Isolation of a cyclic-AMP-independent protein kinase from rat liver and its effect on the enzymic activity of acetyl-CoA carboxylase

ses the first step in this pathway and its activity is regulated allosterically, e.g. citrate (activator) and long-chain fatty acyl-CoA (inhibitor), and by reversible phosphorylation (Hardie, 1980; Hardie et al., 1984). It is thus a prime candidate for such specific inhibition. In the lactating mammary gland, 24 h starvation inhibits lipogenesis by 98% and this is accompanied by an inhibition of acetyl-CoA carboxylase that is the result of increased phosphorylation of the enzyme (Munday & Hardie, 1986). This can be completely reversed by refeeding the rat, but this effect is blocked by streptozotocin treatment, suggesting that insulin stimulates the dephosphorylation and activation of the enzyme in vivo (Munday & Hardie, 1986). In crude extracts of the lactating mammary gland, the V,,, of acetyl-CoA carboxylase was 1.84 f 0.30 (n = 6) and 0.67 0.1 1 (n = 6) pmol/min per g wet wt. tissue for chow fed and high-fat fed rats respectively. After purification by avidin-Sepharose affinity chromatography, the enzyme exhibited an identical inhibition of V,,, (64%) in response to high-fat feeding, accompanied by an increase in the concentration of citrate required to half-maximally activate the enzyme ( A o 5 citrate). This correlated with an increase in its total alkali-labile phosphate content of 1 mol of phosphate/mol of acetyl-CoA carboxylase subunit. The inhibition of acetyl-CoA carboxylase was still evident in isolated mammary acini (Table I), although it is notable that the extent of inhibition was smaller than that measured in the whole tissue. As with lipogenesis, incubation of the cells with insulin completely reversed the inhibition of acetyl-CoA carboxylase by high-fat feeding, but had no effect on activity in chow-fed controls (Table I ) . These changes in activity were observed only in the V,,, of the enzyme since there were no significant differences between the A,,, citrate values for enzyme from any of the acini incubations. Acetyl-CoA carboxylase purified from control and insulin-treated acini from high-fat-fed rats had respective V,,,, values of 1.35 f 0.18 (n = 5 ) and 1.83 & 0.13 ( n = 5 ) nmol/min per mg of enzyme. This increase (36%) was significant and similar to that observed in crude extracts. The A,, citrate of the enzyme was unaltered, but the phosphate content decreased by 0.6 mol of phosphate/mol of enzyme subunit. This is in contrast to the situation in adipocytes where insulin activation of acetyl-CoA carboxylase is accompanied by increased phosphorylation of the enzyme (Browney & Denton, 1982). These results provide further evidence that in the lactating mammary gland the activation of acetyl-CoA carboxylase by insulin is an important component of its stimulation of lipogenesis and, as in the starved-refed transition, appears to occur via a dephosphorylation of the enzyme.