Phosphorylation of the insulin receptor by AMP-activated protein kinase (AMPK) promotes ligand-independent activation of the insulin signalling pathway in rodent muscle

Muscle may experience hypoglycaemia during ischaemia or insulin infusion. During severe hypoglycaemia energy production is blocked, and an increase of AMP:ATP activates the energy sensor and putative insulin-sensitiser AMP-activated protein kinase (AMPK). AMPK promotes energy conservation and survival by shutting down anabolism and activating catabolic pathways. We investigated the molecular mechanism of a unique glucose stress defence pathway involving AMPK-dependent, insulin-independent activation of the insulin signalling pathway. Cardiac or skeletal myocytes were subjected to glucose and insulin-free incubation for increasing intervals up to 20 h. AMPK, and components of the insulin signalling pathway and their targets were quantified by western blot using phosphor-specific antibodies. Phosphomimetics were used to determine the function of IRS-1 Ser789 phosphorylation and in vitro [P-32]ATP kinase assays were used to measure the phosphorylation of the purified insulin receptor by AMPK. Glucose deprivation increased Akt-Thr308 and Akt-Ser473 phosphorylation by almost tenfold. Phosphorylation of glycogen synthase kinase 3 beta increased in parallel, but phosphorylation of ribosomal 70S subunit-S6 protein kinase and mammalian target of rapamycin decreased. AMPK inhibitors blocked and aminoimidazole carboxamide ribonucleotide (AICAR) mimicked the effects of glucose starvation. Glucose deprivation increased the phosphorylation of IRS-1 on serine-789, but phosphomimetics revealed that this conferred negative regulation. Glucose deprivation enhanced tyrosine phosphorylation of IRS-1 and the insulin receptor, effects that were blocked by AMPK inhibition and mimicked by AICAR. In vitro kinase assays using purified proteins confirmed that the insulin receptor is a direct target of AMPK. AMPK phosphorylates and activates the insulin receptor, providing a direct link between AMPK and the insulin signalling pathway; this pathway promotes energy conservation and survival of muscle exposed to severe glucose deprivation.