Phospholipid-Mediated Metabolic Control in Liver and Brown Adipose Tissue

肝脏和棕色脂肪组织中磷脂介导的代谢控制

• 批准号: 8452126
• 负责人: DAVID E. COHEN
• 金额: $ 46.21万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8452126
• 关键词: Acyl Coenzyme A; Address; Adipocytes; Binding; Biochemical; Biological Assay; Biological Models; Brown Fat; Carbon; Cells; Data; Diabetes Mellitus; Disease; Esters; Euglycemic Clamping; Eukaryotic Cell; Event; Exhibits; Fasting; Fatty Acids; Fatty Liver; Glean; Glucose; Glucose Clamp; Glycerol; Goals; Grant; Head; Health; Hepatic; Hepatocyte; Homeostasis; In Vitro; Indirect Calorimetry; Individual; Insulin; Insulin Resistance; Intervention; Intracellular Membranes; Lead; Lecithin; Link; Lipids; Liver; Liver diseases; Mediating; Medication Management; Membrane; Metabolic; Metabolic Control; Metabolic Diseases; Metabolic Pathway; Metabolism; Mission; Mitochondria; Molecular; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Non-Insulin-Dependent Diabetes Mellitus; Norepinephrine; Nutrient; Obesity; Outcome Study; Oxidative Stress; Oxygen Consumption; Pathogenesis; Pathway interactions; Phospholipids; Phosphorylcholine; Physiological; Play; Positioning Attribute; Protein Binding; Proteins; Public Health; Recombinant Proteins; Regulation; Research; Research Proposals; Resistance; Role; Signal Transduction; Specificity; Stress; Surface Plasmon Resonance; System; Testing; Therapeutic; Thermogenesis; Tissues; Translating; Triglycerides; Two-Hybrid System Techniques; Variant; base; blood glucose regulation; design; fatty acid oxidation; glucose metabolism; glucose production; improved; insight; insulin sensitivity; lipid metabolism; member; new therapeutic target; non-alcoholic fatty liver; novel; phosphatidylcholine transfer protein; physical property; protein function; public health relevance; research study; response; sensor

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.

描述（由申请人提供）：脂质和葡萄糖稳态调节的改变，最常见的是在胰岛素抵抗和肥胖的情况下，是包括非酒精性脂肪肝病（NAFLD）在内的常见疾病的发病机制的核心。由于当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。这项研究提案解决了膜磷脂是否调节营养稳态这一尚未解答的问题。我们的长期目标是了解如何利用磷脂介导的代谢控制来达到治疗目的。本研究的目的是确定通过磷脂酰胆碱转移蛋白 (PC-TP) 对膜磷脂酰胆碱成分的感知转化为肝脏和氧化组织内的代谢控制的分子机制。中心假设是，当 PC-TP 结合特定的膜磷脂酰胆碱，然后激活硫酯酶超家族成员 2 (Them2) 时，就会发生关键的调节事件。其基本原理是，磷脂酰胆碱传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。在大量初步数据的指导下，中心假设将在三个具体目标上得到检验：1）证明Them2在PC-TP介导的肝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) 定义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，高胰岛素正常血糖钳夹以及新创建的 Them2-/- 小鼠中的甘油三酯周转研究将测试 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以及 HEK 293T 细胞的实验中收集，其中使用 siRNA 沉默内源 Them2 和 PC-TP 的表达。目标 2 将利用间接量热法以及培养的原代棕色脂肪细胞来评估 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传导。在目标 3 中，将通过 Pulldown 测定、表面等离振子共振和 Them2 的脂肪酰辅酶 A 硫酯酶活性来量化各个磷脂酰胆碱分子种类对 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采用哺乳动物双杂交检测系统的突变分析来鉴定。总体而言，该提案将阐明磷脂酰胆碱介导的脂质和葡萄糖代谢调节机制，这一点很重要，因为膜磷脂酰胆碱的脂肪酰基组成在健康和疾病中存在差异。这些研究有望确定 NAFLD、2 型糖尿病和其他肥胖相关疾病的新治疗靶点。