There is evidence that increasing carbohydrate (CHO) availability during exercise by raising preexercise muscle glycogen levels attenuates the activation of AMPKα2 during exercise in humans. Similarly, increasing glucose levels decreases AMPKα2 activity in rat skeletal muscle in vitro. We examined the effect of CHO ingestion on skeletal muscle AMPK signaling during exercise in nine active male subjects who completed two 120-min bouts of cycling exercise at 65 ± 1% V̇o2 peak. In a randomized, counterbalanced order, subjects ingested either an 8% CHO solution or a placebo solution during exercise. Compared with the placebo trial, CHO ingestion significantly (P < 0.05) increased plasma glucose levels and tracer-determined glucose disappearance. Exercise-induced increases in muscle-calculated free AMP (17.7- vs. 11.8-fold), muscle lactate (3.3- vs. 1.8-fold), and plasma epinephrine were reduced by CHO ingestion. However, the exercise-induced increases in skeletal muscle AMPKα2 activity, AMPKα2 Thr172 phosphorylation and acetyl-CoA Ser222 phosphorylation, were essentially identical in the two trials. These findings indicate that AMPK activation in skeletal muscle during exercise in humans is not sensitive to changes in plasma glucose levels in the normal range. Furthermore, the rise in plasma epinephrine levels in response to exercise was greatly suppressed by CHO ingestion without altering AMPK signaling, raising the possibility that epinephrine does not directly control AMPK activity during muscle contraction under these conditions in vivo.
- adenosine monophosphate-activated protein kinase
- acetyl-coenzyme A carboxylase
- glucose uptake
AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that has been implicated as an important regulator of energy metabolism (19, 41). The α-subunit of AMPK contains the catalytic domain that is the mammalian homolog of the yeast metabolic stress-sensing kinase snf1p (19). Both AMPKα1 and -α2 are expressed in skeletal muscle and are activated by phosphorylation at Thr172 by the AMPK kinase LKB1 (13, 32, 33, 44). Once phosphorylated, AMPK can be further activated allosterically by increases in the free AMP-to-ATP ratio (10, 32). In contracting skeletal muscle of animals and humans, the AMPKα2 isoform is predominantly activated (4, 26, 37, 42, 43). The AMPKβ subunit acts as a targeting subunit anchoring both the α- and γ-subunits (16). The β-subunit also contains a glycogen-binding domain that can target the AMPK to glycogen (29).
Initially, it was thought that muscle glycogen levels controlled AMPK activity because low muscle glycogen content in rodents and humans was associated with increased AMPK activation (24, 31, 39, 42). However, although starting exercise with very low muscle glycogen levels appears to result in greater activation of muscle AMPK during exercise (31, 39, 42), there is no effect of muscle glycogen on AMPK activation during exercise when exercise is started with normal or high muscle glycogen levels after short-term exercise training (24). Although it is possible that muscle glycogen levels may partially regulate AMPK during an acute bout of exercise, it has been shown that when purified AMPK is bound to glycogen there is no effect on enzyme activity (28), and thus other factors appear to be involved.
Aside from manipulating muscle glycogen levels, another way to alter carbohydrate (CHO) availability during exercise is to ingest CHO, which raises plasma glucose levels. In yeast, glucose deprivation results in the rapid activation of snf1p (40); similarly, in cultured mammalian cells, glucose deprivation enhances AMPK activation (7, 34). In contrast, high glucose suppresses AMPK activation, as seen with the progressive decreases in basal AMPK activity in rat muscle in vitro with increasing glucose levels from 0 to 3 to 6 to 25 mmol/l (17). On the basis of these findings, it could be speculated that increasing plasma glucose levels by CHO supplementation may attenuate the increases in skeletal muscle AMPK activity during exercise. In humans, CHO ingestion before and during exercise does not alter the exercise-induced increases in AMPKα Thr172 or acetyl-CoA carboxlylase-β (ACCβ) Ser222 phosphorylation during 2 h of exercise in well-trained individuals (8). However, in that study, preexercise CHO ingestion was associated with a significant elevation in blood glucose and insulin levels before exercise commenced (8). Furthermore, the exercise-induced increase in AMPKα Thr172 phosphorylation appeared to be less during exercise with CHO supplementation, although this was not statistically significant (8). No study has examined whether altering glucose levels during exercise affects AMPK signaling in humans.
CHO ingestion during exercise significantly increases skeletal muscle glucose uptake compared with ingesting a placebo solution that contains no CHO (1, 22). Furthermore, CHO ingestion during cycling exercise has no effect on muscle glycogen use during exercise (6, 12, 23). As discussed above, given that AMPK activation during exercise may potentially be regulated by muscle glycogen levels (9, 42), CHO supplementation represents a model in which glucose uptake can be significantly increased without altering muscle glycogen levels. CHO ingestion also raises plasma insulin levels during exercise (22), but this is unlikely to affect muscle AMPK activity, since insulin has no effect on rat skeletal muscle AMPK activity in vitro (2, 14). Similarly, a hyperinsulinemic clamp has no effect on basal skeletal muscle AMPK activity in humans (15).
Aside from glucose, it has also been postulated that skeletal muscle AMPKα2 is regulated by epinephrine, since in the soleus muscle of male mice, phenylephrine, an α-adrenergic agonist, increases AMPKα2 activity (25). Because CHO ingestion attenuates the increase in plasma epinephrine during prolonged exercise (22), it is possible that there is less activation of AMPK during exercise due to this mechanism.
The effect of CHO ingestion during prolonged cycling exercise on the skeletal muscle energy imbalance is small, with most studies showing either no effect or a small significant attenuation of the increase in muscle energy imbalance (23, 35). Because skeletal muscle free AMP (AMPfree) content is the major regulator of AMPK during exercise, if CHO ingestion attenuates the increase in AMPfree, then it may also reduce the activation of skeletal muscle AMPK during exercise. Therefore, the aim of this study was to determine whether CHO ingestion during exercise attenuates the increase in skeletal muscle AMPK activity during exercise in humans. We hypothesized that CHO ingestion would reduce the increase in skeletal muscle AMPK activity during exercise (based on higher plasma glucose concentration, lower plasma epinephrine, and perhaps lower muscle AMPfree).
Nine healthy, active, but not specifically exercise-trained, nonsmoker males provided informed written consent to participate in this study, which was approved by the Human Research Ethics Committee of The University of Melbourne and conducted in accordance with the Declaration of Helsinki. Participant characteristics were 21 ± 1 yr, 72.6 ± 4.0 kg, 1.80 ± 0.03 m, and peak O2 consumption (V̇o2 peak) of 3.51 ± 0.25 l/min, which equated to 48.5 ± 2.6 ml·kg−1·min−1.
Participants were required to attend the laboratory on four separate occasions. The first visit involved a peak pulmonary oxygen consumption test during cycling (V̇o2 peak), followed by a familiarization session. Subjects then returned to the laboratory for two experimental trials, which involved cycling for 120 min at a workload requiring ∼65% V̇o2 peak. Over the 24-h period prior to the experimental trials, all participants were instructed to refrain from drinking alcohol and caffeine and not to perform any formal exercise. Between trials, participants were asked to maintain their regular exercise patterns. To ensure that food intake was similar between each experimental trial, the participants were given a food diary to record all food consumed over the 24 h before the first experimental trial and asked to match as closely as possible this dietary intake over the 24 h before the second experimental trial.
On the morning of each experimental trial, participants reported to the laboratory in the morning (same time for both experimental trials), having fasted overnight. For five participants, a catheter was inserted into an antecubital vein of both forearms. One catheter was used for the infusion of the nonradioactive glucose tracer [6,6-2H]glucose (Cambridge Isotope Laboratories, Cambridge, MA), with the catheter in the contralateral forearm used for blood sampling. The remaining four participants had only one catheter inserted into an antecubital vein for blood sampling. For glucose kinetics measurements, a bolus of 44.3 ± 2.8 μmol/kg of the glucose tracer was administered intravenously immediately before commencement of a 120-min preexercise constant infusion (0.58 ± 0.03 μmol·kg−1·min−1), which was continued throughout the exercise bout. Blood samples were obtained immediately prior to the commencement of the infusion and at 30 and 10 min and immediately prior to the start of exercise. The exercise protocol consisted of cycling for 120 min at 65 ± 1% of V̇o2 peak (143 ± 11 W). During both experimental trials, the comfort of the participants was helped by fan cooling, and they ingested either a CHO solution or an artificially flavored and sweetened placebo. Participants received 8 ml/kg body wt at the start of exercise followed by an additional 2 ml/kg body wt every 15 min of exercise. For the CHO solution, 8% (wt/vol) of dextrose (d-glucose) was added to Kool-Aid (Kraft Foods, Glenview, IL). Kool-Aid is an unsweetened, caffeine-free powder that, when added to water with sugar, provides a flavored drink. For the placebo solution, Equal (NutraSweet, Mt. Prospect, IL), an artificial sweetener containing aspartame and acesulfame potassium, was added to the Kool-Aid in place of dextrose. Each participant received identical volumes of either solution in a single-blinded, randomized, counterbalanced order.
In the experimental trials, blood was sampled every 15 min during exercise for the measurement of plasma glucose, plasma lactate, and glucose kinetics. For plasma catecholamines and insulin, blood was collected every 30 min. All blood samples were centrifuged at 3,000 g for 20 min with plasma stored at −80°C prior to analysis. Expired air was sampled into Douglas bags for ∼15 min at rest and for 3 min each 15 min of exercise. Heart rate was monitored throughout exercise by use of a heart rate monitor (Polar Favor, Oulu, Finland).
Muscle tissue was obtained from the vastus lateralis muscle under local anesthesia by use of the percutaneous needle biopsy technique with suction. Prior to commencement of exercise three separate incisions were made ∼1 cm apart, and muscle was sampled (in a distal-to-proximal order) at rest and immediately following 60 and 120 min of exercise. One leg was used for each of the experimental trials (chosen at random). After the 60-min muscle sample, a standard 60-s period was allowed for completion of the biopsy and taping of the area prior to recommencing exercise. Samples were frozen in liquid nitrogen (LN2) within 6 ± 0 s of inserting the needle at rest and within 9 ± 1 s of the subject stopping exercise (60- and 120-min samples). Muscle samples were subsequently stored in LN2 for later analysis of AMPKα1 and -α2 activity, AMPKα2 Thr172 phosphorylation, ACCβ Ser222 phosphorylation, and muscle metabolites.
Plasma glucose and lactate were determined using an automated glucose oxidase and l-lactate oxidase method, respectively (EML 105; Radiometer Pacific, Melbourne, Australia), 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 were estimated, as previously described (4), using a modified one-pool, non-steady-state model (36) that has been validated (30). Rates of plasma glucose appearance (glucose Ra) and glucose disappearance (glucose Rd) were determined from the changes in percent enrichment of [6,6-2H]glucose and plasma glucose concentration. Glucose clearance rate (glucose CR) was calculated by dividing the glucose Rd by the plasma glucose concentration. During exercise at ∼60% of V̇o2 max, 90–95% of tracer-determined glucose Rd is oxidized (5, 18).
A portion (∼20 mg) of each muscle sample was freeze-dried and then analyzed for muscle glycogen, ATP, creatine phosphate (PCr), creatine (Cr), and lactate, as previously described (4). The estimated free concentrations of ADP and AMP were based on the near-equilibrium nature of the creatine phosphokinase and adenylate kinase reactions, respectively. Free ADP (ADPfree) was estimated from the measured ATP, Cr, and PCr contents, and H+ concentration was estimated using the measured muscle lactate content according to the formula presented by Mannion et al. (21) for dry muscle. The observed equilibrium constant (Kobs) value employed was 1.66 × 109 M (20). AMPfree was estimated from the measured ATP and the estimated ADPfree by using a Kobs of 1.05 (20).
AMPKα1 activity, AMPKα2 activity, and ACCβ phosphorylation at Ser222 were measured as previously described (3, 4), using ∼70 mg of each frozen muscle sample. The polyclonal antipeptide antibodies to AMPKα1 and -α2 were raised to nonconserved regions of the AMPK isoforms α1 (amino acid sequence 373–390 of rat AMPKα1) and α2 (351–366 and 490–516 of rat AMPKα2). 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 [ACCα-(73–87)A77]·min−1·mg total protein−1 subjected to immunoprecipitation. For AMPKα2 Thr172 phosphorylation, AMPKα2 was immunoprecipitated, subjected to SDS-PAGE, and then immunoblotted for anti-phospho-AMPK-Thr172 [with affinity-purified anti-phospho-AMPK Thr172 antibody raised against AMPKα peptide (KDGEFLRpTSCGSPNY)] or anti-AMPKα2 and subsequently detected using an anti-rabbit IRDye 800-labeled secondary antibody (LI-COR Biosciences, Lincoln, NE).
The two experimental trials were compared using two-factor (treatment × time) repeated-measures analysis of variance (ANOVA) utilizing the statistical package SPSS. If the ANOVA was significant (P < 0.05), specific differences were located using the Fisher's least significant difference test.
Pulmonary Gas Measurements and Substrate Oxidation
V̇o2 averaged 65 ± 1% of V̇o2 peak during the placebo trial and 64 ± 2% of V̇o2 peak during the CHO trial. CHO supplementation significantly increased RER and CHO oxidation during exercise compared with placebo (P < 0.05; Table 1). Ventilation and fat oxidation increased similarly during exercise in the two trials, although fat oxidation tended to be reduced during exercise with CHO supplementation (P = 0.09).
Plasma Glucose, Lactate, and Insulin
Plasma glucose, lactate, and insulin concentrations were not different in the two trials before exercise. Plasma glucose concentration was significantly higher in CHO than in placebo throughout exercise (Fig. 1A). Plasma lactate increased to a similar extent in the two trials during exercise but was significantly lower at 120 min of exercise in CHO than in placebo (Fig. 1B). Plasma insulin was significantly higher throughout exercise in the CHO trial (Fig. 1C).
Glucose Ra (Fig. 2A), glucose Rd (Fig. 2B), and glucose CR (Fig. 2C) were all similar at rest in the two trials, and all significantly increased during exercise in both trials. CHO ingestion significantly increased glucose Ra and glucose Rd above that of placebo throughout the exercise period. Glucose CR was significantly higher in CHO than in placebo during the last hour of exercise.
Plasma epinephrine increased during exercise in both trials, but plasma epinephrine was significantly lower at the end of exercise in CHO (P < 0.01; Fig. 3A). Plasma norepinephrine increased significantly over time in both trials (P < 0.001), with no differences observed between the trials (P = 0.65; Fig. 3).
There was no difference between the two trials in any measured muscle metabolite before exercise (Table 2 and Figs. 4 and 5). As shown in Fig. 4, no differences were observed in muscle glycogen levels between CHO and placebo, with both decreasing significantly during exercise. Net muscle glycogen utilization during exercise did not differ between the placebo and CHO trials (399 ± 38 vs. 344 ± 72 mmol/kg dm, respectively, P = 0.45). Muscle PCr and PCr/(PCr + Cr) ratio significantly decreased, and muscle Cr significantly increased, during exercise to a similar extent in the two trials (Table 2). Muscle ATP levels were unchanged by exercise in both trials (Table 2). In line with the plasma lactate results, muscle lactate content increased during exercise in both trials and was significantly lower at 120 min of exercise in CHO compared with placebo (Table 2). Muscle ADPfree increased during exercise in both trials and tended to be lower during the CHO trial; however, this was not statistically significant (P = 0.06). AMPfree and AMPfree/ATP ratio increased during exercise in both trials, with a significant main effect indicating that both AMPfree and AMPfree/ATP ratio were lower in CHO than placebo (P < 0.05).
AMPKα1 activity (Fig. 5A) and AMPKα2 activity (Fig. 5B) increased during exercise performed with or without CHO supplementation, with no differences observed between the trials (P = 0.6 and P = 0.8 for AMPKα1 and -α2 activity, respectively). In line with the response to exercise of AMPKα1 and -α2 activity, AMPKα2 Thr172 phosphorylation (Fig. 6A) and ACCβ Ser222 phosphorylation (Fig. 6B) both increased during exercise in both trials, with no differences observed between the trials.
The major finding of this study was that CHO ingestion during exercise in humans did not alter skeletal muscle AMPK signaling (i.e., AMPKα1 and -α2 activity, AMPKα2 Thr172 phosphorylation, and ACCβ Ser222 phosphorylation) during exercise. This was despite the fact that CHO supplementation significantly increased plasma glucose levels and glucose Rd and also attenuated the exercise-induced increase in calculated muscle AMPfree.
On the basis of the results of Itani et al. (17), we anticipated that AMPK activity would increase less during exercise performed with CHO supplementation compared with placebo because of the higher plasma glucose levels. However, the significant increase in plasma glucose concentration during exercise with CHO ingestion did not influence AMPK activation during exercise. It should be noted, though, that the 1–2 mmol/l (Fig. 1) increase in plasma glucose concentration with CHO ingestion during exercise was confined to the physiological range. In the study by Itani et al., it was found that increasing incubation glucose levels over a larger range from 0 to 3 to 6 to 25 mmol/l in vitro progressively decreased basal AMPK activity in rat muscle.
Before the present study, only one other group had examined the effect of CHO ingestion on AMPK signaling during exercise. In that study (8), the CHO supplementation protocol involved a CHO-rich meal 2 h before exercise, as well as CHO ingestion during the exercise bout. Although not significantly different, the exercise-induced increase in AMPKα Thr172 phosphorylation appeared to be approximately one-half as great in the CHO-treated experiment compared with exercise performed in the fasted state (8). Furthermore, CHO ingestion prior to exercise resulted in significantly higher blood insulin (∼8-fold), blood glucose, RER, and muscle glycogen synthase activity, and lower uncoupling protein-3 mRNA before the commencement of the exercise bout compared with the fasted state (8). Given that these alterations in preexercise physiological status could have affected metabolism during exercise, it was necessary to examine the effects on AMPK signaling under conditions whereby plasma glucose and glucose kinetics were manipulated exclusively during the exercise bout. Therefore, the findings from the present study, as well as those of De Bock et al. (8), suggest that CHO ingestion, either before or during exercise or in combination, is not sufficient to alter skeletal muscle AMPK signaling during exercise.
In mouse soleus muscle, the α-adrenergic agonist phenylephrine increases AMPKα2 activity ex vivo (25), suggesting a possible regulatory role for plasma epinephrine in the regulation of AMPKα2 activity. In the present study, the increases in plasma epinephrine levels became more pronounced (∼6-fold) over time during exercise with a placebo compared with CHO supplementation (Fig. 3). Despite this, no differences were observed in AMPKα2 activity or AMPKα2 Thr172 phosphorylation during exercise, suggesting that in human skeletal muscle epinephrine is not a regulator of AMPKα2 during exercise. Similarly, we (38) also recently found that plasma epinephrine levels did not correlate with AMPK activation during exercise in humans performed under hypoxic conditions compared with during normoxic conditions at the same relative intensity. However, it is possible that only a small increase in plasma epinephrine is required during exercise to exert a regulatory effect on AMPK; thus further research is required to examine the role, if any, of epinephrine in the regulation of AMPK activity during exercise.
In contracting skeletal muscle, the AMPfree/ATP ratio is considered to be the major regulator of AMPK activity (11, 32), as the activity of the upstream kinase (LKB1) is not changed (32). Indeed, a number of studies have observed parallel changes in skeletal muscle AMPfree and AMPKα2 activity during exercise (3, 4, 38). However, there appeared to be some uncoupling between skeletal muscle calculated AMPfree and AMPK activation in the present study. In agreement with this, we have also found that, during 30 min of exercise at 65% V̇o2 peak, there was a progressive increase in AMPKα2 activity but that muscle AMPfree did not change after the first 5 min of exercise (37). In addition, we recently found no increase in AMPK activity during prolonged exercise at ∼60% V̇o2 peak after short-term exercise training despite four- to eightfold increases in AMPfree during exercise (24). It should be noted, however, that, although the increase in skeletal muscle AMPfree during exercise was significantly attenuated with CHO ingestion (Table 2), there was nevertheless an ∼12-fold increase in AMPfree with exercise. Hardie et al. (11) have proposed that AMPK activation involves substantial amplification due to both AMP binding facilitating AMPK kinase (LKB1) phosphorylation and inhibiting the phosphatase (PP2C)-dependent inactivation of AMPK. Thus, where there is sufficient amplification of the AMP signal, a relatively small reduction in AMP levels may not translate into a reduction in the AMPK activity. It is also possible that the apparent uncoupling between AMPfree and AMPK activation may be due merely to assumptions within the AMPfree calculations. As such, it has been suggested that the PCr/(PCr + Cr) ratio is a better indicator of cellular stress (27), and indeed the decreases in PCr/(PCr + Cr) during exercise with placebo and CHO supplementation appeared to couple the increases in AMPK signaling. However, we have also observed a dissociation between changes in PCr/(PCr + Cr) and AMPK signaling during exercise performed after 10 days of exercise training, since substantial changes in PCr/(PCr + Cr) were observed during exercise after training, but no increases in AMPK activity were observed (24).
In conclusion, CHO supplementation during exercise has no effect on the activation of skeletal muscle AMPK during prolonged exercise in humans. This was despite the fact that CHO ingestion partially reduced the exercise-induced increases in skeletal muscle AMPfree. It appears that the modest increase in plasma glucose concentration when CHO is ingested during exercise is insufficient to alter AMPK signaling during exercise. Furthermore, our results in conjunction with previous findings from our laboratory raise the possibility that plasma epinephrine concentration does not regulate skeletal muscle AMPKα2 activity during exercise in humans, since the reduction in plasma epinephrine levels during CHO ingestion did not alter skeletal muscle AMPK activation/signaling.
This work was supported by grants from the National Health and Medical Research Council of Australia (G. K. McConell, B. J. Canny, M. Hargreaves, and B. E. Kemp), Diabetes Australia (G. K. McConell), and the National Heart Foundation of Australia (B. E. Kemp). B. E. Kemp is an Australian Research Council Federation Fellow.
We thank the participants for taking part in this study and also Drs. Rodney Snow and Vince Murone for technical assistance.
Current address for R. Lee-Young: Dept. of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN (e-mail: email@example.com).
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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