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Am J Physiol Endocrinol Metab 290: E694-E702, 2006. First published November 1, 2005; doi:10.1152/ajpendo.00464.2005
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Effect of exercise intensity and hypoxia on skeletal muscle AMPK signaling and substrate metabolism in humans

G. D. Wadley,1 R. S. Lee-Young,1 B. J. Canny,2 C. Wasuntarawat,3 Z. P. Chen,4 M. Hargreaves,1 B. E. Kemp,4 and G. K. McConell1

Departments of Physiology, 1The University of Melbourne, Parkville, Victoria; 2Monash University, Clayton, Victoria; 3Naresuan University, Phitsanulok, Thailand; and 4St. Vincent's Institute and Commonwealth Scientific and Industrial Research Organisation Molecular and Health Sciences, Fitzroy, Victoria, Australia

Submitted 23 September 2005 ; accepted in final form 26 October 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We compared in human skeletal muscle the effect of absolute vs. relative exercise intensity on AMP-activated protein kinase (AMPK) signaling and substrate metabolism under normoxic and hypoxic conditions. Eight untrained males cycled for 30 min under hypoxic conditions (11.5% O2, 111 ± 12 W, 72 ± 3% hypoxia VO2 peak; 72% Hypoxia) or under normoxic conditions (20.9% O2) matched to the same absolute (111 ± 12 W, 51 ± 1% normoxia VO2 peak; 51% Normoxia) or relative (to VO2 peak) intensity (171 ± 18 W, 73 ± 1% normoxia VO2 peak; 73% Normoxia). Increases (P < 0.05) in AMPK activity, AMPK{alpha} Thr172 phosphorylation, ACCbeta Ser221 phosphorylation, free AMP content, and glucose clearance were more influenced by the absolute than by the relative exercise intensity, being greatest in 73% Normoxia with no difference between 51% Normoxia and 72% Hypoxia. In contrast to this, increases in muscle glycogen use, muscle lactate content, and plasma catecholamine concentration were more influenced by the relative than by the absolute exercise intensity, being similar in 72% Hypoxia and 73% Normoxia, with both trials higher than in 51% Normoxia. In conclusion, increases in muscle AMPK signaling, free AMP content, and glucose disposal during exercise are largely determined by the absolute exercise intensity, whereas increases in plasma catecholamine levels, muscle glycogen use, and muscle lactate levels are more closely associated with the relative exercise intensity.

metabolic regulation; glucose kinetics; contraction

AMP-ACTIVATED PROTEIN KINASE (AMPK) is a serine/threonine protein kinase that responds to cellular energy status by inhibiting ATP-consuming pathways and activating ATP-producing pathways (27). The catalytic {alpha}-subunits of AMPK ({alpha}1 and {alpha}2) are expressed in skeletal muscle and are activated allosterically by increased AMP levels following phosphorylation of the upstream kinase LKB1 (17, 27, 48, 53, 58). Skeletal muscle AMPK activity is increased during contractions and exercise in rodents (18, 19, 38, 50) and humans (8, 9, 14, 57).

It has been suggested that skeletal muscle glucose uptake during exercise is regulated by AMPK (53). This is largely based on studies using the nucleoside intermediate 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR). AICAR activates AMPK in rat skeletal muscle and increases glucose uptake (2, 19, 54) and GLUT4 translocation (32). Further support for AMPK in the regulation of glucose uptake during exercise is the finding that AICAR and contraction stimulation of glucose uptake are not additive (2, 19). However, in some circumstances, AMPK activation and glucose uptake during exercise are not tightly coupled. For example, during low-intensity exercise (9, 56) and during moderate-intensity exercise following short-term exercise training (35), skeletal muscle AMPK{alpha}2 activity does not increase despite large increases in glucose disposal during exercise. In addition, AMPK dominant negative mice and AMPK{alpha}2 null mice have partially reduced and normal contraction-stimulated glucose uptake, respectively (24, 37).

Hypoxic exercise may be a useful experimental model to tease out the role of AMPK in skeletal muscle glucose uptake during exercise, as some studies suggest that hypoxia and contractions involve the same pathway to activate skeletal muscle glucose uptake (6). Indeed, both hypoxia and muscle contraction increase glucose uptake in isolated rat muscle, alter muscle energy status, and activate AMPK (18). However, others find that hypoxia has an additive effect on glucose uptake during contractions in perfused rat hindlimbs (12, 13). In addition, hypoxia-stimulated glucose uptake is absent in mice expressing a dominant negative mutant AMPK, but contraction-stimulated glucose uptake is only partially reduced (~50%) in these mice (37). No study has examined the effect of hypoxia on skeletal muscle AMPK activity, muscle energy balance, and glucose disposal during exercise in humans.

It is critical, when comparing metabolism between hypoxic and normoxic exercise, that the intensity be normalized to both the absolute and relative workload, since the maximal rate of oxygen uptake (VO2 peak) is reduced in hypoxia (5, 28, 34). In other words, when exercise is performed under hypoxic conditions at the same absolute workload (with respect to power output) as normoxic conditions, the relative intensity of exercise (with respect to VO2 peak) is much higher compared with normoxic conditions. During normoxic exercise, the activation of skeletal muscle AMPK increases with exercise intensity (8, 9, 14, 57), but it is not known whether this activation is due to the increased absolute and/or relative intensity, since they both increase. Furthermore, the relative importance of absolute compared with relative exercise intensity is not clear for many of the regulators involved in muscle metabolism. The hormonal response to hypoxic exercise, particularly catecholamines, is largely related to the relative rather than the absolute exercise intensity (28, 34). At the same absolute intensity, hypoxic exercise increases glucose uptake (5, 30, 43), muscle glycogen breakdown (40), and plasma and muscle lactate levels (5, 28, 30, 34) and increases the extent of muscle energy imbalance (25, 40, 47). However, it is not known whether the hypoxia effect on these metabolic variables is due to the hypoxic exercise being performed at a higher relative intensity.

Therefore, the first aim of this study was to determine whether the activation of AMPK in human skeletal muscle during hypoxic exercise is more closely related to the absolute or the relative exercise intensity. The second aim was to determine whether the greater glucose disposal, muscle glycogen breakdown, muscle lactate, and muscle energy imbalance during hypoxic exercise are more closely related to the greater absolute or relative exercise intensity.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Participants

Eight active but not specifically trained, nonsmoking, healthy males (23 ± 1 yr, 81 ± 2 kg, 179 ± 1 cm, means ± SE) volunteered for the study. Participants gave written consent after all procedures and possible risks of participation were explained. The experimental protocol and consent form were approved by The University of Melbourne Human Research Ethics Committee and conducted in accordance with the Declaration of Helsinki. Due to technical problems with the glucose tracer infusion, only the glucose kinetics results from six participants are presented. Also, skeletal muscle metabolites were analyzed on seven participants due to a lack of muscle sampled from one participant.

Administration of Hypoxia

An inspired oxygen concentration of 11.5% was chosen on the basis of previous studies in humans that have observed increases in glucose disposal and skeletal muscle lactate and creatine levels and decreases in creatine phosphate (PCr) levels compared with normoxic exercise at the same absolute intensity (25, 30, 47). Inspired air was made hypoxic by continuously combining room air with nitrogen gas at predetermined flow rates into a 150-liter Douglas bag, from which the subject inspired. The inspired gas mixture was continuously sampled and recorded each minute by O2 and CO2 analyzers and the flow rate of nitrogen was adjusted where necessary to maintain the gas mixture at 11.5% (11.52 ± 0.01% O2). Prior to all hypoxic exercise, participants inspired the hypoxic air for 20 min while resting.

Preliminary Testing

Participants first visited the Department of Physiology at The University of Melbourne for determination of VO2 peak under normoxic conditions (inspired O2 20.9%; normoxia VO2 peak) by an incremental exercise test to volitional fatigue on an electromagnetically braked cycle ergometer (Lode, Groningen, The Netherlands). The second visit involved an identical exercise test as performed in the first visit, except that it was performed under hypoxic conditions (inspired O2 11.5%; hypoxia VO2 peak). Expired gas for all preliminary testing and experimental trials was collected into Douglas bags.

VO2 peak and maximum heart rate (HRmax) were both significantly lower for the hypoxia VO2 peak test (VO2 peak 2.34 ± 0.16 l/min, HRmax 181 ± 3 beats/min) compared with the normoxia VO2 peak test (VO2 peak 3.56 ± 0.30 l/min, HRmax 189 ± 2 beats/min), in accord with previous findings (1, 31).

During the third visit, participants completed a familiarization trial, involving cycling for 15 min at 3 sequential workloads (51% Normoxia, 73% Hypoxia, 73% Normoxia), calculated from the VO2 peak tests and separated by ≥20 min of rest. During these bouts, VO2 was measured, and the workloads were adjusted if required to ensure that all workloads were at the desired intensities for each subject prior to the experimental trials.

Experimental Trials

The final three visits to the laboratory involved cycling for 30 min at each of the following: 1) 51% of normoxia VO2 peak (51% Normoxia; 111 ± 12 W, 51 ± 1% normoxia VO2 peak), 2) the same absolute intensity as 51% Normoxia, but under hypoxic conditions (72% Hypoxia; 11.5% inspired O2, 111 ± 12 W, 72 ± 3% hypoxia VO2 peak), or 3) under normoxic conditions at the same relative intensity to 72% Hypoxia (73% Normoxia; 171 ± 18 W, 73 ± 1% normoxia VO2 peak). These were administered in a randomized, counterbalanced order.

For each experimental trial, participants presented to the laboratory overnight fasted and having abstained from exercise, alcohol, and caffeine for 24 h. Dietary records were kept for 2 days before the first experimental trial and replicated (photocopied and given back) for the following two trials. Additionally, participants consumed 1.125 liters (2,385 kJ) of soft drink containing 140 g of carbohydrate each day for 2 days before each trial to ensure that muscle glycogen levels were relatively uniform within each subject before each trial.

One catheter was inserted into an antecubital forearm vein for infusion of a glucose stable isotope tracer ([6,6-2H]glucose; Cambridge Isotope Laboratories, Cambridge, MA) and another into a contralateral forearm vein for blood sampling. A blood sample was obtained, and then a bolus of 44.4 ± 0.6 µmol/kg of the tracer was administered prior to a 140-min preexercise constant infusion (0.73 ± 0.03 µmol·kg–1·min–1), which was continued throughout exercise (Fig. 1). Expired air was collected into Douglas bags for 15 min at rest and for 2–3 min every 10 min of exercise (Fig. 1). Energy expenditure was estimated from indirect calorimetry. HR was monitored throughout exercise (Polar Favor, Oulu, Finland). Rating of perceived exertion (RPE) was assessed using the Borg scale (ranging from 6: very easy to 19: very, very hard) (3).


Figure 1
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  		Fig. 1. Schematic diagram showing the experimental protocol. Blood samples (Blood), Muscle samples (Biopsy), and expired air samples (Air) were collected, and rating of perceived exertion (RPE) and heart rate (HR) were recorded at the prescribed times during the experiment.

 
Blood was sampled at –140, –50, –30, –20, –10, and 0 min and then every 5 min during exercise (Fig. 1). Blood for catecholamines was added to a tube containing EGTA and reduced glutathione. Blood for nonesterified fatty acids (NEFA) was placed in tubes containing EDTA. Blood for glucose, [6,6-2H]glucose enrichment, lactate, glycerol, and insulin was placed in tubes containing lithium-heparin. All blood was spun, and the plasma was stored at –80°C until analysis.

Muscle samples were obtained from the vastus lateralis by the percutaneous needle biopsy technique under local anesthesia. Before commencement of exercise, two separate incisions were made ~1 cm apart, and then muscle was sampled immediately before and after 30 min of exercise (Fig. 1). Muscle samples were frozen in liquid nitrogen while still in the biopsy needle within 4–6 s of needle insertion at rest and within 8–12 s of exercise cessation. The resting-muscle biopsy for the 72% Hypoxia trial was obtained after 20 min of hypoxia administration (–20 to 0 min; Fig. 1). All muscle samples were stored in liquid nitrogen for later analysis.

Analytic Techniques

Indirect calorimetry. Expired O2 and CO2 were measured with zirconia cell and infrared analyzers, respectively (AEI Technologies, Pittsburgh, PA), which were calibrated with standard commercial gases. Gas volume was measured using a dry gas meter (American Meter Vacumed, Ventura, CA) calibrated against a Tissot spirometer.

Blood analysis. Plasma glucose and lactate were determined using the glucose oxidase and L-lactate oxidase methods, respectively (EML105; Radiometer Pacific, Melbourne, Australia), plasma NEFA by an enzymatic colorimetric procedure (NEFA-C test; Wako, Osaka, Japan), plasma glycerol by an enzymatic fluorometric method (10), plasma epinephrine and norepinephrine by an enzyme immunoassay kit (Labor Diagnostika Nord, Nordhorn, Germany), and plasma insulin using a specific human insulin radioimmunoassay kit (Linco Research, St. Charles, MO). Glucose kinetics at rest and during exercise were estimated, as previously described (9), using a modified one-pool, non-steady-state model (49) that has been validated (42). We assumed 0.65 as the rapidly mixing portion of the glucose pool and estimated the apparent glucose space as 25% of body weight. Rates of plasma glucose appearance (Ra) and disappearance (Rd) were determined from the changes in percent enrichment of [6,6-2H]glucose and the plasma glucose concentration. Glucose clearance rate (Glucose CR) was calculated by dividing the glucose Rd by the plasma glucose concentration. The muscles of the legs account for 80–85% of tracer-determined, whole body glucose uptake during exercise at 55–60% VO2 max and probably a greater proportion during more intense exercise (23). During exercise at 50% of VO2 max workload, >95% of tracer-determined glucose uptake is oxidized (23).

Muscle analysis. A portion (~20 mg) of each muscle sample was freeze-dried and then crushed to a powder with any visible connective tissue removed. Muscle glycogen was extracted by incubating the sample in HCl, then NaOH, and then analyzed for glucose units using an enzymatic fluorometric method (41). Muscle metabolites (ATP, PCr, creatine, and lactate) were extracted using the procedure of Harris et al. (16) and analyzed using enzymatic fluorometric techniques (33). Muscle metabolites were corrected to the highest muscle total creatine content for each subject to account for any nonmuscle contamination of the muscle samples. Free ADP and free AMP were calculated as outlined previously (8).

For AMPK{alpha}1 and AMPK{alpha}2 activity measurement, ~70mg of each frozen muscle biopsy sample (non-freeze dried) was homogenized as described previously (9). The homogenates were incubated with AMPK{alpha}1 or AMPK{alpha}2 antibody-bound protein A beads for 2 h at 4°C. Immunocomplexes were washed with PBS and suspended in 50 mM Tris·HCl buffer (pH 7.5) for AMPK activity assay (7). The polyclonal antipeptide antibodies to AMPK{alpha}1 and -{alpha}2 were raised to nonconserved regions of the AMPK isoforms {alpha}1 (373–390 of rat AMPK{alpha}1) and {alpha}2 (351–366 and 490–516 of rat AMPK{alpha}2). The AMPK activities in the immune complexes were measured in the presence of 200 µM AMP. Activities were calculated as picomoles of phosphate incorporated into the SAMS peptide [acetyl-CoA carboxylase (ACC){alpha} (73–87)A77]/min/mg total protein subjected to immunoprecipitate.

ACCbeta was affinity purified from the muscle homogenates by use of monomeric Avidin-agarose beads. Affinity-purified proteins for determination of ACCbeta or 120 µg of total protein for determination of AMPK{alpha} Thr172 phosphorylation were subjected to SDS-PAGE. Binding of ACCbeta was detected by immunoblotting with either anti-phospho-ACCbeta Ser221 polyclonal antibody (8) or IRDye 800-labeled streptavidin (LI-COR Biosciences, Lincoln, NB). AMPK{alpha} Thr172 phosphorylation was detected in the homogenate with affinity-purified anti-phospho-AMPK Thr172 antibody raised against AMPK{alpha} peptide (KDGEFLRpTSCGSPNY). Binding was detected with anti-rabbit IRDye 800-labeled secondary antibody (LI-COR Biosciences). Direct fluorescence was detected and quantified using the Odyssey infrared imaging system (LI-COR Biosciences).

Statistical Analysis

Results were analyzed using two-factor repeated-measures analysis of variance. If this analysis revealed a significant interaction, specific differences between mean values were located using the Fisher's least significance difference test. All data are presented as means ± SE. The level of significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Respiratory Measurements, RPE, and HR

There was no significant effect (P > 0.05) of hypoxia in 72% Hypoxia on resting VO2 (Table 1). During exercise, VO2 and energy expenditure were not significantly different between 51% Normoxia and 72% Hypoxia (Table 1). However, HR and RPE during exercise were significantly higher during 72% Hypoxia compared with 51% Normoxia (P < 0.05; Table 1). Energy expenditure, VO2, and HR were all significantly higher during 73% Normoxia (P < 0.05; Table 1) compared with 72% Hypoxia and 51% Normoxia. However, there were no significant differences between 72% Hypoxia and 73% Normoxia for exercise VO2 and HR when expressed as a percentage of the maximum values obtained during the respective VO2 peak tests (Table 1), nor was RPE significantly different between these two trials (Table 1).


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  		Table 1. Mean physiological responses at rest and during 30 min of exercise under 2 normoxic (inspired room air) conditions and hypoxic (11.5% inspired oxygen) condition

 
Muscle Metabolites

Exposure to hypoxia for 20 min at rest prior to exercise in 72% Hypoxia had no effect on any measured muscle metabolite (Table 2 and Fig. 2). Exercise during 51% Normoxia resulted in significant decreases in muscle PCr, muscle glycogen, and increases in creatine and calculated free ADP levels (P < 0.05; Table 2) but no significant increase in calculated free AMP levels (P > 0.05; Fig. 2). Exercise during 72% Hypoxia resulted in significantly increased creatine, free ADP, free AMP, and decreased PCr from rest (P < 0.05; Table 2 and Fig. 2), but there was no significant difference in free ADP and free AMP between 51% Normoxia and 72% Hypoxia. Exercise during 73% Normoxia resulted in significant increases in creatine, free ADP, free AMP, and decreased PCr from rest that were significantly greater than in the other two trials (P < 0.05; Table 2 and Fig. 2). There was a main effect for exercise on skeletal muscle free AMP-to-ATP ratio (P < 0.05; Table 2). Muscle lactate levels were not significantly altered from rest following exercise at 51% Normoxia (P > 0.05; Table 2). However, muscle lactate increased significantly, whereas muscle glycogen decreased significantly following exercise at 72% Hypoxia and 73% Normoxia (P < 0.05; Table 2), with no significant difference between these two trials (P > 0.05; Table 2). The changes in muscle lactate and muscle glycogen with exercise in 72% Hypoxia and 73% Normoxia were greater than those observed in 51% Normoxia (P < 0.05; Table 2). Muscle glycogen utilization also tended to be greater in 72% Hypoxia and 73% Normoxia than in 51% Normoxia (105.2 ± 35.4, 253.6 ± 52.5, 259.0 ± 37.7 mmol/kg dry wt, 51% Normoxia, 72% Hypoxia and 73% Normoxia, respectively, P = 0.06). Muscle ATP levels were unaltered following exercise in all trials (P > 0.05; Table 2).


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  		Table 2. Muscle metabolites at rest and immediately following 30 min of exercise under the 2 normoxic conditions and the hypoxic condition

 

Figure 2
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  		Fig. 2. Calculated free AMP levels at rest and following 30 min of exercise under the 2 normoxic (inspired room air) conditions and the hypoxic (11.5% inspired oxygen) condition. Values are means ± SE; n = 7. Open bars, rest; filled bars, exercise. *P < 0.05 vs. rest; {dagger}P < 0.05 vs. 51% Normoxia; {ddagger}P < 0.05 vs. 72% Hypoxia.

 
Muscle AMPK Activity, AMPK{alpha} Thr172 Phosphorylation, and ACCbeta Ser221 Phosphorylation

The administration of hypoxia during the rest period prior to exercise in 72% Hypoxia did not alter either AMPK{alpha}1 or AMPK{alpha}2 activity (P > 0.05; Fig. 3, A and B, respectively). Exercise significantly increased AMPK{alpha}1 activity from rest only during exercise at 73% Normoxia (P < 0.05; Fig. 3A), and at the end of exercise AMPK{alpha}1 activity was significantly higher at 73% Normoxia than at 51% Normoxia and 72% Hypoxia (P < 0.05; Fig. 3A). There was no significant increase in AMPK{alpha}2 activity (Fig. 3B) or AMPK{alpha} Thr172 phosphorylation (Fig. 4A) following 51% Normoxia. Also, similar to the findings for free AMP, there was no significant difference in AMPK{alpha}2 activity or AMPK{alpha} Thr172 phosphorylation between 51% Normoxia and 72% Hypoxia following exercise (Figs. 3B and 4A, respectively). However, AMPK{alpha}2 activity and AMPK{alpha} Thr172 phosphorylation increased with exercise in 72% Hypoxia (P < 0.05; Figs. 3B and 4A, respectively). There was a significantly greater increase in AMPK{alpha}2 activity and AMPK{alpha} Thr172 phosphorylation during exercise in 73% Normoxia compared with the other trials (P < 0.05; Figs. 3B and 4A, respectively). Also, exercise significantly increased ACCbeta Ser221 phosphorylation (P < 0.05; Fig. 4B) from rest during all trials, with the 73% Normoxia trial being significantly greater than the other two trials (P < 0.05; Fig. 4B).


Figure 3
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  		Fig. 3. AMP-activated protein kinase (AMPK){alpha}1 activity (A) and AMPK{alpha}2 activity (B) at rest and following 30 min of exercise under the 2 normoxic (inspired room air) conditions and the hypoxic (11.5% inspired oxygen) condition. Values are means ± SE; n = 8. Open bars, rest; filled bars, exercise. *P < 0.05 vs. rest; {dagger}P < 0.05 vs. 51% Normoxia; {ddagger}P < 0.05 vs. 72% Hypoxia.

 

Figure 4
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  		Fig. 4. AMPK{alpha} Thr172 phosphorylation (A) and acetyl-CoA carboxylase (ACC)beta Ser221 phosphorylation (B) at rest and following 30 min of exercise under the 2 normoxic (inspired room air) conditions and the hypoxic (11.5% inspired oxygen) condition. Western blots are representative from 1 subject. Values are means ± SE; n = 8, expressed relative to ACCbeta protein content. Open bars, rest; filled bars, exercise. *P < 0.05 vs. rest; {dagger}P < 0.05 vs. 51% Normoxia; {ddagger}P < 0.05 vs. 72% Hypoxia.

 
Plasma Glucose, Lactate, Catecholamines, Insulin, Glycerol, and NEFA

Twenty minutes of hypoxia at rest in 72% Hypoxia did not alter any measured blood parameter. Plasma glucose concentration increased significantly during exercise in 72% Hypoxia (P < 0.05; Fig. 5A) but did not change significantly from rest in either normoxia trial. Plasma lactate, glycerol, epinephrine, and norepinephrine concentrations increased significantly during exercise in all trials (P < 0.05, respectively; Figs. 5, B and C and 6, A and B) and were significantly lower during 51% Normoxia compared with the other trials. In general plasma lactate, glycerol, epinephrine, and norepinephrine concentrations were not significantly different between 72% Hypoxia and 73% Normoxia (except for one time point in plasma lactate and norepinephrine) (P < 0.05, respectively; Figs. 5, B and C and 6, A and B). Plasma NEFA concentration decreased significantly during exercise, with no significant difference between trials (P < 0.05, data not shown). Plasma insulin concentration did not change significantly with exercise in any trial (P > 0.05, data not shown).


Figure 5
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  		Fig. 5. Plasma glucose (A), lactate (B), and glycerol levels (C) during 30 min of exercise under the 2 normoxic (inspired room air) conditions and the hypoxic (11.5% inspired oxygen) condition. Values are means ± SE; n = 6 for plasma glucose and n = 8 for plasma lactate and glycerol. {dagger}P < 0.05 vs. 51% Normoxia; {ddagger}P < 0.05 vs. 72% Hypoxia.

 

Figure 6
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  		Fig. 6. Plasma epinephrine (A) and norepinephrine (B) levels during 30 min of exercise under the 2 normoxic (inspired room air) conditions and the hypoxic (11.5% inspired oxygen) condition. Values are means ± SE; n = 8. {dagger}P < 0.05 vs. 51% Normoxia; {ddagger}P < 0.05 vs. 72% Hypoxia.

 
Glucose Kinetics

The administration of hypoxia during the rest period in 72% Hypoxia did not alter glucose Ra, glucose Rd, or glucose CR (P > 0.05; Fig. 7). Glucose Ra was significantly higher toward the end of exercise in 72% Hypoxia compared with 51% Normoxia (P < 0.05; Fig. 7A) with both trials significantly lower than 73% Normoxia (P < 0.05; Fig. 7A). Glucose Rd showed a similar pattern to glucose Ra and was significantly higher following 25 min of cycling (P < 0.05; Fig. 7B) and tended to be higher at the end of exercise in 72% Hypoxia compared with 51% Normoxia (P > 0.05; Fig. 7B), with both trials significantly lower than 73% Normoxia (P < 0.05; Fig. 7B). Glucose CR was similar during exercise in 72% Hypoxia and 51% Normoxia (P > 0.05; Fig. 7C) with both trials significantly lower than 73% Normoxia (P < 0.05; Fig. 7C).


Figure 7
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  		Fig. 7. Glucose rate of appearance (Ra; A), glucose rate of disappearance (Rd; B), and glucose clearance rate (CR; C) during 30 min of exercise under the 2 normoxic (inspired room air) conditions and the hypoxic (11.5% inspired oxygen) condition. Values are means ± SE; n = 6. {dagger}P < 0.05 vs. 51% Normoxia; {ddagger}P < 0.05 vs. 72% Hypoxia.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of this study was that the activation of skeletal muscle AMPK during exercise was dictated predominantly by the absolute rather than the relative exercise intensity. At the same relative intensity (with respect to VO2 peak), the present study observed significantly higher activation of AMPK during normoxia than during hypoxia (73% Normoxia vs. 72% Hypoxia), and this matched the increases in skeletal muscle free AMP levels. This can probably best be explained in terms of ATP turnover between the two trials, since energy expenditure (and, hence, ATP turnover) was ~50% higher during normoxia compared with hypoxia at the same relative intensity (Table 1). The second major finding of this study was that glucose Rd during exercise was also largely dictated by the absolute rather than the relative exercise intensity. On the other hand, muscle glycogen breakdown and increases in muscle lactate and plasma catecholamine levels were dictated by the relative rather than the absolute exercise intensity.

The ACCbeta Ser221 phosphorylation data reinforce the finding that there was no greater AMPK activation during hypoxic exercise compared with exercise at the same absolute intensity in normoxia (72% Hypoxia vs. 51% Normoxia). Although there are some situations during exercise when AMPK activity and ACCbeta Ser221 phosphorylation are not tightly coupled (44, 56), ACCbeta Ser221 phosphorylation is generally considered a sensitive indicator of skeletal muscle AMPK activity in vivo (9, 39). Because allosteric regulators are lost during the assay, the AMPK activity assay in essence indicates the extent of AMPK phosphorylation by the upstream kinase LKB1 but not in vivo activity that is subject to additional allosteric activation by AMP. Indeed, the AMPK{alpha} Thr172 phosphorylation data very closely matched the AMPK activity data in the present study (Figs. 3B and 4A). The ACCbeta Ser221 phosphorylation data suggest that there may have been increases in AMPK activity during 51% Normoxia exercise in vivo. Similarly, we have previously reported that ACCbeta Ser221 phosphorylation increases during low-intensity exercise at 40% VO2 peak despite there being no detectable increase in AMPK activity (9), as has been observed during low-intensity running in rodents (39). The ACCbeta Ser221 phosphorylation data, as well as the AMPK activity and AMPK{alpha} Thr172 phosphorylation data, all clearly show that there was greater AMPK activation during the 73% Normoxia trial than during the 51% Normoxia and 72% Hypoxia trials, indicating the importance of the absolute intensity over the relative intensity in the activation of skeletal muscle AMPK during exercise.

Hypoxic exercise at the same absolute intensity as normoxic exercise has been shown previously to augment the increases in glucose Ra during exercise (5, 30, 43), and the findings of the present study support this (51% Normoxia vs. 72% Hypoxia; Fig. 7A). The greater glucose Ra during hypoxic exercise at the same absolute workload could be due to a direct effect of hypoxia on the liver (45) or to the higher plasma epinephrine levels. Epinephrine infusion during exercise in humans to levels above those seen in the present study has been shown to increase glucose Ra during exercise in some (21, 29) but not all studies (51, 52). Although several regulatory mechanisms, such as higher plasma epinephrine concentration or hypoxia per se, could be increasing glucose Ra during exercise in 72% Hypoxia compared with 51% Normoxia, the major influence on glucose Ra appears to be the absolute workload, since glucose Ra was the highest during exercise in 73% Normoxia (Fig. 7A).

Although hypoxic exercise has been shown previously to increase glucose disposal compared with normoxic exercise at the same absolute intensity, it is has until now been unclear whether this was due to the hypoxia per se or to the higher relative exercise intensity. In accord with most (5, 30, 43) but not all (4) previous research, we found a significant increase in glucose Rd after 25 min of exercise in 72% Hypoxia compared with 51% Normoxia (Fig. 7B). There are several potential stimulatory and inhibitory influences on glucose Rd during exercise in hypoxic conditions that make it difficult to tease out likely mechanism(s) for the greater glucose Rd in 72% Hypoxia than in 51% Normoxia. In terms of stimulatory effects, the higher plasma glucose concentration, and therefore the higher glucose concentration gradient, would be expected to increase glucose disposal into skeletal muscle (60). Supporting this, we observed no difference in glucose CR between 72% Hypoxia and 51% Normoxia, suggesting that the greater glucose Rd might have been due to the higher plasma glucose levels per se. Hypoxia has also been shown to increase blood flow during exercise (30, 46), which increases glucose delivery to the muscle and would be expected to increase glucose disposal (20). In terms of inhibitory effects, the greater muscle glycogen use during exercise may have raised glucose 6-phosphate content (26, 40), which would be expected to inhibit glucose uptake into muscle during exercise. In addition, the higher plasma epinephrine concentrations may have an inhibitory effect on glucose disposal, since infusion of epinephrine during moderate-intensity exercise inhibits glucose disposal (22, 51, 52) independently of muscle glucose 6-phosphate levels (51). Because plasma insulin and NEFA levels were similar during exercise in all trials, it is not likely that they influenced glucose disposal. Regardless of the mechanism for the increased glucose Rd during exercise in 72% Hypoxia compared with 51% Normoxia, the increase was modest compared with the increase in glucose Rd observed during 73% Normoxia, highlighting the importance of the absolute workload over the relative workload in the regulation of glucose Rd during exercise.

AMPK has been proposed as an important regulator of contraction-stimulated glucose uptake. This is supported by recent studies in LKB1 knockout mice that have close to abolished increases in contraction-stimulated glucose uptake (48). In support of this, in the present study the response of glucose Rd during the three exercise trials was similar to the response observed in skeletal muscle AMPK activation (AMPK{alpha}2 activity, AMPK{alpha} Thr172 phosphorylation, and ACCbeta Ser221 phosphorylation). However, other studies suggest that AMPK may mediate only a component of the exercise-induced increase in skeletal muscle glucose uptake. AMPK dominant negative mice, which have reduced skeletal muscle AMPK{alpha}1 and -{alpha}2 activity, have only partially reduced contraction-stimulated glucose uptake (37), and AMPK{alpha}1 and -{alpha}2 null mice have essentially normal contraction-stimulated glucose uptake (24). In addition, in humans, leg glucose uptake is increased from rest during low-intensity exercise despite there being no increase in AMPK{alpha}2 activity (56), and we have found that there is substantial glucose disposal during exercise following short-term training despite there being no activation of AMPK during exercise after training (35). Therefore, the importance of AMPK in the regulation of glucose uptake during exercise remains uncertain.

Potential regulators of AMPK during exercise include free AMP content (27, 53), muscle glycogen levels (11, 55), and plasma epinephrine levels (36). The present study found that changes in AMPK{alpha}2 activity during exercise matched the changes in free AMP levels (Figs. 2 and 3B). The increases in calculated free AMP levels in the present study were largely related to the absolute workload, since energy expenditure (and therefore ATP turnover) was ~50% higher following 73% Normoxia compared with the other two trials (72% Hypoxia and 51% Normoxia; Table 1). AMPK was activated to a greater extent during 73% Normoxia than during 72% Hypoxia (Fig. 3) despite similar levels of net muscle glycogen use between the two trials (Table 2). Furthermore, at the same absolute intensity (51% Normoxia vs. 72% Hypoxia), there was no significant difference between AMPK{alpha}1 or AMPK{alpha}2 activity (Fig. 3) despite significantly different levels of net muscle glycogen use (Table 2). These findings indicate that when preexercise glycogen levels are similar the rate of muscle glycogen use does not directly regulate the extent of AMPK activation during exercise in humans. Minokoshi et al. (36) found that incubation of rodent skeletal muscle with the {alpha}-adrenergic agonist phenylephrine increased AMPK activity. However, in the present study we observed almost identical increases in plasma epinephrine levels during hypoxic and normoxic exercise at the same relative intensity (72% Hypoxia vs. 73% Normoxia; Fig. 6A), but AMPK signaling was much greater in 73% Normoxia than in 72% Hypoxia (Figs. 3, A and B and 4, A and B), suggesting that plasma epinephrine is not a major regulator of AMPK activity during exercise in humans.

Hypoxia potently activates AMPK in noncontracting isolated rodent skeletal muscle (18, 59), although the same effect is not apparent in vivo in rats under resting conditions (15). The findings of the present study in human skeletal muscle support these rodent in vivo findings (15), since exposure to hypoxia at rest did not alter skeletal muscle AMPK{alpha}1 activity, AMPK{alpha}2 activity, AMPK{alpha} Thr172 phosphorylation, or ACCbeta Ser221 phosphorylation, nor were there any changes in the allosteric AMPK regulators such as the calculated free AMP levels or the PCr content. At rest, the lower arterial blood oxygen content (46) induced by hypoxia probably does not cause hypoxia at the muscle level, since leg muscle oxygen consumption is not reduced at rest during hypoxia in humans (46).

In conclusion, we have demonstrated, by manipulating exercise workload and hypoxia, that the absolute exercise intensity is more important than the relative exercise intensity in terms of both activation of skeletal muscle AMPK and the increases in glucose disposal during exercise. When normoxic and hypoxic exercise was performed at the same relative intensity (to VO2 peak, 72% Hypoxia vs. 73% Normoxia), AMPK activity, AMPK{alpha} Thr172 phosphorylation, ACCbeta Ser221 phosphorylation, glucose Ra, glucose Rd, and glucose CR were all significantly higher in normoxia, concomitant with a greater muscle energy imbalance as indicated by higher free AMP levels and lower PCr levels. Finally, the present study indicates that, even though hypoxia at the same relative exercise intensity increases catecholamines and muscle glycogen use to the same extent as normoxic exercise, the degree of AMPK activation is less, indicating that catecholamine release and muscle glycogen use during exercise are not direct determinants of AMPK signaling during exercise.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the National Health and Medical Research Council of Australia, Diabetes Australia, the Australian Research Council (ARC), and the National Heart Foundation of Australia. C. Wasuntarawat was supported by a Research Fellow Grant from the Collaborative Research Network in Physiology, Ministry of Education, Thailand. B. E. Kemp is an ARC Federation Fellow.


   ACKNOWLEDGMENTS
 
We thank the participants for taking part in this study and also Dr. Rodney Snow, Dr. Domenic Caredi, Vince Murone, and Kelly Linden for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. McConell, Dept. of Physiology, Univ. of Melbourne, Parkville, 3010 Australia (e-mail: mcconell{at}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.


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