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

Simon A Hawley1, Fiona A Ross, Cyrille Chevtzoff

  • 1Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

Cell Metabolism
|June 4, 2010
PubMed

Insights

AMP-activated protein kinase (AMPK) activation mechanisms were investigated using AMP-sensitive and insensitive variants. Metformin activates AMPK by inhibiting mitochondrial respiration, revealing distinct activation pathways.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Metabolic Regulation

Background:

  • AMP-activated protein kinase (AMPK) is a crucial energy sensor, but its activation mechanisms by various agents are not fully understood.
  • Identifying these mechanisms is vital for understanding cellular energy homeostasis and developing targeted therapeutics.

Purpose of the Study:

  • To elucidate the diverse mechanisms underlying AMPK activation by different pharmacological and physiological stimuli.
  • To differentiate between AMP-dependent and AMP-independent AMPK activation pathways.

Main Methods:

  • Generation of isogenic cell lines expressing AMP-sensitive (wild-type) and AMP-insensitive (R531G) gamma2 variants of AMPK.
  • Treatment with various agents including mitochondrial poisons, metabolic inhibitors, Ca(2+) ionophores, and osmotic stress.
  • Measurement of cellular energy metabolism using ADP:ATP ratios and extracellular flux analysis (oxygen uptake).
  • Assessment of cytoplasmic Ca(2+) levels and inhibition by CaMKK inhibitors.

Main Results:

  • Mitochondrial poisons and several other agents (e.g., metformin, resveratrol) activated AMPK only in AMP-sensitive cells, correlating with inhibited cellular energy metabolism.
  • Agents like A769662, Ca(2+) ionophore A23187, osmotic stress, and quercetin activated both AMP-sensitive and insensitive AMPK variants to varying degrees.
  • Ca(2+) ionophore and osmotic stress-induced AMPK activation were dependent on Ca(2+) and inhibited by a CaMKK inhibitor.
  • At least six distinct mechanisms for AMPK activation were identified.

Conclusions:

  • AMPK activation involves multiple distinct pathways, including AMP-dependent and AMP-independent mechanisms.
  • Metformin, a widely used antidiabetic drug, activates AMPK by inhibiting mitochondrial respiration.
  • Calcium/calmodulin-dependent protein kinase kinase (CaMKK) mediates AMPK activation by certain stimuli like A23187 and osmotic stress.

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