Lipid Transport, Bile Acid Synthesis, and Cholesterol Homeostasis

描述(由申请人提供)： 建议摘要/摘要肝脂代谢调节紊乱可见于多种重要疾病。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都积聚在肝细胞中。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变的主要原因。NAFLD中的肝脏脂肪堆积似乎部分通过诱导内质网中的未折叠蛋白反应(UPR)而产生损伤；这一过程与动脉粥样硬化的发展过程相似。与动脉粥样硬化一样，减少细胞内脂质堆积的治疗可能通过抑制UPR来减轻NAFLD的肝损伤。最近，我们发现线粒体胆固醇的传递和氧化是调节肝脏脂肪代谢的关键步骤。研究发现，线粒体胆固醇传递蛋白StARD1在肝细胞中的表达增加，下调了胆固醇的生物合成途径，而上调了胆固醇的降解和分泌途径。StARD1过表达的后果包括显著降低细胞内中性脂质(胆固醇和甘油三酯)，增加在脂质稳态中重要的核受体水平，以及降低HMG CoA还原酶、乙酰辅酶A羧基酶和脂肪酸合成酶(胆固醇和脂肪酸生物合成中的速率决定酶)的表达。我们现在已经证明，肝脏StARD1的过表达通过线粒体CYP27A1启动的途径增加胆固醇氧化，由此产生的氧固醇产物是能够调节由此导致的脂代谢变化的调节分子。CYP27A1是一种普遍存在的线粒体酶，我们的初步研究表明，在许多肝外组织中，StARD1/CYP27A1途径也可能调节脂质稳态。在由CYP27A1基因缺失引起的遗传性疾病CTX中，这些调节性氧固醇的缺乏与各种组织(包括肝脏)中脂质的积累、加速的动脉粥样硬化和神经功能障碍有关。这一更新应用的目的是进一步阐明胆固醇氧化的StARD1/CyP27A1途径在调节肝脏脂质平衡中的作用。我们假设StARD1可以作为细胞内胆固醇可利用性的传感器。当胆固醇过多时，线粒体胆固醇的输送增加，导致CYP27A1衍生的氧化甾醇产量增加。由此产生的氧化甾醇通过与核受体结合来调节脂质代谢。我们进一步假设，刺激肝脏中的这一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。具体目标1将利用完整和敲除模型中选择性的StARD1过表达来确定StARD1/CYP27A1途径衍生的氧固醇是否负责激活关键的核受体，这些核受体控制参与胆固醇、脂肪酸和胆汁酸稳态调节的基因的表达；并随后确定编码通路的表达如何与各自的核受体激活相关。在特定的目标2中，我们建议表征和评估参与脂质稳态的StARD1/CyP27A1途径衍生的氧甾醇激活核受体的机制。特异性目标3将确定胆固醇代谢的StarD1/CyP27A1通路在减弱肝细胞中未折叠蛋白反应(UPR)中的作用。特定目标4将首次测试StARD1表达增加的药理潜力，以防止或逆转代表人类疾病的动物模型中肝脏脂肪堆积的障碍。 公共卫生相关性： 非酒精性脂肪性肝病(NAFLD)和动脉粥样硬化是影响美国人，更具体地说，是退伍军人群体的主要脂代谢紊乱。NAFLD现在被认为是美国肝硬变的主要原因。NAFLD中的肝脏脂肪堆积导致类似于动脉粥样硬化发展的事件的进展。与动脉粥样硬化一样，减少细胞内脂肪堆积的治疗可能会减轻NAFLD的肝损伤。本申请中提出的研究代表了基于我们最近的发现的新方法，以更清楚地定义肝细胞中脂代谢的调节。为了开发更有效的治疗脂质相关疾病的方法，更好地了解细胞胆固醇、脂肪酸和胆汁酸代谢至关重要。

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. PUBLIC HEALTH RELEVANCE: Significance/Relevance to VA Patient Care Misssion Nonalcoholic fatty liver disease (NAFLD) and atherosclerosis are major disorders of lipid metabolism affecting the American, and, more specifically, the Veterans population. NAFLD is now recognized as a leading cause of cirrhosis in the U.S. Hepatic lipid accumulation in NAFLD leads to 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. The studies proposed in this application represent novel approaches based upon our recent findings to more clearly define the regulation of lipid metabolism in hepatocytes. A better understanding of cell cholesterol, fatty acid, and bile acid metabolism is crucial in order to develop more effective therapies for lipid related disorders.