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

Summary

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) γ2 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.

Abstract

Graphical Abstract

Highlights

► AMPK is activated by a wide variety of different stresses, drugs, and xenobiotics ► We have developed a sensitive method to test which activators are AMP mediated ► Most, e.g., metformin and resveratrol, inhibit mitochondrial function and elevate AMP ► However, we can now distinguish six different mechanisms for AMPK activation