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

描述（由申请方提供）：本提案的总体目标是检查受损的胰岛素信号传导改变HDL介导的胆固醇逆向转运（RCT）的程度。HDL的一个关键心脏保护功能是将过量的胆固醇从血浆和组织输送到肝脏，通过RCT从体内消除。SR-BI是主要的肝脏HDL受体，介导HDL胆固醇酯（HDL-CE）的选择性摄取。与其他循环脂蛋白相比，HDL颗粒上携带的胆固醇以SR-BI依赖性方式优先分泌到胆汁中。除了SR-BI在经肝固醇转运中的作用外，功能获得和功能丧失研究均已确定SR-BI是肝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胆固醇掺入VLDL。已显示HDL-CE摄取和胆汁胆固醇分泌均受到胰岛素信号传导受损的负面影响，胰岛素信号传导受损是代谢综合征（MetS）和心血管疾病风险增加的关键因素。我们发表的和初步的数据表明，胰岛素信号调节SR-BI的一种新的翻译后机制，促进SR-BI的质膜上的表达。我们假设胰岛素抵抗降低HDL-CE摄取，并通过包括SR-BI重新分布到细胞内室的机制改变SR-BI依赖性胆固醇向肝脏调节和分泌池的递送。具体目标1将确定肝脏胰岛素信号传导和胰岛素抵抗对SR-BI介导的固醇运输的影响 分泌和调节肝脏胆固醇池。通过胰岛素受体的选择性耗竭或通过显性负性磷脂酰肌醇-3激酶（AdnilPI 3 K）的表达破坏肝脏胰岛素信号传导，并确定肝脏胰岛素抵抗降低SR-BI介导的HDL-C选择性胆固醇酯摄取的程度。研究还将确定在胰岛素信号传导受损的情况下，SR-BI功能降低如何改变稳态肝脏胆固醇合成和分泌？具体目标2将检验以下假设：在肝胰岛素抵抗和高胆固醇血症的情况下，胆汁中HDL胆固醇的经皮（THCE）消除减少。这将通过a）确定降低的SR-BI功能是否降低HDL-C的THCE，B）确定肝IR是否以SR-BI依赖性方式降低巨噬细胞对粪便的RCT，和c）检查HDL颗粒组成的变化是否破坏HDL-C的THCE并与SR-BI功能障碍协同作用以降低HDL RCT功能来实现。具体目标3将确定胰岛素信号调节SR-BI细胞表面定位在肝细胞中的分子机制。将在培养的肝细胞中检查胰岛素依赖性PI 3 K信号传导对SR-BI亚细胞分布的影响，以及由于胰岛素信号传导受损导致SR-BI蓄积的细胞内隔室的鉴别。将定义负责的胰岛素依赖性信号通路，并进行研究以确定胰岛素信号调节SR-BI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.