Recent evidence suggested muscular free fatty acids (FFA) accumulation might be responsible for mitochondrial dysfunction in type 2 diabetes mellitus (T2DM). Reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) directly oxidize and damage DNA, proteins, and lipids and play a key role in the pathogenesis of T2DM. ROS/RNS may cause various types of oxidative modifications on specific proteins;such modifications, if irreversible, may lead to severe failure of biological functions, accumulation of damaged proteins and cell death. Identification of oxidatively modified proteins in mitochondria is important for understanding the relationship between protein oxidation, protein aggregation and development of T2DM. Recently, we have reported that targeting the DNA repair protein hOGG1 in mitochondria augments mitochondrial DNA (mtDNA) repair and enhances cellular survival following oxidative stress. The hypothesis to be tested is that mtDNA damage is involved in mitochondrial dysfunction and insulin resistance (IR) following FFA exposure in skeletal muscle. Improving mtDNA repair by expressing hOGG1 in mitochondria in skeletal muscle will diminish the generation of secondary ROS and thus reduce the proportion of oxidative modifications of specific mitochondrial proteins. This will increase mitochondrial protein function, and ultimately lead to enhanced mitochondrial function, increased insulin sensitivity, and cellular survival following exposure to FFA. This hypothesis will be tested through the pursuit of three specific aims. The first is to test using rat L6 skeletal muscle cells that FFA induced mtDNA damage, and consequently caused mitochondrial dysfunction, IR and apoptosis. Additionally, evaluate mtDNA damage and mitochondrial dysfunction in skeletal muscles isolated from a mouse model of T2DM (C57BL6 mice fed a high fat diet). The second is to determine whether targeting of hOGG1 into rat L6 skeletal cell mitochondria will decrease FFA- induced mtDNA damage and thus lead to enhanced insulin sensitivity, cellular survival and proliferation. The third aim is to determine the effect of reducing FFA-induced mtDNA damage through the overexpression of hOGG1 in mitochondria on ROS production and on subsequent oxidation of mitochondrial proteins following FFA-exposure to these cells. To complete this study we propose to make use of a new liquid chromatography tandem mass spectrometry (LC-MS/MS) approach.