EFFECT OF ACUTE ELEVATION OF FFA ON MITOCHONDRIAL FUNCTION IN SKELETAL MUSCLE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 背景：胰岛素抵抗是2型糖尿病（T2 DM）的主要组成部分，并且在糖尿病发展之前很久就存在。 血浆游离脂肪酸（FFA）浓度升高和FFA细胞内代谢产物增加被认为有助于胰岛素抵抗的发病机制。 最近，也有人提出，血浆FFA和细胞内FFA代谢产物升高是线粒体功能障碍的结果。 然而，目前尚不清楚在2型糖尿病和其他胰岛素抵抗状态中观察到的FFA浓度升高是否是线粒体功能障碍的结果，或者它们是否通过损害线粒体功能引起胰岛素抵抗。 为了解决这个问题，我们建议研究健康受试者血浆FFA浓度的短期升高是否会诱导线粒体功能障碍。 研究假设：细胞内脂质增加抑制骨骼肌ATP合成并诱导活性氧簇的形成。 研究设计：没有2型糖尿病家族史的健康葡萄糖耐受受试者将参与两项以4-6周的间隔进行的正葡萄糖胰岛素钳夹研究：（研究1）将以60 ml/小时的速率输注20% Liposyn 7小时以提高血浆FFA浓度，并在4-7小时期间进行正葡萄糖胰岛素钳夹;（研究2）以60 ml/小时输注生理盐水7小时，并在4-7小时期间进行正常血糖胰岛素钳夹。 在脂质/盐水输注前以及输注后4和7小时，将根据骨骼肌活检评估线粒体功能，以检查胰岛素钳夹前后血脂升高对骨骼肌线粒体功能的影响。 将通过共聚焦显微镜和酶法评估线粒体ATP合成、氧化酶活性、电子传递链能力和活性氧（ROS）生成，并通过电子显微镜确定线粒体形态。 主要终点-胰岛素刺激的ATP合成速率和ROS生成。 统计分析-将使用方差分析（ANOVA）或重复测量的混合线性模型，比较基线与盐水和脂质输注4和7小时之间的线粒体膜电位（反映ATP合成速率和活性氧产生），其中重复测量是指4和7小时后的测量以及有脂质输注和无脂质输注（盐水）的测量。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. BACKGROUND: Insulin resistance is a primary component of type 2 diabetes mellitus (T2DM) and is present long before the development of diabetes. Elevated plasma free fatty acid concentrations (FFA) and increased intracellular metabolites of FFA are believed to contribute to the pathogenesis of insulin resistance. Recently, it has also been proposed that elevated plasma FFA and intracellular FFA metabolites are a result of mitochondrial dysfunction. However, it is not clear whether elevated FFA concentrations seen in type 2 diabetes and other insulin resistant states are a result of mitochondrial dysfunction or whether they cause insulin resistance by impairing mitochondrial function. To address this issue, we propose to study whether short term elevation of the plasma FFA concentration in healthy subjects induces mitochondrial dysfunction. STUDY HYPOTHESIS: Increased intracellular lipids inhibit ATP synthesis in the skeletal muscle and induce the formation of reactive oxygen species. STUDY DESIGN: Healthy glucose tolerant subjects without family history of type 2 diabetes will participate in two euglycemic insulin clamp studies performed with an interval of 4-6 weeks: (Study 1) 20% Liposyn will be infused at a rate of 60 ml/hour for 7 hours to elevate the plasma FFA concentration and a euglycemic insulin clamp will be performed during hours 4-7; (Study 2) saline infusion at 60 ml/hour for 7 hours and a euglycemic insulin clamp will be performed during hours 4-7. Mitochondrial function will be assessed from skeletal muscle biopsies performed before, and at 4 and 7 hours after lipid/saline infusion to examine the effect of lipids elevated plasma lipid on skeletal muscle mitochondrial function before and after insulin clamp. Mitochondrial ATP synthesis, oxidative enzyme activity, electron transport chain capacity, and reactive oxygen species (ROS) generation will be assessed by confocal microscopy and enzymatic methods and mitochondrial morphology will be determined by electron microscopy. Primary Endpoint - Insulin stimulated ATP synthesis rate and ROS generation. Statistical Analysis - Mitochondrial membrane potential which reflects the rate of ATP synthesis, and ROS generation will be compared between baseline versus 4 and 7 hours of saline and lipid infusion, using the analysis of variance (ANOVA) or mixed linear model with repeated measures, where the repeated measures are those after 4 and 7 hours as well as those with lipid infusion and without lipid infusion (saline).