1Exercise Metabolism Group, School of Medical Sciences, RMIT University, Victoria 3083; 2St. Vincent's Institute of Medical Research, and Department of Medicine, University of Melbourne, Fitzroy, Victoria 3065; and 3Muscle, Ions and Exercise Group, School of Human Movement, Recreation and Performance, Centre for Rehabilitation, Exercise and Sports Science, Melbourne, Victoria University of Technology, Victoria 3011, Australia
Submitted 14 October 2003 ; accepted in final form 14 December 2003
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ABSTRACT |
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adenosine 5'-monophosphate-activated protein kinase; acetyl-coenzyme A carboxylase; glycogen; fat metabolism
A single bout of low- to moderate-intensity exercise [4070% of maximal O2 uptake (O2 max)] in untrained subjects is predominantly associated with an isoform-specific and intensity-dependent increase in AMPK
2 but not AMPK
1 activity (5, 8, 31, 40). AMPK activity associated with the
2 isoform is also increased during prolonged, low-intensity (45% of
O2 peak) exercise to exhaustion (39), although such low-intensity exercise for a shorter duration does not induce activation of AMPK
2 (8, 40). It appears that AMPK
1 activity is increased only in response to maximal sprint exercise (3), consistent with the observation that the rate rather than the magnitude of fuel depletion increases the activity of both AMPK
1 and -
2 isoforms (15). Activation of AMPK in response to acute exercise is diminished in skeletal muscle of trained individuals compared with untrained subjects during intense (8085% of
O2 peak) cycling undertaken at the same relative intensity (22, 42). This is probably due to a better maintenance of the energy charge as a result of a less pronounced exercise-induced acidification in exercise-trained muscle (22).
Chronic pharmacological activation of AMPK has been reported to mimic several of the classical exercise training-mediated responses on gene expression, such as increased GLUT4, hexokinase, and citrate synthase, and also increases in mitochondrial density and muscle glycogen content (1, 16, 37, 43). However, the effects of exercise training on AMPK and downstream metabolic enzymes such as ACC have received less attention. Langfort et al. (18) showed that 4 wk of endurance training significantly increased basal AMPK1 (27%) but not -
2 protein expression, whereas a training-induced attenuation of AMPK
1 and a complete suppression of -
2 activity during prolonged submaximal (64%
O2 peak) cycling have also been reported (20). The findings of training-induced changes in the AMPK
1 subunit (18) are particularly intriguing, as AMPK
1 mRNA and protein abundance are both markedly higher in well-trained compared with untrained individuals (22). This raises the possibility that the AMPK
1 subunit may facilitate some of the beneficial training-induced adaptations in skeletal muscle (22). On the other hand, there are significant training-induced increases in basal and/or preexercise muscle glycogen content in both cross-sectional (22) and interventional studies (18, 20). Wojtaszewski et al. (38) reported that the degree of AMPK activation both at rest and during subsequent exercise is strongly influenced by the fuel status of the muscle cells (i.e., AMPK activity and ACC
Ser221 phosphorylation are lower when glycogen content is elevated). Accordingly, some of the differences observed in these previous studies may be due, in part, to training-induced increases in muscle glycogen concentration rather than a training effect alone. As noted (22), longitudinal training studies are needed to clarify the influence of glycogen on regulation of AMPK.
In the present study, the regulation of AMPK was determined in skeletal muscle from well-trained individuals during standardized exercise before and after 3 wk of intensified training. By use of an identical experimental protocol, such a regimen has previously been reported to increase whole body rates of fat oxidation and reduce plasma lactate concentration during exercise (33). Accordingly, this design was chosen, since intensified training in already well-trained athletes would not be expected to result in an increase in resting muscle glycogen content or influence glycogen utilization during standardized exercise. Furthermore, using a similar protocol, Weston et al. (34) reported training-induced improvements in muscle acidosis, a factor previously shown to influence AMPK activity (26). Accordingly, this training model might provide a useful means to elucidate the independent influence of training on AMPK signaling. On the basis of previous findings (33, 34), we hypothesized that intensified training would improve muscle acidosis, resulting in attenuation of AMPK activity while increasing whole body rates of fat oxidation, thereby increasing ACC Ser221 phosphorylation.
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METHODS |
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Eight highly trained endurance cyclists/triathletes who were riding 360 ± 40 km/wk (mean ± SE) and who had not undertaken any high-intensity interval training in the 4 wk before the investigation were recruited to participate in this study. Subjects were fully informed of the potential risks involved before providing their written consent. The study was approved by the Human Research Ethics Committee of RMIT University.
Preliminary Testing
O2 peak test.
Peak pulmonary oxygen uptake (
O2 peak) and peak sustained power (PPO) were measured for each subject during incremental cycling exercise to volitional fatigue on an electrically braked ergometer (Lode, Groningen, The Netherlands). The test protocol has been described in detail previously (11). Throughout the maximal exercise test and the subsequently described prolonged, steady-state rides (SS), subjects inspired air through a two-way valve (model R2700; Hans Rudolph, Kansas City, KS), with expirate directed to a CardioO2 Cardiorespiratory Diagnostic Systems metabolic cart (Medical Graphics, St. Paul, MN). Expired gas was passed through a flowmeter, an O2 analyzer, and a CO2 analyzer. The flowmeter was calibrated with a 3-L Hans Rudolph syringe. The gas analyzers were calibrated with gases of known concentrations (4.00% CO2 and 16.00% O2). The flowmeter and gas analyzers were connected to a computer, which calculated minute ventilation (VE), O2 consumption (
O2), CO2 production (
CO2), and respiratory exchange ratio. The results of the maximal test were used to determine the power outputs (W) that corresponded to 65 and 85% of
O2 peak, which were the work rates to be used for the subsequently described SS and high-intensity interval training (HIT) sessions.
SS.
Before the training intervention (described subsequently), all subjects completed a 90-min SS for the determination of whole body rates of substrate oxidation. Thirty-six hours before a ride, subjects performed a supervised training bout in the laboratory (1 h at 65% O2 peak), and then, over the following 36 h, subjects consumed a standard diet consisting of 55 kcal/kg body mass, composed of 57% carbohydrate (CHO; 8 g/kg body mass), 29% fat, and 14% protein and refrained from all further exercise. On the morning of each ride, subjects reported to the laboratory between 0600 and 0700, after an overnight fast. Subjects were then fed a standard breakfast (2.2 g CHO/kg). After 3 h of rest, subjects began the 90-min ride. During the first 60 min, a power output was selected that elicited
65% of
O2 peak, and for the final 30 min subjects rode at a power output that elicited
85% of
O2 peak. At regular intervals throughout SS, respiratory gas was collected. Rates of whole body CHO and fat oxidation (g/min) were calculated by indirect calorimetry, assuming a nonprotein RER (25). These equations are based on the assumption that
CO2 and
O2 accurately reflect tissue O2 consumption and CO2 production. In well-trained subjects similar to those employed in the present investigation, indirect calorimetry was previously shown to be a valid method for quantifying rates of substrate oxidation during steady-state strenuous exercise at 85% of
O2 max (27, 32). Rates of CHO oxidation (µmol·kg1·min1) were subsequently determined by converting the rate of CHO oxidation (g/min) to its molar equivalent and assuming that 6 mol of O2 are consumed and 6 mol of CO2 produced for each mole (180 g) oxidized. Rates of fat oxidation (µmol·kg1·min1) were determined by converting the rate of triglyceride oxidation (g·kg1·min1) to its molar equivalent assuming the average molecular weight of human triglyceride to be 855.3 g/mol and multiplying the molar rate of triglyceride oxidation by 3, because each molecule contains 3 mol of fatty acid (FA). At the end of the training program and after similar diet/exercise control, subjects completed another SS at the same (pretraining) absolute work rate.
HIT and Experimental Trials
Each subject performed seven HIT sessions in the laboratory over a 3-wk period. Each HIT session consisted of a 20-min warm-up at 65% of O2 peak followed by 8 x 5 min work bouts at 85% of
O2 peak with 60 s of recovery during which subjects pedaled at a power output of 100 W. After three HIT sessions, subjects performed a further maximal test, and the intensity of the subsequent three HIT sessions was then adjusted to each subject's (higher) PPO value. Subjects maintained their normal aerobic training throughout the 3-wk intervention period. We previously described the metabolic demands (32) and mitogenic responses (42) to a single bout of HIT in well-trained and untrained subjects.
On the morning of the first and seventh HIT sessions, subjects reported to the laboratory between 0600 and 0800 in a fasted state. In the preceding 24 h, subjects had been fed a controlled diet (previously described) and upon arrival at the laboratory consumed a standardized breakfast (described previously). Thirty minutes after breakfast, a resting muscle biopsy was obtained from the vastus lateralis muscle via an incision made under local anesthesia (1% xylocaine; Astra Pharmaceuticals, Sydney, Australia). The sample was rapidly (<10 s) frozen in liquid N2 and stored at 80°C for subsequent analyses. At the same time, a separate site on the same leg (5 cm distal) was prepared for the second biopsy, to be taken immediately after the HIT session. The power output during the seventh HIT session was performed at the same absolute intensity as for the initial experimental trial (i.e., the first HIT session).
Analyses
Muscle metabolites.
Muscle samples (20 mg) were freeze-dried, dissected free from all nonmuscle contaminants, and powdered. An aliquot of freeze-dried muscle was extracted with 0.5 M HCIO4 (1 mM EDTA) and neutralized with 2.2 M KHCO3. The extract was used for the subsequent determination of ATP, creatine, phosphocreatine (PCr), and lactate by use of enzymatic fluorometric methods (19). All metabolite measurements were normalized to the highest total creatine content from the four samples obtained for each subject to correct for nonmuscle contamination. A second aliquot was extracted in 2 N HCl, boiled for 2 h, and neutralized with 0.67 N NaOH. The homogenate was analyzed for glucose units with the use of an enzymatic fluorometric method (23).
Free ADP and AMP concentrations were calculated with the assumption of equilibrium of the adenylate kinase and creatine kinase reactions (6). Free ADP was calculated using the measured ATP, creatine, and PCr values and an estimated H+ concentration (27) and a creatine kinase equilibrium constant of 1.66 x 109. Free AMP concentration was calculated from the estimated free ADP and measured ATP with the adenylate kinase constant of 1.05.
AMPK activity and AMPK subunit protein expression.
Approximately 80 mg of wet muscle 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 pyrophospahte, 10% glycerol, 1% Triton X-100, 10 µg/ml trypsin inhibitor, 2 µg/ml aprotinin, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride). Homogenates were centrifuged at 20,000 g for 25 min. The supernatant was removed and used for the measurement of AMPK subunit expression following the determination of protein content by the Lowry method (Bio-Rad). AMPK1 and -
2 immunoprecipitation and activity assay, as well as ACC expression and phosphorylation, were also measured (described below).
Approximately 5 mg of protein from the supernatants were incubated with the AMPK1 or AMPK
2 antibody-bound protein A beads for 2 h at 4°C. The polyclonal anti-peptide antibodies to AMPK
1 and -
2 were raised to nonconserved regions of the AMPK isoforms
1 [rat 373390, CARHTLDELNPQKSKHQG (human and rat share this peptide sequence)] and
2 [rat 352366, CMDDSAMHIPPALKPH (there is 1 amino acid difference in this peptide sequence between human and rat) and rat 490514, CSAAGLHRPRSSVDSSTAENHSLSG (there are 3 amino acid differences in this peptide sequence between human and rat)]. Immunocomplexes were washed with PBS and suspended in 50 mM Tris (pH 7.5) buffer for AMPK activity assay (4). The AMPK activities in the immune complexes were measured in the presence of 150 µM AMP. Activities were calculated as picomoles of phosphate incorporated into the SAMS peptide (HMRSAMSGLHLVKRR) per minute per milligram (pmol·mg1·min1) of protein subjected to immunoprecipitate. The post-AMPK immunoprecipitaton supernatants were incubated with 2030 µl of streptavidin-Sepharose beads (Amersham Biosciences, Uppsla, Sweden) for 1 h at 4°C to affinity-purify ACC. The beads were then washed in PBS three times. The ACC fraction was electrophoresed on 7.5% SDS-PAGE and detected by immunoblotting with anti-phospho-ACC
-79 (rat 7385, HMRSSMpSGLHLVK) polyclonal antibody followed by horseradish peroxidase (HRP)-conjugated streptavidin (Amersham Pharmacia Biotech UK, Little Chalfont, UK) for total ACC. The local phosphorylation site sequence is conserved in ACC
around Ser221.
The expression level of the AMPK subunit was determined using antibodies raised against AMPK peptides. AMPK1 and -
2 antibodies were described above. AMPK
consensus sequence peptide (rat 257271)-raised antibodies were used for detecting both
1 and
2. AMPK
1 antibody was raised against the peptide (rat 319331, CQALVLTGGEKKP; human and rat share the same sequence in this region). Two hundred micrograms of protein in the supernatant were loaded onto a 12% acrylamide gel, and Western blotting techniques were used. Briefly, proteins on SDS gel were transferred for 2 h at 60 V to PVDF membranes. Membranes were then blocked for 60 min in 5% skim milk in PBS and probed using
1 and
1 antibodies at a dilution of 1:500 in PBS for 60 min. Membranes were then washed three times for 5 min each in PBS and then exposed to the secondary antibody (1:1,000 in PBS) protein G-HRP (Bio-Rad, Hercules, CA) for 60 min and visualized using ECL. Membranes were then stripped for 20 min, washed in PBS, blocked for 60 min as described above, and reprobed for
2 and
2/
1. The densities of immunoblot signals were quantitated using ImageQuant software.
Statistics
The two exercise trials were compared using two-factor repeated-measures analysis of variance (ANOVA) with Statistica software (version 5; Statsoft, Tulsa, OK). Statistical significance was established at the P < 0.05 level. All values are reported as means ± SE. When main effects or interactions reached significance, the Newman-Keuls post hoc statistics were used to determine the location.
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RESULTS |
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Body mass (75.9 ± 4.8 vs. 76.6 ± 4.7 kg) and sum of seven skinfolds (68.2 ± 12.2, vs. 70.2 ± 12.6 mm) did not change as a result of the training intervention.
PPO, Intensity of Training Sessions, and O2 peak
PPO increased (from 362 ± 33 to 376 ± 39 W, P < 0.05) after the first three HIT sessions but did not increase further by the end of the training intervention. Accordingly, the work rate at which the first three HIT sessions were performed was lower than for the final three sessions (292 ± 31 vs. 303 ± 30 W, P < 0.05). O2 peak was not significantly different after the training intervention (4.9 ± 0.3 vs. 5.0 ± 0.3 l/min).
Whole Body Rates of Substrate Oxidation
The average rate of CHO oxidation during exercise at 65% of O2 peak was similar pre- and posttraining intervention (240 ± 5 vs. 238 ± 7 µmol·kg1·min1). Accordingly, the average rate of fat oxidation at this exercise intensity was unchanged by HIT (14 ± 2 vs. 16 ± 2 µmol·kg1·min1). During exercise at 85% of
O2 peak, there was a significant decrease in the rate of CHO oxidation (from 319 ± 9 to 292 ± 9 µmol·kg1·min1, P < 0.05; Fig. 1A) and a concomitant increase (27%) in the rate of fat oxidation (from 17.6 ± 2.6 to 22.4 ± 3.0 µmol·kg1·min1, P < 0.05; Fig. 1B).
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Muscle glycogen and muscle lactate.
Resting (preexercise) muscle glycogen content was unaffected by the HIT intervention (514 ± 36 vs. 484 ± 43 mmol/kg dry wt; Fig. 2A). Glycogen utilization decreased during exercise to a similar extent in both the pre- and the post-HIT trials (253 ± 30 vs. 272 ± 29 mmol/kg dry wt; 50% Fig. 2A). Resting muscle lactate concentration was unaffected by the training intervention (5.9 ± 0.7 vs. 4.1 ± 0.5 mmol/kg dry wt) but was increased in response to exercise (P < 0.05; Fig. 2B). At the end of exercise, muscle lactate was significantly lower post- than preintervention (13.7 ± 2.3 vs. 26.1 ± 4.3 mmol/kg dry wt, P < 0.05; Fig. 2B).
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Muscle nucleotides, creatine, and PCr. Table 1 displays the muscle nucleotides, creatine, and PCr concentrations at rest and at the end of exercise before and after the training intervention. Creatine levels were similar at rest and increased to a similar extent during exercise after training. PCr concentration was not different at rest, and the magnitude of decline during exercise was unaffected by training. Resting PCr/(PCr + Cr) was similar pre- and posttraining intervention, with the exercise-induced decrease being greater before (0.67 ± 0.05 vs. 0.44 ± 0.05; P < 0.05) than after [0.64 ± 0.03 vs. 0.53 ± 0.05, not significant (NS)] training. ATP concentrations were unaffected by exercise or training. ADP content was not different at rest, with the increase in response to exercise being similar before and after training. Resting AMPfree concentrations were not different, but the exercise-induced increase was greater before (0.97 ± 0.36 vs. 3.49 ± 1.05; P < 0.05) than after (0.78 ± 0.18 vs. 2.80 ± 1.20, NS) training intervention. The calculated concentration of cytosolic AMPfree/ATP was similar at rest, but the exercise-induced increase was greater before (0.03 ± 0.01 vs. 0.19 ± 0.05, P < 0.05) than after (0.04 ± 0.01 vs. 0.14 ± 0.06, NS) training.
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DISCUSSION |
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Our first major finding was that exercise at 85% of O2 peak increased AMPK
1 activity both before and after the training intervention. In contrast, by use of a protocol identical to that employed in the present study, it was previously reported that AMPK
1 was not increased in response to a single bout of intense exercise (42). It is difficult to explain the difference in results, since in both studies the subjects were highly trained and followed similar dietary control before the intense bout of exercise. It was originally thought that AMPK
1 activity was increased only during high-intensity (30-s all-out) sprint exercise (3). However, recent reports indicate that AMPK
1 activity is also increased in response to moderate-intensity exercise. By use of a progressive incremental exercise protocol in untrained subjects, a small but significant increase in AMPK
1 has been reported when the intensity increased from 40 to 60% of
O2 peak (5). Increases in AMPK
1 activity during low- to moderate-intensity exercise (5, 20) are in contrast to previous results showing no change in this isoform at similar intensities (8, 39). On the other hand, AMPK
1 protein content is increased in trained subjects (22), suggesting that this subunit may be playing an important role in the adaptation to exercise training. In this regard, the results of a recent study demonstrated that 3 wk of endurance training in previously untrained subjects increased not only
1 but also
2 and
1 protein content (7). In the present study, intensified training in previously well-trained subjects had little effect on the AMPK isoforms that we measured (Fig. 5).
In accord with the results from previous studies, AMPK2 increased in response to exercise (8, 40). We found a lack of attenuation in the rise of AMPK
2 after the 3-wk training intervention during HIT conducted at 85% of (pretraining)
O2 peak. Because training resulted in a twofold decrease in muscle lactate concentration and reduced acidosis during exercise, we anticipated that there might be an attenuation of AMPK
2 activation, since AMPK activity had previously been found to be pH sensitive (26). Our finding that AMPK
2 was not attenuated after training is novel and somewhat surprising given the large change in muscle lactate concentration. It suggests that other factors besides muscle acidosis play an important role in the regulation of AMPK
2 activity during exercise.
In the present study, ACC phosphorylation increased at the end of exercise. This result is in agreement with results of others (31, 42), who have shown similar increases over a wide range of exercise intensities. The increase in ACC
phosphorylation followed a similar pattern to that of AMPK
2 and was not attenuated by exercise training, thus providing further evidence that ACC
phosphorylation and AMPK
2 activity are tightly coupled to the metabolic needs of contracting muscle.
AMPK-regulated ACC activity is important for the regulation of fat metabolism in rodent muscle (21, 24). Phosphorylation of ACC
results in its deactivation and a corresponding disinhibition of carnitine palmitoyltransferase I, thus increasing the potential for mitochondrial fatty acid uptake and oxidation (28). However, the precise processes regulating the substrate oxidation mix (i.e., CHO and fat) in human skeletal muscle are poorly understood. To the best of our knowledge, this is the first investigation to report the effect of intensified exercise training in well-trained subjects on ACC
phosphorylation. Previously, we reported significant increases in whole body rates of fat oxidation after an identical training intervention in well-trained subjects (33). In the present study, rates of fat oxidation were increased during intense (85%
O2 peak), but not moderate (65%
O2 peak), exercise after intensified training. Yet despite a 27% increase in fat oxidation during exercise of a similar intensity to that employed in the HIT sessions, ACC
phosphorylation was not further increased after training. This finding is in agreement with Chen et al. (5), who reported a progressive increase in ACC
phosphorylation in the face of a decrease in fat oxidation as exercise intensity was increased from 40 to 80% of
O2 peak. On the other hand, ACC
phosphorylation has been shown to decrease during prolonged, low-intensity exercise at a time when glucose delivery and glycogen content were low, and muscle FA uptake was increasing (39). These results (39) clearly demonstrate that persistent elevation and activation of AMPK via phosphorylation are not sufficient to maintain elevated ACC
phosphorylation toward the end of prolonged exercise. Collectively, they suggest that there is dissociation between ACC
phosphorylation and fat oxidation in humans during exercise.
In conclusion, we found that 3 wk of intensified training in previously well-trained individuals decreased muscle lactate concentration and increased rates of whole body fat oxidation during intense (85% O2 peak) cycle exercise. Yet despite these training-induced metabolic changes, there was no attenuation of AMPK
1 and -
2 signaling in response to exercise. Furthermore, ACC
phosphorylation was unaffected by training in the face of an increased rate of whole body fat oxidation. Our data provide evidence to demonstrate that regulation by AMPK is functional in skeletal muscle from individuals with a prolonged history of endurance training.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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