Abstract

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.

Highlights

  • It has long been recognized that in adipocytes, liver cells, hepatoma cells, and intact liver insulin rapidly leads to the activation ofACC [12,13,14,15,16,17,18,19,20,21,22,23,24].investigation of the nature of the phosphorylation changes and of the mechanisms underlying the functional changes in ACC in response to vation display similar insulin sensitivity (Aso0.3 nM). insulin have given mixed results in different laboratories

  • Preservation of this insulin-induced kinase inhibition adipose tissue and adipocytes, insulin appearsto increase the requires thepresence of protein phosphatase inhibitors phosphorylation of ACC at unique phosphorylation sites that in the cell lysis buffer, suggesting that AMPactivated protein kinase (AMPK) itself maybe substrates for casein kinase 11, the multifunctional might be regulated by insulin-stimulated changes in calmodulin-dependent protein kinase, astarfish oocyte p44 kinase phosphorylation

  • Incubation of Fao cells with M insulin leads to atimedependent decrease in AMPK activity, as measured in the presence of 200 p~ 5’-AMP (Fig. 2, top).The insulin-induced inhibition is relatively slow, emerging between 5 and 10 min

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Summary

Introduction

In panel A, 1.5 pg of both control and insulin fractions was assayed over time in the presence of 200 p~ 5'-AMP In panel B , varying amounts of fraction protein (0.65-1.65 pg) of both control (0)and insulin (0)fractions were assayed for 12 min in the presence of 200 p~ 5'-AMP.

Results
Conclusion
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