CONTRACTION-INDUCED CHANGES IN ACETYL-COA CARBOXYLASE AND 5'-AMP-ACTIVATED KINASE IN SKELETAL-MUSCLE

The concentration of malonyl-CoA, a negative regulator of fatty acid o xidation, diminishes acutely in contracting skeletal muscle, To determ ine how this occurs, the activity and properties of acetyl-CoA carboxy lase beta (ACC-beta), the skeletal muscle isozyme that catalyzes malon yl-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 ex tract by mitochondrial carboxylases, an assay was developed in which A CC-beta was first purified by immunoprecipitation with a monoclonal an tibody, ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a K-m for acetyl-CoA (120 mu M) similar to that reported for ACC-beta purif ied by other means, After 5 min of contraction, ACC-beta activity was decreased by 90% despite an apparent increase in the cytosolic concent ration of citrate, a positive regulator of ACC, SDS-polyacrylamide gel electrophoresis of both homogenates and immunopellets from these musc les showed a decrease in the electrophoretic mobility of ACC, suggesti ng 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, homogenizatio n of the muscles in a buffer free of phosphatase inhibitors and contai ning the phosphatase activators glutamate and MgCl2 or treatment of im munoprecipitated ACC-beta with purified protein phosphatase 2A abolish ed the decreases in both ACC-beta activity and electrophoretic mobilit y caused by contraction, The rapid decrease in ACC-beta activity after the onset of contractions (50% by 20 s) and its slow restoration to i nitial values during recovery (60-90 min) were paralleled temporally b y reciprocal changes in the activity of the alpha 2 but not the alpha 1 isoform of 5'-AMP-activated protein kinase (AMPK). In conclusion, th e results suggest that the decrease in ACC activity during muscle cont raction is caused by an increase in its phosphorylation, most probably due, at least in part, to activation of the alpha 2 isoform of AMPK. They also suggest a dual mechanism for ACC regulation in muscle in whi ch inhibition by phosphorylation takes precedence over activation by c itrate, These alterations in ACC and AMPK activity, by diminishing the concentration of malonyl-CoA, could be responsible for the increase i n fatty acid oxidation observed in skeletal muscle during exercise.