This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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).
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