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Insulin Action in Muscle and Fat Cells

胰岛素在肌肉和脂肪细胞中的作用

基本信息

批准号：
8001406
负责人：
JAMES Carlton GARRISON
金额：
$ 25.14万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-15 至 2011-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8001406
关键词：
Actins Adipocytes Affect Animals Binding Biochemical Biological Assay Cells Chromatography Complex Cytoskeleton Defect Enzymes Exhibits Fatty Liver Fiber GLUT4 gene Gene Proteins Genes Genetic Transcription Glucose Intolerance Glycogen Goals Growth Heart Diseases Hepatic Homologous Gene Insulin Insulin Resistance Kidney Diseases Knock-out Knockout Mice Label Lipids Mass Spectrum Analysis Measures Mediator of activation protein Metabolic Metabolism Mus Muscle Muscle Fibers Mutate Neuropathy Non-Insulin-Dependent Diabetes Mellitus Peptide Mapping Peripheral Vascular Diseases Phosphopeptides Phosphorylation Phosphorylation Site Phosphotransferases Physiological Processes Plant Resins Promoter Regions Protein Isoforms Protein Kinase Proteins Reporter Research Research Personnel Role Sepharose Signal Transduction Sirolimus Site Site-Directed Mutagenesis Skeletal Muscle Subfamily lentivirinae System Techniques Testing Tissues Trypsin Vision Disorders Wild Type Mouse Yeasts blood glucose regulation glucose metabolism glucose transport glycogen metabolism human FRAP1 protein in vitro Model in vivo insulin signaling knockout animal lipid biosynthesis lipine overexpression oxidation programs research study response small hairpin RNA trafficking transcription factor yeast two hybrid system

项目摘要

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.

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、周围血管疾病和心脏病的主要原因。该提案旨在研究脂质和 mTORC2，这两个胰岛素作用的新靶点。 Lipin 是 Lpn1fld/fld 小鼠中突变的基因的蛋白质产物，mTORC2 是新发现的雷帕霉素不敏感信号复合物，可控制肌动蛋白细胞骨架和 Akt 磷酸化。 Lpn1fld/fld 小鼠表现出脂肪肝、脂肪生成缺陷、葡萄糖不耐受和胰岛素抵抗。从这些异常情况可以清楚地看出，脂质对于正常的胰岛素作用至关重要。然而，脂质的生化功能尚不清楚。定义这个函数将是一个主要目标。脂质响应胰岛素而磷酸化。在 AIM 1 中，提出了一个涉及肽图谱、定点诱变和质谱分析的计划来确定磷酸化位点。还描述了鉴定磷酸化脂质的激酶的实验。目的2是确定脂质的作用机制。初步结果表明，脂质与 NFAT3 相互作用，NFAT3 是一种转录因子，与 PPAR?2 表达和脂肪生成的控制有关。其他发现，包括酿酒酵母脂质 Smp2 的结果，为检验脂质抑制 ChREBP 的假设提供了强有力的理由，ChREBP 是一种转录因子，可促进编码参与脂肪生成的多种酶基因的表达。将研究脂质磷酸化对脂质与 ChREBP 和 NFAT3 相互作用的作用。提出 ChIP 分析以确定脂质是否与 NFAT3 或 ChREBP 控制的基因启动子区域相关，并描述了报告基因测定以确定脂质是否增强或抑制这些转录因子的活性。由于与脂质相互作用的其他蛋白质可能是脂质功能的关键，因此我们将通过使用多种方法来寻找新的相互作用蛋白质，包括鉴定与脂质共纯化或与脂质琼脂糖树脂结合的蛋白质。目标 3 是检验 mTORC2 是胰岛素代谢作用中介者的假设。在增加或减少 mTORC2 的定义亚基 rictor 水平后，将测量葡萄糖转运和氧化、脂质和糖原合成、GLUT4 易位以及胰岛素信号传导的几个参数。慢病毒将用于在 3T3-L1 脂肪细胞中过表达 rictor 或表达 shRNA 以敲低 rictor。为了研究 mTORC2 的体内功能，我们建议敲除小鼠脂肪细胞和骨骼肌中的 rictor。

项目成果

期刊论文数量（24）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ras signaling in the activation of glucose transport by insulin.

Ras 信号传导通过胰岛素激活葡萄糖转运。

DOI：
10.1073/pnas.91.11.4644
发表时间：
1994
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Manchester,J;Kong,X;Lowry,OH;LawrenceJr,JC
通讯作者：
LawrenceJr,JC

Insulin stimulates dephosphorylation of phosphorylase in rat epitrochlearis muscles.

胰岛素刺激大鼠滑车上肌中磷酸化酶的去磷酸化。

DOI：
发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Zhang,JN;Hiken,J;Davis,AE;LawrenceJr,JC
通讯作者：
LawrenceJr,JC

Effects of insulin and transgenic overexpression of UDP-glucose pyrophosphorylase on UDP-glucose and glycogen accumulation in skeletal muscle fibers.

胰岛素和 UDP-葡萄糖焦磷酸化酶转基因过表达对骨骼肌纤维中 UDP-葡萄糖和糖原积累的影响。

DOI：
10.1074/jbc.m413614200
发表时间：
2005
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Reynolds4th,ThomasH;Pak,Yunbae;Harris,ThurlE;Manchester,Jill;Barrett,EugeneJ;LawrenceJr,JohnC
通讯作者：
LawrenceJr,JohnC

Control of glycogen synthase and phosphorylase by amylin in rat skeletal muscle. Hormonal effects on the phosphorylation of phosphorylase and on the distribution of phosphate in the synthase subunit.

大鼠骨骼肌中胰淀素对糖原合成酶和磷酸化酶的控制。