Use of Cells Expressing gamma Subunit Variants to Identify Diverse Mechanisms of AMPK Activation

S. A. Hawley, F. A. Ross, C. Chevtzoff, K. A. Green, A. Evans, S. Fogarty, M. C. Towler, Laura J. Brown, O. A. Ogunbayo, A. M. Evans, D. G. Hardie

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Abstract / Description of output

A wide variety of agents activate AMPK, but in many cases the mechanisms remain unclear. We generated isogenic cell lines stably expressing AMPK complexes containing AMP-sensitive (wild-type, WT) or AMP-insensitive (R531G) gamma 2 variants. Mitochondrial poisons such as oligomycin and dinitrophenol only activated AMPK in WT cells, as did AICAR, 2-deoxyglucose, hydrogen peroxide, metformin, phenformin, galegine, troglitazone, phenobarbital, resveratrol, and berberine. Excluding AICAR, all of these also inhibited cellular energy metabolism, shown by increases in ADP:ATP ratio and/or by decreases in cellular oxygen uptake measured using an extracellular flux analyzer. By contrast, A769662, the Ca2+ ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca2+, and their effects were inhibited by ST0609, a CaMKK inhibitor. Our approaches distinguish at least six different mechanisms for AMPK activation and confirm that the widely used antidiabetic drug metformin activates AMPK by inhibiting mitochondrial respiration.
Original languageEnglish
Pages (from-to)554-565
Number of pages12
JournalCell Metabolism
Issue number6
Publication statusPublished - Jun 2010

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