Inhibition of AMPK activity in response to insulin in adipocytes - involvement of AMPK pS485, PDEs and cellular energy levels.

Insulin resistance in obesity and type 2 diabetes (T2D) has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes, however the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, if the S485 phosphorylation is required for insulin-induced inhibition of AMPK, or if other mechanisms underlie the reduced kinase activity, is not known. In order to investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a non-phosphorylatable AMPK S485A mutant. AMPK activity measurements by western blotting and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP/ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.