Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase, the rate limiting enzyme for oxidation of fatty acids in skeletal muscle. At submaximal work rates, the rate of fat utilization increases during exercise. Malonyl-CoA, this fat oxidation inhibitor, decreases in the working muscle within minutes after the beginning of exercise. It is increased in muscle of fed rats and isolated muscles exposed to high concentrations of glucose and insulin. A unique isoform (272 kDa) of acetyl-CoA carboxylase (ACC) (the enzyme that synthesizes malonyl-CoA) has been identified in skeletal muscle. The activity of this enzyme has been found to decrease in muscle during exercise concurrent with an increase in the activity of a 5'-AMP-activated protein kinase (AMPK). Purified AMPK has been shown to be effective in phosphorylating and inactivating muscle ACC. In the proposed experiments the short-term mechanisms of regulation of ACC in contracting skeletal muscle will be investigated. These will include studies on correlation between malonyl-CoA and fatty acid oxidation in isolated perfused rat hindlimb, studies on the effect of phosphorylation of ACC by AMPK and calmodulin kinase Ia on activity of ACC in the presence of various concentrations of substrates, activators, and inhibitors, and studies on the effect of different phosphatases on ACC previously inactivated by exercise or muscle stimulation. The mechanisms of activation of AMPK during muscle contraction and exercise will be studied, using precursors of an analogue of AMP and calmodulin antagonists. A putative AMPK kinase will be isolated from skeletal muscle and characterized with respect to activation by calcium/calmodulin and AMP. These studies will provide new information on the basic mechanisms of regulation of malonyl-CoA, the putative modulator of fat oxidation in muscle. These studies will contribute to our understanding of the mechanisms responsible for increasing fat utilization during exercise.
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