Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia

Background: The role of protein phosphorylation in the Pasteur effect - the phenomenon whereby anaerobic conditions stimulate glycolysis - has not bee n addressed. The AMP-activated protein kinase (AMPK) is activated when the oxygen supply is restricted. AMPK acts as an energy-state sensor and inhibi ts key biosynthetic pathways, thus conserving ATP. Here, we studied whether AMPK is involved in the Pasteur effect in the heart by phosphorylating and activating 6-phosphofructo-2-kinase (PFK-2), the enzyme responsible for th e synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis . Results: Heart PFK-2 was phosphorylated on Ser466 and activated by AMPK in vitro. In perfused rat hearts, anaerobic conditions or inhibitors of oxidat ive phosphorylation (oligomycin and antimycin) induced AMPK activation, whi ch correlated with PFK-2 activation and with an increase in fructose 2,6-bi sphosphate concentration. Moreover, in cultured cells transfected with hear t PFK-2, oligomycin treatment resulted in a parallel activation of endogeno us AMPK and PFK-2. In these cells, the activation of PFK-2 was due to the p hosphorylation of Ser466. A dominant-negative construct of AMPK abolished t he activation of endogenous and cotransfected AMPK, and prevented both the activation and phosphorylation of transfected PFK-2 by oligomycin. Conclusions: AMPK phosphorylates and activates heart PFK-2 in vitro and in intact cells. AMPK-mediated PFK-2 activation is likely to be involved in th e stimulation of heart glycolysis during ischaemia.