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.
Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.
Simon A
SA
Hawley
Fiona A
FA
Ross
Cyrille
C
Chevtzoff
Kevin A
KA
Green
Ashleigh
A
Evans
Sarah
S
Fogarty
Mhairi C
MC
Towler
Laura J
LJ
Brown
Oluseye A
OA
Ogunbayo
A Mark
AM
Evans
D Grahame
DG
Hardie
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