Abstract 15679: Phosphorylation Of The Insulin Receptor By AMPK Promotes Ligand-independent Activation Of The Insulin-signaling Pathway And Promotes Survival Of Glucose-stressed Cardiac Myocytes
Introduction: Muscle may experience hypoglycemia during ischemia or insulin infusion. During severe hypoglycemia energy production is blocked and increased AMP activates the energy sensor and putative insulin-sensitizer AMP-dependent kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique hypoglycemia-defense pathway involving AMPK-dependent, insulin-independent activation of the insulin-signaling pathway.
Methods: Cardiac or skeletal myocytes were subjected to glucose and insulin-free incubations for increasing intervals up to 24h. AMPK and components of the insulin signaling pathway and their targets were quantified by western blot using phosphor-specific antibodies. Phosphomimetics was used to determine the function of IRS-1 Ser-789 phosphorylation and in vitro [32-P]-ATP kinase assays were used to measure phosphorylation of IR by AMPK.
Results: Hypoglycemia increased the phosphorylation of Akt-Thr308, Akt-Ser473 and GSK3β-Ser9 each by >10-fold (n=8; p<0.01). P70S6-kinase and mTOR phosphorylation were significantly decreased in parallel. AMPK inhibitors blocked and AICAR mimicked these effects of hypoglycemia. Hypoglycemia increased the phosphorylation of IRS-1 on Ser-789, the only known AMPK target, but phosphomimetics revealed that this conferred negative regulation. Hypoglycemia enhanced tyrosine phosphorylation of IRS-1 (Y612 and Y632) and IR (Y-1162), effects that were again blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified AMPK and immunoprecipitated IR revealed 32-P-incorporation into IR that was blocked by the selective AMPK inhibitor Compound C, confirming that IR is a direct target for AMPK. The consequences of AMPK-mediated enhancement of insulin signaling included preservation of intracellular ATP and enhanced potential for glucose uptake that were AMPK, PI3-kinase and Akt-dependent.
Conclusions: AMPK phosphorylates and activates the IR providing a novel mechanism for AMPK-mediated enhanced insulin sensitivity; the pathway promotes energy conservation and survival of cardiac and skeletal muscle exposed to severe hypoglycemia.
- © 2011 by American Heart Association, Inc.