TY - JOUR
T1 - Use of Cells Expressing gamma Subunit Variants to Identify Diverse Mechanisms of AMPK Activation
AU - Hawley, Simon A.
AU - Ross, Fiona A.
AU - Chevtzoff, Cyrille
AU - Green, Kevin A.
AU - Evans, Ashleigh
AU - Fogarty, Sarah
AU - Towler, Mhairi C.
AU - Brown, Laura J.
AU - Ogunbayo, Oluseye A.
AU - Evans, A. Mark
AU - Hardie, D. Grahame
PY - 2010/6/9
Y1 - 2010/6/9
N2 - 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.
AB - 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.
KW - DEPENDENT PROTEIN-KINASE
KW - RESPIRATORY COMPLEX-I
KW - SIGNALING PATHWAYS
KW - ENDOTHELIAL-CELLS
KW - GLYCOGEN-STORAGE
KW - METFORMIN ACTION
KW - MAMMALIAN-CELLS
KW - CELLULAR-ENERGY
KW - UPSTREAM KINASE
KW - SKELETAL-MUSCLE
UR - http://www.scopus.com/inward/record.url?scp=77956410464&partnerID=8YFLogxK
U2 - 10.1016/j.cmet.2010.04.001
DO - 10.1016/j.cmet.2010.04.001
M3 - Article
C2 - 20519126
SN - 1550-4131
VL - 11
SP - 554
EP - 565
JO - Cell Metabolism
JF - Cell Metabolism
IS - 6
ER -