RAPID COMMUNICATION
AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation

Zhi-Ping Chen1,*, Glenn K. McConell2,*, Belinda J. Michell1, Rodney J. Snow3, Benedict J. Canny2, and Bruce E. Kemp1

1 St. Vincent's Institute of Medical Research, St. Vincent's Hospital, Fitzroy, Victoria 3065; 2 Department of Physiology, Monash University, Clayton, Victoria 3800; and 3 School of Health Sciences, Deakin University, Burwood, Victoria 3025, Australia


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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AMP-activated protein kinase (AMPK) is a metabolic stress-sensing protein kinase responsible for coordinating metabolism and energy demand. In rodents, exercise accelerates fatty acid metabolism, enhances glucose uptake, and stimulates nitric oxide (NO) production in skeletal muscle. AMPK phosphorylates and inhibits acetyl-coenzyme A (CoA) carboxylase (ACC) and enhances GLUT-4 translocation. It has been reported that human skeletal muscle malonyl-CoA levels do not change in response to exercise, suggesting that other mechanisms besides inhibition of ACC may be operating to accelerate fatty acid oxidation. Here, we show that a 30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK-alpha 1 and -alpha 2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser79 (AMPK phosphorylation site) ~8.5-fold. Under these conditions, there is also an ~5.5-fold increase in phosphorylation of neuronal NO synthase-µ (nNOSµ) at Ser1451. These observations support the concept that inhibition of ACC is an important component in stimulating fatty acid oxidation in response to exercise and that there is coordinated regulation of nNOSµ to protect the muscle from ischemia/metabolic stress.

AMP-activated protein kinase; nitric oxide synthase; exercise


    INTRODUCTION
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INTRODUCTION
METHODS
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MALONYL-COENZYME A (CoA) is an allosteric inhibitor of carnitine palmitoyltransferase I (CPT-I), which transports long-chain fatty acids into the mitochondria for oxidation (24). In rats, glucose starvation or energy demand through muscle contraction reduces malonyl-CoA, whereas insulin or inactivity promotes increased levels (24). During exercise or electrical stimulation of rat skeletal muscle, the AMP-activated protein kinase (AMPK) is responsible for phosphorylation and inhibition of acetyl-CoA carboxylase (ACC) (29, 31) and enhanced GLUT-4 translocation (2, 12, 13). Although there is increased fat utilization with exercise in human skeletal muscle, no changes in malonyl-CoA have been detected (20). Alternative mechanisms for controlling CPT-I have been proposed, involving phosphorylation of the cytoskeletal proteins, cytokeratins 8 and 18, by the AMPK (30). However, because acetyl-CoA increases rapidly in human skeletal muscle with vigorous exercise (20), we considered that regulation of fatty acid oxidation via phosphorylation of ACC could be important despite the inability to detect malonyl-CoA changes. It has recently been reported that malonyl-CoA decarboxylase, an enzyme involved in malonyl-CoA catabolism, is activated during rat muscle contraction due to phosphorylation by the AMPK (26), and if a similar mechanism operates in human muscle, it would indicate an important role for malonyl-CoA in exercise. There is a relatively modest increase in muscle AMP concentrations in rodent skeletal muscle during exercise due to rapid metabolism to IMP; yet AMPK activity is readily increased, presumably as a result of increased free AMP. For this reason, we sought to test directly whether ACC phosphorylation at Ser79, the AMPK site, was altered by exercise. An anti-phosphopeptide polyclonal antibody was raised that was specific for ACC phosphorylated on Ser79. In addition, because AMPK phosphorylates and activates endothelial nitric oxide synthase (eNOS) (5), it was of interest to test whether the skeletal muscle NO synthase (NOS) isoform, neuronal NOS (nNOS, or NOS type I)-µ, was also phosphorylated during vigorous exercise. The nNOSµ isoform present in skeletal muscle is an alternative-spliced form of nNOS with a 34-residue insert (27). There is preliminary evidence (1, 3) that NO plays a role in the regulation of skeletal muscle glucose uptake during exercise.


    METHODS
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Subjects. Eleven individuals (seven males, four females) took part in this study (n = 8 for metabolites, n = 5 for ACC, and n = 5 for nNOSµ), which was approved by the Monash University Standing Committee for Research in Humans in accordance with National Health and Medical Research Council of Australia guidelines. Each subject was informed of the risks and stresses associated with participation, and each provided informed written consent. Their mean age, weight, and height were 22 ± 1.6 yr, 73.8 ± 4.8 kg, and 174 ± 4 cm (means ± SE), respectively.

Experimental procedures. Peak pulmonary oxygen uptake (VO2 peak) was measured for each subject during incremental cycling exercise to volitional fatigue on an electrically braked cycle ergometer (Lode, Groningen, The Netherlands) and averaged 3.0 ± 0.28 l/min (40.8 ± 3.0 ml · min-1 · kg-1). At least three days later, the subjects returned to the laboratory for a familiarization ride. This involved completing a single 30-s sprint on an electrically braked cycle ergometer (peak power 834 ± 89 W). The subjects were instructed to cycle as hard and as fast as they could from the very first pedal stroke. This meant that a progressive fatiguing took place over the 30 s. Three to ten days later, the subjects returned to the laboratory for the experimental trial, having been instructed to refrain from strenuous exercise, alcohol, and caffeine for 24 h. A muscle sample was obtained under anesthesia from the vastus lateralis, using the percutaneous needle biopsy technique with suction, and the sample was quickly frozen (within 10-15 s) in liquid nitrogen for later metabolite analysis. Subjects then completed the 30-s sprint, and another muscle sample was obtained through a second incision on the same leg while the subjects were still positioned on the ergometer. This muscle sample was frozen within 15-20 s of the completion of exercise. The muscle samples were analyzed for AMPK activity, ACC, and nNOSµ phosphorylation, as well as for metabolites.

Analytical techniques. Expired air samples were measured for oxygen and carbon dioxide content with Exerstress OX21 and CO21 electronic analyzers (Clinical Engineering Solutions, Sydney, Australia). These analyzers were calibrated with the use of commercial gases of known composition. Expired air volume was measured using a dry gas meter (American Meter, Vacumed) calibrated against a Tissot spirometer. A portion (~20 mg) of the muscle samples was freeze-dried and then crushed to a powder, with any visible connective tissue removed. For muscle glycogen determination, ~1 mg of muscle was added to HCl, incubated at 100°C, and then neutralized with NaOH and analyzed for glucose units with an enzymatic fluorometric method (21). Muscle metabolites were analyzed by extraction of ~2 mg of muscle according to the procedure of Harris et al. (11). Muscle lactate, phosphocreatine (PCr) and creatine (Cr) were analyzed using enzymatic fluorometric techniques (15), whereas muscle ATP, ADP, AMP, and IMP were measured by HPLC, as described previously (28). The contents of ATP, ADP, AMP, IMP, PCr, and Cr were corrected to the peak total Cr (PCr + Cr) content for each subject to account for any nonmuscle contamination of the muscle samples. The estimated free concentrations of ADP and AMP were based on the near-equilibrium nature of the creatine phosphokinase and adenylate kinase reactions, respectively. Free ADP was estimated from the measured ATP, Cr, and PCr contents, and H+ concentration was estimated using the measured muscle lactate content according to the formula presented by Mannion et al. (16) for dry muscle. The observed equilibrium constant (Kobs) value employed was 1.66 × 109 M (14). Free AMP was estimated from the measured ATP and the estimated free ADP using a Kobs of 1.05 (14).

Muscle biopsies (~100 mg, pre- and postexercise) were homogenized in buffer A (50 mM Tris · HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 50 mM NaF, 5 mM Na pyrophosphate, 10% glycerol, 1% Triton X-100, 10 µg/ml trypsin inhibitor, 2 µg/ml aprotinin, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride). The homogenates were incubated with the AMPK-alpha 1 or AMPK-alpha 2 antibody-bound protein A beads for 2 h at 4°C. Immunocomplexes were washed with PBS and suspended in 50 mM Tris buffer (pH 7.5) for AMPK activity assay (5). Rabbit polyclonal antibodies against phosphopeptides based on the amino acid sequence of rat acetyl-CoA carboxylase around Ser79 CHMRSSMSpGLHLVK (pACC) and human nNOSµ around Ser1451 (RLRSESpIAFIE) were purified using the corresponding phosphopeptide affinity columns after precleaning with dephosphopeptide affinity columns. The specificity of the purified antibodies was assessed by enzyme immunoassays and immunoblotting, and they did not recognize the corresponding dephosphoenzyme. Results were analyzed using Student's paired t-tests. All data are presented as means ± SE. The level of significance was set at P < 0.05.


    RESULTS
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We observed significant activation of skeletal muscle AMPK in response to the 30-s sprint (Fig. 1). During this vigorous exercise, the lactate content of the skeletal muscle increased (P < 0.05) from 4.3 ± 0.5 to 92.1 ± 4.8 mmol/kg dry muscle, and IMP increased (P < 0.05) from 0.02 ± 0.01 to 6.3 ± 1.0 mmol/kg dry muscle. PCr (from 85.8 ± 1.8 to 34.7 ± 2.6 mmol/kg dry muscle), glycogen (from 500 ± 38 to 389 ± 33 mmol/kg dry muscle), and ATP (from 22.10 ± 0.8 to 16.0 ± 1.5 mmol/kg dry muscle) all decreased (P < 0.05) as expected (n = 8). The calculated skeletal muscle free ADP (rest, 86 ± 11; 30 s, 123 ± 20 µmol/kg dry muscle) and free AMP (rest, 0.37 ± 0.08; 30 s, 1.11 ± 0.33 µmol/kg dry muscle) increased (P < 0.05) after exercise. The ratio of free AMP to ATP, which is important in the activation of AMPK (10), increased significantly (P < 0.05) from 0.016 ± 0.003 at rest to 0.051 ±0.016 after exercise. Both isoforms of the AMPK, AMPK-alpha 1 and AMPK-alpha 2, were activated during the 30-s sprint exercise (Fig. 1). Previously in rat heart and rat skeletal muscle, activation of the alpha 2-isoform has been more dominant in response to either ischemia (5) or electrical stimulation (29). The immunoprecipitated AMPK isoforms were stimulated approximately twofold (P < 0.05) when measured in the absence of AMP. There was an approximately twofold increase (P < 0.05) in activity measured in the presence of AMP for AMPK-alpha 1 but a lesser (70%) increase for AMPK-alpha 2, indicating a reduction of AMP dependence of AMPK-alpha 2 with activation. There was insufficient AMPK in the biopsy samples to measure the state of phosphorylation of Thr172 in the activation loop of the AMPK, but in electrically stimulated rat skeletal muscle, increases in AMPK activity parallel increases in Thr172 phoshorylation by the upstream AMPK kinase (Z. P. Chen, P. Gregorevic, G. S. Lynch, B. J. Michell, and B. E. Kemp, unpublished observations). It has now been reported (7) that exercise at 70% VO2 max in humans increased the activity of only the AMPK-alpha 2 isoform.


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Fig. 1.   Effect of exercise on AMP protein-activated kinase (AMPK) activity for isoforms AMPK-alpha 1 and -alpha 2. The AMPK activities in the immune complexes were measured in either the presence or absence of AMP (150 µM). Activities were calculated as pmol of phosphate incorporated into the ACC(73-87)A77 peptide · min-1 · mg total protein subjected to immunoprecipitate-1. Values are means ± SE; n = 5. * Difference from rest, P < 0.05.

Because ACC is a biotinylated enzyme, we used Avidin-immobilized agarose beads to extract it from biopsy samples. Immunoblotting with anti-phosphopeptide antibody to the Ser79 site showed that exercise increased the phosphorylation of ACC dramatically at this site by ~8.5-fold (Fig. 2). ACC phosphorylation is unlikely to be due to postextraction events, because NaF present in the extraction buffer inhibits the AMPK. Our results demonstrate that the phosphorylation of ACC in response to exercise is both rapid and dramatic. Phosphorylation of ACC at Ser79 reduces ACC activity (10). We infer that exercise (~200% VO2 max, 30 s) causes rapid inhibition of ACC in human muscle, resulting in local changes in malonyl-CoA that are not readily detected. It should be noted that previous human studies (20) examining malonyl-CoA during exercise used lower exercise intensities (<= 90% VO2 max, 10 min) and did not measure ACC phosphorylation. The increase in ACC phosphorylation was surprisingly large, given that anaerobic metabolism predominates in intense exercise as used in the present study, and, therefore, the contribution of fat oxidation is small.


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Fig. 2.   Effect of exercise on acetyl-coenzyme A (CoA) carboxylase (ACC). Muscle homogenates were incubated with immobilized monomeric Avidin agarose beads to affinity-purify ACC. A: fractions were subjected to SDS-PAGE, and ACC was detected by immunoblotting with either anti-phospho-ACC-79 polyclonal antibody, the amino acid sequence around Ser79 CHMRSSMSpGLHLVK (pACC), or horseradish peroxidase-conjugated streptavidin (DAKO). C, control sample; E, exercised sample. B: quantitative densitometric data are shown. Values are means ± SE; n = 5. * Different from control/rest (P = 0.0007, paired t-test, log transformed).

NO has been implicated as a modulator of skeletal muscle contractility (17), mitochondrial respiration, glucose uptake, glycolysis, and neuromuscular transmission (23). NO is also important in regulating skeletal muscle blood flow both in resting muscle and during recovery from exercise (22). There is compelling evidence in rat skeletal muscle that AMPK is also involved in the regulation of glucose uptake during exercise (12, 13, 18). We have shown that inhibition of NOS reduces glucose uptake during exercise in humans (3). Therefore, it is possible that AMPK increases glucose uptake via NOS. Previously, we found that endothelial NOS (eNOS, or type III) was phosphorylated and activated by the AMPK in rat heart in response to ischemia (5), and there is also increased glucose uptake in the heart with ischemia (25). Activation of eNOS was dependent on phosphorylation at Ser1177 in the COOH terminus, and this site is also phosphorylated by other protein kinases, including Akt/protein kinase B (PKB) (6, 8, 9, 19) and protein kinase A (PKA) (4) (Fig. 3). Because nNOSµ contains a Ser1451 at the position corresponding to Ser1177 in eNOS, we tested it as a substrate for AMPK and PKA and raised an antibody to the phosphopeptide. The nNOSµ was isolated from the biopsy extracts by ADP-Sepharose precipitation and analyzed using the Ser1451 anti-phosphopeptide antibody. The 30-s exercise sprint increased phosphorylation of nNOSµ at Ser1451 by ~5.5-fold (Fig. 4). We detected only nNOSµ in the human skeletal muscle samples, although both eNOS and nNOSµ are present in rat skeletal muscle. Although Akt/PKB phosphorylates eNOS, it does not phosphorylate nNOS (8), so that, in the exercised muscle, Akt/PKB cannot catalyze phosphorylation of Ser1451. Whereas phosphorylation of eNOS at Ser1177 is associated with increased NOS activity, we do not yet know whether phosphorylation of Ser1451 in nNOSµ has the same effect.


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Fig. 3.   Comparison of nitric oxide synthase (NOS) phosphorylation site sequences. Schematic model of NOs showing sequences for the COOH-terminal tails around the Ser1451 phosphorylation site in human neuronal NOS isoform-µ (nNOSµ) and corresponding Ser1177 site in human endothelial NOS (eNOS). PKA, protein kinase A; PKB, protein kinase B.



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Fig. 4.   Effect of exercise on nNOSµ phosphorylation. Human muscle homogenates were incubated with 2',5'-ADP Sepharose beads to affinity purify nNOSµ. A: nNOSµ fraction was subjected to SDS-PAGE, and nNOSµ was detected by immunoblotting with either anti-phospho-nNOS polyclonal antibody, phospho-nNOS, or human nNOS antibody (N31020, Transduction Laboratory). B: quantitative densitometric data are shown. Values are means ± SE; n = 5. *Different from control/rest (P = 0.003, paired t-test, log transformed).

In conclusion, the activation of the AMPK in response to exercise now appears central to upregulation of metabolism through effects on glucose uptake and fatty acid oxidation (phosphorylation of ACC) as well as regulation of skeletal muscle nNOSµ.


    ACKNOWLEDGEMENTS

This work was supported by grants from the National Health and Medical Research Council of Australia (G. K. McConell and B. E. Kemp) and the National Heart Foundation and Diabetes Australia. B. E. Kemp is a NHMRC Fellow, and G. K. McConell is a NHMRC Senior Research Officer.


    FOOTNOTES

* Authors contributed equally to the study.

Address for reprint requests and other correspondence: B. E. Kemp, St. Vincent's Inst. of Medical Research, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia (E-mail: kemp{at}ariel.ucs.unimelb.edu.au).

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.

Received 2 June 2000; accepted in final form 2 August 2000.


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