Aging is a major risk factor for the development of type 2 diabetes (T2DM). Skeletal muscle is the main site of insulin-stimulated glucose disposal and aging is characterized by muscle insulin resistance. It has been suggested that the insulin resistance of aging results from an age-related accumulation of intramyocellular lipids which impair insulin action. However, the molecular basis for the accumulation of intramyocellular fat remains unknown. AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activation results in increased fatty acid oxidation. Recently, it has been established that aging leads to reduced AMPK activity in muscle. Using the insulin clamp technique with muscle biopsies, and a primary human muscle cell culture system, we plan to test the hypothesis that age-related declines in AMPK signaling are responsible for the decreases in fat oxidation, excessive intramyocellular lipid accumulation, and insulin resistance that occur in aging human muscle. The following Specific Aims are proposed: Aim 1) To determine whether reduced AMPK signaling in muscle from older subjects, in vivo, is associated with lower fat oxidation rates and insulin resistance, and whether physical activity improves glucose homeostasis in older subjects by upregulating AMPK signaling in muscle; Aim 2) To determine whether age-related declines in AMPK signaling in old myotubes cultured in vitro increases the susceptibility to fat-induced insulin resistance; and Aim 3) To examine whether the age-related reductions in fat oxidation and insulin action in old myotubes can be reversed by upregulating AMPK activity. PUBLIC HEALTH RELEVANCE: Aging is associated with a high risk for developing type 2 diabetes and impaired glucose tolerance, a pre- diabetic state. However, the reason why older subjects are at high risk for developing these abnormalities in glucose (sugar) metabolism is not known. In this study we will test whether reduced activity of a protein called AMPK is responsible for the abnormal glucose metabolism that occurs in older subjects, and whether restoring the function of this protein improves glucose metabolism in these individuals. If positive, our findings could help design new ways to prevent type 2 diabetes in the elderly.