The role of phosphorylation/dephosphorylation of acetyl-CoA carboxylase in the regulation of mammalian fatty acid biosynthesis.

The main lipogenic tissues of the rat are the liver, white adipose tissue and, during lactation, the mammary gland. The rate of fatty acid synthesis de novo in these tissues must be regulated according to the nutritional and physiological status of the animal. Such short-term regulation is primarily achieved through the action of hormones. Acetyl-CoA carboxyulase is a prime target for such hormonal regulation as it catalyses the rate-limiting step in the conversion of cytoplasmic acetyl-CoA to fatty acid. While acetyl-CoA carboxylase can be regulated allosterically by citrate (activator) and fatty acyl-CoA (inhibitor), it is becoming clear that phosphorylation and dephosphorylation, leading to inactivation and activation respectively, play a major part in the hormonal regulation of this enzyme activity. To examine the effects of hormones on phosphorylation of acetyl-CoA carboxylase, we have labelled isolated hepatocytes or adipocytes to steady state with ['*P]phosphate, treated the cells with or without hormone, and have then purified the enzyme to homogeneity using affinity chromatography on immobilized avidin in the presence of protein kinase and phosphatase inhibitors (NaF and EDTA). We simultaneously make perchloric acid extracts of the cells and analyse the specific radioactivity of cellular adenine nucleotides in these extracts using a h.p.1.c. method (Sharps & McCarl, 1982) in order that the stoichiometry of phosphorylation of acetyl-CoA carboxylase in the intact cell may be estimated. The validity of these estimations has been confirmed in the case of hepatocytes by making spectrophotometric determinations (Ames, 1966) of alkali-labile phosphate for enzyme purified from hepatocytes or intact liver (Holland et al., 1984). In vivo, glucagon inhibits hepatic fatty acid synthesis by approx. 60% (Cook et al., 1977), and a similar inhi-