Phospholipid-Mediated Metabolic Control in the Pathogenesis of NAFLD

项目摘要/摘要 脂肪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背景下， 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。这项研究提案解决了一个悬而未决的问题：膜 磷脂调节营养动态平衡。我们的长期目标是了解磷脂是如何 代谢控制可以用于治疗目的。这项研究的目的是确定 磷脂酰胆碱转移感测膜磷脂酰胆碱的分子机制 蛋白质(PC-TP)由硫代酯酶超家族成员2(Them2)翻译成代谢控制蛋白，a 线粒体相关的长链酰辅酶A硫代酯酶。中心假设是关键的监管事件 发生在骨骼肌中，当PC-TP与特定的膜磷脂酰胆碱分子物种结合并 然后激活Them2。其基本原理是，Them2对骨骼肌代谢的调节应该会产生 对肝脏胰岛素抵抗和脂肪变性的新见解。在广泛的初步数据指导下，中央银行 假设将在三个特定的目标中进行检验：1)证明Them2控制血脂和血糖 骨骼肌代谢；2)确定骨骼肌中Them2促进肝脏的机制 脂肪变性；3)阐明Them2活性的分子决定因素和PC-TP的调节。在目标1中， 将使用小鼠模型来确定Them2调节脂肪酸和葡萄糖的机制 在骨骼肌中的代谢，并促进胰岛素抵抗对营养过剩的反应。细胞自主性 Them2的功能将通过对培养的肌管进行系统研究来收集。目标2将建立 高脂饮食小鼠骨骼肌中Them2促进肝脏胰岛素抵抗的机制 脂肪变性。培养的肝细胞将被用来确定Them2依赖的肌动蛋白或细胞外 肌管释放的囊泡控制肝脏的脂肪和葡萄糖代谢。目标3将确定结构 使Them2能够对膜磷脂酰胆碱组成的变化做出反应的特性。小的 设计用来结合和抑制Them2的分子将被用作探针来表征Them2的功能和 使用一系列生物物理技术与PC-TP相互作用。磷脂酰胆碱依赖 将利用PC-TP的构象变化来确定对Them2活性至关重要的特定基序 和稳定性。Them2和PC-TP的相互作用结构域将通过突变进行鉴定和确认 分析。总体而言，这一提议将阐明磷脂介导的代谢调节的新机制。 控制肝脏的营养代谢，这一点很重要，因为膜的脂肪酰基组成 磷脂酰胆碱在健康和疾病中的作用各不相同。这些研究有望将Them2确立为一种 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.