Mechanisms of Human Insulin Resistance

描述(申请人提供)：胰岛素抵抗是2型糖尿病(T2 DM)、代谢综合征和心血管疾病发病机制的核心，并给患者带来沉重的痛苦和医疗费用负担。直接原因是骨骼肌对葡萄糖摄取的刺激有缺陷。胰岛素抵抗也与脂肪氧化异常和心肌细胞内脂质(IMCL)蓄积有关，尽管分子机制尚不清楚。由于线粒体负责脂质氧化，因此很容易将IMCL的增加解释为线粒体功能障碍，最近的证据支持这一观点。在过去的资助周期中，我们使用基因芯片检测了人类肌肉中的差异基因表达，发现编码线粒体蛋白的多个基因在胰岛素抵抗中下调。这些数据，再加上胰岛素抵抗涉及复合IV蛋白选择性耗尽的观察，使我们对骨骼肌中的线粒体蛋白质组和功能进行了更详细的检查。目前的建议结合人类生理学和基本方法来检验假设：1)线粒体呼吸复合体蛋白的特异性耗尽和线粒体质量的减少损害底物氧化并促进胰岛素抵抗和T2 DM中IMCL的积累；2)这些异常增加ROS的产生，从而进一步损害与氧化蛋白修饰相关的功能，特别是在不受控制的T2 DM中。为了验证这些假设，我们将研究一系列体重范围内具有代谢特征的胰岛素敏感和抵抗的正常血糖受试者，以及处于未治疗状态和正常血糖治疗后的T2 DM患者，以逆转胰岛素抵抗的“葡萄糖毒性”成分。将在胰岛素增敏的噻唑烷二酮以及低热量喂养和减肥前后进行扰动研究，以探索与肌肉线粒体质量和形态的关系，并使用2D Blue天然凝胶电泳法和质谱仪研究呼吸链蛋白质组学。为了评估线粒体的功能，我们将通过高分辨率呼吸验光测量碳水化合物和脂肪底物的氧化，单个呼吸链复合体I-IV的活性，偶联状态，ROS/RNS的产生，以及影响线粒体蛋白质的翻译后氧化修饰。我们还将研究核和线粒体基因组之间的不协调调节是否可能导致线粒体功能障碍。这些研究将首次描述肌肉线粒体的功能和分子缺陷，这些缺陷与底物氧化缺陷和人类胰岛素抵抗中的IMCL有关。

DESCRIPTION (provided by applicant): Insulin resistance is central to the pathogenesis of Type 2 diabetes (T2DM), the Metabolic Syndrome, and cardiovascular disease, and presents a heavy burden of patient suffering and health care costs. The immediate cause involves defective stimulation of glucose uptake into skeletal muscle. Insulin resistance is also associated with abnormalities in fat oxidation and accumulation of intramyocellular lipid (IMCL), although molecular mechanisms are unknown. Since mitochondria are responsible for lipid oxidation, it is tempting to explain increased IMCL on the basis of mitochondrial dysfunction, and there is recent evidence to support this idea. Over the past grant cycle, we have examined differential gene expression in human muscle using cDNA microarrays and found that multiple genes encoding mitochondrial proteins are down-regulated in insulin resistance. This data, together with the observation that insulin resistance involves selective depletion of complex IV proteins, have led us to a more detailed examination of the mitochondrial proteome and function in skeletal muscle. The current proposal combines human physiology and basic methods to test the hypotheses that: 1) specific depletion of respiratory complex proteins per mitochondrion and a decrease in mitochondrial mass impair substrate oxidation and promote IMCL accumulation in insulin resistance and T2DM; 2) these abnormalities increase ROS production which further compromises function in association with oxidative protein modifications, particularly in uncontrolled T2DM. To test these hypotheses, we will study metabolically-characterized insulin sensitive and resistant normoglycemic subjects over a range of body weight, and T2DM patients both in the untreated state and after euglycemic therapy to reverse the "glucose toxicity" component of insulin resistance. Perturbation studies will be performed before and after an insulin-sensitizing thiazolidinedione, and hypocaloric feeding & weight loss, in order to probe relationships with muscle mitochrondrial mass and morphology, and to study respiratory chain proteomics using 2D blue native gel electrophoresis and mass spectrometry. To assess mitochondrial function, we will measure carbohydrate and lipid substrate oxidation by high resolution respirotometry, activity of individual respiratory chain complexes I-IV, coupling status, ROS/RNS generation, and post-translational oxidative modifications affecting mitochondrial proteins. We will also examine whether discordant regulation between nuclear and mitochondrial genomes could underlie mitochondrial dysfunction. These studies will for the first time delineate functional and molecular defects in muscle mitochondria related to defects in substrate oxidation and IMCL in human insulin resistance.