Mechanisms involved in the short-term regulation of acetyl-CoA carboxylase by insulin.

Acetyl-CoA carboxylase is generally considered to have an important role in the regulation of fatty acid synthesis and overall there is good evidence that short-term control of this pathway by insulin and other hormones involves changes in activity of this enzyme (for recent reviews see Brownsey & Denton, 1985, 1986). The first demonstration of the short-term hormonal regulation of this enzyme was the finding of persistent increases in activity in extracts of rat epididymal adipose tissue previously exposed to insulin, by Halestrap & Denton ( 1 973, 1974). Subsequently, evidence that the stimulation of fatty acid synthesis by insulin involves activation of acetyl-CoA carboxylase has also been obtained in brown adipose tissue (McCormack & Denton, 1977), mammary tissue (Munday & Williamson, 1982) and liver (Witters et al., 1979; Buechler et al., 1984). In contrast, hormones which increase cyclic AMP concentrations (such as /I-adrenergic agonists and glucagon) lead to inhibition of fatty acid synthesis and persistent diminutions in acetylCoA carboxylase activity (Brownsey et al., 1979; Witters et al., 1979; Gibbins et al., 1985 Holland et al., 1984, 1985). The activity of acetyl-CoA carboxylase can be altered, at least in vitro, by citrate and fatty acyl-CoA esters. Citrate activates the enzyme and this is associated with polymerization of the dimeric form of the enzyme. In contrast, fatty acyl-CoA esters are potent inhibitors of the enzyme and also cause depolymerization (see Numa & Tanabe, 1984; Brownsey & Denton, 1986). At first sight, the effects of citrate and fatty acyl-CoA esters would appear to be examples of feed-forward activation and end-product inhibition respectively. However, it has proved difficult to establish irrevocably whether alterations in the cytoplasmic concentration of these are important in the hormonal regulation of acetyl-CoA carboxylase activity (Brownsey & Denton, 1986). However, acetyl-CoA carboxylase is phosphorylated in cells (Brownsey et al., 1977). Moreover, both insulin and hormones which increase cell cyclic AMP cause modest increases (I(r-40%) in the overall level of phosphorylation of the enzyme in fat and liver cells although these hormones have contrasting effects on catalytic activity and rates of fatty acid synthesis (Brownsey et al., 1979; Witters et al., 1979; Witters, 1981; Brownsey & Denton, 1982; Holland et al., 1984, 1985). However, two-dimensional thin-layer separation of tryptic peptides derived from labelled acetyl-CoA carboxylase isolated from fat cells by immunoprecipitation showed that the principal sites exhibiting increased phosphorylation in epididymal fat cells exposed to insulin were quite distinct from those in cells exposed to adrenaline (Brownsey & Denton, 1982). The major effect of insulin was to cause a marked increase in the phosphorylation of a peptide (designated the I-peptide), whereas adrenaline resulted in increased phosphorylation of a separate group of two to three peptides with rather similar mobility (the Apeptides). Broadly similar findings were obtained subsequently by Witters et al. (1983) and Holland & Hardie (1985) using fat and liver cells respectively. In these studies, 32 P-labelled acetyl-CoA carboxylase was iso-