Activated macrophages utilize glycolytic ATP to maintain mitochondrial membrane potential and prevent apoptotic cell death

Abstract

We have previously analysed the bioenergetic consequences of activating J774.A1 macrophages (M) with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) and found that there is a nitric oxide (NO)-dependent mitochondrial impairment and stabilization of hypoxia-inducible factor (HIF)-1α, which synergize to activate glycolysis and generate large quantities of ATP. We now show, using tetramethylrhodamine methyl ester (TMRM) fluorescence and time-lapse confocal microscopy, that these cells maintain a high mitochondrial membrane potential (Δψm) despite the complete inhibition of respiration. The maintenance of high Δψm is due to the use of a significant proportion of glycolytically generated ATP as a defence mechanism against cell death. This is achieved by the reverse functioning of FoF1-ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated M with inhibitors of either of these enzymes, but not with inhibitors of the respiratory chain complexes I to IV, led to a collapse in Δψm and to an immediate increase in intracellular ATP, due to the prevention of ATP hydrolysis by the FoF1-ATP synthase. This collapse in Δψm was followed by translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspases 3 and 9 and subsequent apoptotic cell death. Our results indicate that during inflammatory activation glycolytically competent cells such as M use significant amounts of the glycolytically generated ATP to maintain Δψ m and thereby prevent apoptosis. © 2010 Macmillan Publishers Limited All rights reserved.