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

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

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Authors

  • Simon A. Hawley
  • Fiona A. Ross
  • Cyrille Chevtzoff
  • Kevin A. Green
  • Ashleigh Evans
  • Sarah Fogarty
  • Mhairi C. Towler
  • Laura J. Brown
  • Oluseye A. Ogunbayo
  • A. Mark Evans
  • D. Grahame Hardie (Lead / Corresponding author)

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Info

Original languageEnglish
Pages554-565
Number of pages12
JournalCell Metabolism
Journal publication date9 Jun 2010
Journal number6
Volume11
DOIs
StatePublished

Abstract

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.

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