Hyperinsulinemia and insulin resistance are prevalent metabolic characteristics frequently observed in obese individuals and are usually associated with defects in oxidative metabolism. Although the mechanism of the glucose-fatty acid cycle is traditionally used to explain substrate interactions, definitive evidence of its primacy in the regulation of muscle oxidative metabolism is lacking. From evidence collected in different tissues, an alternative mechanism was recently proposed which suggests that reciprocal regulation of glucose and FFA oxidation may be linked through changes in malonyl-CoA levels. Malonyl-CoA, a potent inhibitor of carnitine palmitoyl transferase, is formed from acetyl-CoA in a carboxylation reaction catalyzed by acetyl-CoA carboxylase. The investigators propose to investigate cellular factors that will ultimately impact on malonyl-CoA levels by modulating its rate of production. Muscle contractions, insulin, and long chain acylCoA availability will be used as factors to modify malonyl-CoA levels through covalent modification of acetyl-CoA carboxylase. Glucose availability and exercise-induced changes in glucose metabolism will be used as factors to modify malonyl-CoA levels through changes in substrate supply for acetyl-CoA carboxylase. In the current proposal the investigators plan to employ the in situ perfused hindlimb preparation to address the following hypotheses: (1) that intracellular glucose availability determines the rate of oxidative metabolism in resting and contracting muscles through a cellular mechanism that implicates malonyl-CoA, (2) that carbohydrate and lipid fuel sources impact on the proposed cellular mechanism of regulation by modulating acetyl-CoA availability, and 3) that insulin directly impacts on the proposed cellular mechanism of regulation by modulating both glucose and fatty acid metabolism. We will use tracer methodology in conjunction with arteriovenous balance measurements, analysis of CoA ester levels and of enzyme activities to quantify carbohydrate and fatty acid metabolism in hindlimbs of rats perfused at predetermined rates of glucose uptake during rest and muscle contractions. For the first time, malonyl-CoA levels will be correlated to the rate of fatty acid oxidation in skeletal muscle.
