1 Department of Physiology, Monash University, Clayton, Victoria 3800; and 2 St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
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ABSTRACT |
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The effect of prolonged
moderate-intensity exercise on human skeletal muscle AMP-activated
protein kinase (AMPK)1 and -
2 activity and acetyl-CoA carboxylase
(ACC
) and neuronal nitric oxide synthase (nNOSµ) phosphorylation
was investigated. Seven active healthy individuals cycled for 30 min at
a workload requiring 62.8 ± 1.3% of peak O2
consumption (
O2 peak) with muscle biopsies obtained from the vastus lateralis at rest and at 5 and 30 min
of exercise. AMPK
1 activity was not altered by exercise; however,
AMPK
2 activity was significantly (P < 0.05)
elevated after 5 min (~2-fold), and further elevated
(P < 0.05) after 30 min (~3-fold) of exercise.
ACC
phosphorylation was increased (P < 0.05) after
5 min (~18-fold compared with rest) and increased (P < 0.05) further after 30 min of exercise (~36-fold compared with
rest). Increases in AMPK
2 activity were significantly correlated with both increases in ACC
phosphorylation and reductions in muscle
glycogen content. Fat oxidation tended (P = 0.058) to
increase progressively during exercise. Muscle creatine phosphate was
lower (P < 0.05), and muscle creatine, calculated free
AMP, and free AMP-to-ATP ratio were higher (P < 0.05)
at both 5 and 30 min of exercise compared with those at rest. At 30 min
of exercise, the values of these metabolites were not significantly
different from those at 5 min of exercise. Phosphorylation of nNOSµ
was variable, and despite the mean doubling with exercise,
statistically significance was not achieved (P = 0.304). Western blots indicated that AMPK
2 was associated with both
nNOSµ and ACC
consistent with them both being substrates of
AMPK
2 in vivo. In conclusion, AMPK
2 activity and ACC
phosphorylation increase progressively during moderate exercise at
~60% of
O2 peak in humans, with
these responses more closely coupled to muscle glycogen content than
muscle AMP/ATP ratio.
adenosine monophosphate-activated protein kinase; acetyl-coenzyme A
carboxylase-; neuronal nitric oxide synthase; prolonged exercise; humans
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INTRODUCTION |
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AMP-ACTIVATED PROTEIN KINASE (AMPK)
plays an important role in coordinating metabolism with a number of
physiological processes (16, 22). In rat skeletal muscle,
activation of AMPK appears to lead to enhanced fat oxidation and
glucose uptake (3, 19, 25). In transgenic mice expressing
a dominant negative mutant of AMPK, there is impaired glucose uptake in
response to both contraction and hypoxia (26). There are
two isoforms of AMPK, AMPK1 and -
2, expressed in skeletal muscle.
Maximal sprint exercise [~200% peak O2 consumption
(
O2 peak)] over 30 s in humans causes a large increase in the calculated free AMP and free AMP-to-ATP ratio (AMP/ATP) (28) and activates both AMPK
1 and -
2
(6). AMPK
1 is not activated during lower intensity
exercise such as at 50 or 70%
O2 peak,
or after 5 min of exercise at 90%
O2 peak after 55 min of cycling at 75%
O2 peak (75-90%
O2 peak) (14, 39).
AMPK
2 activity is increased after exercise at 70 and 75-90%
O2 peak, but not after exercise at 50%
O2 peak (14, 39). There
are smaller increases in free AMP and the free AMP/ATP ratio during
low-intensity submaximal exercise that may be insufficient to activate
AMPK (21, 28). During exercise at ~60%
O2 peak, the level of muscle energy
imbalance increases (21), and glucose uptake (4) and fat oxidation (12) increase from
rest. If AMPK
2 activity is important for activation of fat oxidation
and glucose uptake, one would expect increases in AMPK
2 activity and
phosphorylation of acetyl-CoA carboxylase (ACC
) at this moderate
exercise intensity.
Skeletal muscle fat oxidation is dependent on fatty acids being
transported into the mitochondria via carnitine palmitoyltransferase I
(CPT I) (32). Fatty acid transport via CPT I is
allosterically inhibited by malonyl-CoA (32). It has been
shown in rat skeletal muscle that AMPK phosphorylates and inhibits
ACC (36), which is responsible for converting
acetyl-CoA to malonyl-CoA. Indeed, treadmill exercise in rats causes an
increase in AMPK activity and reductions in ACC
activity and
malonyl-CoA content (37). In addition, activation of AMPK
via 5'-aminoimidazole-4-carboxyamide-1-
-D-ribofuranoside (AICAR) causes decreases in ACC
activity and malonyl-CoA
levels as well as an increase in fat oxidation in perfused rat hindlimb (25). In humans, malonyl-CoA decreases (12-17%)
significantly during exercise at intensities between 85 and 100%
O2 peak (8, 27), but not
during exercise at 60-65%
O2 peak (8, 27). However, Dean et al. (8) found
decreased ACC
activity at 60%
O2 peak as well as at the higher
exercise intensities despite no change in malonyl-CoA at 60%
O2 peak. Because ACC
activity is
reduced at 60%
O2 peak, it is likely
that ACC
phosphorylation by AMPK is increased at this intensity. We
found that ACC
phosphorylation by AMPK increases 8.5-fold during
sprint exercise in humans (6). Therefore, the first aim of
this study was to determine whether prolonged exercise at ~60%
O2 peak increased AMPK
2 activity and
ACC
phosphorylation in human skeletal muscle.
Activation of AMPK in rat skeletal muscle by AICAR has been shown to increase skeletal muscle glucose uptake (19) by increasing the translocation of GLUT-4 to the sarcolemma (23). AICAR appears to act via an insulin-independent mechanism that is believed to be similar to contraction-stimulated glucose uptake (19). Hayashi et al. (19) have shown that the combination of AICAR and contraction is not additive on glucose uptake, whereas both contraction with insulin and AICAR with insulin have additive effects on glucose uptake. Exercise-stimulated glucose uptake appears to involve both AMPK-dependent and AMPK-independent mechanisms (26). The mechanism by which AMPK increases GLUT-4 translocation and glucose uptake is unknown. Previously we found infusion of a nitric oxide synthase (NOS) inhibitor during cycling in humans reduces leg glucose uptake without affecting leg blood flow (4). Furthermore, we recently found an eightfold increase in neuronal NOS (nNOSµ) phosphorylation by AMPK immediately after 30-s maximal sprint exercise in humans (6). Although there is evidence that NO (produced by nNOSµ) is a signaling intermediate in stimulation of GLUT-4 translocation (31) and glucose uptake during rat skeletal muscle contraction (2), other studies do not support a role for NO in contraction-stimulated glucose transport (11, 20).
Glucose uptake and fat oxidation increase from 5 to 30 min of exercise
in humans (12), but a study in rats found that there was
no change in AMPK activity and ACC activity from 5 to 30 min of
treadmill running (37). It is therefore important to determine whether AMPK
2 activity, ACC
phosphorylation, and
nNOSµ phosphorylation increase progressively during prolonged
moderate exercise in humans and whether AMPK is associated with these
enzymes in human skeletal muscle.
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METHODS |
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Subjects. Seven active, healthy individuals (3 males and 4 females) volunteered for this study, which was approved by the Monash University Standing Committee on Ethics in Research involving Humans, in accordance with National Health and Medical Research Council of Australia guidelines. Before commencing the study, they completed a medical questionnaire and provided informed, written consent. Their mean age, weight, and height were 20 ± 1 yr, 67.2 ± 4.5 kg, and 168.3 ± 4.8 cm, respectively (means ± SE). None of the females was using oral contraceptives.
Experimental procedures.
Peak pulmonary oxygen consumption during cycling
(O2 peak) was determined using a graded
exercise test to volitional exhaustion on an electrically braked cycle
ergometer (Lode, Gronignen, The Netherlands) and averaged 2.4 ± 0.2 l/min (35.9 ± 2.3 ml · kg
1 · min
1). On a
separate day, subjects completed a familiarization trial, which
involved cycling continuously for 30 min at a workload calculated from
the
O2 peak test to be equivalent to
60% of their
O2 peak. Approximately 10 days later, subjects returned to the laboratory having abstained for
the previous 24 h from alcohol, caffeine, and strenuous exercise
and from food overnight. The experimental trial consisted of 30 min of
cycling at 62.8 ± 1.3% of
O2 peak (98 ± 11 W) with muscle
biopsies conducted at rest and after 5 and 30 min of exercise. Muscle
biopsies were obtained from three separate sites of the vastus
lateralis muscle under local anesthetic by use of the percutaneous
needle biopsy technique, with suction. Once removed, muscle samples
were frozen in liquid nitrogen. Muscle samples obtained at rest were
frozen in liquid nitrogen 7 ± 1 s after the biopsying of the
muscle. The muscle samples obtained at 5 and 30 min of exercise were
frozen in liquid nitrogen 17 ± 2 s after the cessation of
exercise. AMPK
1 and -
2 activity, ACC
phosphorylation, nNOSµ
phosphorylation, and muscle metabolites were later analyzed in the
muscle samples. Expired air was collected into Douglas bags for 3 min
after 7, 17, and 27 min of exercise for calculation of oxygen
consumption, carbon dioxide production, respiratory exchange ratio, fat
oxidation, and carbohydrate oxidation. Heart rate was noted at 10, 20, and 30 min of exercise.
Analytical techniques. Oxygen and carbon dioxide content in the expired air was analyzed using Exerstress OX21 and CO21 electronic analyzers (Clinical Engineering Solutions, Sydney, Australia) calibrated with gases of known composition. Gas volume was measured using a dry gas meter (American Meter, Vacumed, Ventura, CA) calibrated against a Tissot spirometer.
Approximately 20 mg of each muscle sample were freeze-dried for analysis of muscle metabolites. Each sample was crushed to a fine powder, with any visible connective tissue being removed. Muscle glycogen was extracted from ~1 mg of freeze-dried muscle by incubating the sample in HCl for 2 h at 100°C, and after neutralization with NaOH, the sample was analyzed for glucose units with the use of an enzymatic fluorometric method (29). Muscle metabolites [ATP, creatine phosphate (PCr), creatine (Cr), and lactate] were extracted from ~2 mg of freeze-dried muscle by use of the procedure of Harris et al. (17) and analyzed using enzymatic fluorometric techniques (24). The contents of ATP, 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. Free ADP and AMP were calculated as outlined previously (6). Approximately 80 mg of wet muscle was 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 AMPKStatistical analysis. Results were analyzed using one-way or two-way repeated-measures analysis of variance utilizing the statistical package SPSS. Specific differences were located using the least significant difference (LSD) test. A significance level of P < 0.05 was set.
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RESULTS |
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Muscle ATP concentration at 5 min of exercise was not
significantly different from the value at rest, whereas muscle PCr
concentration was lower (P < 0.05), and muscle Cr
concentration was higher (P < 0.05), at 5 min of
exercise compared with those at rest (Table 1). In addition, calculated free ADP,
calculated free AMP, and the calculated free AMP/ATP ratio were higher
(P < 0.05) at 5 min of exercise than at rest (Table
1). There was no significant difference in the values of these muscle
metabolites at 5 min compared with 30 min of exercise. Muscle glycogen
concentration tended to decrease progressively during exercise, with
the concentration at 30 min being significantly (P < 0.05) less than both the concentration at rest and that at 5 min of
exercise (Table 1). Muscle lactate concentration tended to increase
during exercise (P = 0.097; Table 1). AMPK1 activity
measured without added AMP did not change during exercise (Fig.
1A). In contrast, AMPK
2
activity measured without added AMP increased after 5 min (~2-fold)
of exercise and then increased further after 30 min (~3-fold) of
exercise (P < 0.05; Fig. 1B). Addition of
150 µM AMP increased (P < 0.05) AMPK
1 and -
2
activity to a similar extent in the rest and exercised muscle samples,
and the responses of the AMPK
1 and -
2 isoforms to AMP were
similar (Fig. 1). ACC
phosphorylation increased greatly and
progressively with exercise, being ~18-fold greater than rest at 5 min (P < 0.05) and 36-fold greater than rest at 30 min
of exercise (P < 0.05; Fig.
2). There was a significant correlation across the trial between AMPK
2 activity and ACC
phosphorylation (r = 0.805, P < 0.001 without AMP;
r = 0.637, P = 0.004 with AMP). There
was also a significant negative correlation across the trial between
AMPK
2 activity and muscle glycogen (r =
0.574,
P = 0.01 without AMP; r =
0.533,
P = 0.02 with AMP). Although the level of nNOSµ
phosphorylation approximately doubled with exercise, this was not a
statistically significant increase (P = 0.304; Fig.
3). Previously, we found that more
intense sprint exercise was associated with a 5.5-fold increase in
nNOSµ phosphorylation at Ser1451 (6). It was
of interest to test whether nNOSµ is associated with AMPK
2 in
vivo. We detected AMPK
2 associated with 2',5'-ADP-Sepharose affinity-purified nNOSµ in immunoblots (Fig.
4). This indicated that nNOSµ is
associated with AMPK
2 in vivo, supporting the idea that
it is a direct substrate. Partially purified AMPK
2 does not bind
2',5'-ADP-Sepharose beads. Similarly, we found that AMPK
2 was
associated with avidin affinity-purified ACC
(Fig. 4), indicating that ACC
is a direct substrate of AMPK
2 in vivo. AMPK
2 is not bound by immobilized monomeric avidin agarose beads. The amount of
AMPK
2 associated with either ACC
or nNOSµ was the same for rest
and exercise samples (Fig. 4). Oxygen consumption and heart rate over
the 30-min cycling period averaged 1.52 ± 0.08 l/min and 147 ± 3 beats/min, respectively. The respiratory exchange ratio decreased
(P < 0.05) significantly during the trial, averaging 0.97 ± 0.03, 0.95 ± 0.03, and 0.94 ± 0.03, at 10, 20, and 30 min of exercise, respectively. Calculated fat oxidation tended
(P = 0.058) to increase during exercise, and calculated
carbohydrate oxidation tended (P = 0.123) to decrease
during exercise (Fig. 5).
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DISCUSSION |
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The major finding of this study was that there is a large,
progressive increase in ACC phosphorylation during
moderate-intensity exercise at ~60%
O2 peak in humans, which is coupled with the progressive increase in AMPK
2 activity and fat oxidation. We found that the progressive increase in AMPK
2 activity during exercise was more closely coupled to changes in muscle glycogen content
during exercise than changes in muscle energy balance, since muscle
glycogen content was the only metabolic parameter measured whose change
between 5 and 30 min was statistically significant. These results
indicate that changes in AMPK activity may occur independently of
detectable increases in the AMP/ATP ratio. Enhanced activation of
skeletal muscle AMPK is seen with reduced muscle glycogen content in
both contracting rat muscle (10) and during exercise in
humans (30). We interpret our results as indicating that
the initial increase in AMPK activity from rest depends on the AMP/ATP
ratio but that the subsequent increase between 5 and 30 min is
dependent on the reduction in muscle glycogen content.
Maximal sprint exercise (~200%
O2 peak) in humans results in an
increase in the activity of both skeletal muscle AMPK
1 and -
2
(6). However, in several studies, including this study
(Fig. 1A), AMPK
1 activity does not increase during submaximal exercise in humans (14, 39). It has now been
shown during exercise in humans that AMPK
1 activity is not increased above the resting levels at 50 (14), ~60 (Fig. 1A), 70 (14), and 75-90% (39)
O2 peak. This suggests that AMPK
1
activation may not be required during prolonged exercise and is not
responsible for the increase in ACC
phosphorylation in the present
study (Fig. 2). Two previous studies in humans have found no increase in AMPK
2 activity during exercise at 50%
O2 peak but an increase at 70 (14), or 75-90% (39) of
O2 peak. Our results indicate that
there is a threshold for activation of AMPK
2 that occurs at ~60%
O2 max within the moderate exercise range.
Hypoxia is a very strong stimulator of AMPK (18), and it
is possible that a level of hypoxia is required before AMPK1 is activated in skeletal muscle. As discussed earlier, we found that both
AMPK
1 and -
2 were increased after sprint cycling in humans (6). Such exercise is likely to be associated with a large decrease in intramuscular PO2. Intense
electrical stimulation of rat skeletal muscle in vitro also increases
both AMPK
1 and -
2 (18), and it has been suggested
that a level of hypoxia exists under such conditions (39).
In isolated rat muscle, hypoxia causes much greater increases in both
AMPK
1 and -
2 than does either in vitro electrical stimulation or
exposure to AICAR (18). In situ stimulation of rat muscle,
where the circulation is still intact, only activates AMPK
2
(36).
Skeletal muscle ACC phosphorylation by AMPK increased progressively
during exercise (Fig. 2), in line with the progressive increase in
AMPK
2 activity (Fig. 1B) and fat oxidation (Fig. 5). Our
results differ from those obtained in rat muscle, where it was found
that there was no change in AMPK activity and ACC activity from 5 to 30 min of treadmill running (37). The reason for this
discrepancy is unclear but may reflect species differences. We have
found that AMPK
2 and ACC
are associated in human skeletal muscle
in vivo (Fig. 4), consistent with ACC
being a direct substrate for
AMPK. Previously, we found that ACC
phosphorylation increased ~8.5-fold after a maximal 30-s sprint (6). There is a
greater degree of increase in ACC
phosphorylation (18-fold) during
moderate-exercise intensity; however, this was measured at 5 min (Fig.
2), providing a 10-fold increase in exercise duration to allow for the
accumulation of more phosphorylated ACC
compared with the sprint
exercise study (6).
In line with the progressive elevation in ACC phosphorylation during
exercise, we found a trend (P = 0.058) for fat
oxidation to increase progressively during the exercise bout. Although
it is well documented that skeletal muscle malonyl-CoA decreases during
exercise in rats (37), the findings in humans are less clear. Two groups have shown that skeletal muscle malonyl-CoA levels
are not reduced during exercise at 60-65%
O2 peak, but there is a small
(12-17%) reduction during exercise at 85-100%
O2 peak (8, 27). The
smaller change in measured malonyl-CoA during exercise in humans than
in rats has been suggested to be due to the much lower levels in human
skeletal muscle, therefore making it difficult to detect small changes
(32). It has therefore been suggested that measurement of
AMPK activity and ACC
activity in humans is an alternative to
measuring malonyl-CoA, because reductions in ACC
activity lead to
reductions in malonyl-CoA (32). Phosphorylation of ACC
at the serine79 AMPK-specific site has been shown to be a
potent inhibitor of ACC
activity in rat skeletal muscle (36,
38). Indeed, Dean et al. (8) found decreased ACC
activity compared with rest after exercise at 60%
O2 peak in humans, but there was no
detectable change in malonyl-CoA. It seems reasonable that the
progressive increase in fat oxidation (P < 0.058)
observed during exercise in the present study reflects the progressive increase in ACC
phosphorylation. The possibility that AMPK may have
a dual role in regulating malonyl-CoA has been raised by the report
that AMPK phosphorylates and activates malonyl-CoA decarboxylase
(33). It should also be noted that there is some evidence
that factors other than malonyl-CoA, such as muscle pH (34) and muscle carnitine levels, influence fat oxidation
in skeletal muscle during exercise.
Glucose uptake increases progressively during prolonged submaximal
exercise (12). If AMPK activity is indeed playing a role in glucose uptake during exercise, one would expect that it would also
increase progressively with exercise duration. This was found to be the
case with regard to AMPK2 activity, which increased progressively,
whereas AMPK
1 activity did not change during exercise (Fig. 1). The
downstream substrate(s) of AMPK responsible for activating glucose
uptake is not known. We (4) and others (1) have found that NOS inhibition reduces skeletal muscle glucose uptake
in response to exercise. Fryer et al. (13) found that AICAR increased both NOS activity and glucose uptake in rat soleus and
extensor digitorum longus skeletal muscle strips and that both of these
effects of AICAR were abolished with a NOS inhibitor. However, other
studies do not support the concept that NO is directly involved in the
signaling pathway for contraction-stimulated glucose uptake (11,
20). Dean et al. (8) recently reported that NOS
inhibition has little effect on AICAR- or hypoxia-stimulated glucose
transport in rat skeletal muscle (9). The reason for these
differing results is unclear. Because AMPK stimulates glucose uptake
into skeletal muscle (3, 19) and AMPK has been shown to
phosphorylate nNOSµ during intense sprint exercise (6), we tested whether nNOSµ phosphorylation increases during
moderate-intensity exercise. Although we found that nNOSµ
phosphorylation, on average, doubled during exercise (Fig. 3), this was
not statistically significant and was much smaller than the 5.5-fold
increase we observed after a maximal 30-s sprint (6). In
addition, the increase in nNOSµ phosphorylation was much less than
the level of phosphorylation of ACC
(36-fold) during exercise in the
present study. Nevertheless, we find that nNOSµ is associated with
AMPK
2, supporting the view that nNOSµ is a direct substrate for
AMPK
2 in vivo (Fig. 4). At this stage, it is not clear what the
physiological function of nNOSµ phosphorylation at
Ser1451 is in exercising skeletal muscle. In view of the
modest phosphorylation during moderate exercise and the conflicting
results regarding the role of NO in glucose transport, it would seem
unlikely that Ser1451 phosphorylation is essential for
exercise-induced glucose transport.
In conclusion, skeletal muscle AMPK2, but not AMPK
1, activity
increased progressively during 30 min of moderate-intensity exercise at
~60%
O2 peak. There was
also a large, progressive increase in ACC
phosphorylation during
exercise, and the increase in AMPK
2 activity correlated with the
increase in ACC
phosphorylation. Fat oxidation also tended to
increase progressively during exercise. After the first 5 min of
exercise, the increases in AMPK
2 activity and ACC
phosphorylation
were more closely related to changes in muscle glycogen content than
muscle AMP/ATP levels.
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ACKNOWLEDGEMENTS |
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We thank the subjects for taking part in this study and also thank Dr. Rodney Snow for technical assistance.
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FOOTNOTES |
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* T. J. Stephens and Z.-P. Chen contributed equally to this study.
This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia (G. K. McConell and B. E. Kemp), the National Heart Foundation, and Diabetes Australia. B. E. Kemp is an NHMRC Fellow, and G. K. McConell is an NHMRC Senior Research Officer.
Address for reprint requests and other correspondence: G. McConell, Dept. of Physiology, Monash Univ., Clayton, Victoria, Australia 3800 (E-mail: g.mcconell{at}med.monash.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.
10.1152/ajpendo.00101.2001
Received 7 March 2001; accepted in final form 30 October 2001.
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