Role and Regulation of Skeletal Muscle Mitochondrial Dynamics in Type 2 Diabetes

 描述（由申请人提供）：线粒体作为孤立的、球形的、产生能量的细胞器的传统观点正在经历革命性的转变。新出现的数据表明，线粒体形成一个动态的网状结构，受裂变和融合周期的调节。许多调节这些活动的蛋白质的发现导致了 在了解人类疾病方面取得了重大进展。我们已经证明，发动蛋白相关蛋白1（Drp 1），一种控制线粒体分裂的蛋白质，在糖尿病前期运动后活化减少，这种减少与胰岛素敏感性增加和脂肪氧化有关。我们现在建议在这项研究的基础上，验证线粒体动力学是2型糖尿病胰岛素抵抗的关键机制这一假设。我们的中心假设是，在糖尿病中，线粒体脂质代谢升高导致Drp 1的募集和激活-可能是通过增加活性氧，导致线粒体碎片化增加和线粒体渗透性转换孔的开放。在目标1a中，我们将在体内和体外研究人类骨骼肌线粒体动力学的代谢表型，从2型糖尿病患者，肥胖，瘦健康对照。转化性首次人体研究将使用急性脂质挑战（Aim 1b）和运动训练（目的1c），以研究人类骨骼肌线粒体动力学改变对胰岛素敏感性的生理意义。将使用正常血糖高胰岛素钳夹评估胰岛素抵抗，并使用间接量热法测量体内底物代谢。将从肌肉活检组织和透化肌纤维中评估线粒体分裂/融合、断裂、功能、膜电位、线粒体活性氧和脂质中间体蓄积。在目标2中，我们将使用抑制和表达克隆实验来直接检查在完整的离体培养的人骨骼肌细胞中操纵线粒体片段化的影响。这项研究将提供骨骼肌线粒体动力学的全面和补充分析，并将产生关于运动与营养调节2型糖尿病线粒体动力学和功能之间联系的新数据。该实验方法利用创新的分子和细胞工具，与生理学上重要的人类研究相结合，以获得有关胰岛素抵抗的有意义的数据，并有可能产生见解，为后代带来新的糖尿病治疗方法。

 DESCRIPTION (provided by applicant): The traditional view of mitochondria as isolated, spherical, energy producing organelles is undergoing a revolutionary transformation. Emerging data show that mitochondria form a dynamic networked reticulum that is regulated by cycles of fission and fusion. The discovery of a number of proteins that regulate these activities has led to important advances in understanding human disease. We have demonstrated that activation of dynamin related protein 1 (Drp1), a protein that controls mitochondrial fission, is reduced following exercise in prediabetes, and the decrease is linked to increased insulin sensitivity and fat oxidation. We now propose to build on this research and test the hypothesis that mitochondrial dynamics is a key mechanism of insulin resistance in type 2 diabetes. Our central hypothesis is that in diabetes elevated mitochondrial lipid metabolism causes recruitment and activation of Drp1 - likely through increased reactive oxygen species, leading to increased mitochondrial fragmentation and opening of the mitochondrial permeability transition pore. In Aim 1a we will perform in vivo and in vitro studies of human skeletal muscle mitochondrial dynamics across the metabolic phenotype ranging from patients with type 2 diabetes, to obese, to lean healthy controls. Translational first-in-man studies will use an acute lipid challenge (Aim 1b) and exercise training (Aim 1c) to investigate the physiological significance of altered skeleta muscle mitochondrial dynamics on insulin sensitivity in humans. Insulin resistance will be assessed using euglycemic hyperinsulinemic clamps, and in vivo substrate metabolism will be measured using indirect calorimetry. Mitochondrial fission/fusion, fragmentation, function, membrane potential, mitochondrial reactive oxygen species, and the accumulation of lipid intermediates will be assessed from muscle biopsy tissue and permeabilized muscle fibers. In Aim 2, we will use inhibition and expression cloning experiments to directly examine the impact of manipulating mitochondrial fragmentation in intact ex vivo cultured human skeletal muscle cells. This research will provide a comprehensive and complementary analysis of skeletal muscle mitochondrial dynamics, and will also generate novel data on the link between exercise and nutrient regulation of mitochondrial dynamics and function in type 2 diabetes. The experimental approach harnesses innovative molecular and cellular tools, interfaced with physiologically significant human studies to obtain meaningful data on insulin resistance, and has the potential to generate insights that will lead to new diabetes therapies for future generations.