PROJECT SUMMARY The prevalence of obesity (Body Mass Index ≥ 30) in the U.S. adults has reached nearly 40%. Obesity is accompanied by many metabolic problems that contribute to a significantly higher risk for chronic diseases, such as type 2 diabetes (T2D), insulin resistance and cardiovascular diseases. One of the key features of obesity and a forerunner of T2D is the impaired insulin action in skeletal muscle, resulting in insulin resistance. Therefore, the discovery of new targets to regulate skeletal muscle insulin sensitivity and whole-body glucose homeostasis is urgently needed. Such new targets could be exploited for new therapeutic treatments for insulin resistance and T2D. Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission. Excessive activation of Drp1 leads to aberrant mitochondrial fission causing imbalanced mitochondrial dynamics and dysfunction. We have recently reported that Drp1 is overactivated in skeletal muscle from obese insulin resistant humans and this overactivation is adversely correlated to impaired insulin action in skeletal muscle. In addition, in animals rendered insulin resistance with high-fat diet, pharmacological or genetic inhibition of Drp1 improves skeletal muscle insulin action and whole-body glucose homeostasis. However, more studies are still needed in order to understand the causal contribution of Drp1-mediated mitochondrial fission to insulin resistance. We hypothesize that excessive Drp1-mediated mitochondrial fission contributes to obesity-induced skeletal muscle insulin resistance and inhibition of skeletal muscle Drp1 is sufficient to improve mitochondrial integrity, reduce mtROS production, and improve insulin signaling, thereby ameliorating skeletal muscle insulin resistance and improving whole-body glucose homeostasis. In Aim 1, we will utilize skeletal muscle-specific Drp1 knockout mouse and clinical-relevant human skeletal muscle cell culture models, along with novel mitochondrial quality-monitoring tool (pMitoTimer) to define the role of Drp1-mediated mitochondrial fission in the regulation of obesity-induced skeletal muscle insulin resistance. These studies will determine the causal role of Drp1 in regulating skeletal muscle insulin resistance in obesity in vivo, and its underling mechanism of action. In Aim 2, we will utilize a pharmacological inhibitor of Drp1 to determine the effectiveness and therapeutic potential of targeting Drp1-mediated mitochondrial fission in alleviating obesity-induced insulin resistance. The successful completion of the proposed study will provide fundamental insights on contribution of Drp1-mediated mitochondrial fission to the development of obesity-induced insulin resistance and T2D. In addition, undergraduate students will gain extensive experience in both human and animal research using a variety of biomedical techniques while exploring the contribution of mitochondrial dynamics to the development of metabolic diseases to explore novel t...