Regulation of Hepatic Glucose Homeostasis by Phosphatidylcholine Transfer Protein

描述(申请人提供)：肥胖引起的胰岛素抵抗是导致2型糖尿病和非酒精性脂肪肝(NAFLD)的主要病理生理缺陷。由于目前的管理选择仍然有限，识别管理胰岛素代谢反应的新调节机制应该有助于识别药物干预的新机会。本研究的目的是探讨膜磷脂酰胆碱(PC)成分在调节肝脏脂肪和糖代谢中的作用。其基本原理是，对调节肝脏胰岛素敏感性的新机制的识别可能导致识别治疗NAFLD和2型糖尿病的新治疗靶点。在大量前期数据的指导下，本研究计划的中心假设是磷脂酰胆碱转移蛋白(PC-TP)作为PC分子种类的感受器，通过与硫酯酶超家族成员2(THEM2)和结节性硬化症复合体2(TSC2)形成调节复合体，通过内质网(ER)应激调节胰岛素信号转导能力，从而控制血糖的动态平衡。这将在两个特定的目标中进行测试：1)确定PC-TP和THEM2通过TSC2相互作用调节胰岛素信号的机制；2)确定PC分子物种在PC-TP结合和激活THEM2和TSC2中的作用。在目标1中，将通过测量关键效应分子的活性来确定siRNA介导的PC-TP和THEM2表达沉默对细胞培养系统中胰岛素信号转导的影响。TSC2在PC-TP和THEM2介导的胰岛素信号调节中的作用将在缺乏TSC2表达的细胞系中进行探讨。将使用化学试剂衣霉素在培养细胞中以及在Pctp-/-和Them2-/-小鼠长期高脂饮食中研究PC-TP和THEM2在内质网应激诱导中的潜在作用。预计PC-TP和THEM2将通过与TSC2形成新的蛋白质复合体和通过调节内质网应激来直接调节肝细胞胰岛素信号转导。这一点很重要，因为内质网应激增加与肥胖相关的胰岛素抵抗的发展有关。在AIM2中，PC分子种类对纯化的重组PC-TP与THEM2或TSC2相互作用的影响将通过体外下拉实验和表面等离子体共振来检测。结合PC-TP的PC分子物种对THEM2和TCS2活性的调节作用将在体外通过分别测定它们的脂肪酰辅酶A硫酯酶和GAP活性来检测。我们预计，与PC-TP结合的多不饱和PC分子物种将促进与THEM2和TSC2的相互作用，并刺激这些蛋白的活性。总体而言，这项建议将阐明膜磷脂成分调节葡萄糖代谢的机制，这一点具有重要意义，因为膜磷脂酰胆碱的脂肪酰基组成在健康和疾病中各不相同。这些研究有望为2型糖尿病和非酒精性脂肪肝的治疗确定新的治疗目标。

DESCRIPTION (provided by applicant): Obesity-induced resistance to insulin action is the primary pathophysiological defect that predisposes to type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, identification of new regulatory mechanisms that govern the metabolic response to insulin should serve to identify novel opportunities for pharmacologic intervention. The objective of this research is to explore the role of membrane phosphatidylcholine (PC) composition in the regulation of hepatic lipid and glucose metabolism. The rationale is that the identification of a novel mechanism that regulates hepatic insulin sensitivity could lead to the identification of new therapeutic targets for the treatment of NAFLD and type 2 diabetes. Guided by extensive preliminary data, the central hypothesis of this research plan is that phosphatidylcholine transfer protein (PC-TP) functions as a sensor of PC molecular species and controls glucose homeostasis by forming a regulatory complex together with thioesterase superfamily member 2 (THEM2) and tuberous sclerosis complex 2 (TSC2), which modulates insulin signaling potentialy via endoplasmic reticulum (ER) stress. This will be tested in two specific aims: 1) Define the mechanisms by which PC-TP and THEM2 regulate insulin signaling through TSC2 interactions; 2) Determine the role of PC molecular species on binding and activation of THEM2 and TSC2 by PC-TP. In aim 1, the effect of siRNA-mediated silencing of PC-TP and THEM2 expression on insulin signaling will be determined in cell culture systems by measuring the activity of key effectors. The role of TSC2 in the PC-TP- and THEM2-mediated regulation of insulin signaling will be probed in cell lines lacking TSC2 expression. The potential roles of PC-TP and THEM2 in the induction of ER stress will be studied using chemical reagent tunicamycin in cultured cells and prolonged high-fat diet in Pctp-/- and Them2-/- mice. It is anticipated that PC-TP and THEM2 will regulate hepatocellular insulin signaling directly both by forming a novel protein complex with TSC2 and by modulating ER stress. This is important because increased ER stress is associated with development of insulin resistance in association with obesity. In aim2, the influence of PC molecular species on the interactions between purified recombinant PC-TP and THEM2 or TSC2 will be examined in vitro by puldown assays and by surface plasmon resonance. The efect of the PC molecular species bound to PC-TP on the activity regulation of THEM2 and TCS2 will be detected in vitro by measuring their fatty acyl-CoA thioesterase and GAP activity, respectively. We expect that polyunsaturated PC molecular species bound to PC-TP will promote interactions with THEM2 and TSC2 and stimulate the activity of these proteins. Overall, this proposal will elucidate mechanisms by which membrane phospholipid composition regulates glucose metabolism, which is significant because the faty acyl composition of the membrane phosphatidylcholines varies in health and disease. These studies are expected to identify new therapeutic targets for the management of type 2 diabetes and NAFLD.