PGC-1 & Mitichondrial Dysfunction in Diabetes

描述（由申请人提供）：我们在本项目中的研究重点是确定预测骨骼肌胰岛素敏感性变化的核编码线粒体基因的表达变化，目的是确定骨骼肌线粒体功能障碍与胰岛素抵抗之间联系的分子机制。我们发现脂质输注引起的脂质供过于需要降低PGC-11、NRF-1和核编码线粒体基因的mRNA表达并诱导胰岛素抵抗。这与我们最初的假设一致，即PGC-11表达降低会导致核编码线粒体基因表达降低，导致与胰岛素抵抗相关的线粒体功能障碍。进一步的研究表明，线粒体功能障碍、胰岛素敏感性和基因表达之间存在复杂的关系。其他数据表明，胰岛素抵抗的人可能会抵抗运动。我们还发现ATP合酶2在体内被磷酸化，这可能受到胰岛素的调节，并在胰岛素抵抗中发生改变。线粒体蛋白丰度的变化或胰岛素抵抗中的翻译后修饰，或它们对运动或脂质供应过剩的反应，所知相对较少。本提案的总体目标是了解线粒体呼吸，蛋白质丰度和磷酸化调节的潜在变化如何导致线粒体功能障碍。为了实现这一目标，我们将使用葡萄糖钳，肌肉活检，用于定量蛋白质丰度变化的新型蛋白质组学技术，以及使用能量钳进行体外线粒体呼吸测量。我们建议：1。确定在肌酸激酶“能量钳”模拟能量需求增加的条件下，从胰岛素抵抗的人体肌肉中分离的线粒体是否会减少呼吸。2. 为了确定胰岛素抵抗如何改变线粒体蛋白丰度的模式。3. 确定胰岛素抵抗如何改变ETC蛋白的磷酸化。我们将使用免疫沉淀和质谱分析来量化ETC蛋白磷酸化的位点特异性变化。4. 确定胰岛素抵抗是否伴随着线粒体生物发生方面的“运动抵抗”。5. 确定实验性脂质供过于求是否会降低线粒体呼吸功能。公共卫生相关性：肥胖症和2型糖尿病的流行比例正在上升。在美国，他们的并发症占医疗保健费用的40%。尽管如此，导致其发展的机制仍不清楚。该项目将有助于在分子水平上阐明这些机制。鉴于有明确证据表明肥胖和胰岛素抵抗的病因与线粒体功能障碍有关，这些研究具有很高的临床意义，并可能为线粒体生物能量学的控制提供有用的新见解

DESCRIPTION (provided by applicant): Our research in this project has focused on defining the changes in expression of nuclear encoded mitochondrial genes that predict changes in insulin sensitivity in skeletal muscle, with the goal of defining the molecular mechanisms underlying the connection between mitochondrial dysfunction and insulin resistance in skeletal muscle. We have found that lipid oversupply produced by a lipid infusion decreases mRNA expression of PGC-11, NRF-1, and nuclear encoded mitochondrial genes and induces insulin resistance. This was consistent with our original hypothesis that decreasing PGC-11 expression would result in lower expression of nuclear encoded mitochondrial genes and lead to mitochondrial dysfunction associated with insulin resistance. Additional studies showed a complex relationship between mitochondrial dysfunction, insulin sensitivity, and gene expression. Other data suggested that insulin resistant individuals could be exercise resistant. We also have shown that ATP synthase 2 is phosphorylated in vivo, and this may be regulated by insulin and altered in insulin resistance. Relatively little is known about changes in mitochondrial protein abundance or post-translational modification in insulin resistance, or their response to exercise or lipid oversupply. The overall goal of this proposal is to understand how underlying changes in regulation of mitochondrial respiration, protein abundance and phosphorylation contribute to mitochondrial dysfunction. To accomplish this goal, we will use glucose clamps, muscle biopsies, novel proteomics techniques for quantification of protein abundance changes, and in vitro mitochondrial respiration measurements using an energy clamp. We propose: 1. To determine whether mitochondrial isolated from insulin resistant human muscle have decreased respiration during conditions of increased energy demand simulated by a creatine kinase "energy clamp". 2. To determine how insulin resistance alters the pattern of abundance of mitochondrial proteins. 3. To determine how insulin resistance alters phosphorylation of proteins in the ETC. We will use immunoprecipitation and mass spectrometry analysis to quantify site-specific changes in phosphorylation of ETC proteins. 4. To determine whether insulin resistance is accompanied by "exercise resistance" with regard to mitochondrial biogenesis. 5. To determine whether experimental lipid oversupply decreases mitochondrial respiratory function. PUBLIC HEALTH RELEVANCE: Obesity and type 2 diabetes mellitus are increasing in epidemic proportion. Their complications account for up to forty percent of health care costs in the U.S. Despite this, the mechanisms responsible for their development remain unclear. This project will help to clarify these mechanisms on a molecular level. Given the clear evidence of a link to mitochondrial dysfunction in the etiology of obesity and insulin resistance, the studies are of high clinical significance and may provide useful new insights into the control of mitochondrial bioenergetics