The role of hepatic insulin resistance on SR-BI dependant HDL cholesterol uptake

描述(由申请人提供)：这项建议的总体目标是检查胰岛素信号受损改变高密度脂蛋白介导的反向胆固醇运输(RCT)的程度。高密度脂蛋白的一个关键的心脏保护功能是将多余的胆固醇从血浆和组织输送到肝脏，然后通过RCT从体内消除。SR-BI是肝脏主要的高密度脂蛋白受体，参与高密度脂蛋白胆固醇酯的选择性摄取。与其他循环脂蛋白相比，高密度脂蛋白颗粒携带的胆固醇优先在胆汁中以SR-BI依赖的方式分泌。除了SR-BI在通过肝脏的类固醇转运中的作用外，研究已经证实SR-BI是肝脏胆固醇代谢的决定因素，包括胆固醇合成、胆汁胆固醇分泌速率和将胆固醇掺入极低密度脂蛋白。高密度脂蛋白-CE的摄取和胆汁胆固醇的分泌都被证明受到胰岛素信号受损的负面影响，胰岛素信号是代谢综合征(METS)的一个关键因素，并增加了心血管疾病的风险。我们已发表的和初步的数据表明，胰岛素信号通过一种新的翻译后机制来调节SR-BI，这种机制促进了SR-BI在质膜上的表达。我们假设，胰岛素抵抗降低了高密度脂蛋白-CE的摄取，并通过包括将SR-BI重新分配到细胞内隔室的机制改变了依赖SR-BI的胆固醇向肝脏调节池和分泌池的输送。特异性目标1将确定肝脏胰岛素信号和胰岛素抵抗对SR-BI介导的类固醇转运的影响 分泌和调节肝脏胆固醇池。选择性地耗尽胰岛素受体或表达显性负磷脂酰肌醇-3K(AdnilPI3K)将干扰肝脏胰岛素信号转导，并确定肝脏胰岛素抵抗降低SR-BI介导的高密度脂蛋白胆固醇选择性摄取的程度。研究还将确定在胰岛素信号受损的情况下，SR-BI功能降低如何改变稳态肝脏胆固醇的合成和分泌？特异性目标2将测试假设，即在肝脏胰岛素抵抗和高甘油三酯血症的情况下，经肝(THCE)对胆汁中高密度脂蛋白胆固醇的清除减少。这将通过a)确定SR-BI功能降低是否降低了HDL-C的THCE，b)确定肝脏IR是否以SR-BI依赖的方式减少巨噬细胞到粪便的RCT，以及c)检查高密度脂蛋白颗粒组成的变化是否扰乱了HDLC的THCE，并与SR-BI功能障碍协同作用来降低HDLRCT功能。特异靶3将确定胰岛素信号调节SR-BI细胞在肝细胞表面定位的分子机制。胰岛素依赖的PI3K信号对SR-BI亚细胞分布的影响将在培养的肝细胞中进行检测，以及由于胰岛素信号受损而聚集SR-BI的细胞内隔室(S)的身份。负责的胰岛素依赖信号通路将被定义，并将进行研究，以确定SR-BI上通过胰岛素信号调节其所需的决定因素。

DESCRIPTION (provided by applicant): The overall objective of this proposal is to examine the extent to which impaired insulin signaling alters HDL- mediated reverse cholesterol transport (RCT). A key cardioprotective function of HDL is the delivery of excess cholesterol from plasma and tissues to the liver for elimination from the body via RCT. SR-BI is the major hepatic HDL receptor and mediates the selective uptake of HDL cholesterol esters (HDL-CE). Compared to other circulating lipoproteins, cholesterol carried on HDL particles is preferentially secreted int bile in an SR-BI dependent manner. In addition to SR-BI's role in trans-hepatic sterol transport, both gain and loss-of-function studies have established that SR-BI is a determinant of hepatic cholesterol metabolism including cholesterol synthesis, biliary cholesterol secretion rates and the incorporation of cholesterol into VLDL. Both HDL-CE uptake and biliary cholesterol secretion have been shown to be negatively affected by impaired insulin signaling, a key contributing element to the Metabolic Syndrome (MetS) and the increased risk for cardiovascular disease. Our published and preliminary data indicate that insulin signaling regulates SR-BI by a novel post-translational mechanism that promotes SR-BI expression on the plasma membrane. We hypothesize that insulin resistance reduces HDL-CE uptake and alters SR-BI-dependent delivery of cholesterol to hepatic regulatory and secretory pools through mechanisms that include the redistribution of SR-BI to an intracellular compartment. Specific Aim 1 will determine the effect of hepatic insulin signaling and insulin resistance on SR-BI mediated sterol trafficking to secretory and regulatory hepatic cholesterol pools. Hepatic insulin signaling will be disrupted by selective depletion of insulin receptors or by expression of a dominant negative phosphatidylinositol-3 kinase (AdnilPI3K) and the extent to which hepatic insulin resistance reduces SR-BI mediated HDL-C selective cholesterol ester uptake determined. Studies will also determine how reduced SR-BI function alters steady-state hepatic cholesterol synthesis and secretion in the setting of impaired insulin signaling? Specific Aim 2 will test the hypothesis tha transhepatic (THCE) elimination of HDL cholesterol in bile is reduced in the setting of hepatic insulin resistance and hypertriglyceridemia. This will be accomplished by a) determining whether reduced SR-BI function diminishes THCE of HDL-C, b) determining whether hepatic IR decreases macrophage to feces RCT in an SR-BI dependent manner, and c) examining whether changes in HDL particle composition disrupt THCE of HDL-C and synergize with SR-BI dysfunction to reduce HDL RCT function. Specific Aim 3 will determine the molecular mechanisms by which insulin signaling regulates SR-BI cell surface localization in hepatocytes. The effect of insulin-dependent PI3K signaling on SR- BI subcellular distribution will be examined in cultured hepatocytes, together with the identity of the intracellular compartment(s) in which SR-BI accumulates as a result of impaired insulin signaling. The responsible insulin- dependent signaling pathways will be defined and studies will be performed to identify the determinants on SR- BI necessary for its regulation by insulin signaling.