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Am J Physiol Endocrinol Metab 290: E583-E590, 2006. First published October 25, 2005; doi:10.1152/ajpendo.00395.2005
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Low-intensity contraction activates the {alpha}1-isoform of 5'-AMP-activated protein kinase in rat skeletal muscle

Taro Toyoda,1 Satsuki Tanaka,2 Ken Ebihara,2 Hiroaki Masuzaki,2 Kiminori Hosoda,2 Kenji Sato,3 Tohru Fushiki,1 Kazuwa Nakao,2 and Tatsuya Hayashi4

1Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University; 2Department of Medicine and Clinical Science, Graduate School of Medicine, Kyoto University; 3Laboratory of Food Science, Department of Food Sciences and Nutritional Health, Kyoto Prefectural University; and 4Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Japan

Submitted 23 August 2005 ; accepted in final form 20 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Skeletal muscle expresses two catalytic subunits, {alpha}1 and {alpha}2, of the 5'-AMP-activated protein kinase (AMPK), which has been implicated in contraction-stimulated glucose transport and fatty acid oxidation. Muscle contraction activates the {alpha}2-containing AMPK complex (AMPK{alpha}2), but this activation may occur with or without activation of the {alpha}1-containing AMPK complex (AMPK{alpha}1), suggesting that AMPK{alpha}2 is the major isoform responsible for contraction-induced metabolic events in skeletal muscle. We report for the first time that AMPK{alpha}1, but not AMPK{alpha}2, can be activated in contracting skeletal muscle. Rat epitrochlearis muscles were isolated and incubated in Krebs-Ringer bicarbonate buffer containing pyruvate. In muscles stimulated to contract at a frequency of 1 and 2 Hz during the last 2 min of incubation, AMPK{alpha}1 activity increased twofold and AMPK{alpha}2 activity remained unchanged. Muscle stimulation did not change the muscle AMP concentration or the AMP-to-ATP ratio. AMPK activation was associated with increased phosphorylation of Thr172 of the {alpha}-subunit, the primary activation site. Muscle stimulation increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, and the rate of 3-O-methyl-D-glucose transport. In contrast, increasing the frequency (≥5 Hz) or duration (≥5 min) of contraction activated AMPK{alpha}1 and AMPK{alpha}2 and increased AMP concentration and the AMP/ATP ratio. These results suggest that 1) AMPK{alpha}1 is the predominant isoform activated by AMP-independent phosphorylation in low-intensity contracting muscle, 2) AMPK{alpha}2 is activated by an AMP-dependent mechanism in high-intensity contracting muscle, and 3) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle.

exercise; twitch; glucose transport; acetyl-coenzyme A carboxylase; beta-oxidation

EXERCISE TRIGGERS AN INCREASE in glucose and fatty acid utilization in contracting muscle. 5'-AMP-activated protein kinase (AMPK) is an important signaling intermediary in this increased use of substrates. AMPK activation leads to contraction-stimulated glucose transport in skeletal muscle (21, 34) and is thought to be involved in the regulation of fatty acid oxidation by phosphorylation of acetyl-CoA carboxylase (ACC) (49). Phosphorylation of AMPK by its upstream kinase AMPK kinase (AMPKK) increases the activity of AMPK (26, 47). AMPK activity also increases in response to an increase in AMP concentration or in the AMP-to-ATP ratio (1, 2, 9, 33). Binding of AMP directly activates AMPK through allosteric modification and makes AMPK a better substrate for AMPKK (33). AMPK is a heterotrimeric kinase, consisting of a catalytic {alpha}-subunit (1) and two regulatory subunits, beta and {gamma} (6). Two distinct {alpha}-isoforms ({alpha}1, {alpha}2) exist in mammals (42), and the {alpha}-isoform determines the enzyme characteristics, such as different conditions of activation in contracting muscle.

The {alpha}2-containing AMPK complex (AMPK{alpha}2) is considered the major AMPK isoform responsible for the metabolic changes in contracting skeletal muscle. A single bout of moderate-intensity exercise at ~70% of maximal O2 uptake (VO2max), which increases glucose transport and ACC phosphorylation, significantly activates AMPK{alpha}2, but not the {alpha}1-containing AMPK complex (AMPK{alpha}1), in human vastus lateralis muscle (12, 44, 50). In rat skeletal muscle, electrical stimulation (ES) of the sciatic nerve to produce periodic muscle contractions (46) and voluntary treadmill running exercise (35) increase only AMPK{alpha}2 activity, which is accompanied by increased glucose transport and ACC phosphorylation. Whole body AMPK{alpha}2 knockout mice exhibit impaired whole body insulin sensitivity and abolished stimulation of glucose transport into skeletal muscle induced by 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMPK activator (27). These results suggest that lower-intensity exercise or muscle contraction activates AMPK{alpha}2 more than AMPK{alpha}1 and that activation of AMPK{alpha}2 alone is sufficient to increase glucose transport and ACC phosphorylation. An acute bout of exercise increases the nuclear content of AMPK{alpha}2 but not of AMPK{alpha}1 (31). These findings indicate the importance of AMPK{alpha}2 in contraction-induced metabolic changes in skeletal muscle.

AMPK{alpha}1 has also been implicated in skeletal muscle metabolism. Earlier studies suggested that AMPK{alpha}1 activity increases only in response to high-intensity exercise, such as a 30-s all-out sprint exercise in humans (3). High-intensity contractions, such as electrically induced tetanic contractions, increase AMPK{alpha}1 activity in isolated rat skeletal muscle (35). In contrast, recent reports indicate that AMPK{alpha}1 activity also increases in response to moderate-intensity exercise (4, 5). One study reported a small but significant increase in AMPK{alpha}1 when exercise intensity increased from 40 to 60% of VO2max in a progressive incremental exercise protocol (4). Repeated higher-intensity exercise at 85% of VO2max also increases AMPK{alpha}1 activity in humans (5). These reports suggest that AMPK{alpha}1 can be activated by exercise at a lower intensity than previously thought necessary.

The pharmacological activation of AMPK{alpha}1 increases glucose transport and ACC phosphorylation, suggesting that AMPK{alpha}1 is also involved in glucose and fatty acid metabolism (22, 45). We (45) have previously shown that AMPK{alpha}1 is activated by hydrogen peroxide (H2O2) in isolated rat epitrochlearis muscle and that this is accompanied by increased glucose transport and ACC phosphorylation. Sodium nitroprusside (SNP) stimulates predominantly AMPK{alpha}1 activity and enhances glucose transport in isolated rat skeletal muscle (22). In whole body AMPK{alpha}1 knockout mice, contraction-induced glucose transport decreases slightly but significantly in the soleus muscle (27), a muscle that normally has an abundance of {alpha}1-subunit in wild-type animals (48). Moreover, the protein content of {alpha}1-subunit, but not {alpha}2-subunit, increases after 3–8 wk of endurance training in human (10, 29) and rat (38) skeletal muscle, and the protein content of {alpha}1-subunit is markedly higher in well-trained than in untrained individuals (36). These findings raise the possibility that AMPK{alpha}1 may play an important role in exercise-induced metabolic changes in skeletal muscle.

To our knowledge, no one has studied whether increased AMPK{alpha}1 activation increases glucose transport and ACC phosphorylation in wild-type skeletal muscle during contraction. In most previous studies, coactivation of AMPK{alpha}2 might have obscured the role of AMPK{alpha}1 in contracting skeletal muscle. The purpose of this study was to clarify the metabolic role of AMPK{alpha}1 in contracting skeletal muscle. We first determined the stimulation parameters that selectively increase the kinase activity of AMPK{alpha}1 but not AMPK{alpha}2. Because tetanic contractions stimulate both AMPK{alpha}1 and AMPK{alpha}2, even when the stimulation duration is for 10 s (35), we used "twitch" contractions to precisely manipulate the contraction intensity.


   MATERIALS AND METHODS
TOP
 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 fed a 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. Pyruvate was purchased from Nacalai Tesque (Kyoto, Japan). The SAMS peptide (HMRSAMSGLHLVKRR) was provided by A. Otaka (Graduate School of Pharmaceutical Sciences, Kyoto University) (45). 3-O-[methyl-3H]-D-glucose (3-MG) was purchased from American Radiolabeled Chemicals (St. Louis, MO). [{gamma}-32P]ATP and D-[1-14C]mannitol were obtained from NEN Life Science Products (Boston, MA). P81 filter paper was obtained from Whatman International (Maidstone, UK). 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) (45). Peptides used for immunization were provided by A. Otaka. Immunized sera were used as antibodies.

Muscle treatment. Rat epitrochlearis muscles were treated as described previously, with modifications (21, 45). Rats were killed by cervical dislocation, and the muscles were rapidly removed. When anesthetized, rats were treated with pentobarbital sodium (50 mg/kg body wt ip), and the muscles were isolated either with or without cervical dislocation. Isolated muscles were frozen immediately after isolation or incubated as follows. 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, 24.6 NaHCO3) containing 2 mM pyruvate (KRBP) for 40 min. The muscles were then incubated for 60 min in KRBP. For the twitch contraction treatments, muscles were stimulated during the last 0.5, 1, 2, 5, and 8 min of the incubation period at various frequencies (0.5–8 Hz) with 0.1-ms square-wave 50-V pulses. For the tetanic 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 3-MG transport), or immediately frozen in liquid nitrogen and subsequently analyzed for AMP, ATP (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 centrifuged at 20,000 g for 40 min at 4°C. For Western blot analysis, denatured lysates (10 µg of protein) were separated on either 7% polyacrylamide gel for phosphorylated AMPK or 5% gel for phosphorylated 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 (21, 45). Immunoprecipitates were washed twice both in lysis buffer and in wash buffer (240 mM HEPES and 480 mM NaCl). Kinase reactions were performed in (in mM) 40 HEPES (pH 7.0), 0.1 SAMS peptide, 0.2 AMP, 80 NaCl, 0.8 dithiothreitol, 5 MgCl2, 0.2 ATP (2 µCi of [{gamma}-32P]ATP), in a final volume of 40 µl for 20 min at 30°C. 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 (3:25 wt/vol) in an ethanol-dry ice bath and centrifuged at 20,000 g for 2 min at –9°C. 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 2 min at –9°C. The supernatant was filtered through a 0.45-µm pore Cosmonice filter W (Nacalai Tesque) 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 COSMOSIL 5PE-MS Packed Column (4.6 x 250 mm; Nacalai Tesque) equilibrated with 20 mM sodium phosphate containing 25 mM N,N-diethylethanolamine at 1 ml/min. Elution was monitored at 254 nm.

3-MG transport. To assay 3-MG transport, muscles were transferred to 2 ml of KRB containing 1 mM [3H]3-MG (1.5 µCi/ml) and 7 mM D-[1-14C]mannitol (0.3 µCi/ml) at 30°C and further incubated for 10 min (21). 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 followed by post hoc comparison with Dunnett's or Scheffé's test as appropriate. Unpaired t-tests were used for comparison as appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
AMPK{alpha}1 activity was elevated immediately after isolation and decreased during incubation. To allow the epitrochlearis muscles to recover from the isolating procedure, we first determined the incubation period that stabilizes AMPK activity, as measured in anti-{alpha}1 and anti-{alpha}2 immunoprecipitates from muscles that had been either frozen immediately after isolation or incubated in KRBP for 60, 100, or 120 min and then frozen. AMPK{alpha}1 kinase activity was lower in the incubated muscle than in muscle frozen immediately after isolation (Fig. 1A). AMPK{alpha}1 activity attained a constant level at 60 min of incubation and did not change afterwards, whereas AMPK{alpha}2 activity remained unchanged throughout the incubation period (Fig. 1A). We also investigated whether AMPK{alpha}1 activity differs between muscles frozen immediately after isolation under anesthesia and muscles incubated for 100 min. Regardless of whether cervical dislocation was or was not performed, AMPK{alpha}1 activity was higher in the muscles frozen immediately after isolation under anesthesia than in the incubated muscles; AMPK{alpha}2 activity did not differ between conditions (Fig. 1B).


Figure 1
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  		Fig. 1. Incubation decreases 5'-AMP-activated protein kinase (AMPK){alpha}1 activity to a constant level in isolated rat epitrochlearis muscles. A: rats were killed by cervical dislocation, and isolated muscles were either frozen immediately after isolation or incubated in Krebs-Ringer bicarbonate buffer containing 2 mM pyruvate (KRBP) for 60, 100, or 120 min and then frozen in liquid nitrogen. B: rats were randomly assigned to an anesthetized or a nonanesthetized group. In the anesthetized group, rats were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), and muscles were isolated with or without cervical dislocation. Isolated muscles were frozen immediately after isolation. In the nonanesthetized group, rats were killed by cervical dislocation, and isolated muscles were frozen immediately after isolation or incubated in KRBP for 100 min and then frozen in liquid nitrogen. Isoform-specific AMPK activity was determined in the anti-{alpha}1 or anti-{alpha}2 subunit of AMPK immunoprecipitates. Fold increases are expressed relative to the activity of muscles incubated for 100 min. Values are means ± SE; n = 6 per group. **P < 0.01 vs. 100-min incubation group.

 
ES increased AMPK{alpha}1activity in a time- and frequency-dependent manner. Tetanic contraction is a standard procedure to stimulate AMPK activity in isolated skeletal muscle (21, 35). Even a single 10-s tetanic contraction is enough to activate both AMPK{alpha}1 and AMPK{alpha}2 (35). To investigate the time dependency of isoform-specific AMPK activity stimulated by ES at frequencies lower than the tetanic stimulus (100 Hz), isolated epitrochlearis muscles were incubated to stabilize AMPK activity and then stimulated at 1 Hz for various periods. ES at 1 Hz rapidly activated AMPK{alpha}1 twofold within 2 min and maintained maximal activity for 2–8 min (Fig. 2A). In contrast, AMPK{alpha}2 activation required ≥5 min of ES, and the 1.5-fold increase was smaller than that of AMPK{alpha}1 (P < 0.05; Fig. 2A). To determine whether this effect was dependent on stimulation frequency, isolated epitrochlearis muscles were stimulated for 2 min at various frequencies. ES at 1 and 2 Hz activated predominantly AMPK{alpha}1, whereas ES at 5 and 8 Hz activated both AMPK{alpha}1 and AMPK{alpha}2 (Fig. 2B). ES at 5 and 8 Hz activated AMPK{alpha}1 maximally and to the same degree as tetanic contraction did, about four times the basal level, whereas the activity of AMPK{alpha}2 was less than the maximal level achieved at 8 Hz (Fig. 2B).


Figure 2
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  		Fig. 2. Low-frequency electrical stimulation (ES) increases predominantly AMPK{alpha}1 activity in a time- and contraction frequency-dependent manner in rat epitrochlearis muscles. Isolated muscles were electrically stimulated (50 V) to contract at 1 Hz for the indicated periods (A) or at the frequencies indicated for 2 min (B). To tetanically contract the muscles, muscles were stimulated for 10 s/min, repeated 10 times. Isoform-specific AMPK activity was determined in anti-{alpha}1 or anti-{alpha}2 subunit of AMPK immunoprecipitates. Values are means ± SE; n = 9–29 per group. **P < 0.01 vs. the 0-min (A) or vs. the 0-Hz group (B).

 
The primary site responsible for AMPK activation is the Thr172 residue in both the {alpha}1 and {alpha}2 catalytic subunits (17, 43). To determine whether the ES-induced activation at 1 and 2 Hz of predominantly AMPK{alpha}1 was accompanied by the phosphorylation of Thr172, we measured the degree of phosphorylation of Thr172 by use of a phosphospecific antibody in homogenates from muscles that had been stimulated by ES at the two frequencies. Compared with sham-operated muscles, muscles simulated with ES at 1 and 2 Hz exhibited markedly increased phosphorylation of Thr172 (Fig. 3).


Figure 3
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  		Fig. 3. Low-frequency ES increases phosphorylation of Thr172 in the AMPK {alpha}-subunit. Isolated muscles were electrically stimulated (50 V) to contract at 1 and 2 Hz for 2 min, and cell lysates were subjected to Western blot analysis with an anti-phosphorylated AMPK antibody.

 
ES at 1 and 2 Hz changed neither AMP nor the AMP/ATP ratio. To determine whether ES-induced activation of AMPK{alpha}1 results from the conventional changes associated with increased AMPK activity (33), we measured AMP concentration and the AMP/ATP ratio after stimulation. ES at 1 and 2 Hz, which activated AMPK{alpha}1 but not AMPK{alpha}2, did not increase AMP concentration or the AMP/ATP ratio (Fig. 4, A and B). In contrast, ES at 5 and 8 Hz, and tetanic stimulation, which activated both AMPK{alpha}1 and AMPK{alpha}2, markedly increased AMP concentration and the AMP/ATP ratio (Fig. 4, A and B).


Figure 4
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  		Fig. 4. Adenine nucleotide levels in epitrochlearis muscles treated with low-frequency ES. Isolated muscles were electrically stimulated (50 V) to contract for 2 min at the frequencies indicated. To tetanically contract the muscles, muscles were stimulated for 10 s/min, repeated 10 times. Intracellular AMP concentration (A) and ATP concentration were determined by HPLC, and the AMP/ATP ratio (B) were calculated. Values are means ± SE; n = 8–9 per group. **P < 0.01 vs. basal values.

 
Predominant activation of AMPK{alpha}1 by ES at 1 and 2 Hz was associated with increased 3-MG transport and ACC phosphorylation. We next investigated whether ES at 1 and 2 Hz acutely affects glucose transport activity and the phosphorylation state of ACC, a downstream target of AMPK, in skeletal muscle. ES at 1 and 2 Hz increased the transport of the nonmetabolizable glucose analog 3-MG in a frequency-dependent manner (Fig. 5). We used a phosphospecific antibody that recognizes rat ACC1 phosphorylated at Ser79 to assess the specific phosphorylation of skeletal muscle ACC, because this site is equivalent to Ser79 of ACC1 (15, 37). ES at 1 and 2 Hz increased glucose transport and ACC phosphorylation (Fig. 6).


Figure 5
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  		Fig. 5. Electrical stimulation increases 3-O-methyl-D-glucose (3-MG) transport in rat epitrochlearis muscles. Isolated muscles were electrically stimulated (1 or 2 Hz at 50 V) to contract for 2 min. Values are means ± SE; n = 6–10 per group. **P < 0.01 vs. basal values.

 

Figure 6
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  		Fig. 6. Low-frequency ES increases phosphorylation of serine in acetyl-CoA carboxylase (ACC). Isolated muscles were electrically stimulated (50 V) to contract at 1 or 2 Hz for 2 min, and cell lysates were subjected to Western blot analysis with an anti-phosphorylated ACC antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our data show three novel findings relating to the isoform-specific AMPK profile in skeletal muscle. First, immediately after isolation, the activity of AMPK{alpha}1 increased greatly but the activity of AMPK{alpha}2 did not change (Fig. 1, A and B). Second, muscle contraction induced by ES at 1 and 2 Hz for 2 min activated AMPK{alpha}1 but not AMPK{alpha}2 (Fig. 2B), suggesting that AMPK{alpha}1 is more sensitive to low-intensity contraction than AMPK{alpha}2 is. Third, contraction-induced AMPK{alpha}1 activation was associated with increased glucose transport and ACC phosphorylation (Figs. 5 and 6).

We believe that the marked activation of AMPK{alpha}1 immediately after isolation is a post mortem artifact, on the basis of previous studies of liver AMPK (8, 14, 33) showing that, in liver dissected at ambient temperature, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, one of the downstream targets of AMPK, is highly phosphorylated and inactivated (8). In contrast, in liver dissected after cold clamping and homogenized within 10 s after dissection, most HMG-CoA reductase is in an unphosphorylated active form (8), indicating that liver AMPK remains inactive in vivo and is easily activated as a post mortem artifact. Moreover, in freshly isolated rat hepatocytes, AMPK activity increases and then decreases to the expected in vivo level during incubation in oxygenated medium for 60 min (33). Hardie and Carling (14) proposed that rapid cooling is required to preserve the in vivo activation state of AMPK and that incubation in oxygenated medium restores AMPK activity to in vivo levels. Consistent with this proposal, AMPK{alpha}1 activation is not observed in muscle after electrically stimulated in situ contraction followed by muscle isolation (40, 46). In contrast, AMPK{alpha}1 activation occurs in muscle after electrically stimulated in situ contraction followed by freeze clamping (24).

We have not identified the critical step of AMPK{alpha}1 activation induced by our isolation procedure. Ischemia limits oxygen supply and leads to reduced ATP generation and can activate AMPK (20), and stopping or reducing the blood or oxygen supply to muscles during isolation is one possible cause of AMPK{alpha}1 activation. However, hypoxic energy deprivation should activate both AMPK{alpha}1 and AMPK{alpha}2 (20). In addition, the time needed to freeze the muscle after cervical dislocation was about 30 s, which was about the same amount of time needed to freeze the tissue incubated in oxygenated buffer and then exposed to room air. Furthermore, the activation of AMPK{alpha}1 by muscle isolation under anesthesia without cervical dislocation (Fig. 1B) shows clearly that cervical dislocation is not a critical step in the activation of AMPK{alpha}1. The freezing procedure after incubation is nearly identical to that used after isolation. Thus changes in unidentified factors, such as nervous system input or a soluble factor, might cause the post mortem artifact and increase skeletal muscle AMPK{alpha}1 activation during the isolation procedures.

Our finding that low-intensity contraction of skeletal muscle activated mainly AMPK{alpha}1 differs from that of previous studies showing predominantly AMPK{alpha}2 activation (12, 44, 50). Our use of a different muscle preparation might explain these differences. We measured the AMPK activity of muscles that had been isolated, incubated in KRBP, and then stimulated to contract, whereas previous studies measured AMPK activity of muscles isolated after contraction (12, 44, 50). Because isolation can activate AMPK{alpha}1, the actual contraction-induced increase of AMPK{alpha}1 activity would not be apparent in the latter protocol. AMPK{alpha}1 could also be activated by isolation of both contracted and noncontracted muscles. Our incubation for more than 60 min decreased AMPK{alpha}1 activity to a constant level and allowed us to observe the activation of AMPK{alpha}1 in low-intensity stimulated contracted muscle.

The finding that AMPK{alpha}1 activation was not accompanied by an increase in AMP concentration and the AMP/ATP ratio (Fig. 4, A and B) suggests that AMPK{alpha}1 activation induced by low-intensity contraction is regulated by an AMP-independent mechanism. Although intracellular energy status is an important determinant of AMPK activity (1, 2, 9, 33, 39), recent studies have shown that AMPK is activated by phosphorylation in the absence of changes in the concentration of AMP or ATP or in ADP/ATP or AMP/ATP ratios (11, 22, 32, 45, 51). Our data (Fig. 4) and those of others (22, 45) are consistent in showing that the predominant activation of AMPK{alpha}1 is not accompanied by a decrease in intracellular energy status in skeletal muscle. SNP activates AMPK{alpha}1 in extensor digitorum longus muscle without depleting ATP (22), and H2O2 activates AMPK{alpha}1 in rat epitrochlearis muscle without increasing AMP concentration and the AMP/ATP ratio (45). We cannot exclude the possibility that AMPK{alpha}1 is activated by the increase in free AMP content, which comprises 0.11–0.50% of the total AMP content (13) and increases markedly in response to muscle contraction (7, 25). Importantly, however, a study using purified AMPK showed that AMPK{alpha}2 depends more on AMP activation by the upstream kinase than does AMPK{alpha}1 (41). In this context, the absence of AMPK{alpha}2 activation in our study also suggests that AMP does not accumulate in muscle and that AMPK{alpha}1 activation involves an AMP-independent mechanism (Fig. 2, A and B). Moreover, we detected parallel increases in AMP concentration and AMPK{alpha}2 activity in muscles stimulated by ES at ≥5 Hz (Figs. 2B and 4A).

Our finding that the predominant AMPK{alpha}1 activation is accompanied by phosphorylation of the {alpha}-subunit in Thr172 suggests that an upstream kinase is involved in AMPK{alpha}1 activation by low-intensity contracted muscle (Fig. 3). Two mammalian AMPKKs have been identified: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) (19, 23) and the LKB1 complex consisting of LKB1 and two regulatory proteins, called Ste20-related adaptor protein and mouse protein 25 (16). In vitro studies have demonstrated that CaMKK is activated by Ca2+ and calmodulin but not by AMP (19). In an LKB1-deficient cell line, AMPK can be activated by treatment with mannitol, 2-deoxyglucose, and the Ca2+ ionophore ionomycin, but not by the AMP analog AICAR (23). In rat brain slices, depolarization induced by increasing K+ concentration increases intracellular Ca2+ concentration by opening the voltage-gated Ca2+ channel and activates AMPK without elevating the AMP/ATP ratio; this activation is blocked by the CaMKK inhibitor STO-609 (18). AMP concentration did not increase in muscles stimulated by ES at 1 and 2 Hz (Fig. 4), suggesting that CaMKK might be responsible for the predominant activation of AMPK{alpha}1. In contrast, the LKB1 complex is constitutively active and is not directly activated by AMP, but the binding of AMP to AMPK facilitates the phosphorylation of AMPK by the LKB1 complex (16, 40). Thus the LKB1 complex may depend more on AMP to phosphorylate AMPK than does CaMKK. We cannot discount the possible involvement of yet-to-be-characterized AMPKKs, and further study is needed to clarify the AMPKK that activates predominantly AMPK{alpha}1 in low-intensity contracting skeletal muscle.

Our results show that ES at 1 and 2 Hz activates AMPK{alpha}1 and increases in the rate of glucose transport and ACC phosphorylation, indicating that AMPK{alpha}1, as well as AMPK{alpha}2, is involved in contraction-stimulated glucose transport and ACC phosphorylation. Our results are consistent with previous studies showing that low-intensity muscle contraction increases glucose transport and ACC phosphorylation (30, 37). ES at 1 and 2.5 Hz for 5 min increases glucose transport in isolated rat soleus muscles (30). ACC activity decreases after in situ contraction of rat gastrocnemius muscle stimulated by ES at 0.2 and 1 Hz for 5 min via the tibial nerve, and this decreased activity is inversely correlated with the concentration of phosphorylated ACC (37). Moreover, H2O2 activates increased glucose transport and ACC phosphorylation associated with AMPK{alpha}1 activation (45). SNP increases glucose transport associated with AMPK{alpha}1 activation (22). In support of our observations, contraction-induced glucose transport is not reduced by knockout of the {alpha}1- or {alpha}2-subunit of AMPK but is inhibited by dominant mutants of both isoforms (27, 34).

Our results lead us to hypothesize that 1) AMPK{alpha}1 regulation is more sensitive to physical or physiological stress than AMPK{alpha}2 is; 2) AMPK{alpha}1 is the predominant isoform activated by low-intensity contractions; 3) AMPK{alpha}1 activation induced by low-intensity contractions is regulated by an AMP-independent phosphorylation, whereas AMPK{alpha}2 activation induced by high-intensity contraction is regulated by AMP-dependent mechanism; and 4) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle. Previous data show that low-intensity exercise, even at a level (e.g., 30–40% of VO2max) previously believed not to activate AMPK, increases skeletal muscle glucose transport and ACC phosphorylation (4, 28), suggesting the involvement of an AMPK-independent pathway. Our results suggest that AMPK{alpha}1 may be activated in these muscles by low-intensity exercise and that measuring the activity may be disturbed by additional activation during isolation. Only during very-high-intensity exercise, when the activation by muscle contraction may exceed that of the isolating stimuli, would AMPK{alpha}1 activity be detectable.

In summary, we have demonstrated for the first time that muscle AMPK{alpha}1, but not AMPK{alpha}2, is activated immediately after isolation. Stabilizing muscle in KRBP followed by low-intensity contraction activates AMPK{alpha}1 via phosphorylation without increasing AMP concentration, and it increases glucose transport and ACC phosphorylation. We conclude that low-intensity muscle contraction activates AMPK{alpha}1 and leads to enhanced glucose transport and ACC phosphorylation in rat skeletal muscle.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by research grants from the Japanese Ministry of Education, Science, Sports, and Culture (to T. Hayashi). T. Toyoda was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.


   ACKNOWLEDGMENTS
 
We thank Akira Otaka for providing the purified SAMS peptide and isoform-specific antigen peptides. We are grateful to Kazuo Inoue and Teruo Kawada for suggestions, and to Chikako Wada for secretarial assistance. We also thank the Radioisotope Research Center, Kyoto University, for instrumental support in radioisotope experiments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Hayashi, Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-nihonmatsu-cho, Sakyo-ku, Kyoto, 606-8501, 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
 

  1. Carling D, Clarke PR, Zammit VA, and Hardie DG. Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities. Eur J Biochem 186: 129–136, 1989.[ISI][Medline]
  2. Carling D, Zammit VA, and Hardie DG. A common bicyclic protein kinase cascade inactivates the regulatory enzymes of fatty acid and cholesterol biosynthesis. FEBS Lett 223: 217–222, 1987.[CrossRef][ISI][Medline]
  3. Chen ZP, McConell GK, Michell BJ, Snow RJ, Canny BJ, and Kemp BE. AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation. Am J Physiol Endocrinol Metab 279: E1202–E1206, 2000.[Abstract/Free Full Text]
  4. Chen ZP, Stephens TJ, Murthy S, Canny BJ, Hargreaves M, Witters LA, Kemp BE, and McConell GK. Effect of exercise intensity on skeletal muscle AMPK signaling in humans. Diabetes 52: 2205–2212, 2003.[Abstract/Free Full Text]
  5. Clark SA, Chen ZP, Murphy KT, Aughey RJ, McKenna MJ, Kemp BE, and Hawley JA. Intensified exercise training does not alter AMPK signaling in human skeletal muscle. Am J Physiol Endocrinol Metab 286: E737–E743, 2004.[Abstract/Free Full Text]
  6. Davies SP, Hawley SA, Woods A, Carling D, Haystead TA, and Hardie DG. Purification of the AMP-activated protein kinase on ATP-gamma-sepharose and analysis of its subunit structure. Eur J Biochem 223: 351–357, 1994.[ISI][Medline]
  7. Dudley GA and Terjung RL. Influence of acidosis on AMP deaminase activity in contracting fast-twitch muscle. Am J Physiol Cell Physiol 248: C43–C50, 1985.[Abstract/Free Full Text]
  8. Easom RA and Zammit VA. A cold-clamping technique for the rapid sampling of rat liver for studies on enzymes in separate cell fractions. Suitability for the study of enzymes regulated by reversible phosphorylation-dephosphorylation. Biochem J 220: 733–738, 1984.[ISI][Medline]
  9. Ferrer A, Caelles C, Massot N, and Hegardt FG. Activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by adenosine 5' -monophosphate. Biochem Biophys Res Commun 132: 497–504, 1985.[CrossRef][ISI][Medline]
  10. Frosig C, Jorgensen SB, Hardie DG, Richter EA, and Wojtaszewski JF. 5'-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle. Am J Physiol Endocrinol Metab 286: E411–E417, 2004.[Abstract/Free Full Text]
  11. Fryer LG, Parbu-Patel A, and Carling D. The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem 277: 25226–25232, 2002.[Abstract/Free Full Text]
  12. Fujii N, Hayashi T, Hirshman MF, Smith JT, Habinowski SA, Kaijser L, Mu J, Ljungqvist O, Birnbaum MJ, Witters LA, Thorell A, and Goodyear LJ. Exercise induces isoform-specific increase in 5' AMP-activated protein kinase activity in human skeletal muscle. Biochem Biophys Res Commun 273: 1150–1155, 2000.[CrossRef][ISI][Medline]
  13. Goodman MN and Lowenstein JM. The purine nucleotide cycle. Studies of ammonia production by skeletal muscle in situ and in perfused preparations. J Biol Chem 252: 5054–5060, 1977.[Abstract/Free Full Text]
  14. Hardie DG and Carling D. The AMP-activated protein kinase—fuel gauge of the mammalian cell? Eur J Biochem 246: 259–273, 1997.[ISI][Medline]
  15. Hardie DG and Pan DA. Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase. Biochem Soc Trans 30: 1064–1070, 2002.[CrossRef][ISI][Medline]
  16. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, Alessi DR, and Hardie DG. Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2: 28.1–28.16, 2003.
  17. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, and Hardie DG. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271: 27879–27887, 1996.[Abstract/Free Full Text]
  18. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM, Frenguelli BG, and Hardie DG. Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2: 9–19, 2005.[CrossRef][ISI][Medline]
  19. Hawley SA, Selbert MA, Goldstein EG, Edelman AM, Carling D, and Hardie DG. 5' -AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms. J Biol Chem 270: 27186–27191, 1995.[Abstract/Free Full Text]
  20. Hayashi T, Hirshman MF, Fujii N, Habinowski SA, Witters LA, and Goodyear LJ. Metabolic stress and altered glucose transport: activation of AMP-activated protein kinase as a unifying coupling mechanism. Diabetes 49: 527–531, 2000.[Abstract]
  21. Hayashi T, Hirshman MF, Kurth EJ, Winder WW, and Goodyear LJ. Evidence for 5' AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport. Diabetes 47: 1369–1373, 1998.[Abstract]
  22. Higaki Y, Hirshman MF, Fujii N, and Goodyear LJ. Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle. Diabetes 50: 241–247, 2001.[Abstract/Free Full Text]
  23. Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, and Witters LA. The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280: 29060–29066, 2005.[Abstract/Free Full Text]
  24. Hurst D, Taylor EB, Cline TD, Greenwood LJ, Compton CL, Lamb JD, and Winder WW. AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance-trained rats. Am J Physiol Endocrinol Metab 289: E710–E715, 2005.[Abstract/Free Full Text]
  25. Hutber CA, Hardie DG, and Winder WW. Electrical stimulation inactivates muscle acetyl-CoA carboxylase and increases AMP-activated protein kinase. Am J Physiol Endocrinol Metab 272: E262–E266, 1997.[Abstract/Free Full Text]
  26. Ingebritsen TS, Lee HS, Parker RA, and Gibson DM. Reversible modulation of the activities of both liver microsomal hydroxymethylglutaryl coenzyme A reductase and its inactivating enzyme. Evidence for regulation by phosphorylation-dephosphorylation. Biochem Biophys Res Commun 81: 1268–1277, 1978.[CrossRef][ISI][Medline]
  27. Jorgensen SB, Viollet B, Andreelli F, Frosig C, Birk JB, Schjerling P, Vaulont S, Richter EA, and Wojtaszewski JF. Knockout of the alpha2 but not alpha1 5' -AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside but not contraction-induced glucose uptake in skeletal muscle. J Biol Chem 279: 1070–1079, 2004.[Abstract/Free Full Text]
  28. Kemppainen J, Fujimoto T, Kalliokoski KK, Viljanen T, Nuutila P, and Knuuti J. Myocardial and skeletal muscle glucose uptake during exercise in humans. J Physiol 542: 403–412, 2002.[Abstract/Free Full Text]
  29. Langfort J, Viese M, Ploug T, and Dela F. Time course of GLUT4 and AMPK protein expression in human skeletal muscle during one month of physical training. Scand J Med Sci Sports 13: 169–174, 2003.[CrossRef][ISI][Medline]
  30. Lund S, Holman GD, Schmitz O, and Pedersen O. Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin. Proc Natl Acad Sci USA 92: 5817–5821, 1995.[Abstract/Free Full Text]
  31. McGee SL, Howlett KF, Starkie RL, Cameron-Smith D, Kemp BE, and Hargreaves M. Exercise increases nuclear AMPK alpha2 in human skeletal muscle. Diabetes 52: 926–928, 2003.[Abstract/Free Full Text]
  32. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, and Kahn BB. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415: 339–343, 2002.[CrossRef][Medline]
  33. Moore F, Weekes J, and Hardie DG. Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP-activated protein kinase. A sensitive mechanism to protect the cell against ATP depletion. Eur J Biochem 199: 691–697, 1991.[ISI][Medline]
  34. Mu J, Brozinick JT Jr, Valladares O, Bucan M, and Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 7: 1085–1094, 2001.[CrossRef][ISI][Medline]
  35. Musi N, Hayashi T, Fujii N, Hirshman MF, Witters LA, and Goodyear LJ. AMP-activated protein kinase activity and glucose uptake in rat skeletal muscle. Am J Physiol Endocrinol Metab 280: E677–E684, 2001.[Abstract/Free Full Text]
  36. Nielsen JN, Mustard KJ, Graham DA, Yu H, MacDonald CS, Pilegaard H, Goodyear LJ, Hardie DG, Richter EA, and Wojtaszewski JF. 5' -AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle. J Appl Physiol 94: 631–641, 2003.[Abstract/Free Full Text]
  37. Park SH, Gammon SR, Knippers JD, Paulsen SR, Rubink DS, and Winder WW. Phosphorylation-activity relationships of AMPK and acetyl-CoA carboxylase in muscle. J Appl Physiol 92: 2475–2482, 2002.[Abstract/Free Full Text]
  38. Pold R, Jensen LS, Jessen N, Buhl ES, Schmitz O, Flyvbjerg A, Fujii N, Goodyear LJ, Gotfredsen CF, Brand CL, and Lund S. Long-term AICAR administration and exercise prevents diabetes in ZDF rats. Diabetes 54: 928–934, 2005.[Abstract/Free Full Text]
  39. Ponticos M, Lu QL, Morgan JE, Hardie DG, Partridge TA, and Carling D. Dual regulation of the AMP-activated protein kinase provides a novel mechanism for the control of creatine kinase in skeletal muscle. EMBO J 17: 1688–1699, 1998.[CrossRef][ISI][Medline]
  40. Sakamoto K, Goransson O, Hardie DG, and Alessi DR. Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR. Am J Physiol Endocrinol Metab 287: E310–E317, 2004.[Abstract/Free Full Text]
  41. Salt I, Celler JW, Hawley SA, Prescott A, Woods A, Carling D, and Hardie DG. AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform. Biochem J 334: 177–187, 1998.[ISI][Medline]
  42. Stapleton D, Mitchelhill KI, Gao G, Widmer J, Michell BJ, Teh T, House CM, Fernandez CS, Cox T, Witters LA, and Kemp BE. Mammalian AMP-activated protein kinase subfamily. J Biol Chem 271: 611–614, 1996.[Abstract/Free Full Text]
  43. Stein SC, Woods A, Jones NA, Davison MD, and Carling D. The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345: 437–443, 2000.[CrossRef][ISI][Medline]
  44. Stephens TJ, Chen ZP, Canny BJ, Michell BJ, Kemp BE, and McConell GK. Progressive increase in human skeletal muscle AMPKalpha2 activity and ACC phosphorylation during exercise. Am J Physiol Endocrinol Metab 282: E688–E694, 2002.[Abstract/Free Full Text]
  45. Toyoda T, Hayashi T, Miyamoto L, Yonemitsu S, Nakano M, Tanaka S, Ebihara K, Masuzaki H, Hosoda K, Inoue G, Otaka A, Sato K, Fushiki T, and Nakao K. Possible involvement of the {alpha}1-isoform of 5' -AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle. Am J Physiol Endocrinol Metab 287: E166–E173, 2004.[Abstract/Free Full Text]
  46. Vavvas D, Apazidis A, Saha AK, Gamble J, Patel A, Kemp BE, Witters LA, and Ruderman NB. Contraction-induced changes in acetyl-CoA carboxylase and 5' -AMP-activated kinase in skeletal muscle. J Biol Chem 272: 13255–13261, 1997.[Abstract/Free Full Text]
  47. Weekes J, Hawley SA, Corton J, Shugar D, and Hardie DG. Activation of rat liver AMP-activated protein kinase by kinase kinase in a purified, reconstituted system. Effects of AMP and AMP analogues. Eur J Biochem 219: 751–757, 1994.[ISI][Medline]
  48. Winder WW, Hardie DG, Mustard KJ, Greenwood LJ, Paxton BE, Park SH, Rubink DS, and Taylor EB. Long-term regulation of AMP-activated protein kinase and acetyl-CoA carboxylase in skeletal muscle. Biochem Soc Trans 31: 182–185, 2003.[ISI][Medline]
  49. Winder WW, Wilson HA, Hardie DG, Rasmussen BB, Hutber CA, Call GB, Clayton RD, Conley LM, Yoon S, and Zhou B. Phosphorylation of rat muscle acetyl-CoA carboxylase by AMP-activated protein kinase and protein kinase A. J Appl Physiol 82: 219–225, 1997.[Abstract/Free Full Text]
  50. Wojtaszewski JF, Nielsen P, Hansen BF, Richter EA, and Kiens B. Isoform-specific and exercise intensity-dependent activation of 5' -AMP-activated protein kinase in human skeletal muscle. J Physiol 528: 221–226, 2000.[Abstract/Free Full Text]
  51. Zou MH, Hou XY, Shi CM, Kirkpatick S, Liu F, Goldman MH, and Cohen RA. Activation of 5' -AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite. J Biol Chem 278: 34003–34010, 2003.[Abstract/Free Full Text]



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