Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase*

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H2O2 was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H2O2 to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H2O2 on kinase activity. Similar to the results obtained with H2O2-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H2O2 are increased. These results demonstrate that physiologically relevant concentrations of H2O2 can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.

and the ␤ and ␥ subunits have regulatory function. Formation of the ␣␤␥ complex is required for optimal allosteric activation of AMPK, which is induced by binding of AMP to the ␥ subunit (1)(2)(3)(4). In addition to activation by AMP, phosphorylation of the Thr 172 residue of the ␣ subunit enhances kinase activity (5,6). Recent studies have shown that the autoinhibitory domain (AID), located between amino acids 312 and 335 of the AMPK␣ subunit, is responsible for the lack of kinase activity under basal conditions (7)(8)(9), whereas AMP-induced conformational changes within the ␣␤␥ complex diminish function of the AID and lead to kinase activation.
The regulation of AMPK activity is primarily thought to result from alterations in the intracellular AMP/ATP ratio, arising from diminished ATP generation due to hypoxia, glucose deprivation, heat shock, or reduction in mitochondrial oxidative phosphorylation or from increased ATP consumption, such as occurs during strenuous exercise (2, 10 -12). Once activated, AMPK can phosphorylate and modulate the function of essential metabolic pathways participating in the regulation of glucose and lipid homeostasis (13)(14)(15). A major effect of AMPK activation is in preserving energy for use under conditions where ATP is limiting (4,16). AMPK activation appears to prevent or diminish inflammation-associated organ injury, including the development of atherosclerotic cardiovascular disease in diabetes (17), ischemia-induced cardiac dysfunction (18 -20), and hepatic dysfunction in animal models of nonalcoholic steatohepatitis as well as in humans with this condition (21,22). Our studies have also suggested that therapeutic approaches to increase AMPK activity diminish the severity of LPS-induced acute lung injury in mice (23,24).
Although increased formation of reactive oxygen species (ROS) is generally thought to be associated with pathophysiological situations leading to cellular injury and organ dysfunction, recent studies have shown beneficial effects of ROS in modulating inflammation, including TLR4-induced neutrophil activation and LPS-associated acute lung injury (24 -27). Several studies have demonstrated that increased intracellular concentrations of H 2 O 2 result in activation of AMPK and enhancement of AMPK-mediated cellular adaptation (28 -30), including maintenance of redox homeostasis (31,32). In cardiac preconditioning studies, antioxidants diminished H 2 O 2 -associated activation of AMPK and resulted in increased severity of ischemia-reperfusion-induced cardiac heart injury (33).
Despite the ability of increased intracellular concentrations of H 2 O 2 to induce AMPK activation in many cell types, the mechanism for this effect has not been well characterized. Whereas initial reports showed that H 2 O 2 -dependent activation of AMPK resulted from ATP depletion and increased AMP/ATP ratios (34), other studies demonstrated that increased intracellular concentrations of H 2 O 2 were associated with activation of AMPK before or without alteration in the ATP/AMP ratio (35)(36)(37).
H 2 O 2 can affect redox-sensitive signaling pathways as a result of oxidative modification of cysteine residues in proteins (38 -40). We therefore hypothesized that a potential mechanism by which increased intracellular concentrations of H 2 O 2 can activate AMPK is through oxidation of cysteines in one or more AMPK subunits. Our present experiments demonstrate that exposure to H 2 O 2 is associated with cysteine oxidation in the AMPK␣ subunit and is able to directly activate AMPK.

EXPERIMENTAL PROCEDURES
Mice-Male C57BL/6, C3HeB/FeJ, or acatalasemic C3Ga.Cg-Cat B/J mice, 8 -12 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, ME). The mice were kept on a 12 h/12 h light/dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols (University of Alabama at Birmingham Institutional Animal Care and Use Committee).
Acute Lung Injury Model-Acute lung injury was induced by intratracheal administration of 1 mg/kg LPS in 50 l of PBS as described previously (23)(24)(25)41). Briefly, mice were anesthetized with isoflurane and then suspended by their upper incisors on a 60°incline board. The tongue was gently extended, and LPS solution was deposited into the pharynx (24,42,43). Mice were pretreated with saline or ATZ (500 mg/kg body weight dissolved in 0.9% saline) i.p., and 4 h later LPS (1 mg/kg) was administered intratracheally. Lungs were harvested 24 h after LPS administration.
Culture of Human Embryonic Kidney Cells-HEK 293 cells were maintained at 37°C in 5% CO 2 in RPMI 1640 growth medium (Invitrogen) that contained 8% fetal bovine serum (Atlanta Biologicals; Norcross, GA), L-glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 ng/ml) (Sigma). Prior to use in experiments, the cells were washed twice and incubated with RPMI 1640 medium (FBS, 0.5%) for 1 h and then treated as described in the figure legends.
Measurement of AMPK Activity-AMPK activity was determined using a radiometric assay and SAMS peptide substrate, as described previously (44) with minor modifications. Briefly, recombinant AMPK␣␤␥ complex (25 ng/sample) or purified FLAG-AMPK␣ was incubated in kinase buffer (60 l/sample) (MOPS (5 mM), ␤-glycerol-phosphatase (2.5 mM), MgCl 2 (5 mM), 1 mM EGTA, and diethylene triamine pentaacetic acid (100 M)) and SAMS peptide (5 g/sample) for 10 min at room temperature. The phosphorylation of SAMS peptide was initiated by the inclusion of 0.2 l of [ 32 P]ATP and cold ATP (20 M) mix, and samples were incubated at 30°C. The reaction mix (2 l) was transferred to phosphocellulose P81 at the times indicated in the figure legends. Air-dried phosphocellulose P81 was washed three times (10 min each wash) in phosphoric acid (1%) solution. The phosphocellulose P81 was then subjected to autoradiography, and dot density was determined using Alpha-Innotech software (Santa Clara, CA).
Western Blot Analysis of AMPK Subunits-Lung homogenates or extracts from HEK 293 cells were prepared in lysis buffer (Tris, pH 7.4 (50 mM), NaCl (150 mM), Nonidet P-40 (0.5% v/v), EDTA (1 mM), EGTA (1 mM), Na 3 VO 4 (1 mM), NaF (50 mM), and protease inhibitors) and then sonicated and centrifuged at 10,000 ϫ g for 15 min at 4°C. The protein concentration in supernatants was determined using Bradford reagent (Bio-Rad) with BSA as a standard (24,45). Samples were mixed with Laemmli sample buffer and boiled for 5 min. Equal amounts of protein were resolved by 8% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Immobilon P, Millipore, Billerica, MA). The membranes were probed with specific antibodies to AMPK subunits, followed by detection with horseradish peroxidase-conjugated anti-mouse or goat anti-rabbit IgG. Bands were visualized by enhanced chemiluminescence (SuperSignal; Pierce). Each experiment was carried out two or more times using HEK 293 cells or with lung homogenates obtained from separate groups of mice.
Imaging of DCF Fluorescence-Intracellular levels of ROS, including H 2 O 2 , were measured using the redox-sensitive probe DCFH-DA (46) in conjunction with fluorescent microscopy (24,26,38,45,47,48). Briefly, HEK 293 cells (ϳ80% confluent) were incubated in a 4-well chambered coverglass (Nalge; Naperville, IL) with DCFH-DA (10 M) for 60 min, followed by treatment with H 2 O 2 (0 or 200 M) or glucose oxidase (10 milliunits/ml) at 37°C. Images were acquired at the indicated time periods (as described in the figure legends) by single bidirectional scans of live cells using a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT laser confocal optics. The pinhole setting was 0.2 Airy units, and laser excitation was set for 5% to avoid dye photo-oxidation. The levels of fluorescence were averaged using SimplePCI software (Compix, Cranberry Township, PA). Images were processed using IPLab Spectrum and Adobe Photoshop (Adobe Systems) software.
S-Glutathionylation of AMPK-Purified AMPK (100 ng) in kinase buffer containing diethylene triamine pentaacetic acid (100 M) was incubated with GSH-biotin (0 or 100 M) for 15 min, followed by exposure to H 2 O 2 (200 M) for an additional 5 min. Samples were then boiled in Laemmli sample buffer (without DTT) for 5 min, resolved in non-reducing SDS-PAGE, followed by Western blot analysis with streptavidin-HRP. Mem-branes were subsequently reprobed with antibodies specific for AMPK subunits.
Detection of GSS-AMPK Adduct Formation in HEK 293 Cells-HEK 293 cells (2 ϫ 10 6 /ml) were incubated with ethyl ester GSH-biotin (6 mM) for 1.5 h. The cells were then washed twice with culture buffer to remove the excess of GSH and treated with H 2 O 2 (0 or 300 M) for 15 or 30 min. Cell lyses were prepared in the presence of N-ethylmaleimide (5 mM) and then passed through Bio-Gel P10 to remove free GSH-biotin and N-ethylmaleimide. The level of GSS-protein conjugates was determined using non-reducing Western blot analysis with streptavidin-HRP, whereas GSS-AMPK subunit levels were measured after pull-down with streptavidin-agarose (60 min at 4°C), followed by reducing SDS-PAGE and Western blot analysis with antibodies to the AMPK␣, AMPK␤, or AMPK␥ subunits.
Labeling of AMPK Free Cysteine Thiols-The extent of free (unoxidized) cysteine residues within AMPK subunits was determined using the BIAM labeling assay (49 -52). Briefly, cell extracts or lung extracts (0.4 mg/sample) obtained from control, acatalasemic, or ATZ-treated mice were incubated with BIAM (200 M) for 30 min at room temperature, and then excess BIAM was removed by passing the extracts through Bio-Gel P10. Next, BIAM-protein conjugates were precipitated with streptavidin-agarose for 1 h at 4°C. Samples were washed four times with lysis buffer containing 0.5% SDS to obtain specific pull-down of biotinylated proteins and to avoid potential contamination with unlabeled proteins. BIAM-protein adducts were extracted from streptavidin agarose by boiling in Laemmli sample buffer for 10 min and then subjected to reducing SDS-PAGE and Western blot analysis with antibodies to the AMPK␣, AMPK␤, or AMPK␥ subunits.
Metal-catalyzed Oxidation of Purified AMPK-Human purified AMPK␣␤␥ complex (25 ng) was incubated with H 2 O 2 (0 or 100 M) in the presence or absence of Fe 2ϩ (100 M) or Cu 1ϩ (100 M) in kinase buffer (without diethylene triamine pentaacetic acid) for 10 min at 25°C, followed by measurement of AMPK activity over the next 30 min.
Autophosphorylation of AMPK-Human purified recombinant AMPK␣␤␥ complex (100 ng) was incubated in kinase buffer with H 2 O 2 (0, 100, or 200 M) for 10 min at room temperature, and then 0.2 l of [ 32 P]ATP and ATP (20 M) were added to the cultures for an additional 30 min at 37°C. Proteins were then subjected to SDS-PAGE and autoradiography.
Co-immunoprecipitation-Cells expressing FLAG-tagged AMPK␣ (WT) or AMPK␣ (amino acids 1-335) truncation were lysed in immunoprecipitation buffer (53) that preserves protein-protein interactions, followed by incubation of cell extracts with anti-FLAGM2 beads for 60 min at 4°C. Beads were washed with immunoprecipitation buffer four times. The amount of AMPK␤ subunit associated with AMPK␣ or AMPK␣ 1-335 was then determined by subsequent probing of the Western blot membrane with anti-FLAG and anti-AMPK␤ antibodies.

Construction of Expression Plasmids and Recombinant
Protein Expression-Full-length human AMPK␣ cDNA was purchased from Open Biosystems and cloned into 3XFLAG-CMV10 (Sigma) for mammalian expression. FLAG-tagged AMPK␣ and the C-terminal truncation mutants AMPK␣ 1-335 and AMPK␣ 1-312 were obtained by insertion of PCR products into 3XFLAG-CMV10. Mutation of cysteine 299, 304, or 312 to alanine within FLAG-tagged full-length AMPK␣ (WT) or truncated AMPK␣ (1-312 was performed using standard mutagenesis techniques. Transiently expressed FLAG-AMPK␣, WT, or truncation mutants were purified using anti-M2 FLAG-agarose beads as described previously (54) and then subjected to Western blot analysis with anti-FLAGM2 or anti-phospho-Thr 172 -AMPK antibodies. In parallel experiments, beads containing FLAG-AMPK␣ (e.g. WT or truncation mutants) were washed twice with kinase buffer, and AMPK activity was determined using a radiometric assay and SAMS peptide as a substrate.
Measurement of Cellular Nucleotides-The levels of ATP, ADP, and AMP in HEK 293 cells were determined by etheno derivatization and subsequent HPLC analysis of the resulting fluorescent species, as described previously (55,56).
Statistical Analyses-Experiments with purified AMPK or HEK 293 cells were each performed two or more times. Student's t test was used for comparisons between two groups, whereas Tukey's test was performed for comparisons between more than two groups, with p Ͻ 0.05 considered to be statistically significant. Mouse lung homogenates were obtained from two separate groups of control or acatalasemic mice or mice treated with ATZ (n ϭ 3 mice in each group).

Exposure of HEK 293 Cells to H 2 O 2 Results in Activation of
AMPK-ROS, including H 2 O 2 , have been shown to activate AMPK in macrophages, neutrophils, and other cell populations. As shown in Fig. 1, A and B, exposure of HEK 293 cells to H 2 O 2 resulted in activation of AMPK that was dose-and timedependent (Fig. 1, A and B). Activation of AMPK occurred rapidly after cellular exposure to H 2 O 2 , with increased levels of  Fig. 2A, exposure to H 2 O 2 resulted in an increase in the levels of GSS-protein adduct formation in HEK 293 cells.
We next determined whether H 2 O 2 exposure produced S-glutathionylation of AMPK subunits. In these experiments, GSS-protein conjugates were precipitated using streptavidin-agarose, followed by Western blot analysis of AMPK subunits. As shown in Fig. 2B, there were increased amounts of GSSphospho-Thr 172 -AMPK␣ and GSS-AMPK␤, but not of GSS-AMPK␥, in cells treated with H 2 O 2 . Similarly, increased oxidation of cysteine residues within the AMPK␣ and AMPK␤ subunits was detected when cell extracts were incubated with BIAM and H 2 O 2 . Under these conditions, a decrease in BIAM-protein adduct formation, such as was found for the AMPK␣ and AMPK␤ subunits after direct exposure to H 2 O 2 , indicated the presence of oxidized cysteine residues that were unable to react with BIAM (Fig. 2, C and D). These results show that activation of AMPK by H 2 O 2 is associated with enhanced oxidative modification of both the AMPK␣ and AMPK␤ subunits.
Activation of AMPK as Well as Cysteine Oxidation in the AMPK␣ Subunit Precede Decline in ATP levels in H 2 O 2 -exposed HEK 293 Cells-Although the above experiments demonstrated that cellular exposure to H 2 O 2 resulted in activation as well as oxidative modification of AMPK, those studies do not show that oxidative modification precedes activation of AMPK or is responsible for kinase activation. An alternate explanation is that activation of AMPK could have been produced by decreases in cellular levels of ATP and alteration in the ATP/ AMP ratio as a result of incubation of HEK 293 cells with H 2 O 2 , with oxidative modification of AMPK occurring as a subsequent and independent event. Indeed, we found that exposure of HEK 293 cells to H 2 O 2 (250 M) for 30 min resulted in about a 30 -40% decrease in intracellular levels of ATP. Because of the rapidity with which AMPK became activated after incubation of HEK 293 cells with H 2 O 2 , we measured AMPK activation and cellular levels of ATP in HEK 293 cells exposed to glucose oxidase (GO)-generated H 2 O 2 , a methodology that, in contrast to direct cellular exposure to H 2 O 2 , results in a more gradual increase in H 2 O 2 levels in the cell culture media as well as sustained elevations in intracellular steady state concentrations of H 2 O 2 (26,59). As shown in Fig. 3, inclusion of GO (10 milliunits/ml) in the cell cultures time-dependently induced DCF fluorescence and resulted in increased phosphorylation of AMPK␣ and ACC, as well as S-glutathionylation of both AMPK␣ and phospho-Thr 172 -AMPK␣. Importantly, activation and oxidation of the AMPK␣ subunit was present before any changes in intracellular concentrations of ADP and ATP or of the ATP/ADP ratio occurred (Fig. 3B). These results show that the stimulatory effects of H 2 O 2 on AMPK activity are not associated with diminished cellular ATP concentrations.
Assembly of the AMPK␣␤␥ complex can be potentially affected by exposure to increased concentrations of H 2 O 2 . However, incubation of cells with H 2 O 2 did not appear to modify the composition of AMPK␣␤␥ complexes (supplemental Fig. S1A).
Direct Exposure of AMPK to H 2 O 2 Increases Kinase Activity-In order to determine if H 2 O 2 could directly activate AMPK, we incubated recombinant AMPK␣␤␥ complex with H 2 O 2 and then determined kinase activity. As shown in Fig. 4, A and B, and supplemental Fig. S2, A and B, exposure of the AMPK␣␤␥ complex to H 2 O 2 dose-dependently increased AMPK activity, even in the presence of AMP. In additional experiments, we have found that H 2 O 2 -dependent activation of AMPK resulted in autophosphorylation of the AMPK␣ and AMPK␤ subunits (supplemental Fig. S2C). These results are consistent with previous studies showing that activated AMPK undergoes autophosphorylation, with the ␣ and ␤ subunits being affected (60). Cell extracts were subjected to pulldown with streptavidin-agarose, followed by Western blotting with antibodies specific for AMPK␣ or phospho-Thr 172 -AMPK (p-AMPK) (input, levels of AMPK or phospho-Thr 172 in cell extract prior to pull-down assay; Pull down, the amount of AMPK or phospho-Thr 172 -AMPK obtained after precipitation with streptavidin-agarose). Shown is the mean Ϯ S.D. obtained from two experiments. (Fig. 2), the increase in kinase activity produced by direct incubation of the AMPK␣␤␥ complex with H 2 O 2 was associated with oxidative modification of the ␣ and ␤ subunits, as shown by a decrease in BIAM adduct formation as well as increased S-glutathionylation of the AMPK␣ and AMPK␤ subunits (Fig. 4, C and D). These results indicate that H 2 O 2 can directly increase AMPK activity and that such activation is accompanied by oxidative modification of cysteine residues within the AMPK␣ and AMPK␤ subunits.

Consistent with results obtained from experiments that utilized cells cultured with H 2 O 2
Hydrogen Peroxide Can Activate AMPK␣ without Participation of ␤ or ␥ Subunits-To determine whether H 2 O 2 -induced oxidation of the AMPK␣ subunit induces kinase activation independently of the effects of H 2 O 2 on the other AMPK subunits, we transiently expressed AMPK␣ WT as well as AMPK with truncation of the C-terminal region (AMPK␣ 1-335 or AMPK␣ 1-312) in HEK 293 cells (Fig. 5). Deletion of the C-terminal region of the AMPK␣ subunit diminished complex for-mation between the ␣ and ␤ subunits (supplemental Fig. S1B), consistent with previous studies that have shown the importance of the ␤/␥ binding domain located within the amino acids 397-552 region of AMPK␣ (7,61). Exposure of HEK 293 cells to H 2 O 2 resulted in increased activity and Thr 172 phosphorylation of both wild type and truncated 1-335 or 1-312 AMPK␣ (Fig. 5E). Fig. 5, exposure of the AMPK␣ subunit to H 2 O 2 directly increases kinase activity even after elimination of the ␤/␥ binding domain or of the AID. In particular, incubation with H 2 O 2 still resulted in acti-   (Fig. 6). Mutation of Cys 299 had no effect on GSS-AMPK 1-312 adduct formation, suggesting that Cys 304 was a specific target of H 2 O 2 -induced S-glutathionylation. Next, we determined if mutation of Cys 299 and Cys 312 affected the phosphorylation of Thr 172 in AMPK␣. As shown in Fig. 7C, exposure of cells to H 2 O 2 produced similar levels of phosphorylation of AMPK 1-312 as compared with mutant AMPK 1-312 Cys 299/304 . This result suggests that oxidative modification of AMPK␣, subsequent to phosphorylation, is an essential step in kinase activation.

H 2 O 2 -dependent Oxidative Modification of Cysteine Thiols Results in Activation of AMPK␣-As shown in
In additional experiments, we found that mutation of Cys 299 and Cys 304 in full-length AMPK␣ (amino acids 1-552) also diminished the ability of H 2 O 2 but not 5-aminoimidazole-4carboxamide-1-␤-D-ribofuranoside, to induce kinase activity (Fig. 8).
AMPK Activity Is Increased in the Lungs of Acatalasemic Mice or Mice with Pharmacologically Induced Inhibition of Catalase-Previous studies have found that H 2 O 2 at physiologically relevant concentrations can produce activation of AMPK in vivo (33). Recently, we have shown that treatment of neutrophils with ATZ, an inhibitor of catalase, resulted in increased intracellular steady state levels of H 2 O 2 as well as diminished LPS-induced proinflammatory responses, including decreased nuclear translocation of NF-B and expression of proinflammatory cytokines (25). Given the above studies showing that direct exposure of AMPK to H 2 O 2 as well as incubation of HEK 293 cells with H 2 O 2 resulted in oxidation of the AMPK␣ subunit and enhanced kinase activity, we hypothesized that in vivo conditions that produce increased intracellular concentrations of H 2 O 2 would also be associated with oxidative modifications of the AMPK␣ subunit and increased kinase activity.
As shown in Fig. 9, administration of ATZ to mice resulted in increased levels in the lungs of phosphorylated Thr 172 -AMPK  and AMPK activation, as shown by increased levels of the phosphorylated form of ACC (phospho-Ser 79 -ACC), a downstream target of AMPK. Measurement of free cysteine thiols with BIAM labeling showed decreased amounts of BIAM-AMPK␣ and BIAM-AMPK␤ adduct formation in lung homogenates of mice given ATZ as compared with control mice treated with saline. Intratracheal LPS administration had little or no effect on AMPK phosphorylation in the lungs, whereas administration of ATZ before injection of LPS resulted in increased kinase activity. In a similar manner to acute inhibition of catalase by ATZ, increased phosphorylation and oxidation of AMPK were present in lung homogenates from acatalasemic mice (supplemental Fig. S4) compared with control mice with normal catalase function.

DISCUSSION
In this study, we demonstrated that H 2 O 2 can directly activate AMPK in vitro and in vivo through a mechanism associated with enhanced oxidative modification, including S-glutathionylation of cysteine residues, of the AMPK␣ subunit. Previous reports had shown that exposure of various cell populations to H 2 O 2 or to agents that produce increased intracellular concentrations of H 2 O 2 , such as by inhibiting mitochondrial electron transport, result in increased AMPK activity (29,35,37,62). The ability of H 2 O 2 to activate AMPK has been hypothesized to be indirect and to occur through decreasing intracellular levels of ATP and increasing the ratio of AMP to ATP, thereby enhancing binding of AMP to the AMPK␥ subunit with resultant allosteric activation of the AMPK␣ kinase domain (34). A recent study that utilized an AMP-insensitive mutant of the AMPK␥ subunit suggested that the stimulatory effects of H 2 O 2 on AMPK activation are mediated by a diminished ATP/ ADP ratio (63). However, in the present experiments, we found that exposure of cells to increased levels of H 2 O 2 activated and oxidized AMPK before any decrease in ATP levels or in the ATP/ADP ratio occurred. Moreover, we confirmed that H 2 O 2 could directly activate AMPK by demonstrating that incubation of the AMPK␣␤␥ complex with H 2 O 2 increased kinase activity. However, because exposure of HEK 293 and other cell populations to H 2 O 2 also results in diminished intracellular ATP levels and increased AMP/ATP ratios, in addition to increasing intracellular concentrations of H 2 O 2 , the ability of H 2 O 2 to directly activate AMPK does not necessarily imply that this is the major mechanism by which increased generation of H 2 O 2 produces activation of AMPK in vivo. Future experiments will be necessary to delineate the relative importance of direct oxidation of the AMPK␣ subunit as compared with alterations in AMP/ATP ratios in activating AMPK during pathophysiologic conditions, such as ischemia-reperfusion injury, that are associated with increased production of H 2 O 2 .
Although exposure to H 2 O 2 resulted in oxidative modification and S-glutathionylation of both the ␣ and ␤ subunits of AMPK, H 2 O 2 -induced modification of the AMPK␣ subunit alone was sufficient to increase kinase activity. Of note, exposure of truncated AMPK␣ (amino acids 1-335), which lacks the binding domain for interaction with the AMPK␤ and AMPK␥ subunits, to H 2 O 2 still increased AMPK activity, indicating that association with the ␤ and ␥ subunits was not necessary for AMPK activation by H 2 O 2 . Recent studies have shown that interaction between of the AID and ␣-helix C region of the AMPK␣ subunit is responsible for retention of the inactive "open" conformation of the AMPK␣ subunit within the AMPK␣␤␥ complex (7)(8)(9). However, exposure of AMPK␣ or AMPK␣ truncation mutants lacking the AID to H 2 O 2 resulted in enhanced AMPK kinase activity, showing that the AID is unlikely to play an important role in this effect. Mutation of cysteine 299 decreased and mutation of cysteine 304 totally blocked the activation of AMPK␣ by H 2 O 2 , suggesting that oxidative modification of these two cysteines plays an important role in the ability of H 2 O 2 to induce activation of AMPK. Although our results show that direct exposure to H 2 O 2 enhances the kinase activity of AMPK, enhanced phosphorylation of AMPK was also present under such conditions. In particular, incubation of the AMPK␣␤␥ complex with H 2 O 2 dosedependently increased phosphorylation of the AMPK␣ and AMPK␤ subunits. Such findings are consistent with previous studies that showed that activated AMPK undergoes autophosphorylation during activation (60).
Although H 2 O 2 is a relatively weak oxidant, extracellularly generated H 2 O 2 is capable of rapidly crossing cellular membranes to oxidize redox-sensitive cysteines of intracellular proteins and to modulate their activity in signaling pathways (38). The results of the present experiments, and particularly of those showing that direct exposure of the AMPK␣␤␥ complex or of the AMPK␣ subunit to H 2 O 2 increased kinase activity and diminished BIAM adduct formation, suggest that the mechanism by which H 2 O 2 induces such effects is through oxidative modification of vulnerable cysteine residues. The ability of H 2 O 2 to produce S-glutathionylation of the AMPK␣ and AMPK␤ subunits is consistent with this hypothesis. Although the present findings suggest that exposure to H 2 O 2 alone is sufficient to oxidatively modify and activate AMPK, it is possi-ble that other ROS, such as hydroxyl radical, derived from H 2 O 2 contribute to these effects. However, we found that metal-dependent generation of OH ⅐ in vitro diminished the activity of AMPK (supplemental Fig. S3). Such results suggest that H 2 O 2 itself, rather than derived strong oxidants, is responsible for activation of AMPK under pathophysiologic in vivo conditions associated with increased generation of ROS.
Similar to the effects of H 2 O 2 in cell cultures, we found that increased intracellular concentrations of H 2 O 2 in the lungs under in vivo conditions also were associated with AMPK activation (64). A role for H 2 O 2 in modulating AMPK activity in vivo was previously reported after cardiac ischemia, when increased levels of H 2 O 2 in the heart were accompanied by activation of AMPK and protection from a second ischemic event (1,(65)(66)(67)(68). Consistent with our experiments with purified AMPK and with HEK 293 cells, we found activation of AMPK and oxidative modification of the AMPK␣ subunit in the lungs of acatalasemic mice and in mice treated with the catalase inhibitor ATZ, conditions in which intracellular concentrations of H 2 O 2 are elevated (25).
Our experiments demonstrate a novel mechanism for AMPK activation that involves oxidative modification of the AMPK␣ subunit as a result of direct exposure to H 2 O 2 (Fig. 10). Previous studies have shown that activation of AMPK as well as increased intracellular concentrations of H 2 O 2 have potent anti-inflammatory properties, including diminished severity of LPS-induced acute lung injury (25). The present experiments suggest that at least one mechanism by which H 2 O 2 exerts its anti-inflammatory effects in vivo is through directly activating AMPK. Future studies will be necessary to determine if the primary mechanism for the anti-inflammatory effects of FIGURE 9. Mice with pharmacologic inhibition of catalase activity or acatalasemic mice have increased activation and oxidation of AMPK␣ subunit in the lungs. A and B, mice were given 50 l of saline or LPS (1 mg/kg) in 50 l of saline intratracheally and were treated with ATZ i.p. at 500 mg/kg. ATZ was administered 4 h before intratracheal saline or LPS administration. Lungs were harvested 24 h after treatment of the mice with LPS, and the levels of the AMPK␣ or AMPK␤ subunits, phospho-Thr 172 -AMPK␣, ACC, and phospho-Ser 79 -ACC (p-ACC), were determined using Western blot analysis. Representative Western blots are shown in A, whereas quantitative analysis of AMPK phosphorylation in lung homogenates is shown in B (mean Ϯ S.D. values were obtained from 3 mice/group; *, p Ͻ 0.05). C, lung homogenates obtained from control saline-treated mice or mice given LPS, ATZ, or ATZ and LPS were incubated with BIAM, and levels of AMPK-BIAM adduct formation were determined by Western blot analysis with streptavidin-HRP. The mean Ϯ S.D. is shown using results from two experiments. FIGURE 10. Putative mechanism of AMPK activation by H 2 O 2 . The AMPK␣␤␥ complex is in an "open" inactive state, whereas H 2 O 2 induces allosteric rearrangement to the active "closed" conformation as a result of H 2 O 2dependent oxidative modification of cysteine residues (-SOH), including S-glutathionylation (-SSG). Such oxidative modification, followed by dissociation of AID from ␣-helix C and activation of AMPK␣, can be achieved without binding of ␤/␥ subunit. In the heterotrimeric AMPK complex, oxidative modification of the ␣ and ␤ subunits can also facilitate AMP-dependent activation of the kinase domain.