Use of cells expressing gamma subunit variants to identify diverse mechanisms of AMPK activation

Cell Metab. 2010 Jun 9;11(6):554-65. doi: 10.1016/j.cmet.2010.04.001.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / antagonists & inhibitors
  • AMP-Activated Protein Kinases / metabolism*
  • Adenosine Diphosphate / metabolism
  • Adenosine Triphosphate / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Benzimidazoles / pharmacology
  • Calcimycin / pharmacology
  • Calcium / metabolism
  • Cell Line
  • Dinitrophenols / pharmacology
  • Energy Metabolism
  • Enzyme Activation
  • Humans
  • Hypoglycemic Agents / pharmacology
  • Metformin / pharmacology
  • Naphthalimides / pharmacology
  • Oligomycins / pharmacology
  • Phosphorylation
  • Protein Subunits / metabolism


  • Anti-Bacterial Agents
  • Benzimidazoles
  • Dinitrophenols
  • Hypoglycemic Agents
  • Naphthalimides
  • Oligomycins
  • Protein Subunits
  • STO 609
  • Calcimycin
  • Adenosine Diphosphate
  • Adenosine Triphosphate
  • Metformin
  • AMP-Activated Protein Kinases
  • Calcium