Phospholipid-Mediated Metabolic Control in the Pathogenesis of NAFLD

项目概要/摘要 脂质和葡萄糖稳态调节改变，最常见于胰岛素抵抗和肥胖， 是非酒精性脂肪性肝病（NAFLD）发病机制的核心。因为现在的管理层 选择仍然有限，新的代谢途径的发现将有助于确定新的机会， 药物干预这项研究建议解决了一个悬而未决的问题，即膜是否 磷脂调节营养平衡。我们的长期目标是了解磷脂介导的 代谢控制可用于治疗目的。本研究的目的是确定 通过磷脂酰胆碱转移感知膜磷脂酰胆碱的分子机制 蛋白（PC-TP）被硫酯酶超家族成员2（Them 2）翻译成代谢控制， 大豆相关长链酰基辅酶A硫酯酶。核心假设是关键的调控事件 当PC-TP结合特定的膜磷脂酰胆碱分子种类时发生在骨骼肌中， 然后激活主题2。基本原理是Them 2对骨骼肌代谢的调节应该产生 肝脏胰岛素抵抗和脂肪变性的新见解。在大量初步数据的指导下， 我们将在三个特定的目标中检验这一假设：1）证明Them 2控制血脂和血糖 2）确定骨骼肌中Them 2促进肝脏代谢的机制 3）阐明Them 2活性的分子决定因素和PC-TP的调节作用。在目标1中， 小鼠模型将用于确定Them 2调节脂肪酸和葡萄糖的机制 在骨骼肌中的代谢和促进胰岛素抵抗，以响应营养过剩。细胞自主 Them 2的功能将从培养的肌管中的系统研究中收集。目标2将建立 在高脂肪喂养的小鼠中，骨骼肌中的Them 2促进肝脏胰岛素抵抗的机制， 脂肪变性培养的肝细胞将用于确定是否存在Them 2依赖性肌因子或细胞外 从肌管释放的囊泡控制肝脂质和葡萄糖代谢。目标3将确定结构 Them 2能够响应膜磷脂酰胆碱组成的变化。小 设计用于结合和抑制Them 2的分子将用作探针以表征Them 2功能， 使用一系列生物物理学技术研究与PC-TP的相互作用。磷脂酰胆碱依赖性 将利用PC-TP中的构象变化来鉴定对Them 2活性至关重要的特定基序 与稳定Them 2和PC-TP的相互作用结构域将通过突变鉴定和确认。 分析。总的来说，这一建议将阐明磷脂介导的代谢调节的新机制 控制肝脏营养代谢，这是重要的，因为膜的脂肪酰基组成 磷脂酰胆碱在健康和疾病中变化。这些研究有望将Them 2确立为 NAFLD管理的易处理目标。

PROJECT SUMMARY/ABSTRACT Altered regulation of lipid and glucose homeostasis, most often in the setting of insulin resistance and obesity, is central to the pathogenesis of 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 phosphatidylcholines by phosphatidylcholine transfer protein (PC-TP) is translated into metabolic control by thioesterase superfamily member 2 (Them2), a mitochondria-associated long chain acyl-CoA thioesterase. The central hypothesis is that key regulatory events occur in the skeletal muscle when PC-TP binds specific membrane phosphatidylcholine molecular species and then activates Them2. The rationale is that regulation of skeletal muscle metabolism by Them2 should yield new insights into hepatic insulin resistance and steatosis. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To demonstrate that Them2 controls lipid and glucose metabolism in skeletal muscle; 2) To define mechanisms whereby Them2 in skeletal muscle promotes hepatic steatosis; and 3) To elucidate the molecular determinants of Them2 activity and regulation by PC-TP. In Aim 1, mouse models will be used to determine mechanisms whereby Them2 regulates fatty acid and glucose metabolism in skeletal muscle and promotes insulin resistance in response to overnutrition. Cell autonomous functions of Them2 will be gleaned from systematic studies in cultured myotubes. Aim 2 will establish the mechanisms in high fat fed mice whereby Them2 in skeletal muscle promotes hepatic insulin resistance and steatosis. Cultured hepatocytes will be used to determine whether Them2-dependent myokines or extracellular vesicles released from myotubes control hepatic lipid and glucose metabolism. Aim 3 will determine structural characteristics that enable Them2 to respond to changes in membrane phosphatidylcholine composition. Small molecules designed to bind and inhibit Them2 will be used as probes to characterize Them2 function and interactions with PC-TP using an array of biophysical techniques. Phosphatidylcholine-dependent conformational changes in PC-TP will be leveraged to identify specific motifs that are critical for Them2 activity and stability. The interacting domains of Them2 and PC-TP will be identified and confirmed by mutational analyses. Overall, this proposal will elucidate new mechanisms of phospholipid-mediated metabolic regulation that control hepatic nutrient metabolism, which is significant because the fatty acyl composition of membrane phosphatidylcholines varies in health and disease. These studies are expected to establish Them2 as a tractable target for the management of NAFLD.