery protein, StARD1, in hepatocytes was found to down-regulate pathways of cholesterol biosynthesis while up-regulating pathways of cholesterol degradation and secretion. Consequences of StARD1 overexpression include markedly decreased intracellular neutral lipids (cholesterol and triglycerides), increased levels of key nuclear receptors important in lipid homeostasis, and reduced expression of HMG CoA reductase, acetyl CoA carboxylase, and fatty acid synthase (rate-determining enzymes in the biosynthesis of cholesterol and fatty acids). We now have shown that hepatic StARD1 overexpression increases cholesterol oxidation via pathways initiated by mitochondrial CYP27A1, and the resulting oxysterol products are regulatory molecules capable of mediating the resulting changes in lipid metabolism. CYP27A1 is a ubiquitous mitochondrial enzyme, and our preliminary data indicate that the StARD1/CYP27A1 pathway may regulate lipid homeostasis in many extrahepatic tissues as well. In the hereditary disorder CTX, caused by genetic deletion of CYP27A1, absence of these regulatory oxysterols is associated with accumulation of lipids in various tissues (inclusive of the liver), accelerated atherosclerosis, and neurologic impairment. The objective of this renewal application is to further elucidate the role of the StARD1/CYP27A1 pathway of cholesterol oxidation in the regulation of hepatic lipid homeostasis. We hypothesize that StARD1 serves as an intracellular sensor of cholesterol availability. When cholesterol is present in excess, mitochondrial cholesterol delivery increases, leading to increased production of CYP27A1 derived oxysterols. The resulting oxysterols then modulate lipid metabolism by binding to nuclear receptors. We further hypothesize that stimulation of this pathway in the liver could represent a useful strategy for treatment of nonalcoholic fatty liver disease. Four specific aims are proposed to study this hypothesis. Specific aim 1 will use selective StARD1 overexpression in intact and knock-out models to determine if StARD1/CYP27A1 pathway derived oxysterols are responsible for activating key nuclear receptors that control the expression of genes involved in the regulation of cholesterol, fatty acid, and bile acid homeostasis; and, subsequently determining how expression of the encoded pathways correlates with respective nuclear receptor activation. In Specific aim 2, we propose to characterize and assess mechanisms of activation of nuclear receptors by StARD1/CYP27A1 pathway derived oxysterols involved in lipid homeostasis. Specific aim 3 will determine the role of the StarD1/CYP27A1 pathway of cholesterol metabolism in attenuating the unfolded protein response (UPR) in hepatocytes. Specific aim 4 will test for the first time the pharmacologic potential of increased StARD1 expression to prevent or reverse disorders of liver lipid accumulation in animal models representative of human disease.