Mechanisms of Human Insulin Resistance

描述（由申请人提供）：胰岛素抵抗是2型糖尿病（T2DM）、代谢综合征和心血管疾病发病机制的核心，是患者痛苦和医疗费用的沉重负担。直接原因包括对骨骼肌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细胞内脂质积累（IMCL）有关，尽管分子机制尚不清楚。由于线粒体负责脂质氧化，因此在线粒体功能障碍的基础上解释IMCL增加是很有吸引力的，最近有证据支持这一观点。在过去的资助周期中，我们使用cDNA微阵列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，加上胰岛素抵抗涉及复杂IV蛋白的选择性消耗的观察，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研究。目前的建议结合人体生理学和基本方法来验证以下假设：1)每个线粒体呼吸复合物蛋白的特异性消耗和线粒体质量的减少损害底物氧化并促进胰岛素抵抗和T2DM的IMCL积累；2)这些异常增加ROS的产生，进一步损害与氧化蛋白修饰相关的功能，特别是在未控制的T2DM中。为了验证这些假设，我们将在一定体重范围内研究代谢特征的胰岛素敏感和胰岛素抵抗的正常血糖受试者，以及未经治疗和经过正糖治疗的T2DM患者，以逆转胰岛素抵抗的“葡萄糖毒性”成分。摄动研究将在胰岛素致敏噻唑烷二酮、低热量喂养和减肥前后进行，以探索与肌肉线粒体质量和形态的关系，并使用2D蓝色天然凝胶电泳和质谱法研究呼吸链蛋白质组学。为了评估线粒体功能，我们将通过高分辨率呼吸计测量碳水化合物和脂质底物氧化，个体呼吸链复合物I-IV的活性，偶联状态，ROS/RNS生成以及影响线粒体蛋白的翻译后氧化修饰。我们还将研究核基因组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中与底物氧化和IMCL缺陷相关的功能和分子缺陷。

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.