Patients with syndromes of insulin resistance, including obesity, the Metabolic Syndrome, and Type 2 diabetes mellitus, have markedly increased risk for atherosclerosis. A growing body of work has shown that insulin contributes to the maintenance of vascular cell homeostasis and that local insulin resistance has pathological effects including loss of the bioactivity of endothelium-derived nitric oxide (NO) and conversion to a pro-inflammatory phenotype that may promote vascular remodeling and atherosclerosis. Insulin-mediated activation of nitric oxide synthase depends on Akt/PI3 kinase and the availability of mitochondria-derived reactive oxygen species. Recent studies suggest that these signaling mechanisms also depend on the activity of AMP-dependent protein kinase (AMP kinase). Our preliminary data indicate that a period of strict bed rest or short-term lipid infusion produces acute insulin resistance in healthy subjects that is associated with a marked impairment of vasodilator function. Use of this methodology provides a unique opportunity to investigate the vascular consequences of insulin resistance without the confounding factors present in patients with more advanced disease. This project will investigate potential mechanisms accounting for vascular dysfunction in these states of acute insulin resistance in humans. In Aim 1, we will test the hypothesis that activation of AMP kinase will blunt the adverse effects of insulin resistance on vascular function by measuring basal and stimulated AMP kinase activity in leukocytes and muscle and by determining whether vascular dysfunction is prevented by pretreatment with metformin, which activates AMP kinase and increases insulin sensitivity.. In Aim 2, we will investigate the contribution of mitochondrial dysfunction to vascular dysfunction in insulin resistance by measuring systemic markers of oxidative stress and mitochondrial membrane potential and ROS production in leukocytes. In addition, we will determine whether the mitochondria-directed antioxidants lipoic acid and acetyl-L-carnitine blunt vascular dysfunction and insulin resistance. In Aim 3, we will test the hypothesis that activation of NFicB contributes to the development of vascular dysfunction in acute insulin resistance by measuring circulating adhesion molecules, pro-inflammatory cytokines, and adipokines. In addition, we will determine whether vascular dysfunction can be prevented by treatment with sulfasalazine, which inhibits activation of NFkappaB. We suggest that these translational studies will provide new insights into the mechanisms of vascular dysfunction in patients with insulin resistance that will be relevant to the prevention and management of vascular disease in the setting of obesity, the metabolic syndrome, and Type 2 diabetes mellitus.