Phospholipid-Mediated Metabolic Control in Liver and Brown Adipose Tissue

描述（由申请人提供）：脂质和葡萄糖稳态调节的改变，最常发生在胰岛素抵抗和肥胖的情况下，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。由于目前的治疗选择仍然有限，新的代谢途径的发现将有助于确定药物干预的新机会。这项研究计划解决了膜磷脂是否调节营养稳态的悬而未决的问题。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）感知膜磷脂酰胆碱组成转化为肝脏和氧化组织代谢控制的分子机制。核心假设是PC-TP结合特异性膜磷脂酰胆碱并激活硫酯酶超家族成员2 （Them2）时发生关键调控事件。其基本原理是磷脂酰胆碱感应通路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。在大量初步数据的指导下，中心假设将在三个具体目标中进行验证：1)证明Them2在pc - tp介导的肝脏脂质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结合活化Them2的影响。在Aim 1中，在新创建的Them2-/-小鼠中进行高胰岛素正糖钳夹和甘油三酯转换研究将测试PC-TP下游Them2的功能。更多的机制见解将从培养的原代小鼠肝细胞和HEK 293T细胞的实验中收集，其中内源性Them2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-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用于评估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Aim 3中，个体磷脂酰胆碱分子种对PC-TP- Them2相互作用的控制将通过下拉实验、表面等离子体共振和Them2的脂肪酰基辅酶a硫酯酶活性进行量化。PC-TP和Them2的相互作用结构域将通过采用哺乳动物双杂交分析系统的突变分析来确定。总的来说，这一提议将阐明磷脂酰胆碱介导的脂质和糖代谢调节机制，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是不同的。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. 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.