Regulation of Hepatic Lipid and Glucose Metabolism by Phosphatidylcholine Transfe

描述（由申请人提供）：磷脂酰胆碱转移蛋白（PC-TP，又名StarD 2）是一种高度特异性的细胞内脂质结合蛋白，在肝脏和其他氧化组织（包括棕色脂肪、心脏和骨骼肌）中表达增强。使用PC-TP缺陷（Pctp-/-）小鼠，我们已经证明，PC-TP调节胆汁脂质分泌，肝脏胆固醇稳态和高密度脂蛋白（HDL）代谢。此外，缺乏PC-TP表达的小鼠对动脉粥样硬化的发展具有相对抵抗力。与我们最初预测的PC-TP将磷脂酰胆碱转运至质膜以从肝细胞输出相反，本研究表明在肝脏脂质稳态中具有更全面的调节作用。对Pctp-/-小鼠的初步研究还显示，胰岛素介导的肝脏葡萄糖生成大幅减少，以及肝外氧化组织对能量底物的利用发生改变。PC-TP可能通过与线粒体相关蛋白硫酯酶超家族成员2（Them 2）的直接相互作用，潜在地通过控制脂肪酸进入线粒体来调节脂质和葡萄糖代谢。具体目标1将检验PC-TP调节肝脏胰岛素敏感性的假设。我们将研究PC-TP表达的缺乏是否可以防止饮食诱导的胰岛素抵抗，以及PC-TP的肝脏特异性表达是否会降低肝脏胰岛素敏感性。胰岛素信号将使用细胞培养系统，其中PC-TP的表达水平有很大的变化进行研究。我们还将在体内和细胞培养中探索PC-TP-Them 2相互作用有助于控制胰岛素作用的证据。具体目标2将探索PC-TP对表达PC-TP和Them 2的棕色脂肪的分化和功能的影响。将在从Pctp-/-和野生型小鼠培养的棕色前脂肪细胞的分化过程中表征基因表达以及细胞功能和形态。PC-TP影响棕色脂肪细胞成熟的机制将通过在分化过程中系统地重新引入PC-TP（有或没有Them 2）来收集。这些数据将与体内棕色脂肪产热的测量相关联。细胞培养研究将探索PC-TP和Them 2激活棕色脂肪中关键转录因子的机制。具体目标3将研究PC-TP的体外和体内活性之间的机制关系。高通量筛选已经鉴定了PC-TP的磷脂酰胆碱转移活性的小分子抑制剂。这一特定目标将继续开发最有前途的抑制剂。将阐明抑制机制，并在细胞中测试化合物。将与PC-TP表达缺失或敲低的细胞进行比较。这些研究应该为脂质和葡萄糖代谢的调节提供新的见解，并可能导致一种新的方法来管理胰岛素抵抗，这是代谢综合征和非酒精性脂肪肝疾病的标志。公共卫生相关性：胰岛素抵抗是代谢综合征和II型糖尿病的标志，易患非酒精性脂肪肝。拟议的研究将检查磷脂酰胆碱转移蛋白/StarD 2（一种高度特异性的脂质结合蛋白）在胰岛素介导的肝脏和身体能量消耗组织内脂质和葡萄糖代谢调节中的关键作用。预期这些实验可以将磷脂酰胆碱转移蛋白/StarD 2确立为用于管理胰岛素抵抗的分子靶点。

DESCRIPTION (provided by applicant): Phosphatidylcholine transfer protein (PC-TP, a.k.a. StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in liver and other oxidative tissues, including brown fat, heart, and skeletal muscle. Using PC-TP-deficient (Pctp-/-) mice, we have demonstrated that PC-TP regulates biliary lipid secretion, hepatic cholesterol homeostasis and high density lipoprotein (HDL) metabolism. In addition, mice lacking PC-TP expression are relatively resistant to developing atherosclerosis. In contrast to our original prediction that PC-TP transports phosphatidylcholines to the plasma membrane for export from hepatocytes, this research has suggested a more global regulatory role in hepatic lipid homeostasis. Preliminary studies in Pctp-/- mice have also revealed profound insulin-mediated decreases in hepatic glucose production, as well as altered energy substrate utilization by extrahepatic oxidative tissues. PC-TP may regulate lipid and glucose metabolism via direct interactions with the mitochondrial-associated protein thioesterase superfamily member 2 (Them2), potentially by controlling access of fatty acids to mitochondria. Specific Aim 1 will test the hypothesis that PC-TP regulates hepatic insulin sensitivity. We will examine whether lack of PC-TP expression protects against diet-induced insulin resistance and whether liver-specific expression of PC-TP reduces hepatic insulin sensitivity. Insulin signaling will be studied using cell culture systems in which PC-TP expression levels are broadly varied. We will also explore evidence both in vivo and in cell culture that PC-TP-Them2 interactions contribute to the control of insulin action. Specific Aim 2 will explore the influence of PC-TP on the differentiation and function of brown fat, which expresses both PC-TP and Them2. Gene expression, as well as cellular function and morphology will be characterized during the differentiation of brown pre-adipocytes cultured from Pctp-/- and wild type mice. Mechanisms by which PC-TP influences the maturation of brown adipocytes will be gleaned by systematically reintroducing PC-TP with or without Them2 during the process of differentiation. These data will be correlated with measurements of brown fat thermogenesis in vivo. Studies in cell culture will explore mechanisms by which PC-TP and Them2 activate key transcription factors in brown fat. Specific Aim 3 will investigate a mechanistic relationship between the in vitro and in vivo activities of PC-TP. High-throughput screening has identified small molecule inhibitors of the phosphatidylcholine transfer activity of PC-TP. This Specific Aim will continue the development of the most promising inhibitors. Mechanisms of inhibition will be elucidated and compounds will be tested in cells. Comparisons will be made to cells in which PC-TP expression is absent or knocked down. These studies should provide new insights into the regulation of lipid and glucose metabolism and may lead to a novel approach to managing insulin resistance, which constitutes the hallmark of the metabolic syndrome and non-alcoholic fatty liver disease. PUBLIC HEALTH RELEVANCE: Resistance to insulin is a hallmark of the metabolic syndrome and type II diabetes, which predispose to non-alcoholic fatty liver disease. The proposed studies will examine a key role for phosphatidylcholine transfer protein/StarD2, a highly specific lipid binding protein, in insulin- mediated regulation of lipid and glucose metabolism within the liver and energy consuming tissues of the body. It is anticipated that these experiments may establish phosphatidylcholine transfer protein/StarD2 as a molecular target for the management of insulin resistance.