Them1-Mediated Metabolic Regulation and Pathogenic Role in NAFLD

 描述（由申请方提供）：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导致肥胖个体的NAFLD。这项研究的长期目标是了解细胞脂质分子和代谢之间的调节关系，特别是当它们提供治疗机会时。本研究的目的是了解调节能量稳态和营养代谢的基本新机制。核心假设是硫酯酶超家族成员1（Them 1）在棕色脂肪组织（BAT）中作为控制细胞内脂肪酸运输和调节产热基因表达的脂质调节的脂肪酰辅酶A硫酯酶起作用。在肥胖的情况下，我们假设肝脏和白色脂肪组织中Them 1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的过度产生而引起的炎症。其基本原理是Them 1介导的代谢调节机制应该对NAFLD的发病机制产生新的见解。在广泛的初步数据的指导下，中心假设将在三个具体目标中进行测试：1）确定Them 1限制BAT能量消耗的机制; 2）证明Them 1在NAFLD中的主要致病作用; 3）阐明脂质结合类固醇生成急性调节蛋白相关脂质转移（START）结构域对Them 1活性的分子调节。在目标1中，最近开发的Them 1-/-小鼠和培养的棕色脂肪细胞将用于测试BAT中的Them 1限制脂肪酸进入线粒体并降低产热基因表达的假设。目标2将利用新创建的转基因小鼠以及细胞培养系统来探索Them 1是否直接导致肝脂肪变性和胰岛素抵抗。具有肝脏特异性过表达的小鼠将用于确定Them 1在促进肝脏ER应激、线粒体功能障碍和炎症中的作用。具有脂肪特异性过表达的小鼠将揭示Them 1对白色脂肪组织内的炎症和肝脂肪变性的贡献。目的3将通过质谱法确定Them 1 START结构域的脂质配体。X射线晶体学将用于确定Them 1与其配体复合物的结构、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，这将有助于结构-功能研究，并有助于描述Them 1的生物学作用。总的来说，这项提案将阐明Them 1介导的代谢调节，这是重要的，因为在健康中保存能量的机制可能会在营养过剩的条件下促进疾病。这些研究有望为NAFLD的管理确定新的治疗靶点。

 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.