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
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ABSTRACT |
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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-1 and -
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
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INTRODUCTION |
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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.
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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 (O2 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- ![]() |
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-1 and AMPK-
2, were activated during
the 30-s sprint exercise (Fig. 1). Previously in rat heart and rat
skeletal muscle, activation of the
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-
1 but a lesser (70%) increase for
AMPK-
2, indicating a reduction of AMP dependence of AMPK-
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%
O2 max in humans increased the
activity of only the AMPK-
2 isoform.
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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% O2 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%
O2 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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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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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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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