Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation.
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) gamma2 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 Ca(2+) ionophore, A23187, osmotic stress, and quercetin activated both variants to varying extents. A23187 and osmotic stress also increased cytoplasmic Ca(2+), and their effects were inhibited by STO609, 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.
Hawley
Simon A
SA
Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Ross
Fiona A
FA
Chevtzoff
Cyrille
C
Green
Kevin A
KA
Evans
Ashleigh
A
Fogarty
Sarah
S
Towler
Mhairi C
MC
Brown
Laura J
LJ
Ogunbayo
Oluseye A
OA
Evans
A Mark
AM
Hardie
D Grahame
DG
D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004140:Dinitrophenols / Q000494:pharmacology; D004734:Energy Metabolism; D004789:Enzyme Activation; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D008687:Metformin / Q000494:pharmacology; D053644:Naphthalimides / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001562:Benzimidazoles; D004140:Dinitrophenols; D007004:Hypoglycemic Agents; D053644:Naphthalimides; D009840:Oligomycins; D021122:Protein Subunits; C458525:STO 609; D000001:Calcimycin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008687:Metformin; D055372:AMP-Activated Protein Kinases; D002118:Calcium