Hypoxic activation of AMPK is dependent on mitochondrial ROS but independent of an increase in AMP/ATP ratio

Abstract

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status found in metazoans that is known to be activated by stimuli that increase the cellular AMP/ATP ratio. Full activation of AMPK requires specific phosphorylation within the activation loop of the catalytic domain of the alpha-subunit by upstream kinases such as the serine/threonine protein kinase LKB1. Here we show that hypoxia activates AMPK through LKB1 without an increase in the AMP/ATP ratio. Hypoxia increased reactive oxygen species (ROS) levels and the antioxidant EUK-134 abolished the hypoxic activation of AMPK. Cells deficient in mitochondrial DNA (rho(0) cells) failed to activate AMPK during hypoxia but are able to in the presence of exogenous H(2)O(2). Furthermore, we provide genetic evidence that ROS generated within the mitochondrial electron transport chain and not oxidative phosphorylation is required for hypoxic activation of AMPK. Collectively, these data indicate that oxidative stress and not an increase in the AMP/ATP ratio is required for hypoxic activation of AMPK.

Figures

Fig. 1

Hypoxic activation of AMPK is independent of AMP levels. (A) AMP, ADP, and ATP levels were assessed by HPLC in MEFs exposed to 21%O2 or 1.5%O2 or 0% O2 for 10 minutes. (B) AMP/ATP ratio in MEFs exposed to 21%O2 or 1.5%O2 or 0% O2 for 10 minutes. (C) AMP, ADP, and ATP levels were assessed by HPLC in MEFs exposed to 21%O2 or 1.5%O2 or 0% O2 for 60 minutes. (D) AMP/ATP ratio in MEFs exposed to 21%O2 or 1.5%O2 or 0% O2 for 60 minutes.

Fig. 2

LKB1 is required for the hypoxic activation of AMPK. (A) AMPK activation assessed by phospho-ACC protein in Ampkα WT and Ampkα1-/-2-/- cells exposed to 21% O2 or 1.5%O2 for 30 minutes, or to 100 μMH2O2 for 15 minutes. An anti-ACC antibody was used as loading control. (B) Lkb1-/- cells were stably reconstituted with either vector alone or with Lkb1. AMPK activation was then assessed by phospho-ACC protein in both cell types exposed to 21%O2 or 1.5%O2 for 30 minutes, or to 100 μMH2O2 for 15 minutes. An anti-ACC antibody was used as loading control.

Fig. 3

ROS is required for the hypoxic activation of AMPK. (A) Ampkα WT cells were exposed to 21%O2 (0′), 1.5%O2 for 30 minutes and 60 minutes, or to 2 mM AICAR for 15 minutes at 21%O2 ± EUK-134 (10 μM). EUK-134 is a synthetic superoxide dismutase/ catalase mimetic, which scavenges both superoxide and H2O2. AMPK activation was then assessed by phospho-ACC protein and an anti-ACC antibody was used as loading control. (B) Ampkα WT cells were exposed to 21%O2 (0′), or 1.5%O2 for 60 minutes ± EUK-134 (10 μM) and ROS was measured using redox sensitive GFP. (C) Ampkα WT cells were exposed to 21%O2 (0′), or 1.5%O2 for 60 minutes ± EUK-134 (10 μM) and ROS was measured using Amplex Red.

Fig. 4

Hypoxic activation of AMPK requires mitochondrial ROS. (A) WT 143B were exposed to 21%O2 (0′), to 1.5%O2 for 30 and 60 minutes (30′ or 60′), or to a bolus of H2O2 (100 μM) for 15 minutes. H2O2 is used here as a positive control for AMPK activation. An anti-ACC antibody was used as loading control. (B) ρ0 143B cells were exposed to 21%O2 (0′), to 1.5%O2 for 30 and 60 minutes (30′ or 60′), or to a bolus of H2O2 (100 μM) for 15 minutes. An anti-ACC antibody was used as loading control. (C) WT 143B and ρ0 143B cells were exposed to 21%O2 (0′), or 1.5%O2 for 60 minutes ± EUK-134 (10 μM) and ROS was measured using Amplex Red.

Fig. 5

Oxidative phosphorylation is not necessary for the hypoxic activation of AMPK. (A) ΔCytchrome b 143B cells were subjected to 21%O2 (0′), to 1.5%O2 for 30 and 60 minutes (30′ or 60′), or to a bolus of H2O2 (100 μM) for 15 minutes. AMPK activation was then assessed by phospho-ACC protein and an anti-ACC antibody was used as loading control. (B) ΔCytchrome b 143B cells were subjected to 21%O2 (0′) or to 1.5%O2 for 30 and 60 minutes (30′ or 60′) ± EUK-134 (10 μM). AMPK activation was then assessed by phospho-ACC protein and an anti-ACC antibody was used as loading control. (C) WT 143B and ΔCytchrome b 143B cells were exposed to 21%O2 (0′), or 1.5% O2 for 60 minutes ± EUK-134 (10 μM) and ROS was measured using Amplex Red.

Fig. 6

Hydrogen peroxide can activate AMPK without an increase in [AMP]/ [ATP] ratio. (A) WT 143B cells and ρ0 143B cells were exposed to 20 uM H2O2 for 5 minutes and AMPK activation was measured using the phospho-ACC protein and an anti-ACC antibody was used as loading control. (B) ρ0 143B cells were exposed to 20 uM H2O2 for 5 minutes or to AICAR for 30 minutes and AMPK activation was measured using the phospho-AMPK protein and an anti-AMPK antibody was used as loading control. (C) [AMP]/ [ATP] ratio in WT 143B cells and ρ0 143B cells exposed to 20 uM H2O2 for 5 minutes.