The principal purpose of the proposed experiments is to elucidate the mechanisms of regulation of malonyl-CoA (inhibitor of fatty acid oxidation) content in skeletal muscle. This will be accomplished by studying the short-term mechanisms of regulation of acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of malonyl-CoA. Upstream signaling proteins, AMP-activated protein kinase (AMPK) and AMP-activated protein kinase kinase (AMPKK) will be investigated. AMPK and AMPKK are also likely involved in contraction-induced stimulation of glucose uptake and control of expression of genes for GLUT4, hexokinase, and mitochondrial oxidative enzymes. Results will be applicable to these other important processes that appear to be regulated by the AMPKK/AMPK signaling system. Phosphorylation states of the AMPK phosphorylation site of muscle ACC and of the threonine 172 (AMPKK site) site in AMPK will be correlated with activity of purified ACC preparations, activity of ACC from electrically stimulated muscle, and activity of ACC in muscle from rats run on the treadmill. Roles of creatine phosphate (AMPK inhibitor) and 5'-AMP (AMPKK and AMPK activator) will be investigated using in vitro and in situ models. The effect of insulin on the rate of dephosphorylation of the AMPK phosphorylation site of ACC and the AMPKK site of AMPK will characterized. If effects of insulin are noted, the downstream signaling steps will be investigated, using wortmannin and phosphatase inhibitors. A putative AMPK kinase will be isolated from skeletal muscle and characterized with respect to molecular weight and regulation by creatine phosphate, 5'-AMP, and calcium/calmodulin. The question of whether AMPKK can be activated by phosphorylation during muscle contraction similarly to AMPK will be investigated. Adaptations of the AMPK/AMPKK signaling system to endurance training will be further characterized. Type 2 diabetes mellitus and obesity are two interrelated metabolic diseases that are increasing in incidence in the United States at alarming rates. These experiments will provide new information on regulation of AMPK and AMPKK, key enzymes that have now become targets for development of new pharmaceuticals for treatment of Type 2 diabetes and possibly obesity. This information will also provide essential basic information for investigation of the hypothesis that some forms of type 2 diabetes are due to defects in the AMPK/AMPKK signaling pathway. The results will provide additional rationale for the use of regular exercise in preventing and treating type 2 diabetes and obesity.
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