James L. Gamble

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 beta (ACC-beta), 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-beta was first purified by immunoprecipitation with a monoclonal antibody. ACC-beta was quantitatively recovered in the immunopellet and exhibited a high sensitivity to citrate (12-fold activation) and a Km for acetyl-CoA (120 microM) similar to that reported for ACC-beta purified by other means. After 5 min of contraction, ACC-beta 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-beta with purified protein phosphatase 2A abolished the decreases in both ACC-beta activity and electrophoretic mobility caused by contraction. The rapid decrease in ACC-beta 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 alpha2 but not the alpha1 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 alpha2 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.

The role of acetyl-coenzyme A carboxylase (ACC) in regulating fatty acid oxidation was investigated in isolated fatty acid perfused working rat hearts. Overall fatty acid oxidation rates were determined by addition of 1.2 mM [3H]palmitate to the perfusate of hearts in which the endogenous triglyceride pool was prelabeled with [14C]palmitate. Rates of both exogenous and endogenous fatty acid oxidation were measured by simultaneous measurement of 3H2O and 14CO2 production, respectively. A second series of hearts were perfused under similar conditions except that [U-14C]glucose was present in the perfusate for measurement of glucose oxidation rates. Addition of dichloroacetate (DCA, 1 mM) to the perfusate resulted in a dramatic stimulation of glucose oxidation (a 411% increase), with a parallel decrease in fatty acid oxidation (from 305 +/- 51 to 206 +/- 40 nmol/g dry weight.min.unit work). DCA treatment increased the contribution of glucose oxidation to ATP production from 7.1 to 30.6%, while decreasing the contribution of overall fatty acid oxidation from 92.9 to 69.4%. Tissue levels of malonyl-CoA in hearts treated with DCA were higher compared to controls (14.0 +/- 0.6 and 10.0 +/- 0.7 nmol/g dry weight, respectively) and were negatively correlated (r = -0.85) with overall fatty acid oxidation rates. Acetyl-CoA levels were also significantly higher in DCA-treated hearts, and a positive correlation (r = 0.88) was seen between myocardial acetyl-CoA and malonyl-CoA levels. This suggests that DCA treatment increased the supply of acetyl-CoA for ACC. Western blots revealed the presence of both the 280-kDa (ACC-280) and the 265-kDa (ACC-265) isoforms of ACC in cardiac tissue, with a predominance of ACC-280. The activity of ACC extracted from hearts was similar in both groups when assayed under optimal conditions of acetyl-CoA and citrate. However, using affinity purified ACC, it was demonstrated that heart ACC (predominantly ACC-280) had a higher Km for acetyl-CoA than ACC isolated from white adipose tissue (predominantly ACC-265). We conclude that ACC is an important regulator of fatty acid oxidation in the heart and that acetyl-CoA supply is a key determinant of heart ACC-280 activity. As acetyl-CoA levels increase, ACC-280 is activated resulting in an increase in malonyl-CoA inhibition of fatty acid oxidation.

The AMP-activated protein kinase is a heterotrimeric enzyme, important in cellular adaptation to the stress of nutrient starvation, hypoxia, increased ATP utilization, or heat shock. This mammalian enzyme is composed of a catalytic α subunit and noncatalytic β and γ subunits and is a member of a larger protein kinase family that includes the SNF1 kinase of <i>Saccharomyces cerevisiae.</i> In the present study, we have identified by truncation and site-directed mutagenesis several functional domains of the α1 catalytic subunit, which modulate its activity, subunit association, and protein turnover. C-terminal truncation of the 548-amino acid (aa) wild-type α1 protein to aa 312 or 392 abolishes the binding of the β/γ subunits and dramatically increases protein expression. The full-length wild-type α1 subunit is only minimally active in the absence of co-expressed β/γ, and α1(1–392) likewise has little activity. Further truncation to aa 312, however, is associated with a large increase in enzyme specific activity, thus revealing an autoinhibitory sequence between aa 313 and 392. α-1(1–312) still requires the phosphorylation of the activation loop Thr-172 for enzyme activity, yet is now independent of the allosteric activator, AMP. The increased levels of protein expression on transient transfection of either truncated α subunit cDNA are because of a decrease in enzyme turnover by pulse-chase analysis. Taken together, these data indicate that the α1 subunit of AMP-activated protein kinase contains several features that determine enzyme activity and stability. A constitutively active form of the kinase that does not require participation by the noncatalytic subunits provides a unique reagent for exploring the functions of AMP-activated protein kinase.