Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase.
The activity of acetyl-CoA carboxylase (ACC), a rate-limiting enzyme of fatty acid biosynthesis and malonyl-CoA production, can be regulated by several mechanisms, including multisite covalent phosphorylation, both in vitro and in intact cells. Evidence has been presented by others to indicate that a 5'-AMP-activated protein kinase (AMPK) is likely the major regulatory kinase active on ACC. While insulin is known to activate ACC in several cell types, accompanied by changes in ACC phosphorylation, the mechanism underlying this activation has been obscure. In the present study, we have examined, in Fao hepatoma cells, the effects of insulin on ACC and AMPK activity, the latter measured with a synthetic peptide corresponding to one of the phosphorylation sites on ACC for AMPK. Our results show that insulin leads to inhibition of kinase activity prior to the onset of ACC activation; the peak of maximal kinase inhibition (approximately 35% at 10 min) is seen to precede the onset of ACC activation (20 min). The inhibition of kinase activity due to insulin is observed both in the absence and presence of varying stimulating concentrations of added 5'-AMP. Both kinase inhibition and ACC activation display similar insulin sensitivity (A50 0.3 nM). Preservation of this insulin-induced kinase inhibition requires the presence of protein phosphatase inhibitors in the cell lysis buffer, suggesting that AMPK itself might be regulated by insulin-stimulated changes in kinase phosphorylation. Taken together, these data are consistent with the hypothesis that the 5'-AMP-activated protein kinase is a regulated component of the insulin signal transduction pathway and may be the major target for insulin regulation of ACC.
Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire 03756.
L A
LA
Witters
B E
BE
Kemp
D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D013379:Substrate Specificity
D007328:Insulin; D009097:Multienzyme Complexes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase