Them1-Mediated Metabolic Regulation and Pathogenic Role in NAFLD

 描述(由申请人提供)：肥胖引起的肝脏胰岛素抵抗是非酒精性脂肪性肝病(NAFLD)发病机制的核心。这项研究提案解决了一个悬而未决的问题，即通常促进能量守恒的分子机制如何有助于肥胖者的非酒精性脂肪肝。这项研究的长期目标是了解细胞脂分子和新陈代谢之间的调节关系，特别是当它们提供治疗机会时。这项研究的目的是了解能量平衡和营养代谢调节的基本新机制。中心假说是，硫酯酶超家族成员1(Them1)在棕色脂肪组织(BAT)中作为一种脂质调节的脂肪酰辅酶A硫酯酶发挥作用，它控制细胞内脂肪酸的运输，并调节生热基因的表达。在肥胖的背景下，我们假设Them1在肝脏和白色脂肪组织中的上调促进了内质网(ER)应激、线粒体功能障碍和由于游离脂肪酸过量产生而引起的炎症。其基本原理是，Them1介导的代谢调节机制将为NAFLD的发病机制提供新的见解。在大量初步数据的指导下，中心假设将在三个特定目标下进行检验：1)确定Them1限制BAT能量消耗的机制；2)证明Them1在NAFLD中的主要致病作用；3)阐明Them1活性的分子调控通过脂质结合的类固醇激素生成急性调节蛋白相关脂转移(Start)结构域。在目标1中，最近培育的Them1-/-小鼠和培养的棕色脂肪细胞将被用来检验蝙蝠体内Them1限制脂肪酸进入线粒体并减少生热基因表达的假设。Aim 2将利用新创造的转基因小鼠以及细胞培养系统来探索Them1是否直接导致肝脏脂肪变性和胰岛素抵抗。肝脏特异性过表达的小鼠将被用来确定Them1在促进肝脏内质网应激、线粒体功能障碍和炎症方面的作用。脂肪特异性过表达的小鼠将揭示Them1对白色脂肪组织内的炎症和肝脏脂肪变性的贡献。目的3用质谱法测定Them1起始结构域的脂质配体(S)。X射线结晶学将用于确定Them1与其配体的络合物的结构、功能结构域之间的关系以及脂质结合对酶活性的影响。高通量筛选将识别小分子抑制剂，这将促进结构-功能研究，并有助于描述Them1的生物学作用。总体而言，这项提议将阐明Them1介导的代谢调节，这一点非常重要，因为在营养过剩的情况下，保存健康能量的机制可能会促进疾病。这些研究有望确定治疗非酒精性脂肪肝的新靶点。

 DESCRIPTION (provided by applicant): Hepatic insulin resistance due to obesity is central to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). This research proposal addresses the unanswered question of how molecular mechanisms that normally promote energy conservation contribute to NAFLD in obese individuals. The long-term goal of this research is to understand the regulatory relationships between cellular lipid molecules and metabolism, particularly as they present therapeutic opportunities. The objective of this research is to understand fundamental new mechanisms for the regulation of energy homeostasis and nutrient metabolism. The central hypothesis is that thioesterase superfamily member 1 (Them1) functions in brown adipose tissue (BAT) as a lipid-regulated fatty acyl-CoA thioesterase that controls intracellular fatty acid trafficking and that regulates the expression of thermogenic genes. In the setting of obesity, we postulate that upregulation of Them1 in liver and white adipose tissue promotes endoplasmic reticulum (ER) stress, mitochondrial dysfunction and inflammation due to the overproduction of free fatty acids. The rationale is that the mechanisms of Them1-mediated metabolic regulation should yield new insights into the pathogenesis of NAFLD. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To determine the mechanisms whereby Them1 limits energy expenditure in BAT; 2) To demonstrate a primary pathogenic role for Them1 in NAFLD; and 3) To elucidate molecular regulation of Them1 activity by the lipid-binding steroidogenic acute regulatory protein-related lipid transfer (START) domain. In Aim 1, recently developed Them1-/- mice and cultured brown adipocytes will be used to test the hypothesis that Them1 in BAT limits access of fatty acids to mitochondria and reduces the expression of thermogenic genes. Aim 2 will leverage newly created transgenic mice, as well as cell culture systems to explore whether Them1 contributes directly to hepatic steatosis and insulin resistance. Mice with liver-specific overexpression will be used to establish the role of Them1 in promoting hepatic ER stress, mitochondrial dysfunction and inflammation. Mice with adipose-specific overexpression will reveal the contributions of Them1 to inflammation within white adipose tissue and to hepatic steatosis. Aim 3 will determine the lipid ligand(s) of the Them1 START domain by mass spectrometry. X-ray crystallography will be used to determine the structure of Them1 in complex with its ligands, the relationship among functional domains and the influence of lipid binding on enzymatic activity. High throughput screening will identity small molecule inhibitors, which should facilitate structure-function studies and help delineate the biological roles of Them1. Overall, this proposal will elucidate Them1-mediated metabolic regulation, which is significant because mechanisms that conserve energy in health may promote disease under conditions of overnutrition. These studies are expected to identify new therapeutic targets for the management of NAFLD.