Insulin Action in Muscle and Fat Cells

描述（由申请人提供）：本研究的总体目标是阐明肌肉和脂肪细胞中胰岛素控制代谢的机制。这些细胞响应胰岛素的能力缺陷是2型糖尿病的主要原因，而2型糖尿病又是视力障碍、神经病变、肾脏疾病、外周血管疾病和心脏病的主要原因。本研究旨在研究胰岛素作用的两个新靶点lipin和mTORC 2。Lipin是Lpn 1fld/fld小鼠中突变基因的蛋白产物，mTORC 2是一种新发现的雷帕霉素不敏感信号复合物，控制肌动蛋白细胞骨架和Akt的磷酸化。lpn 1fld/fld小鼠存在脂肪肝、脂肪生成缺陷、葡萄糖耐受不良和胰岛素抵抗。从这些异常可以清楚地看出，脂蛋白对正常的胰岛素作用是必不可少的;然而，脂蛋白的生化功能尚不清楚。确定这一职能将是一个主要目标。脂蛋白对胰岛素的反应是磷酸化。在AIM 1中，提出了一个涉及肽图谱、定点诱变和质谱的计划，以确定磷酸化位点。还描述了鉴定使脂蛋白磷酸化的激酶的实验。目的2：探讨Lipin的作用机制.初步结果表明，脂蛋白相互作用与NFAT 3，转录因子，已牵连在控制的过氧化物酶体增殖物激活受体？2表达与脂肪形成。其他发现，包括与S。cerevesiae lipin，Smp 2，提供了一个强有力的理由来检验lipin抑制ChREBP的假设，ChREBP是一种促进编码参与脂肪生成的酶的多个基因表达的转录因子。将研究脂蛋白磷酸化对脂蛋白与ChREBP和NFAT 3相互作用的作用。ChIP分析提出，以确定是否脂蛋白协会与NFAT 3或ChREBP控制的基因的启动子区，和报告分析，以确定脂蛋白是否增强或抑制这些转录因子的活性。由于与脂蛋白相互作用的其他蛋白质可能是脂蛋白功能的关键，因此我们将通过多种方法寻找新的相互作用蛋白质，包括鉴定与脂蛋白共纯化的蛋白质或与脂蛋白-琼脂糖树脂结合的蛋白质。目的3：验证mTORC 2是胰岛素代谢效应的介导因子这一假说。葡萄糖转运和氧化、脂质和糖原合成、GLUT 4易位以及胰岛素信号传导的几个参数将在增加或降低mTORC 2的定义亚基rictor水平后测量。慢病毒将用于在3 T3-L1脂肪细胞中过表达rictor或表达shRNA以敲低rictor。为了研究mTORC 2在体内的功能，我们建议敲除小鼠脂肪细胞和骨骼肌中的Rictor。

DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate mechanisms involved in the control of metabolism by insulin in muscle and fat cells. A defect in the ability of these cells to respond to insulin is a primary cause of Type 2 diabetes mellitus, which in turn is a leading cause of vision disorders, neuropathy, kidney disease, peripheral vascular disease, and heart disease. This proposal is to investigate lipin and mTORC2, two new targets of insulin action. Lipin is the protein product of the gene that is mutated in Lpn1fld/fld mice, and mTORC2 is a newly discovered rapamycin-insensitive signaling complex that controls both the actin cytoskeleton and phosphorylation of Akt. Lpn1fld/fld mice exhibit fatty liver, defective adipogenesis, glucose intolerance and insulin resistance. It is clear from these abnormalities that lipin is essential for normal insulin action; however, the biochemical function of lipin is unknown. Defining this function will be a major objective. Lipin is phosphorylated in response to insulin. In AIM 1 a plan involving peptide mapping, site directed mutagenesis, and mass spectrometry is presented to determine the sites of phosphorylation. Experiments to identify the kinases that phosphorylate lipin are also described. AIM 2 is to determine the mechanism of action of lipin. Preliminary results indicate that lipin interacts with NFAT3, a transcription factor that has been implicated in the control of PPAR?2 expression and adipogenesis. Other findings, including results with the S. cerevesiae lipin, Smp2, provide a strong reason to test the hypothesis that lipin represses ChREBP, a transcription factor that promotes expression of multiple genes encoding enzymes involved in lipogenesis. The role of lipin phosphorylation on lipin interactions with ChREBP and NFAT3 will be investigated. ChIP analyses are proposed to determine whether lipin associates with the promoter regions of genes controlled by NFAT3 or ChREBP, and reporter assays are described to determine whether lipin enhances or represses the activity of these transcription factors. Since other proteins that interact with lipin may hold the key to lipin function, we will search for new interacting proteins by using multiple approaches including identification of proteins that co purify with lipin or that bind to a lipin-agarose resin. AIM 3 is to test the hypothesis that mTORC2 is a mediator of the metabolic effects of insulin. Glucose transport and oxidation, lipid and glycogen synthesis, GLUT4 translocation, and several parameters of insulin signaling will be measured after increasing or decreasing levels of rictor, the defining subunit of mTORC2. Lentivirus will be used to overexpress rictor or to express shRNA to knockdown rictor in 3T3-L1 adipocytes. To investigate mTORC2 function in vivo, we propose to knockout rictor in adipocytes and skeletal muscle of mice.