Contraction-induced Changes in Acetyl-CoA Carboxylase and 5′-AMP-activated Kinase in Skeletal Muscle*

Abstract

The concentration of malonyl-CoA, a negative regulator of fatty acid oxidation, diminishes acutely in contracting skeletal muscle. To determine how this occurs, the activity and properties of acetyl-CoA carboxylase β (ACC-β), the skeletal muscle isozyme that catalyzes malonyl-CoA formation, were examined in rat gastrocnemius-soleus muscles at rest and during contractions induced by electrical stimulation of the sciatic nerve. To avoid the problem of contamination of the muscle extract by mitochondrial carboxylases, an assay was developed in which ACC-β was first purified by immunoprecipitation with a monoclonal antibody. ACC-β was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and aK _m for acetyl-CoA (120 μm) similar to that reported for ACC-β purified by other means. After 5 min of contraction, ACC-β activity was decreased by 90% despite an apparent increase in the cytosolic concentration of citrate, a positive regulator of ACC. SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these muscles showed a decrease in the electrophoretic mobility of ACC, suggesting that phosphorylation could account for the decrease in ACC activity. In keeping with this notion, citrate activation of ACC purified from contracting muscle was markedly depressed. In addition, homogenization of the muscles in a buffer free of phosphatase inhibitors and containing the phosphatase activators glutamate and MgCl₂ or treatment of immunoprecipitated ACC-β with purified protein phosphatase 2A abolished the decreases in both ACC-β activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-β activity after the onset of contractions (50% by 20 s) and its slow restoration to initial values during recovery (60–90 min) were paralleled temporally by reciprocal changes in the activity of the α2 but not the α1 isoform of 5′-AMP-activated protein kinase (AMPK). In conclusion, the results suggest that the decrease in ACC activity during muscle contraction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the α2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in which inhibition by phosphorylation takes precedence over activation by citrate. These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase in fatty acid oxidation observed in skeletal muscle during exercise.