Phospholipid-Mediated Metabolic Control in Liver and Brown Adipose Tissue

描述（由申请人提供）：脂质和葡萄糖稳态的调节改变，最常见于胰岛素抵抗和肥胖症的情况下，是包括非酒精性脂肪性肝病（NAFLD）在内的常见疾病发病机制的核心。由于目前的管理选择仍然有限，新的代谢途径的发现将有助于确定新的药物干预的机会。这项研究建议解决了膜磷脂是否调节营养稳态的未回答的问题。我们的长期目标是了解磷脂介导的代谢控制如何用于治疗目的。本研究的目的是确定磷脂酰胆碱转移蛋白（PC-TP）的膜磷脂酰胆碱组合物的传感被翻译成肝脏和氧化组织内的代谢控制的分子机制。核心假设是，当PC-TP结合特异性膜磷脂酰胆碱，然后激活硫酯酶超家族成员2（Them 2）时，发生关键的调控事件。其基本原理是磷脂酰胆碱敏感途径的机制应该对胰岛素抵抗及其并发症（包括NAFLD）产生新的见解。在广泛的初步数据的指导下，中心假设将在三个特定目标中进行测试：1）证明Them 2在PC-TP介导的肝脏脂质和葡萄糖代谢调节中起核心作用; 2）确定PC-TP和Them 2限制棕色脂肪产热的机制; 3）确定磷脂酰胆碱分子种类对PC-TP结合和激活Them 2的影响。在目标1中，在新创建的Them 2-/-小鼠中的高胰岛素血正葡萄糖钳夹以及甘油三酯周转研究将测试PC-TP下游的Them 2的功能。将从培养的原代小鼠肝细胞以及HEK 293 T细胞中的实验中收集额外的机制见解，其中内源性Them 2和PC-TP的表达使用siRNA沉默。目的2将利用间接量热法以及培养的原代棕色脂肪细胞，以评估PC-TP-Them 2相互作用是否限制线粒体脂肪酸氧化。培养的棕色脂肪细胞也将用于评估PC-TP-Them 2相互作用是否通过增加氧化应激来减少去甲肾上腺素信号传导。在目的3中，通过下拉测定、表面等离子体共振和Them 2的脂肪酰基-CoA硫酯酶活性来定量单个磷脂酰胆碱分子种类对PC-TP-Them 2相互作用的控制。PC-TP和Them 2的相互作用结构域将通过采用哺乳动物双杂交测定系统的突变分析来鉴定。总的来说，这个建议将阐明磷脂酰胆碱介导的调节脂质和葡萄糖代谢的机制，这是重要的，因为膜磷脂酰胆碱的脂肪酰基组成在健康和疾病中变化。这些研究有望为NAFLD、2型糖尿病和其他肥胖相关疾病的管理确定新的治疗靶点。

DESCRIPTION (provided by applicant): Altered regulation of lipid and glucose homeostasis, most often in the setting of insulin resistance and obesity, is central to the pathogenesis of common disorders including non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, the discovery of new metabolic pathways will serve to identify novel opportunities for pharmacologic intervention. This research proposal addresses the unanswered question of whether membrane phospholipids regulate nutrient homeostasis. Our long-term goal is to understand how phospholipid-mediated metabolic control can be leveraged for therapeutic purposes. The objective of this research is to determine the molecular mechanisms whereby sensing of membrane phosphatidylcholine composition by phosphatidylcholine transfer protein (PC-TP) is translated into metabolic control within the liver and oxidative tissues. The central hypothesis is that key regulatory events occur when PC-TP binds specific membrane phosphatidylcholines and then activates thioesterase superfamily member 2 (Them2). The rationale is that the mechanisms of a phosphatidylcholine-sensing pathway should yield new insights into insulin resistance and its complications, including NAFLD. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) Demonstrate that Them2 plays a central role in PC-TP-mediated regulation of hepatic lipid and glucose metabolism; 2) Determine the mechanisms by which PC-TP and Them2 limit thermogenesis in brown fat; and 3) Define the influence of phosphatidylcholine molecular species on binding and activation of Them2 by PC-TP. In Aim 1, hyperinsulinemic euglycemic clamp, as well as triglyceride turnover studies in newly created Them2-/- mice will test functions of Them2 downstream of PC-TP. Additional mechanistic insights will be gleaned from experiments in cultured primary mouse hepatocytes, as wel as HEK 293T cells in which expression of endogenous Them2 and PC-TP are silenced using siRNAs. Aim 2 will utilize indirect calorimetry, as well as cultured primary brown adipocytes in order to assess whether PC-TP-Them2 interactions limit mitochondrial fatty acid oxidation. Cultured brown adipocytes wil also be used to evaluate whether PC-TP-Them2 interactions reduce norepinephrine signaling by increasing oxidative stress. In Aim 3, control of PC-TP- Them2 interactions by individual phosphatidylcholine molecular species will be quantified by pulldown assays, surface plasmon resonance and the fatty acyl-CoA thioesterase activity of Them2. The interacting domains of PC-TP and Them2 will be identified by mutational analysis employing a mammalian two-hybrid assay system. Overall, this proposal will elucidate mechanisms of phosphatidylcholine-mediated regulation of lipid and glucose metabolism, which is significant because the fatty acyl composition of the membrane phosphatidylcholines varies in health and disease. These studies are expected to identify new therapeutic targets for the management of for NAFLD, type 2 diabetes and other obesity-associated disorders.