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. Author manuscript; available in PMC: 2012 Apr 14.
Published in final edited form as: Free Radic Biol Med. 2009 Mar 3;46(10):1386–1391. doi: 10.1016/j.freeradbiomed.2009.02.019

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

Brooke M Emerling a, Frank Weinberg a, Colleen Snyder a, Zach Burgess a, Gökhan M Mutlu a, Benoit Viollet b,c, GR Scott Budinger a, Navdeep S Chandel a,*
PMCID: PMC3326346  NIHMSID: NIHMS365444  PMID: 19268526

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 α-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 (ρ0 cells) failed to activate AMPK during hypoxia but are able to in the presence of exogenous H2O2. 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.

Keywords: AMP-activated kinase, Hypoxia, LKB1, Mitochondria, Reactive oxygen species, Free radicals

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase complex consisting of a catalytic α subunit and two regulatory β and γ subunits [1-3]. AMPK is ubiquitously expressed and functions as an intracellular energy sensor by facilitating ATP production and suppressing unnecessary ATP use in energy-stressed cells. To date conditions that elevate intracellular AMP or decrease ATP levels are known to activate AMPK through the allosteric binding of AMP, which allows AMPK to sense cellular [AMP]/ [ATP] ratios. Full activation of AMPK requires specific phosphorylation (Thr172) within the activation loop of the catalytic domain of the α-subunit by upstream kinases, including LKB1, a serine/threonine protein kinase and tumor suppressor [4,5]. Recently, mammalian Ca2+/calmodulin-dependent kinase kinase (CaMKK) has also been identified as an AMPK kinase [6]. AMPK phosphorylates diverse targets, many of which are directly involved in controlling cellular energy metabolism, such as acetyl-CoA carboxylase (ACC) and glycogen synthase [7,8]. The net effect of AMPK activation is to inhibit lipid and glycogen synthesis concomitant with the activation of fatty acid oxidation and glycolysis. Thus, AMPK switches the cell from energy-storing to releasing energy for use under conditions where ATP is limiting.

Reactive oxygen species (ROS) have been viewed traditionally as damaging to the cell, however, ROS can also function as important activators of key cellular processes, and have been shown to play a vital role in cell signaling networks. For example, the ROS generated within complex III of the mitochondria during hypoxia are both sufficient and required to activate the hypoxia inducible factor-1 (HIF-1) [9-13]. HIF-1 is the key transcription factor that regulates the cellular responses to hypoxia and is critically important for tumor progression and angiogenesis. Hypoxia in the range of 2-20 TORR or 0.3%-5% O2 in mammalian cells does not result in a change in bioenergetics or induce cell death [14]. By contrast, anoxia is considered 0-2 TORR or 0-0.3% O2 and results in a decrease in ATP and induction of cell death (Schroedl et al. 2002). However, multiple reports have indicated that hypoxia indeed activates AMPK [15-19]. Here we show that AMPK activation under hypoxic conditions (1.5% O2) is not associated with an increase in AMP levels, but is dependent on the generation of mitochondrial ROS (mtROS). Furthermore, by using cells deficient in the mitochondrial complex III subunit, cytochrome b, we provide evidence here that the mitochondrial ROS, independent, of oxidative phoshporylation is required for the hypoxic activation of AMPK signaling.

Materials and methods

Cell Culture and Reagents

Ampkα WT and Ampkα1-/-2-/- mouse embryonic fibroblasts (MEFs) were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate, supplemented with 10% heat inactivated fetal bovine serum (Gibco), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, and 20 mM HEPES. WT 143B osteosarcoma cells were cultured as above, whereas the rho0 (ρ0)-143B cells, WT cybrids, and Δ Cytochrome b cybrids were supplemented with 100 μg/ml of Uridine (Sigma). ρ0 cells are generated by culturing cells in ethiduim bromide at low concentrations (25-50 ng/ml). WT and Δ Cytochrome b cybrids, provided by Carlos Moraes, were previously described [20]. Lkb1-/- cells, obtained from R. Depinho and N. Bardeesy, were retrovirally infected with an Lkb1 cDNA or vector control. These cells were selected in DMEM containing puromycin. All cells were cultured at 37 °C in 5% CO2 humidfied incubators for normoxia conditions. The following reagents were used: AICA-Riboside (AICAR) (Calbiochem), Hydrogen peroxide (Sigma), and EUK-134 (Eukarion, Inc.).

Oxygen conditions

Hypoxic conditions (1.5% O2 and 5% CO2 balanced with N2) were achieved in a humidified variable aerobic workstation (INVIVO O2, BioTrace), which contains an oxygen sensor that continuously monitors the chamber oxygen tension.

Immunoblot Analysis

Protein levels were analyzed in whole cell lysates obtained using lysis buffer (Cell Signaling) and 50 μg of samples were resolved on a SDS polyacrylamide gel. Gels were then analyzed by immunoblotting with the following antibodies: phospho-Acetyl CoA Carboxylase (Ser79), phospho-AMPKα (Thr172), Acetyl CoA Carboxylase (ACC), and AMPKα (Cell Signaling Technology, Inc.). A representative blot is shown of three independent experiments.

Measurement of AMP, ADP, and ATP

AMP, ADP, ATP measured using HPLC as previously described [21]. Cells were lysed in 300 μl of media with 20 μl of 1 M HClO4. HC104 was removed by mixed phase extraction employing 11.75:13.25 (vol/vol) of tri-n-octylamine and Freon 11. Lysates were run on a Zorbax Rx C8 column. The data presented are the results from three independent experiments and the error bars indicate the SEM.

Measurement of ROS

MEFs were infected with 100pfu adenovirus encoding a redox sensitive GFP targeted to the cytosol. Cells were harvested for analysis with the CyanADP Flow Cytometry Analyzer (Dako) 24 or 48 hours after being placed in conditions. The mean fluorescent channel for the ratio of violet excitable to blue excitable was determined with Summit v4.2 software (Dako). The percent oxidized probe is determined as the ratio of the sample mean subtracted from the mean from the probe reduced by 1 mM DTT to the mean from probe oxidized by 1 mM H2O2 subtracted from the mean from the probe reduced by 1 mM DTT. Intracellular ROS was also measured using Amplex Red (Molecular Probes) according to manufacturer’s protocol. Briefly, cells were lysed in Amplex Red solution (100 μM) supplemented with horseradish peroxidase (HRP, 2 mUnits/ml) and 200 mUnits/ml of superoxide dismutase (SOD, OXIS International) and incubated in the dark for 30 minutes. Fluorescence was measured in the Spectra Max Gemini plate reader with excitation of 540 nm and emission of 590 nm. The data presented are the result of four independent experiments and the error bars indicate the SEM.

Statistical Analysis

Data are presented as means ± standard error of mean. One-way analysis of variance was performed to determine the presence of significant differences in the data. When analysis of variance indicated that a significant difference was present, two-sample Student’s t-tests were performed to compare experimental data with data gathered at 21% O2. Statistical significance was determined at a value of P < 0.05.

Results and Discussion

In certain oxygen sensitive tissues, it has been proposed that hypoxia inhibits oxidative phosphorylation resulting in an increase in [AMP]/[ATP] ratio and AMPK activation [22]. However, in most tissues mitochondrial respiration is not limited at oxygen levels above 0.3% [23]. Thus, hypoxia in the range of 1-2% O2 does not inhibit mitochondrial respiration nor increase [AMP]/ [ATP] ratio. To assess the levels of ATP, ADP and AMP in wild-type mouse embryonic fibroblasts (MEFs) exposed to normoxia or hypoxia we utilized high-performance liquid chromatography (HPLC). Figs. 1A and C demonstrate that when cells are exposed to hypoxia (1.5%O2) for 10 or 60 minutes, there were no appreciable changes in AMP, ADP, or ATP levels. By contrast cells exposed to anoxia (0% O2) show a decrease in ATP levels. Furthermore, only cells exposed to anoxia show an increase in the [AMP]/ [ATP] ratio (Figs. 1B and D). These data are consistent with previous investigators who have demonstrated no changes in ATP,ADP and AMP in cells acutely exposed to hypoxia [14] To determine whether hypoxia activates AMPK, wild-type cells (Ampkα WT) and cells deficient in both AMPKα1 and α2 (Ampkα1-/-2-/-) were exposed to 21%O2 (0′) or 1.5%O2 for 30 minutes. Lysates were immunoblotted using an antibody that recognizes the phosphorylation site (Ser79) on acetyl-CoA carboxylase (ACC), a direct target of AMPK [2,24] (Fig. 2A). Hypoxia activates AMPK, as assessed by the phosphorylation of ACC, in Ampkα WT cells (Fig. 2A). H2O2 treatment also did not activate ACC in the Ampkα1-/-2-/- cells. These data demonstrate that hypoxia activates AMPK signaling independent of an increase in the [AMP]/ [ATP] ratio.

Fig. 1.

Fig. 1

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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.

Fig. 2

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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.

The tumor suppressor LKB1 has been identified as the predominant upstream kinase responsible for the phosphorylation of the critical Thr172 site on the α-subunit of AMPK. To determine if LKB1 is the upstream regulator of AMPK under hypoxic conditions, we used Lkb1-/- cells that we stably reconstituted with Lkb1 or with a vector control. The reintroduction of Lkb1 rescued AMPK activation under hypoxic conditions. In the Lkb1-/- cells reconstituted with the vector control, there was no detectable AMPK activation when exposed to hypoxia for 30 minutes (Fig. 2B). H2O2 can slightly activate AMPK in the absence of LKB1 most likely through CaMKK [25-27]. Thus, oxidative stress is likely to activate AMPK through LKB1 dependent and independent signaling pathways.

To determine whether ROS are required for the hypoxic activation of AMPK, Ampkα WT cells were exposed to 21%O2 (0′), 1.5%O2 for 30 minutes or one hour (30′ or 60′), or AICA-Riboside (AICAR) (A) in the absence or presence of EUK-134 (10 μM). AICAR mimics AMP, thereby acting as an activator of AMPK. EUK-134 is a synthetic superoxide dismutase/catalase mimetic, which scavenges both superoxide and H2O2. It has been widely used in mammalian models of inflammation and in longevity studies in C. elegans [28,29]. As demonstrated in Fig. 3A, EUK-134 prevents the hypoxic activation of AMPK, but has no effect on the AICAR-induced activation of AMPK. Indeed, as detected by a redox sensitive GFP probe, hypoxic generation of ROS can be scavenged by treatment with EUK-134 (Fig. 3B). The redox sensitive GFP probe contains GFP mutations with two oxidant sensitive surface-exposed cysteine residues placed at positions 147 and 204 (S147C and Q204C) [30]. These results were corroborated by using Amplex Red (Fig. 3C). Together these results illustrate that the hypoxic activation of AMPK is dependent on ROS.

Fig. 3.

Fig. 3

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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.

To test whether mitochondrial ROS (mtROS) are required for the hypoxic activation of AMPK we generated ρ0 143B cells. ρ0 cells are cells depleted of their mitochondrial DNA and are unable to carry out functional electron transport and generate mtROS [31]. WT 143B and ρ0 143B cells were subjected to 21%O2 (0′), 1.5%O2 (30′ or 60′), or to H2O2 (100 μM) for 15 minutes. In contrast to WT 143B cells, ρ0 143B cells fail to activate AMPK under hypoxia, as shown by the phosphorylation of ACC (Figs. 4A and B). As expected both cell types can activate AMPK in response to H2O2. The ρ0 143B cells also failed to increase intracellular hydrogen peroxide levels as determined by Amplex Red (Fig. 4C).

Fig. 4.

Fig. 4

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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.

To further explore the role of mtROS in the hypoxic activation of AMPK we used cells that are deficient in cytochrome b, a subunit of the mitochondrial complex III. These cells are cybrids that were generated by repopulating ρ0 143B cells with mitochondria that contain either wild-type (WT) mitochondria DNA or a 4-base pair deletion of the mitochondrial encoded cytochrome b gene (ΔCytochrome b) [20]. ΔCytochrome b cells are cytochrome b deficient cells that do not consume oxygen, similar to the ρ0 cells, yet can generate ROS at the Qo site of complex III [32]. Interestingly, hypoxia does activate AMPK in ΔCytochrome b cells (Fig. 5A). These data indicate that the ability of cells to conduct oxidative phosphorylation is not related to their ability to activate AMPK signaling. Furthermore, we show that the activation of AMPK can be inhibited in ΔCytochrome b cells by treatment with EUK-134 (Fig. 5B). The hypoxia-induced increase in intracellular hydrogen peroxide levels could be abrogated by EUK-134 in the ΔCytochrome b cells (Fig. 5C). Collectively, our data indicate that the ROS generated by complex III of the mitochondria is the stimulus for the activation of AMPK signaling and not the rise in AMP levels under hypoxic conditions, thereby identifying a novel signaling pathway for the activation of AMPK.

Fig. 5.

Fig. 5

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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.

Our results imply that AMPK can be activated by oxidative stress during hypoxia without an increase in [AMP]/ [ATP] ratio. Previous studies have indicated that H202 increases [AMP]/ [ATP] ratio and activates AMPK [33]. To test whether oxidative stress can activate AMPK independently of a change in [AMP]/ [ATP] ratio, we exposed WT and ρ0 143B cells to 20 uM H2O2, a smaller concentration than utilized in the literature. Both cell types robustly activated AMPK within 5 minutes (Fig. 6A and B). However, only the WT cells displayed an increase [AMP]/ [ATP] ratio after 5 minutes of 20 uM H2O2 (Fig. 6C). The ρ0 cells did not display an increase in [AMP]/ [ATP] ratio, implying that H2O2 increases [AMP]/ [ATP] ratio by inhibiting mitochondrial oxidative phosphorylation. These data further indicate that oxidative stress can activate AMPK without increasing the [AMP]/ [ATP] ratio. Mitochondrial oxidative stress is associated but not limited to aging, cancer and diabetes. Our study suggests that mitochondrial oxidative stress might activate AMPK without altering the [AMP]/ [ATP] ratio in these disease processes.

Fig. 6.

Fig. 6

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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.

These findings indicate that mitochondrial generated ROS can serve as signaling molecules to activate AMPK. We and others have previously shown that complex III generated ROS are required for hypoxic activation of HIF-1 [9-13]. However, AMPK activation during hypoxia is not required for HIF-1 [16]. The exact role of AMPK activation during hypoxia is not fully understood. Hypoxic activation of AMPK can suppress mTOR [19]. A consequence of mTOR inhibition is the induction of autophagy. Indeed, hypoxia-induced autophagy requires AMPK activation [34]. Thus, AMPK activation during hypoxia is likely an adaptive response to activate autophagy as an early signal to cells that likely will be deprived of nutrients. We suggest that as oxygen levels decrease the mitochondrial complex III acts as an oxygen sensor by releasing ROS into the intermembrane space. Subsequently, the ROS diffuse to the cytosol where they activate a variety of signaling pathways resulting in divergent biological responses such as activation of HIF-1 or AMPK.

Acknowledgments

This work is supported in part by National Institutes of Health Grants (CA123067-03) to NSC. BME was supported by a fellowship from the American Heart Association Grant 0610044Z. We thank Carlos Moraes and I.F.M. de Coo for the cytochrome b mutant cells. We thank Dr. Ronald Depinho and Dr. Nabeel Bardeesy for the Lkb1-/- MEFs.

Abbreviations

AICAR

AICA-Riboside

ROS

Reactive Oxygen Species

References

  • [1].Rutter GA, Da Silva Xavier G, Leclerc I. Roles of 5′-AMP-activated protein kinase (AMPK) in mammalian glucose homoeostasis. Biochem. J. 2003;375:1–16. doi: 10.1042/BJ20030048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Hardie DG, Scott JW, Pan DA, Hudson ER. Management of cellular energy by the AMP-activated protein kinase system. FEBS letters. 2003;546:113–120. doi: 10.1016/s0014-5793(03)00560-x. [DOI] [PubMed] [Google Scholar]
  • [3].Carling D. The AMP-activated protein kinase cascade–a unifying system for energy control. Trends Biochem. Sci. 2004;29:18–24. doi: 10.1016/j.tibs.2003.11.005. [DOI] [PubMed] [Google Scholar]
  • [4].Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, Boudeau J, Hawley SA, Udd L, Makela TP, Hardie DG, Alessi DR. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 2004;23:833–843. doi: 10.1038/sj.emboj.7600110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, Cantley LC. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl. Acad. Sci. U. S. A. 2004;101:3329–3335. doi: 10.1073/pnas.0308061100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Birnbaum MJ. Activating AMP-activated protein kinase without. AMP. Mol. Cell. 2005;19:289–290. doi: 10.1016/j.molcel.2005.07.012. [DOI] [PubMed] [Google Scholar]
  • [7].Hue L, Beauloye C, Bertrand L, Horman S, Krause U, Marsin AS, Meisse D, Vertommen D, Rider MH. New targets of AMP-activated protein kinase. Biochem. Soc. Trans. 2003;31:213–215. doi: 10.1042/bst0310213. [DOI] [PubMed] [Google Scholar]
  • [8].Leff T. AMP-activated protein kinase regulates gene expression by direct phosphorylation of nuclear proteins. Biochem. Soc. Trans. 2003;31:224–227. doi: 10.1042/bst0310224. [DOI] [PubMed] [Google Scholar]
  • [9].Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA, Rodriguez AM, Schumacker PT. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J. Biol. Chem. 2000;275:25130–25138. doi: 10.1074/jbc.M001914200. [DOI] [PubMed] [Google Scholar]
  • [10].Brunelle JK, Bell EL, Quesada NM, Vercauteren K, Tiranti V, Zeviani M, Scarpulla RC, Chandel NS. Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. Cell Metab. 2005;1:409–414. doi: 10.1016/j.cmet.2005.05.002. [DOI] [PubMed] [Google Scholar]
  • [11].Guzy RD, Hoyos B, Robin E, Chen H, Liu L, Mansfield KD, Simon MC, Hammerling U, Schumacker PT. Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing. Cell Metab. 2005;1:401–408. doi: 10.1016/j.cmet.2005.05.001. [DOI] [PubMed] [Google Scholar]
  • [12].Mansfield KD, Guzy RD, Pan Y, Young RM, Cash TP, Schumacker PT, Simon MC. Mitochondrial dysfunction resulting from loss of cytochrome c impairs cellular oxygen sensing and hypoxic HIF-alpha activation. Cell Metab. 2005;1:393–399. doi: 10.1016/j.cmet.2005.05.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Schroedl C, McClintock DS, Budinger GR, Chandel NS. Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species. Am. J. Physiol. Lung Cell Mol. Physiol. 2002;283:L922–931. doi: 10.1152/ajplung.00014.2002. [DOI] [PubMed] [Google Scholar]
  • [14].Quintero M, Colombo SL, Godfrey A, Moncada S. Mitochondria as signaling organelles in the vascular endothelium. Proc. Natl. Acad. Sci. U. S. A. 2006;103:5379–5384. doi: 10.1073/pnas.0601026103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Nagata D, Mogi M, Walsh K. AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress. J. Biol. Chem. 2003;278:31000–31006. doi: 10.1074/jbc.M300643200. [DOI] [PubMed] [Google Scholar]
  • [16].Laderoute KR, Amin K, Calaoagan JM, Knapp M, Le T, Orduna J, Foretz M, Viollet B. 5′-AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments. Mol. Cell. Biol. 2006;26:5336–5347. doi: 10.1128/MCB.00166-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Jung SN, Yang WK, Kim J, Kim HS, Kim EJ, Yun H, Park H, Kim SS, Choe W, Kang I, Ha J. Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells. Carcinogenesis. 2008;29:713–721. doi: 10.1093/carcin/bgn032. [DOI] [PubMed] [Google Scholar]
  • [18].Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E, Witters LA, Ellisen LW, Kaelin WG., Jr. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004;18:2893–2904. doi: 10.1101/gad.1256804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Liu L, Cash TP, Jones RG, Keith B, Thompson CB, Simon MC. Hypoxia-induced energy stress regulates mRNA translation and cell growth. Mol. Cell. 2006;21:521–531. doi: 10.1016/j.molcel.2006.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Rana M, de Coo I, Diaz F, Smeets H, Moraes CT. An out-of-frame cytochrome b gene deletion from a patient with parkinsonism is associated with impaired complex III assembly and an increase in free radical production. Ann. Neurol. 2000;48:774–781. [PubMed] [Google Scholar]
  • [21].Budinger GR, Chandel N, Shao ZH, Li CQ, Melmed A, Becker LB, Schumacker PT. Cellular energy utilization and supply during hypoxia in embryonic cardiac myocytes. Am. J. Physiol. 1996;270:L44–53. doi: 10.1152/ajplung.1996.270.1.L44. [DOI] [PubMed] [Google Scholar]
  • [22].Evans AM, Mustard KJ, Wyatt CN, Peers C, Dipp M, Kumar P, Kinnear NP, Hardie DG. Does AMP-activated protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to calcium signaling in O2-sensing cells? J. Biol. Chem. 2005;280:41504–41511. doi: 10.1074/jbc.M510040200. [DOI] [PubMed] [Google Scholar]
  • [23].Jones DP. Intracellular diffusion gradients of O2 and ATP. Am. J. Physiol. 1986;250:C663–675. doi: 10.1152/ajpcell.1986.250.5.C663. [DOI] [PubMed] [Google Scholar]
  • [24].Ha J, Daniel S, Kong IS, Park CK, Tae HJ, Kim KH. Cloning of human acetyl-CoA carboxylase cDNA. European journal of biochemistry / FEBS. 1994;219:297–306. doi: 10.1111/j.1432-1033.1994.tb19941.x. [DOI] [PubMed] [Google Scholar]
  • [25].Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR, Carlson M, Carling D. Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2005;2:21–33. doi: 10.1016/j.cmet.2005.06.005. [DOI] [PubMed] [Google Scholar]
  • [26].Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, Hardie DG. Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab. 2005;2:9–19. doi: 10.1016/j.cmet.2005.05.009. [DOI] [PubMed] [Google Scholar]
  • [27].Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, Witters LA. The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J. Biol. Chem. 2005;280:29060–29066. doi: 10.1074/jbc.M503824200. [DOI] [PubMed] [Google Scholar]
  • [28].Anderson I, Adinolfi C, Doctrow S, Huffman K, Joy KA, Malfroy B, Soden P, Rupniak HT, Barnes JC. Oxidative signalling and inflammatory pathways in Alzheimer’s disease. Biochem. Soc. Symp. 2001:141–149. doi: 10.1042/bss0670141. [DOI] [PubMed] [Google Scholar]
  • [29].Gill MS, Olsen A, Sampayo JN, Lithgow GJ. An automated high-throughput assay for survival of the nematode Caenorhabditis elegans. Free Radic. Res. Med. 2003;35:558–565. doi: 10.1016/s0891-5849(03)00328-9. [DOI] [PubMed] [Google Scholar]
  • [30].Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J. Biol. Chem. 2004;279:22284–22293. doi: 10.1074/jbc.M312847200. [DOI] [PubMed] [Google Scholar]
  • [31].Chandel NS, Schumacker PT. Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms. FEBS lett. 1999;454:173–176. doi: 10.1016/s0014-5793(99)00783-8. [DOI] [PubMed] [Google Scholar]
  • [32].Bell EL, Klimova TA, Eisenbart J, Moraes CT, Murphy MP, Budinger GR, Chandel NS. The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production. J. Cell Biol. 2007;177:1029–1036. doi: 10.1083/jcb.200609074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Choi SL, Kim SJ, Lee KT, Kim J, Mu J, Birnbaum MJ, Kim S. Soo, Ha J. The regulation of AMP-activated protein kinase by H(2)O(2) Biochem. Biophys. Res. Commun. 2001;287:92–97. doi: 10.1006/bbrc.2001.5544. [DOI] [PubMed] [Google Scholar]
  • [34].Papandreou I, Lim AL, Laderoute K, Denko NC. Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3 L. Cell Death Differ. 2008 Oct;15(10):1572–1581. doi: 10.1038/cdd.2008.84. [DOI] [PubMed] [Google Scholar]

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