Lipid Transport, Bile Acid Synthesis, and Cholesterol Homeostasis

描述（由申请人提供）： 建议摘要/摘要肝脏脂质代谢调节紊乱见于多种重要疾病。特别地，非酒精性脂肪性肝病（NAFLD）是肝脂质稳态的障碍，其中胆固醇和甘油三酯都在肝细胞中积累。一部分NAFLD患者进展为肝细胞坏死、炎症和进行性纤维化。NAFLD现在被认为是美国肝硬化的主要原因。NAFLD中的肝脏脂质积聚似乎部分通过诱导内质网中的未折叠蛋白反应（UPR）产生损伤;与动脉粥样硬化发展中发现的事件类似的事件的进展。与动脉粥样硬化形成一样，减少细胞内脂质积聚的治疗可以通过抑制UPR来减轻NAFLD中的肝损伤。 最近，我们已经确定线粒体胆固醇的传递和氧化作为调节肝脏脂质代谢的关键步骤。发现肝细胞中线粒体胆固醇递送蛋白StARD 1的表达增加下调胆固醇生物合成途径，同时上调胆固醇降解和分泌途径。StARD 1过表达的后果包括细胞内中性脂质（胆固醇和甘油三酯）显著降低，脂质稳态中重要的关键核受体水平升高，HMG CoA还原酶、乙酰CoA羧化酶和脂肪酸合酶（胆固醇和脂肪酸生物合成中的速率决定酶）表达降低。我们现在已经表明，肝脏StARD 1过表达通过线粒体CYP 27 A1启动的途径增加胆固醇氧化，并且所产生的氧固醇产物是能够介导脂质代谢变化的调节分子。CYP 27 A1是一种普遍存在的线粒体酶，我们的初步数据表明，StARD 1/CYP 27 A1途径也可以调节许多肝外组织中的脂质稳态。在由CYP 27 A1基因缺失引起的遗传性疾病CTX中，这些调节性氧化固醇的缺乏与脂质在各种组织（包括肝脏）中的蓄积、加速动脉粥样硬化和神经功能缺损相关。 此次更新申请的目的是进一步阐明胆固醇氧化的StARD 1/CYP 27 A1途径在调节肝脏脂质稳态中的作用。我们假设StARD 1作为胆固醇利用率的细胞内传感器。当胆固醇过量存在时，线粒体胆固醇递送增加，导致CYP 27 A1衍生的氧固醇的产生增加。所产生的氧固醇然后通过结合到核受体来调节脂质代谢。我们进一步假设，刺激肝脏中的这一通路可能是治疗非酒精性脂肪肝的一种有用的策略。 提出了四个具体目标来研究这一假设。具体目标1将在完整和敲除模型中使用选择性StARD 1过表达，以确定StARD 1/CYP 27 A1途径衍生的氧固醇是否负责激活控制参与胆固醇、脂肪酸和胆汁酸稳态调节的基因表达的关键核受体;并随后确定编码途径的表达如何与相应核受体激活相关。在具体目标2中，我们提出表征和评估StARD 1/CYP 27 A1途径衍生的参与脂质稳态的氧固醇激活核受体的机制。具体目标3将确定胆固醇代谢的StarD 1/CYP 27 A1途径在减弱肝细胞中未折叠蛋白反应（UPR）中的作用。具体目标4将首次测试增加StARD 1表达的药理学潜力，以预防或逆转代表人类疾病的动物模型中的肝脏脂质蓄积障碍。

DESCRIPTION (provided by applicant): 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.