Progressive increase in human skeletal muscle AMPKalpha 2 activity and ACC phosphorylation during exercise

T. J. Stephens1,*, Z.-P. Chen2,*, B. J. Canny1, B. J. Michell2, B. E. Kemp2, and G. K. McConell1

1 Department of Physiology, Monash University, Clayton, Victoria 3800; and 2 St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of prolonged moderate-intensity exercise on human skeletal muscle AMP-activated protein kinase (AMPK)alpha 1 and -alpha 2 activity and acetyl-CoA carboxylase (ACCbeta ) 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 (VO2 peak) with muscle biopsies obtained from the vastus lateralis at rest and at 5 and 30 min of exercise. AMPKalpha 1 activity was not altered by exercise; however, AMPKalpha 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. ACCbeta 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 AMPKalpha 2 activity were significantly correlated with both increases in ACCbeta 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 AMPKalpha 2 was associated with both nNOSµ and ACCbeta consistent with them both being substrates of AMPKalpha 2 in vivo. In conclusion, AMPKalpha 2 activity and ACCbeta phosphorylation increase progressively during moderate exercise at ~60% of VO2 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-beta ; neuronal nitric oxide synthase; prolonged exercise; humans


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, AMPKalpha 1 and -alpha 2, expressed in skeletal muscle. Maximal sprint exercise [~200% peak O2 consumption (VO2 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 AMPKalpha 1 and -alpha 2 (6). AMPKalpha 1 is not activated during lower intensity exercise such as at 50 or 70% VO2 peak, or after 5 min of exercise at 90% VO2 peak after 55 min of cycling at 75% VO2 peak (75-90% VO2 peak) (14, 39). AMPKalpha 2 activity is increased after exercise at 70 and 75-90% VO2 peak, but not after exercise at 50% VO2 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% VO2 peak, the level of muscle energy imbalance increases (21), and glucose uptake (4) and fat oxidation (12) increase from rest. If AMPKalpha 2 activity is important for activation of fat oxidation and glucose uptake, one would expect increases in AMPKalpha 2 activity and phosphorylation of acetyl-CoA carboxylase (ACCbeta ) 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 ACCbeta (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 ACCbeta activity and malonyl-CoA content (37). In addition, activation of AMPK via 5'-aminoimidazole-4-carboxyamide-1-beta -D-ribofuranoside (AICAR) causes decreases in ACCbeta 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% VO2 peak (8, 27), but not during exercise at 60-65% VO2 peak (8, 27). However, Dean et al. (8) found decreased ACCbeta activity at 60% VO2 peak as well as at the higher exercise intensities despite no change in malonyl-CoA at 60% VO2 peak. Because ACCbeta activity is reduced at 60% VO2 peak, it is likely that ACCbeta phosphorylation by AMPK is increased at this intensity. We found that ACCbeta 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% VO2 peak increased AMPKalpha 2 activity and ACCbeta 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 ACCbeta activity from 5 to 30 min of treadmill running (37). It is therefore important to determine whether AMPKalpha 2 activity, ACCbeta phosphorylation, and nNOSµ phosphorylation increase progressively during prolonged moderate exercise in humans and whether AMPK is associated with these enzymes in human skeletal muscle.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (VO2 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 VO2 peak test to be equivalent to 60% of their VO2 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 VO2 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. AMPKalpha 1 and -alpha 2 activity, ACCbeta 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 AMPKalpha 1 or AMPKalpha 2 antibody-bound protein A beads for 2 h at 4°C. Immunocomplexes were washed with PBS and suspended in 50 mM Tris (pH 7.5) buffer for AMPK activity assay (7). The polyclonal antipeptide antibodies to AMPKalpha 1 and -alpha 2 were raised to nonconserved regions of the AMPK isoforms -alpha 1 (373-390, CARHTLDELNPQKSKHQG) and alpha 2 (490-516, CSAAGLHRPRSSVDSSTAENHSLSG). There is no cross-reactivity between the polyclonal antibodies (5). The AMPK activities in the immune complexes were measured in either the presence or absence of 150 µM AMP. Activities were calculated as picomoles of phosphate incorporated into the ACCbeta (73-87)A77 peptide per minute per milligram of total protein subjected to immunoprecipitate. Muscle homogenates were also incubated either with immobilized monomeric avidin agarose beads or with 2',5'-ADP Sepharose beads to affinity purify ACCbeta and nNOSµ, respectively. Avidin affinity purification of ACC has been used extensively (8, 35), as has 2',5'-ADP-Sepharose for nNOS isolation (15). Partially purified AMPKalpha 2 does not bind to either immobilized monomeric avidin agarose beads or 2',5'-ADP Sepharose beads (results not shown), and the other biotin-containing enzymes pyruvate carboxylase, methylcrotonyl carboxylase, and proprionyl carboxylase are not known to be substrates for the AMPK. The ACCbeta fraction was subjected to SDS-PAGE, and ACCbeta was detected by immunoblotting with either anti-phospho-ACCbeta -79 polyclonal antibody (6) or horseradish peroxidase-conjugated streptavidin (DAKO, Carpinteria, CA) for total ACCbeta . The nNOSµ fraction was subjected to SDS-PAGE, and nNOSµ was detected by immunoblotting with either antiphospho-nNOSµ-1451 polyclonal antibody (6) or human nNOS antibody (N-31020; Transduction Laboratory, Lexington, KY). In an attempt to determine whether ACCbeta and nNOSµ are associated with AMPKalpha 2 in vivo in humans, the immunoblots for ACCbeta and nNOSµ were stripped and then reimmunoblotted with the AMPKalpha 2-specific antibody (7).

Statistical 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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). AMPKalpha 1 activity measured without added AMP did not change during exercise (Fig. 1A). In contrast, AMPKalpha 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) AMPKalpha 1 and -alpha 2 activity to a similar extent in the rest and exercised muscle samples, and the responses of the AMPKalpha 1 and -alpha 2 isoforms to AMP were similar (Fig. 1). ACCbeta 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 AMPKalpha 2 activity and ACCbeta 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 AMPKalpha 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 AMPKalpha 2 in vivo. We detected AMPKalpha 2 associated with 2',5'-ADP-Sepharose affinity-purified nNOSµ in immunoblots (Fig. 4). This indicated that nNOSµ is associated with AMPKalpha 2 in vivo, supporting the idea that it is a direct substrate. Partially purified AMPKalpha 2 does not bind 2',5'-ADP-Sepharose beads. Similarly, we found that AMPKalpha 2 was associated with avidin affinity-purified ACCbeta (Fig. 4), indicating that ACCbeta is a direct substrate of AMPKalpha 2 in vivo. AMPKalpha 2 is not bound by immobilized monomeric avidin agarose beads. The amount of AMPKalpha 2 associated with either ACCbeta 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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Table 1.   Muscle metabolites during prolonged exercise



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Fig. 1.   AMP-activated protein kinase (AMPK)alpha 1 activity (A) and AMPKalpha 2 activity (B) before and during exercise at 62.8 ± 1.3% of peak O2 consumption (VO2 peak; n = 7). The AMPK activity in the immune complex was measured in either the presence or absence of AMP (150 µM). *Different from rest (P < 0.05); and dagger different from 5 min of exercise (P < 0.05).



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Fig. 2.   Acetyl-CoA carboxylase-beta (ACC) phosphorylation (p-ACC) before and during exercise at 62.8 ± 1.3% VO2 peak (n = 7). A: representative blot; B: mean quantitative densitometric data. *Different from rest (P < 0.05); and dagger different from 5 min of exercise (P < 0.05).



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Fig. 3.   Neuronal nitric oxide synthase (nNOS) phosphorylation (p-nNOS) before and during exercise at 63.0 ± 1.4% VO2 peak (n = 6). A: representative blot; B: mean quantitative densitometric data (P = 0.304).



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Fig. 4.   AMPKalpha 2 association with ACCbeta and nNOSµ. Both ACCbeta and nNOSµ were isolated from extracts by use of avidin agarose and 2'5'-ADP-Sepharose, respectively. After SDS-PAGE, ACCbeta and nNOSµ were detected by immunoblotting as described in METHODS. The same membranes were reimmunoblotted for AMPKalpha 2. Lanes 1 and 2 are from 1 subject, lanes 3 and 4 from a 2nd subject; lanes 1 and 3 are at rest and lanes 2 and 4 after exercise. C, positive control.



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Fig. 5.   Carbohydrate (A; P = 0.123) and fat (B; P = 0.058) oxidation during exercise at 62.8 ± 1.3% VO2 peak (n = 7).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major finding of this study was that there is a large, progressive increase in ACCbeta phosphorylation during moderate-intensity exercise at ~60% VO2 peak in humans, which is coupled with the progressive increase in AMPKalpha 2 activity and fat oxidation. We found that the progressive increase in AMPKalpha 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% VO2 peak) in humans results in an increase in the activity of both skeletal muscle AMPKalpha 1 and -alpha 2 (6). However, in several studies, including this study (Fig. 1A), AMPKalpha 1 activity does not increase during submaximal exercise in humans (14, 39). It has now been shown during exercise in humans that AMPKalpha 1 activity is not increased above the resting levels at 50 (14), ~60 (Fig. 1A), 70 (14), and 75-90% (39) VO2 peak. This suggests that AMPKalpha 1 activation may not be required during prolonged exercise and is not responsible for the increase in ACCbeta phosphorylation in the present study (Fig. 2). Two previous studies in humans have found no increase in AMPKalpha 2 activity during exercise at 50% VO2 peak but an increase at 70 (14), or 75-90% (39) of VO2 peak. Our results indicate that there is a threshold for activation of AMPKalpha 2 that occurs at ~60% VO2 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 AMPKalpha 1 is activated in skeletal muscle. As discussed earlier, we found that both AMPKalpha 1 and -alpha 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 AMPKalpha 1 and -alpha 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 AMPKalpha 1 and -alpha 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 AMPKalpha 2 (36).

Skeletal muscle ACCbeta phosphorylation by AMPK increased progressively during exercise (Fig. 2), in line with the progressive increase in AMPKalpha 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 AMPKalpha 2 and ACCbeta are associated in human skeletal muscle in vivo (Fig. 4), consistent with ACCbeta being a direct substrate for AMPK. Previously, we found that ACCbeta phosphorylation increased ~8.5-fold after a maximal 30-s sprint (6). There is a greater degree of increase in ACCbeta 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 ACCbeta compared with the sprint exercise study (6).

In line with the progressive elevation in ACCbeta 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% VO2 peak, but there is a small (12-17%) reduction during exercise at 85-100% VO2 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 ACCbeta activity in humans is an alternative to measuring malonyl-CoA, because reductions in ACCbeta activity lead to reductions in malonyl-CoA (32). Phosphorylation of ACCbeta at the serine79 AMPK-specific site has been shown to be a potent inhibitor of ACCbeta activity in rat skeletal muscle (36, 38). Indeed, Dean et al. (8) found decreased ACCbeta activity compared with rest after exercise at 60% VO2 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 ACCbeta 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 AMPKalpha 2 activity, which increased progressively, whereas AMPKalpha 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 ACCbeta (36-fold) during exercise in the present study. Nevertheless, we find that nNOSµ is associated with AMPKalpha 2, supporting the view that nNOSµ is a direct substrate for AMPKalpha 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 AMPKalpha 2, but not AMPKalpha 1, activity increased progressively during 30 min of moderate-intensity exercise at ~60% VO2 peak. There was also a large, progressive increase in ACCbeta phosphorylation during exercise, and the increase in AMPKalpha 2 activity correlated with the increase in ACCbeta phosphorylation. Fat oxidation also tended to increase progressively during exercise. After the first 5 min of exercise, the increases in AMPKalpha 2 activity and ACCbeta phosphorylation were more closely related to changes in muscle glycogen content than muscle AMP/ATP levels.


    ACKNOWLEDGEMENTS

We thank the subjects for taking part in this study and also thank Dr. Rodney Snow for technical assistance.


    FOOTNOTES

* 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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