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Am J Physiol Endocrinol Metab 287: E166-E173, 2004. First published March 16, 2004; doi:10.1152/ajpendo.00487.2003
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Possible involvement of the {alpha}1 isoform of 5'AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle

Taro Toyoda,1 Tatsuya Hayashi,2 Licht Miyamoto,2 Shin Yonemitsu,2 Masako Nakano,2 Satsuki Tanaka,2 Ken Ebihara,2 Hiroaki Masuzaki,2 Kiminori Hosoda,2 Gen Inoue,2 Akira Otaka,3 Kenji Sato,4 Tohru Fushiki,1 and Kazuwa Nakao2

1Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502; 2Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto 606-8507; 3Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501; and 4Department of Food Sciences and Nutritional Health, Kyoto Prefectural University, Kyoto 606-8522, Japan

Submitted 29 October 2003 ; accepted in final form 9 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H2O2, AMPK{alpha}1 activity increased in a time- and dose-dependent manner, whereas AMPK{alpha}2 activity remained unchanged. The activation of AMPK{alpha}1 was associated with phosphorylation of AMPK Thr172, suggesting that an upstream kinase is involved in the activation process. H2O2-induced AMPK{alpha}1 activation was blocked in the presence of the antioxidant N-acetyl-L-cysteine (NAC), and H2O2 significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H2O2 did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H2O2 increased 3-O-methyl-D-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPK{alpha}1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPK{alpha}1-mediated mechanism.

contraction; epitrochlearis muscle; hydrogen peroxide; hypoxanthine; xanthine oxidase

THERE IS A SIGNIFICANT ELEVATION in the rate of glucose transport and metabolism in contracting skeletal muscle to meet the increased requirement for ATP production. Glucose transport in skeletal muscle occurs by facilitated diffusion with glucose transporter carrier proteins. In rodent and human muscles, the major isoform of the glucose transporter GLUT4 translocates from microvesicles to the sarcolemmal and transverse-tubular membranes in response to contraction, and it promotes glucose uptake into muscle cells (19).

Recent studies have shown that 5'AMP-activated protein kinase (AMPK) is an important signaling intermediary that leads to contraction-stimulated glucose transport in skeletal muscle (18, 33, 42). AMPK is a serine/threonine kinase consisting of a catalytic {alpha} subunit and two regulatory subunits, {beta} and {gamma}. There are two distinct {alpha} isoforms in mammals. AMPK{alpha}1 is widely expressed, whereas the {alpha}2 subunit isoform is expressed predominantly in liver, heart, and skeletal muscle (45). AMPK is activated in response to an increase in AMP concentration or in the AMP/ATP ratio by allosteric modification with AMP and phosphorylation by an AMP-responsive upstream kinase, AMPKK (6, 11, 16, 46). In fact, AMPK is activated in response to energy-depriving stresses in skeletal muscle that induce increased glucose transport, including contraction, hyperosmolarity, hypoxia, and chemical inhibition of oxidative phosphorylation (17).

Skeletal muscle cells continuously produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) (2, 40, 41). Contraction increases the production of oxidants, leading to a shift in the prooxidant-antioxidant balance toward oxidants, i.e., oxidative stress (1, 2, 10, 21, 37, 41). The mitochondrial electron transport system is considered the major intracellular source for the production of ROS (4, 5, 27, 36, 48, 50–52). There are other potential sources in skeletal muscle, including cytosolic NAD(P)H oxidase (3, 22), infiltrating phagocyte cells (24), and endothelial tissue containing xanthine oxidase (26, 28). Nitric oxide (NO), one of the RNS, is also produced in contracting skeletal muscle by NO synthase (23, 32).

One of the major oxidants, hydrogen peroxide (H2O2), stimulates glucose transport in skeletal muscle. Cartee and Holloszy (7) demonstrated that the rate of 3-O-methyl-D-glucose (3MG) uptake is markedly increased in rat epitrochlearis muscle after incubation with 0.3–3 mM H2O2 for 30 min. Recently, Higaki et al. (20) showed that sodium nitroprusside (SNP), one of the NO donors, stimulated 2-deoxyglucose uptake in rat extensor digitorum longus muscle with AMPK activation. In the present study, we hypothesized that oxidative stress increases glucose transport in skeletal muscle via an AMPK-activating mechanism. We have presented, for the first time, evidence suggesting that AMPK{alpha}1 is predominantly activated in response to acute oxidative stress and that it plays a role in enhancing glucose transport in skeletal muscle.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. Male Sprague-Dawley rats weighing 100 g were purchased from Clea Japan (Tokyo, Japan). Animals were housed in an animal room maintained at 23°C with a 12:12-h light-dark cycle and were fed standard laboratory diet (Certified Diet MF; Oriental Koubo, Tokyo, Japan) and water ad libitum. Rats were fasted overnight before the experiments and were randomly assigned to the experimental groups. All protocols for animal use and euthanasia were reviewed and approved by the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Japan.

Materials. H2O2 was purchased from Kanto Chemicals (Tokyo, Japan). Pyruvate was purchased from Nacalai Tesque (Kyoto, Japan). The SAMS peptide (HMRSAMSGLHLVKRR) was synthesized and purified. All radioactive materials, [{gamma}-32P]ATP, 3-O-[methyl-3H]-D-glucose, and [14C]mannitol, were obtained from New England Nuclear (NEN) Life Science Products (Boston, MA). P81 filter paper was obtained from Whatman International (Maidstone, England). Protein A-Sepharose CL-4B was from Amersham Biosciences (Uppsala, Sweden). All other reagents were of analytical grade and obtained from Sigma (St. Louis, MO), unless otherwise stated.

Antibodies. AMPK antibodies were raised in rabbit against isoform-specific peptides derived from the amino acid sequences of rat {alpha}1 (residues 339–358) or {alpha}2 (residues 490–514). Peptides were synthesized with a cysteine residue at the NH2 terminus, coupled to keyhole-limpet hemocyanin, and then used for immunization. Immunized sera were used as antibodies.

Muscle incubation. Rat epitrochlearis muscles were treated, as described previously with modifications (18). Rats were killed by cervical dislocation, and the muscles were rapidly removed. Both ends of each muscle were tied with sutures (silk 3-0, Natsume Seisakusho, Tokyo, Japan), and the muscles were mounted on an incubation apparatus with the resting tension set to 0.5 g. The buffers were continuously gassed with 95% O2-5% CO2 and maintained at 37°C. Muscles were preincubated in 7 ml of Krebs-Ringer bicarbonate buffer (KRB) (in mM: 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, and 24.6 NaHCO3) containing 2 mM pyruvate (KRBP) for 40 min. The muscles were then incubated for 60 min in KRBP in the absence or presence of 20 mM N-acetyl-L-cysteine (NAC), 3,000 U/ml catalase, or 0.1 mM NG-monomethyl-L-arginine (L-NMMA). For oxidant treatments, muscles were stimulated with H2O2 (0.1, 0.5, 1.0, or 3.0 mM) during the last 20 min of the incubation period or with 1 mM hypoxanthine and 0.05 U/ml xanthine oxidase (HXXO) during the last 40 min of the incubation period. For the contraction treatments, muscles were stimulated during the last 10 min of the incubation period (train rate = 1/min, train duration = 10 s, pulse rate = 100 pulses/s, duration = 0.1 ms, volts = 50 V). The muscles were then used for the measurement of glucose uptake (see 3MG transport) or immediately frozen in liquid nitrogen and subsequently analyzed for ATP, ADP, AMP, IMP, glutathione (GSH), oxidized glutathione (GSSG) (see Assays for metabolites), and isoform-specific AMPK activity, or used for Western blot analysis.

Western blotting and isoform-specific AMPK activity assay. Muscles were homogenized in ice-cold lysis buffer (1:40 wt/vol) containing 20 mM Tris·HCl (pH 7.4), 1% Triton X, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM sodium pyrophosphate, 2 mM dithiothreitol, 4 mg/l leupeptin, 50 mg/l trypsin inhibitor, 0.1 mM benzamidine, and 0.5 mM phenylmethylsulfonyl fluoride, and were centrifuged at 20,000 g for 40 min at 4°C. For Western blot analysis, denatured lysates (20 µg protein) were separated on either 10% polyacrylamide gel for phosphorylated AMPK or 7.5% gel for phosphorylated acetyl-CoA carboxylase (ACC). Proteins were then transferred to polyvinylidene difluoride membranes (PolyScreen; NEN Life Science Products) at 100 V for 1 h. Membranes were blocked with Block Ace (Yukijirushi Nyugyo, Sapporo, Japan) overnight at 4°C and were then incubated with phosphospecific antibodies directed against AMPK{alpha} Thr172 (Cell Signaling Technology, Beverly, MA) or against ACC Ser79 (Upstate Biotechnology, Lake Placid, NY). The membranes were then washed, reacted with anti-rabbit IgG coupled to peroxidase, and developed with enhanced chemiluminescence reagents according to the manufacturer's instructions (Amersham, Buckinghamshire, UK). The signal on the blot was detected and quantified with a Lumino-Image Analyzer LAS-1000 System (Fuji Photo Film, Tokyo, Japan). For the AMPK activity assay, the supernatants (100 µg of protein) were immunoprecipitated with isoform-specific antibodies directed against the {alpha}1 or {alpha}2 catalytic subunits of AMPK and protein A-Sepharose beads (18). Immunoprecipitates were washed twice in both lysis buffer and wash buffer (240 mM HEPES and 480 mM NaCl). Kinase reactions were performed in 40 mM HEPES (pH 7.0), 0.1 mM SAMS peptide, 0.2 mM AMP, 80 mM NaCl, 0.8 mM dithiothreitol, 5 mM MgCl2, and 0.2 mM ATP (2 µCi [{gamma}-32P]ATP) in a final volume of 40 µl for 20 min at 30°C. For determining the effect of AMP and IMP on AMPK activity, the reactions were performed in the absence or presence of 0.2 mM AMP and 0.2 mM IMP, respectively. At the end of the reaction, a 15-µl aliquot was removed and spotted onto Whatman P81 paper. The papers were washed six times in 1% phosphoric acid and once in acetone. 32P incorporation was quantitated with a scintillation counter, and kinase activity was expressed as fold increases relative to the basal samples.

Assays for metabolites. Frozen muscles were homogenized in 0.2 M HClO4 (3:80 wt/vol) containing 100 µM EDTA in an ethanol-dry ice bath and were centrifuged at 20,000 g for 10 min at –9°C. To determine GSH and GSSG concentrations, analysis with high-performance liquid chromatography (HPLC; EP-300, Eicom, Kyoto, Japan) equipped with a fluorescence HPLC monitor (EPC-300, Eicom) and postcolumn derivatization with ortho-phthalaldehyde (OPA) was performed, according to the method of Lenton et al. (25) with some modifications. The supernatant was applied to an Eicompak SC-5 ODS (3 x 150 mm; Eicom) equilibrated with 50 mM phosphate buffer containing 100 mg/l octanesulfonic acid sodium salt (pH 3.5) at 0.3 ml/min. The eluate and postcolumn reaction medium, which consisted of 2.5 M NaOH with 0.05% OPA dissolved initially in 1 ml ethanol, were combined in a 5:4 ratio (final pH 12) with a T junction immediately before passage through a 0.25 x 3,000-mm reaction loop maintained at 38°C. After passage through the loop, the reaction was stopped with 2 M H3PO4. The fluorescence was monitored at an excitation wavelength of 340 nm and an emission wavelength of 425 nm. To determine the concentration of ATP and its degradation products, the supernatant of the homogenate was neutralized with a solution of 2 N KOH and 0.4 M imidazole and then centrifuged at 20,000 g for 10 min at –9°C. The supernatant was filtered through a 0.45-µm-pore Cosmonice filter W (Nacalai Tesque, Kyoto, Japan) and then analyzed by HPLC (DX300, Dionex, Sunnyvale, CA) equipped with an SPD-10Ai detector (Shimadzu, Kyoto, Japan) and an AS-8020 autoinjector (Tosho, Tokyo, Japan). The filtrate was applied to a Shodex Asahipack GS-320 HQ (7.6 x 300 mm; Showa Denko, Tokyo, Japan) and equilibrated with 200 mM sodium phosphate buffer (pH 3.0) at 1 ml/min. Elution was monitored at 254 nm. To determine the concentration of phosphocreatine (PCr), the supernatant was analyzed enzymatically (17).

3MG transport. To assay 3MG transport, muscles were transferred to 2 ml KRB containing 1 mM 3-O-[methyl-3H]-D-glucose (1.5 µCi/ml) and 7 mM D-[14C]mannitol (0.3 µCi/ml; NEN) at 30°C and further incubated for 10 min (18). The muscles were then blotted onto filter paper, trimmed, frozen in liquid nitrogen, and stored at –80°C. Frozen muscles were weighed and processed by incubating them in 300 µl of 1 M NaOH at 80°C for 10 min. Digestates were neutralized with 300 µl of 1 M HCl, and particulates were precipitated by centrifugation at 20,000 g for 2 min. Radioactivity in aliquots of the digested protein was determined by liquid scintillation counting for dual labels, and the extracellular and intracellular spaces were calculated.

Statistical analysis. Results are presented as means ± SE. Means were compared by one-way analysis of variance (ANOVA) followed by post hoc comparison with Fisher's protected least significant difference method.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
H2O2 increased phosphorylation of AMPK Thr172 and ACC Ser79. The primary site responsible for AMPK activation is the Thr172 residue in both the {alpha}1 and {alpha}2 catalytic subunits (46). To determine whether oxidative stress increases AMPK activity in skeletal muscle, we measured the degree of phosphorylation of Thr172 with a phosphospecific antibody in homogenates from epitrochlearis muscles incubated in the absence or presence of 3 mM H2O2. H2O2 treatment markedly increased phosphorylation of Thr172 and ACC Ser79 (Fig. 1A). ACC is a downstream target of AMPK, and phosphorylation of Ser79 reflects the total AMPK activity that can be modified by allosteric or covalent mechanisms (12, 38).



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  		Fig. 1. H2O2 acutely activates 5'AMP-activated protein kinase (AMPK){alpha}1 in rat skeletal muscle. Isolated rat epitrochlearis muscles were stimulated with H2O2 (3 mM, 20 min), and cell lysates were subjected to Western blot analysis by use of anti-phosphorylated-AMPK and anti-phosphorylated-acetyl-CoA carboxylase (ACC) antibodies (A). Isoform-specific AMPK activity was determined in anti-AMPK{alpha}1 and -{alpha}2 immunoprecipitates (B). To study muscle contractions, isolated epitrochlearis muscles were electrically stimulated to contract 10 times for 10 s each min. Values are means ± SE; n = 9–17/group. **P < 0.01 vs. basal.

 
H2O2 activated AMPK{alpha}1 in a time- and dose-dependent manner. To identify which catalytic subunit is activated by H2O2, AMPK activity was measured in anti-{alpha}1 and anti-{alpha}2 immunoprecipitates from epitrochlearis muscles after treatment with H2O2. H2O2 increased only AMPK{alpha}1 activity, whereas there was no effect on AMPK{alpha}2 (Fig. 1B). Muscle contraction in vitro increased the activity of both isoforms, as previously reported (17). To determine the dose and time dependency of isoform-specific AMPK activity, isolated epitrochlearis muscles were stimulated with H2O2 for various periods and at various concentrations. In response to 3 mM H2O2, AMPK{alpha}1 activity increased rapidly to 2.5-fold within 10 min and maintained maximal activity (~3-fold above basal) from 20 to 40 min (Fig. 2A). AMPK{alpha}1 was also activated in a dose-dependent manner, with maximal activity observed at 3 mM (Fig. 2B). In contrast to AMPK{alpha}1 activity, AMPK{alpha}2 activity did not change significantly (Fig. 2, A and B).



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  		Fig. 2. H2O2 increases AMPK{alpha}1 activity in a time- and dose-dependent manner in rat skeletal muscle. Isolated epitrochlearis muscles were treated with 3 mM H2O2 for the indicated periods (A) or at the indicated concentrations of H2O2 for 20 min (B). Isoform-specific AMPK activity was determined in anti-AMPK{alpha}1 and -{alpha}2 immunoprecipitates. Values are means ± SE; n = 4–15/group. **P < 0.01 vs. 0 min (A) or 0 mM (B) group.

 
Antioxidant NAC inhibited H2O2- and HXXO-induced AMPK{alpha}1 activation. To demonstrate that AMPK{alpha}1 activation is due to increased intracellular oxidative stress, we examined whether the antioxidant NAC (15) inhibited H2O2-induced AMPK{alpha}1 activation. Treatment with NAC fully inhibited {alpha}1 activation by 1 mM H2O2 and partially inhibited AMPK activity induced by 3 mM H2O2 (Fig. 3A). We also examined the effects of another ROS donor, the superoxide-generating HXXO system (34). Like H2O2 treatment, HXXO activated predominantly AMPK{alpha}1, and the effect was abolished in the presence of NAC (Fig. 3B).



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  		Fig. 3. Antioxidant N-acetyl-L-cysteine (NAC) inhibits H2O2- and hypoxanthine + xanthine oxidase (HXXO)-stimulated AMPK{alpha}1 activity. Isolated rat epitrochlearis muscles were preincubated in the presence or absence of 20 mM NAC and then treated with H2O2 (1 or 3 mM, 20 min; A) or HXXO (1 mM hypoxanthine + 0.05 U/ml xanthine oxidase, 40 min; B). Isoform-specific AMPK activity was determined in anti-AMPK{alpha}1 and -{alpha}2 immunoprecipitates. Values are means ± SE; n = 5–19/group. **P < 0.01.

 
Nucleotide and PCr levels in H2O2- and HXXO-treated muscles. Neither H2O2 nor HXXO treatments increased the conventional parameters associated with increased AMPK activity, including AMP levels and the AMP-to-ATP ratio (AMP/ATP), whereas contraction in vitro markedly increased these parameters (Table 1). Interestingly, as during muscle contraction, ATP levels were decreased in the H2O2- and HXXO-treated groups without any change in ADP concentration, resulting in a decrease in total adenine nucleotides (ATP + ADP + AMP) (Table 1). Correspondingly, IMP levels were increased in response to H2O2, HXXO, and muscle contractions (Table 1). PCr, which has an inhibitory effect on AMPK activity (39), did not change in the H2O2-treated muscles and slightly decreased in the HXXO-treated muscles (Table 1).


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  		Table 1. Nucleotide and PCr levels in epitrochlearis muscles treated with H2O2 and HXXO

 
Both AMPK{alpha}1 and AMPK{alpha}2 were allosterically activated by AMP but not by IMP. To determine whether IMP is an allosteric activator of AMPK, we measured the kinase activity in the absence and presence of 0.2 mM IMP in the reaction mixture. IMP activated neither AMPK{alpha}1 nor AMPK{alpha}2 that was immunoprecipitated from muscles treated with or without 3 mM H2O2. In contrast, AMP increased the kinase activity of both isoforms (Fig. 4), although AMPK{alpha}1 was activated to a lesser extent (a twofold increase) than AMPK{alpha}2 (a sixfold increase) (Fig. 4).



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  		Fig. 4. Both AMPK{alpha}1 and AMPK{alpha}2 are allosterically activated by AMP but not by IMP. Isolated rat epitrochlearis muscles were incubated in the presence or absence of 3 mM H2O2 for 20 min. Isoform-specific AMPK activity was determined in anti-AMPK{alpha}1 and -{alpha}2 immunoprecipitates with or without 0.2 mM IMP or 0.2 mM AMP. Values are means ± SE; n = 5/group. **P < 0.01 vs. corresponding control (–IMP, –AMP).

 
Redox status of H2O2- and HXXO-treated muscles. To determine whether exogenous ROS alter the intracellular redox status of rat epitrochlearis muscles, we measured the ratio of oxidized glutathione to glutathione concentrations (GSSG/GSH), a sensitive marker of oxidative stress (44). Both H2O2 and HXXO treatments increased GSSG/GSH, and the effect was blocked in the presence of NAC (Fig. 5). Catalase, another antioxidant, also blocked the increase in GSSG/GSH in H2O2-treated muscles. The GSSG/GSH values were 16.0 ± 4.9 x 10–3 in the basal state, 31.3 ± 7.2 x10–3 in H2O2-treated muscles, and 10.5 ± 3.1 x 10–3 in H2O2 + catalase-treated muscles (P < 0.01, H2O2 vs. H2O2 + catalase).



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  		Fig. 5. Antioxidant NAC inhibits H2O2- and HXXO-stimulated oxidative stress. Isolated rat epitrochlearis muscles were preincubated in the presence or absence of 20 mM NAC and then treated with H2O2 (1 mM, 20 min) or HXXO (1 mM hypoxanthine + 0.05 U/ml xanthine oxidase, 40 min), and intracellular levels of oxidized glutathione (GSSG) and glutathione (GSH) were determined by HPLC. Values are means ± SE; n = 7–15/group. **P < 0.01 vs. basal.

 
Antioxidant NAC partially inhibited H2O2- and HXXO-induced 3MG uptake. We next investigated whether oxidative stress affects glucose transport activity in skeletal muscle. Both 1 mM H2O2 and HXXO induced a threefold increase in 3MG transport (Fig. 6A). Although NAC fully inhibited AMPK activation by 1 mM H2O2 and HXXO, NAC only partially blocked H2O2- and HXXO-induced 3MG transport (Fig. 6A). The inhibitory effect of NAC was ~50% in both 1 mM H2O2 and HXXO groups (Fig. 6A). We also investigated whether NO accounts for the remaining glucose transport activity that was not blocked by NAC. However, addition of L-NMMA, a NOS inhibitor, to the incubation buffer did not reduce the remaining glucose transport activity (Fig. 6B).



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  		Fig. 6. Antioxidant NAC partially inhibits oxidative stress-induced 3-O-methyl-D-glucose (3MG) transport. Isolated rat epitrochlearis muscles were preincubated in the presence or absence of 20 mM NAC and then treated with H2O2 (1 mM, 20 min; A), or preincubated in the presence or absence of 20 mM NAC and of 0.1 mM NG-monomethyl-L-arginine (L-NMMA) and then treated with H2O2 (1 mM, 20 min; B), after which 3MG transport activity was determined. Values are means ± SE; n = 5–22/group. **P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
An important finding of our study is that AMPK activity increased in H2O2- and HXXO-treated muscles and that this activation was blocked in the presence of NAC (Fig. 3, A and B), suggesting that acute oxidative stress activates AMPK in skeletal muscle. Increased phosphorylation of ACC, an intracellular substrate of AMPK, also confirms an actual increase in kinase activity (12, 38). Moreover, a phosphospecific immunoblot of Thr172, an essential site for full kinase activity (46) (Fig. 1A), suggests covalent modification by upstream kinase(s). In support of our observations, previous studies have shown that H2O2 induces a large increase in AMPK activity in NIH-3T3 cells (9), and peroxynitrite increases the phosphorylation of ACC and AMPK in bovine aortic endothelial cells (BAEC) (58, 59). The RNS donor SNP also stimulates AMPK in rat skeletal muscle (20).

Other novel findings of this study are that the {alpha}1 isoform is predominantly activated in H2O2- and HXXO-treated muscles (Fig. 3, A and B) and that it is activated in the absence of an absolute increase in AMP or an increase in AMP relative to ATP (Table 1). Intracellular AMP and AMP/ATP are both important determinants of AMPK activity, and AMPK{alpha}2, rather than -{alpha}1, has greater AMP dependence in both the allosteric activation by AMP and the covalent activation by upstream kinase (43, 46). However, recent studies suggest that AMPK can be activated by a second mechanism that does not require changes in AMP or AMP/ATP (20, 31, 58). Peroxynitrite activates AMPK in BAEC without affecting cellular AMP/ATP (58), and SNP also activates AMPK{alpha}1 in rat skeletal muscle without depletion of ATP (20). In cultured muscle cells derived from heterozygous H-2Kb tsA58 transgenic mice, hyperosmotic stress and metformin activate AMPK with no increase in AMP/ATP (13). In the soleus muscle of FVB mice, activation of AMPK{alpha}2 by intravenous injection of leptin is not accompanied by changes in intracellular AMP, ADP, or ATP concentrations (31). Therefore, it is reasonable to infer that an AMP-independent mechanism is involved in oxidative stress-stimulated AMPK{alpha}1 activation in skeletal muscle.

Physiological concentrations of PCr inhibit purified AMPK obtained from rat liver (39), suggesting that PCr is a regulator of AMPK activity. In our study, AMPK{alpha}1 was significantly activated in response to H2O2 and HXXO, although PCr concentration did not decrease in response to H2O2 and only slightly decreased in response to HXXO (Table 1). This observation suggests that oxidative stress promotes activation of AMPK{alpha}1 via a mechanism that is independent of muscle PCr concentration. Consistently, SNP activates AMPK{alpha}1 despite no decrease in PCr concentration in rat skeletal muscle (20). Moreover, cycle ergometry exercise at 60–70% of maximal O2 uptake (O2 max) does not activate AMPK{alpha}1 despite a significant 25–70% decrease in PCr concentration in human vastus lateralis muscle (14, 47).

Recent studies of the activation of AMPK{alpha}1 have presented several lines of evidence suggesting that the regulation of the {alpha} isoforms is isoform specific. In INS-1 cells, the subcellular localization of AMPK{alpha}1 and -{alpha}2 differs, with {alpha}1 localized predominantly in the cytosol and {alpha}2 in both the cytosol and nucleus (43). In fact, an acute bout of exercise increased the nuclear AMPK{alpha}2 content in human skeletal muscle (29). Furthermore, the proportion of AMPK{alpha} isoforms is fiber-type specific; type 1 fibers express more {alpha}1 and less {alpha}2 than do type 2 fibers (54). In terms of tissue distribution, AMPK{alpha}1 is expressed ubiquitously, whereas AMPK{alpha}2 is expressed predominantly in skeletal muscle, heart, and liver (45). Two studies of rat liver have shown that AMPK{alpha}1 and -{alpha}2 have different substrate specificities (30, 56). Taken together, these data suggest that there is an oxidative stress-responsive mechanism leading to the specific activation of AMPK{alpha}1 in skeletal muscle.

We also found that increased AMPK{alpha}1 activity is accompanied by an increase in the rate of glucose transport in H2O2- or HXXO-treated skeletal muscle (Fig. 6A). Similarly, a close relationship between AMPK{alpha}1 and glucose transport has recently been documented in rat skeletal muscle stimulated with SNP (20) or metformin (57). On the other hand, H2O2- or HXXO-stimulated glucose transport activity was only partially inhibited by NAC (Fig. 6A), although NAC blocked AMPK{alpha}1 activation by oxidants (Fig. 3, A and B), suggesting that AMPK{alpha}1 is not fully responsible for oxidative stress-stimulated glucose transport. Our observation that addition of L-NMMA failed to further reduce glucose transport activity suggests that NOS is not involved in the remaining transport activity that is not inhibited by NAC (Fig. 6B). We also cannot exclude the possibility that mild oxidative stress that is not detectable by changes in GSSG/GSH is sufficient to stimulate glucose transport via a pathway independent of AMPK{alpha}1. Studies involving the specific inhibition of {alpha}1 activity, with use of genetic disruption or pharmacological manipulation, should provide more direct evidence.

In the present study, we identified slight but significant reductions in ATP and total adenine nucleotides and a corresponding increase in IMP in H2O2- or HXXO-treated muscles (Table 1). The major pathway for IMP production is de novo synthesis, including the deamination of AMP and the conversion of hypoxanthine to IMP (49). IMP can also be used for AMP resynthesis (IMP->adenylosuccinate->AMP) (49). Therefore, adenine nucleotide degradation or the inhibition of AMP resynthesis may be accelerated in oxidative-stressed muscles. It is noteworthy that a similar accumulation of IMP was observed in contracted skeletal muscles (Table 1). Considering the possible role of IMP, we found that neither AMPK{alpha}1 nor AMPK{alpha}2 activities were enhanced in the presence of IMP (Fig. 4), suggesting that, unlike AMP, IMP is not an allosteric effector for AMPK (Fig. 4). This finding is consistent with a previous report showing an absence of allosteric effect of IMP on mixtures of AMPK{alpha}1 and AMPK{alpha}2 (6).

On the basis of our findings, we propose the hypothesis that oxidative stress is an important stimulus of AMPK{alpha}1 activity and glucose transport in contracting skeletal muscle. Although there are two different isoforms of the {alpha} subunit in skeletal muscle, the {alpha}2 isoform seems to be activated preferentially and to be responsible for activating glucose transport during moderate-intensity exercise. Cycle ergometer exercise at 50% of O2 max does not alter {alpha}2 or {alpha}1 activity, but exercise at 60–75% of O2 max significantly increases {alpha}2 activity in human vastus lateralis muscles (14, 47, 55). In rat skeletal muscle, electrical stimulation of the sciatic nerve to produce periodic muscle contractions (53) and voluntary treadmill running exercise (35) significantly increase only {alpha}2 activity. In contrast, {alpha}1 (and also the {alpha}2 isoform) is significantly activated in response to very high-intensity exercise such as 30 s of "all-out" sprint exercise requiring power outputs two- to threefold greater than those attained at maximal aerobic exercise in humans (8). Similarly, high-intensity contractions, such as electrically induced tetanic contractions, increase the activities of {alpha}1 and {alpha}2 isoforms in isolated rat skeletal muscle (17). Because high-intensity exercise results in greater energy consumption and has a higher energy requirement than moderate-intensity exercise, the activation of AMPK{alpha}1 may play a role in boosting glucose transport in response to high-intensity exercise. However, future studies involving the complete inhibition of exercise-stimulated AMPK{alpha}1 and exercise-induced oxidative stress are required to test our hypothesis.

In summary, in the present study we have demonstrated that AMPK{alpha}1, but not AMPK{alpha}2, is activated in response to the acute oxidative stress that is induced by H2O2 and HXXO in isolated rat skeletal muscle, and that the inhibition of AMPK{alpha}1 activity by the antioxidant NAC was accompanied by a significant decrease in H2O2- and HXXO-stimulated glucose transport. We conclude that acute oxidative stress may stimulate glucose transport, at least in part, via AMPK{alpha}1-dependent mechanisms in skeletal muscle.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by Research Grant 15500441 from the Japanese Ministry of Education, Science, Sports and Culture (to T. Hayashi).


   ACKNOWLEDGMENTS
 
We are grateful to Kazuo Inoue, Yasuki Higaki, and Nobuharu Fujii for suggestions. We also thank Yoko Koyama for secretarial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Hayashi, Dept. of Medicine and Clinical Science, Kyoto Univ. Graduate School of Medicine, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan (E-mail: tatsuya{at}kuhp.kyoto-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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