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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 背景：胰岛素抵抗是2型糖尿病(T2 DM)的主要组成部分，早在糖尿病发生之前就已存在。血浆游离脂肪酸浓度(FFA)升高和细胞内FFA代谢产物增多被认为是胰岛素抵抗的发病机制之一。最近，也有人提出血浆FFA升高和细胞内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2型糖尿病和其他胰岛素抵抗状态中FFA浓度的升高是线粒体功能障碍的结果，还是通过损害线粒体功能而导致胰岛素抵抗。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导致线粒体功能障碍。 研究假设：细胞内脂质的增加抑制了骨骼肌中的ATP合成，并诱导了活性氧的形成。 研究设计：无2型糖尿病家族史的健康糖耐量受试者将参加两项正糖胰岛素钳夹试验，研究间隔4-6周：(研究1)以60ml/小时的速度输注20%脂肪酸脂以升高血浆游离脂肪酸浓度7小时，并在4-7小时内进行正血糖胰岛素钳夹试验；(研究2)在4-7小时内以60毫升/小时的速度输注生理盐水7小时，并进行正血糖胰岛素钳夹试验。在胰岛素钳夹前、注射脂肪/生理盐水后4小时和7小时进行骨骼肌活组织检查，观察血脂升高对骨骼肌线粒体功能的影响。线粒体的ATP合成、氧化酶活性、电子传递链能力和活性氧物种(ROS)的产生将通过共聚焦显微镜和酶学方法进行评估，线粒体的形态将通过电子显微镜进行测定。 初级终点-胰岛素刺激ATP合成率和ROS的产生。 统计分析-使用方差分析(ANOVA)或具有重复测量的混合线性模型，比较基线与生理盐水和脂质输注(生理盐水)4和7小时之间的线粒体膜电位(反映ATP合成速率和ROS生成)，其中重复测量是在4和7小时后以及有脂肪输注和没有脂肪输注(生理盐水)的情况下。

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).