Insulin Action in Muscle and Fat Cells

描述(申请人提供)：这项研究的总体目标是阐明肌肉和脂肪细胞中胰岛素控制新陈代谢的机制。这些细胞对胰岛素反应能力的缺陷是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。这项建议是为了研究胰岛素作用的两个新靶点--脂类和mTORC2。Lipin是该基因在Lpn1feld/fld小鼠中突变的蛋白质产物，而mTORC2是新发现的雷帕霉素不敏感的信号复合体，它既控制肌动蛋白的细胞骨架，又控制Akt的磷酸化。Lpn1fLD/fLD小鼠表现为脂肪肝、脂肪生成缺陷、糖耐量异常和胰岛素抵抗。从这些异常中可以清楚地看出，脂类对于正常的胰岛素作用是必不可少的；然而，脂类的生化功能尚不清楚。定义这一功能将是一个主要目标。脂肪被磷酸化是对胰岛素的反应。在AIM 1中，提出了一种包括肽图谱、定点突变和质谱学的方案来确定磷酸化位点。还描述了鉴定使脂类磷酸化的酶的实验。目的2确定脂类的作用机制。初步结果表明，Lipin与NFAT3相互作用，NFAT3是一种转录因子，参与控制PPAR？2的表达和脂肪形成。其他发现，包括对S.cerevesiae Lipin，Smp2的研究结果，提供了一个强有力的理由来检验这样的假设，即脂类抑制ChREBP的假设，ChREBP是一种转录因子，可以促进参与脂肪生成的多个编码酶的基因的表达。脂类磷酸化在脂类与ChREBP和NFAT3相互作用中的作用将被研究。芯片分析被用来确定脂类是否与NFAT3或ChREBP控制的基因的启动子区域相关联，报告分析被用来确定脂类是增强还是抑制这些转录因子的活性。由于与脂类相互作用的其他蛋白质可能掌握着脂类功能的关键，我们将通过多种方法寻找新的相互作用蛋白，包括鉴定与脂类共同纯化的蛋白或与脂类-琼脂糖树脂结合的蛋白。目的3是验证mTORC2是胰岛素代谢效应的中介的假设。葡萄糖转运和氧化、脂类和糖原合成、GLUT4易位以及胰岛素信号传导的几个参数将在增加或降低mTORC2的决定亚基Rictor的水平后进行测量。慢病毒可用于在3T3-L1脂肪细胞中过表达Rictor或表达shRNA以击倒Rictor。为了研究mTORC2在体内的功能，我们建议在小鼠的脂肪细胞和骨骼肌中敲除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.