Phospholipid-Mediated Metabolic Control in Liver and Brown Adipose Tissue

描述(申请人提供)：脂肪和葡萄糖稳态调节的改变，最常发生在胰岛素抵抗和肥胖的背景下，是包括非酒精性脂肪性肝病(NAFLD)在内的常见疾病的发病机制的核心。由于目前的管理选择仍然有限，新代谢途径的发现将有助于确定药物干预的新机会。这项研究方案解决了膜磷脂是否调节营养动态平衡这一悬而未决的问题。我们的长期目标是了解磷脂介导的代谢控制如何被用于治疗目的。本研究的目的是确定磷脂酰胆碱转移蛋白(PC-TP)对膜磷脂酰胆碱组成的感知转化为肝脏和氧化组织内代谢控制的分子机制。中心假说是，当PC-TP与特定的膜磷脂酰胆碱结合，然后激活硫酯酶超家族成员2(Them2)时，关键调控事件发生。其基本原理是，磷脂酰胆碱敏感通路的机制应该能为胰岛素抵抗及其并发症(包括NAFLD)提供新的见解。在大量初步数据的指导下，中心假说将在三个具体目标上进行检验：1)证明Them2在PC-TP介导的肝脏脂肪和葡萄糖代谢调节中发挥核心作用；2)确定PC-TP和Them2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Them2的影响。在目标1中，高胰岛素正血糖钳夹，以及新创建的Them2-/-小鼠的甘油三酯转换研究将测试Pc-TP下游的Them2的功能。在原代培养的小鼠肝细胞以及HEK 293T细胞中，内源性Them2和PC-TP的表达被siRNAs沉默，我们将从实验中收集到更多的机制见解。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-Them2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被用来评估PC-TP-Them2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。在目标3中，将通过下拉试验、表面等离子体共振和Them2的脂酰辅酶A硫酯酶活性来量化个别磷脂酰胆碱分子物种对PC-TP-Them2相互作用的控制。PC-TP和Them2的相互作用结构域将通过使用哺乳动物双杂交检测系统的突变分析来确定。总体而言，这项建议将阐明磷脂酰胆碱介导的调节脂肪和葡萄糖代谢的机制，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有所不同。这些研究有望为NAFLD、2型糖尿病和其他肥胖相关疾病的治疗确定新的治疗目标。 公共卫生相关性：这项拟议的研究与公共健康相关，因为控制脂肪和葡萄糖动态平衡的磷脂依赖途径的发现有望为胰岛素抵抗及其并发症(包括非酒精性脂肪性肝病)的发病机制提供新的见解。拟议的研究与NIDDK的使命相关，因为它们有望确定与糖尿病和肥胖症相关的常见疾病管理的新治疗目标。

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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the discovery of a phospholipid-dependent pathway that controls lipid and glucose homeostasis is expected to provide new insights into the pathogenesis of insulin resistance and its complications, including non-alcoholic fatty liver disease. The proposed studies are relevant to the mission of the NIDDK because they are expected to identify new therapeutic targets for the management of common disorders related to diabetes and obesity.