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Am J Physiol Endocrinol Metab 292: E715-E722, 2007. First published October 31, 2006; doi:10.1152/ajpendo.00380.2006
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AS160 phosphorylation is associated with activation of {alpha}2beta2{gamma}1- but not {alpha}2beta2{gamma}3-AMPK trimeric complex in skeletal muscle during exercise in humans

Jonas T. Treebak, Jesper B. Birk, Adam J. Rose, Bente Kiens, Erik A. Richter, and Jørgen F. P. Wojtaszewski

Department of Human Physiology, Institute of Exercise and Sport Sciences, Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark

Submitted 31 July 2006 ; accepted in final form 31 October 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We investigated time- and intensity-dependent effects of exercise on phosphorylation of Akt substrate of 160 kDa (AS160) in human skeletal muscle. Subjects performed cycle exercise for 90 min (67% VO2 peak, n = 8), 20 min (80% VO2 peak, n = 11), 2 min (110% of peak work rate, n = 9), or 30 s (maximal sprint, n = 10). Muscle biopsies were obtained before, during, and after exercise. In trial 1, AS160 phosphorylation increased at 60 min (60%, P = 0.06) and further at 90 min of exercise (120%, P < 0.05). {alpha}2beta2{gamma}3-AMP-activated protein kinase (AMPK) activity increased significantly to a steady-state level after 30 min, whereas {alpha}2beta2{gamma}1-AMPK activity increased after 60 min of exercise with a further significant increase after 90 min. {alpha}2beta2{gamma}1-AMPK activity and AS160 phosphorylation correlated positively (r2 = 0.55). In exercise trials 2, 3, and 4, {alpha}2beta2{gamma}3-AMPK activity but neither AS160 phosphorylation nor {alpha}2beta2{gamma}1-AMPK activity increased. Akt Ser473 phosphorylation was unchanged in all trials, whereas Akt Thr308 phosphorylation increased significantly in trial 3 and 4 only. These results show that AS160 is phosphorylated in a time-dependent manner during moderate-intensity exercise and suggest that {alpha}2beta2{gamma}1- but not {alpha}2beta2{gamma}3-AMPK may act in a pathway responsible for exercise-induced AS160 phosphorylation. Furthermore, we show that AMPK complexes in skeletal muscle are activated differently depending on exercise intensity and duration.

Akt; Akt substrate of 160 kilodaltons; adenosine 5'-monophosphate-activated protein kinase

EXERCISE IS A POTENT STIMULUS for increasing glucose uptake in skeletal muscle. Although exercise, like insulin, increases the glucose transport capacity at the plasma membrane by recruitment of GLUT4 glucose transporters, the events eliciting this effect are still unclear (15). Both feed-forward Ca2+-dependent (e.g., Ca2+/calmodulin protein kinase) signals and metabolic feedback-dependent [e.g., AMP-activated protein kinase (AMPK)] mechanisms have been hypothesized to be involved (32). It is apparent that recruitment of GLUT4 to the plasma membrane occurs via different proximal signaling pathways when comparing the effects of insulin and exercise. However, it is still a working hypothesis that, at some distal point, common effectors are involved in response to these two stimuli. Interestingly, one such effector may be the recently discovered Akt substrate of 160 kDa (AS160). AS160 links insulin signaling and GLUT4 trafficking (7, 18, 27, 36, 48) and has been characterized as a GTPase-activating protein (GAP) containing a GAP homology domain in the COOH-terminal part of the protein. Thus AS160 is thought to regulate the GTPase activity of certain Rab proteins to inhibit GLUT4 vesicle movement to, and/or fusion with, the plasma membrane (27, 29). Phosphorylation of AS160 on one or more of the six known phosphorylation sites by Akt inhibits its GAP activity such that, in response to insulin, the GTP form of Rab proteins is elevated, leading to an increased number of GLUT4 molecules in the plasma membrane. In patients with type 2 diabetes mellitus, AS160 phosphorylation is impaired in response to insulin stimulation (20), implying a role for AS160 in insulin-stimulated glucose uptake. More conclusive data obtained from murine skeletal muscle have been published recently showing that, in tibialis anterior muscles expressing a mutated form of AS160 in which four phosphorylation sites on AS160 are mutated, insulin-stimulated glucose uptake is reduced by 33% (24).

AMPK is a heterotrimeric serine/threonine protein kinase consisting of three subunits of which one ({alpha}) is catalytic and two (beta, {gamma}) have regulatory functions. Multiple mammalian isoforms of the three subunits exist. Two {alpha}-subunits ({alpha}1, {alpha}2), two beta-subunits (beta1, beta2) and three {gamma}-subunits ({gamma}1, {gamma}2, and {gamma}3) have been characterized. However, only three AMPK complexes ({alpha}1beta2{gamma}1, {alpha}2beta2{gamma}1, and {alpha}2beta2{gamma}3) appear to exist in human skeletal muscle (44). AMPK is allosterically activated by AMP in response to an increase in the AMP-to-ATP ratio, and the binding of AMP to AMPK {gamma}-subunits makes AMPK a better substrate for upstream kinases that phosphorylate {alpha}-AMPK on Thr172 in the activation loop and a worse substrate for protein phosphatases (5, 12). AMPK has been shown to be activated in skeletal muscle by acute exercise in both rodents (17, 31, 43) and humans (8, 46), and exercise intensity seems to play an important role for the activity of AMPK as exercise at 60–75% of VO2 peak appears to be necessary for AMPK activation in humans (8, 38, 46). At these intensities, complexes containing {alpha}2-AMPK are regulated to a higher extent than complexes containing {alpha}1-AMPK. Exercise at a more moderate intensity (~50% of VO2 peak) does not elicit any detectable AMPK activity except if exercise is continued until exhaustion (45).

There is now evidence that AS160 may also be targeted by AMPK in skeletal muscle of rodents as 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an activator of AMPK, is able to induce phosphorylation of AS160 in epitrochlearis muscles from rats and extensor digitorum longus muscles from mice (3, 23, 40). Furthermore, skeletal muscle AS160 can be phosphorylated in response to contractions in vitro, and complexes of AMPK containing the {alpha}2-subunit but not the {gamma}3-subunit appear to be mandatory for complete contraction-induced AS160 phosphorylation (23, 40). Both AICAR and contraction increase glucose transport in rodent muscle, and the possibility therefore exists that AS160 is also, like insulin, a mediator for these stimuli to increase GLUT4 recruitment to the plasma membrane. Indeed, contraction-induced glucose transport was recently found to be partially impaired in tibialis anterior muscle expressing an AS160 mutant, unable to be phosphorylated (24). Thus, in accordance with a role for AS160 in insulin-stimulated glucose transport, AS160 may also partially mediate contraction-induced glucose transport.

Recent papers have shown that AS160 can be phosphorylated in human skeletal muscle in response to both insulin (2, 19–21) and moderate-intensity (~70% of VO2 peak) exercise (6). However, for AS160 to play a role in glucose uptake in human skeletal muscle during moderate-intensity exercise, we hypothesize AS160 to be phosphorylated in a time-dependent manner corresponding that of glucose uptake (11, 22). In addition, based on our observations in mouse muscle (40), we hypothesize that regulation of AS160 would be associated with {alpha}2beta2{gamma}1-AMPK trimer activation and not {alpha}2beta2{gamma}3. To answer some of these questions, we conducted four different exercise experiments investigating the time- and intensity-dependent phosphorylation of AS160 in healthy human skeletal muscle, and we measured phosphorylation of Akt and activity of the three different trimer complexes of AMPK in an attempt to correlate these activities with AS160 phosphorylation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Experimental protocol. This study is comprised of two experiments which in the following will be referred to as "the time course study" and "the intensity study." Both experiments conformed to the Declaration of Helsinki and were approved by the ethics committee of Copenhagen and Frederiksberg (nos. KF01277313 and KF11149/99).

The time course study. Eight healthy young men [25 ± 1 yr, VO2 peak 55 ± 1 ml·min–1·kg–1, body mass index (BMI) 22.8 ± 0.4 kg/m2, mean ± SE] gave their written informed consent to participate in the study. A detailed description of the experimental protocol and the participants in this study has been given previously (33). Subjects were rested in the supine position for 1 h after which a resting biopsy was taken from the vastus lateralis muscle. Next, subjects initiated cycle exercise for 90 min with a workload corresponding to 67 ± 2% of their VO2 peak. After 1, 10, 30, 60, and 90 min of exercise, biopsies were taken under local anesthesia (2–3 ml of 2% Lidocaine) and frozen in liquid nitrogen. Samples were afterwards stored at –80°C.

The intensity study. Thirty subjects (26 ± 1 yr, VO2 peak 52 ± 1 ml·min–1·kg–1, BMI 23.9 ± 0.4 kg/m2, mean ± SE) gave their written informed consent and were randomly assigned to one of three exercise interventions. They all performed one test trial before the experiment. After 1 h of rest, a needle biopsy from the vastus lateralis muscle was obtained. The subjects then performed cycle exercise in accordance to one of the following three protocols: 1) 11 subjects performed 20 min of cycling at 80% VO2 peak (77 ± 3% VO2 peak, work rate = 222 ± 8 watts, total work performed ~266 kJ); 2) 9 subjects performed a 2-min cycle test at a work rate (376 ± 18 watts) corresponding to 110% of peak work rate, which was defined as the highest work intensity maintained for a whole minute during an incremental VO2 peak test. Within the first 30 s, the subject increased the pedal frequency to the range of 100–120 min–1 before resistance was applied to the bike (total work performed ~43 kJ). When occurring, fatigue quickly developed into a state where pedal frequency dropped markedly, at which time the test was terminated; 3) 10 subjects performed a 30-s "all out" sprint exercise trail. Without resistance on the bike, the subject increased the pedal frequency to ~140 ± 8 min–1, and, after 10 s, a workload corresponding to 7.5 N/kg body wt was applied. On average, the test lasted 30.5 ± 0.5 s, and in this period the average work rate was 666 ± 59 W (total work performed ~20 kJ). Independent of exercise protocol, the subject was placed in the supine position immediately after exercise, and a second biopsy was obtained from the vastus lateralis muscle. One incision was made in each leg, and the pre- and postexercise biopsies were randomly taken in the dominant and the nondominant leg. The biopsies were frozen in liquid nitrogen within 15 s after the termination of the exercise. The biopsies were stored at –80°C.

Muscle homogenate and lysate preparation. All materials were purchased from Sigma-Aldrich unless otherwise stated. For extraction of tissue protein for analyses, a portion of the muscle biopsy from each sample was taken and freeze-dried, after which the tissue was dissected free of connective tissue and blood. The samples were then homogenized in ice-cold buffer [10% glycerol, 20 mM sodium pyrophosphate, 150 mM NaCl, 50 mM HEPES (pH 7.5), 1% Nonidet P-40, 20 mM beta-glycerophosphate, 10 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM sodium orthovanadate, and 3 mM benzamidine, pH 7.5] for 20 s using a homogenizer (PT 3100; Brinkman Instruments). Homogenates were rotated end-over-end for 1 h at 4°C, and part of the homogenate was then subjected to centrifugation (17,500 g, 20 min, 4°C). Homogenates or lysates were quickly frozen in liquid nitrogen and stored at –80°C. Protein content in lysates was measured by the bicinchoninic acid method (Pierce, Rockford, IL).

Immunoprecipitation of AS160. Using a TBC1D4 (Abcam, Cambridge, UK) antibody and 20 µl of protein G agarose beads in 50% slurry with PBS, AS160 protein was immunoprecipitated (gentle rotation, 12 h, 4°C) from 300 µg of protein from human muscle samples. The supernatants from the samples were removed, and the immunocomplexes were subsequently washed two times in homogenization buffer and three times in PBS. The immunocomplexes were boiled in Laemmli buffer and subjected to SDS-PAGE probing for AS160 protein using the TBC1D4 antibody and AS160 phosphorylation using a phospho-(Ser/Thr) Akt substrate (PAS) antibody (Cell Signaling Technology, Danvers, MA).

Test of phosphospecificity of the PAS antibody. The phosphospecificity of the PAS antibody was tested as previously described (34) with minor modifications. In brief, this was done by extracting protein from a powdered human skeletal muscle sample using the homogenization buffer described above with or without phosphatase inhibitors and incubating proteins from these extracts with or without 2 U/µg protein {lambda}-protein phosphatase (New England Biolabs, Herts, UK) at 30°C for 1 h. The incubation was stopped by addition of Laemmli buffer, and samples were subjected to immunoblotting procedures.

Immunoblot analyses. Phosphorylation of AS160 was measured using the PAS antibody (Cell Signaling Technology). Total AS160 protein level was determined using an antibody recognizing the COOH-terminal amino acids 1287–1299 of human TBC1D4 (Abcam). Phosphorylation level of Akt on Ser473 and Thr308 was measured with phospho-specific antibodies (Cell Signaling Technology) and adjusted to total Akt using an Akt total antibody (Cell Signaling Technology). Muscle lysates were adjusted to equal protein concentration and boiled in Laemmli buffer (5 min, 96°C). To compare muscle lysates with muscle homogenates for AS160 phosphorylation and total AS160 protein, equal volumes of lysate and homogenates were boiled in Laemmli buffer (5 min, 96°C). Samples were loaded on either 5% Tris·HCl Criterion gels (Bio-Rad Denmark) or self-cast 7.5% gels and transferred to polyvinylidene difluoride membrane (Immobilon Transfer Membrane; Millipore Denmark). Membranes were blocked in washing buffer (10 mM Tris base, 150 mM NaCl, 0.25% Tween 20) containing 2% low-fat milk protein for 1 h at room temperature. Membranes were then incubated with primary antibodies overnight at 4°C, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibody (DAKO; Denmark) for 1 h at room temperature. Bands were visualized using a Kodak Image Station (2000MM; Kodak Denmark) and enhanced chemiluminescence (Millipore). Immunoreactive proteins were quantified (Kodak 1D 3.6 software), and results were expressed in relative units in comparison with control samples loaded on each gel. For all Western blots performed, it was ensured that the signal obtained was within the dynamic range of the analysis.

AMPK activity. Isoform-specific AMPK activity was measured in the presence of 200 µM AMP in immunoprecipitates from 200 µg of muscle lysate protein using anti-{alpha}1, -{alpha}2, -beta2, and -{gamma}3 antibodies described previously (44) and the AMARA peptide (HAMARAASAAAIARRR; 100 µM) as substrate, as previously described (13). The {alpha}2beta2{gamma}1 activity was analyzed by immunodepleting lysates for {alpha}2beta2{gamma}3 heterotrimers by an overnight {gamma}3 immunoprecipitation followed by another overnight {alpha}2 immunoprecipitation on which the {alpha}2beta2{gamma}1 activity was measured. The remaining lysates were then incubated with the {alpha}1 antibody to pull down the {alpha}1beta2{gamma}1 complex. The {gamma}3, {alpha}2, and the {alpha}1 antibody immunoprecipitates ~100% of total {gamma}3, {alpha}2, or {alpha}1 protein and neither one, two, nor three overnight incubations at 4°C had any influence on the phosphorylation state of {alpha}-AMPK subunits (data not shown). Furthermore, the activity associated with each of the three complexes was unaffected by either one or two overnight immunoprecipitations compared with a 4-h immunoprecipitation (data not shown).

Statistics. Data are expressed as means ± SE. Levene's test of equality of variance was used to test if groups had equal variances. This was the case in all data sets. Statistical evaluation was performed either by paired t-tests or by one- or two-way ANOVA with repeated measures using Tukey's honest significant difference post hoc analysis to identify significant differences between groups. In data sets where more than two levels of a given factor were analyzed, Mauchly's test of sphericity was applied. If the test was significant, the Greenhouse-Geisser corrected P value was used. Correlation analyses were done using Pearson's product-moment correlation coefficient. All statistical analyses were performed using SPSS 14.0. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Recovery of AS160 protein. To ensure that no AS160 protein was lost during the preparation of the muscle lysate, this was tested by comparing AS160 protein expression and phosphorylation in muscle homogenate and lysate. We were not able to detect any difference in AS160 expression/phosphorylation between muscle homogenate and lysate (data not shown).

Phosphospecificity of the PAS antibody. The phosphospecificity of the PAS antibody was tested in a buffer with or without phosphatase inhibitors and with or without the addition of the {lambda}-phosphatase. The PAS signals normally obtained in a basal and in an exercised muscle sample were completely removed in the dephosphorylation conditions. Total AS160 protein expression was not different in any of the samples. These data show that the PAS antibody detects phosphorylation of a protein migrating at 160 kDa (Fig. 1A).


Figure 1
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  		Fig. 1. Verification of antibody specificity. A: samples homogenized in a buffer with or without phosphatase inhibitors and incubated with or without {lambda}-phosphatase at 30°C for 1 h. No phospho-(Ser/Thr) Akt substrate (PAS) staining was seen in samples where endogenous phosphatases were not inhibited. Addition of {lambda}-phosphatase to these samples did not elicit any further dephosphorylation of the samples. B: using an antibody made against the COOH-terminal part of Akt substrate of 160 kDa (AS160), we were able to completely deplete exercise- and insulin-treated samples from the protein recognized by the PAS antibody migrating at 160 kDa. This indicates that the antibody we used when blotting for AS160 likely recognizes AS160.

 
Specificity of the TBC1D4 and PAS antibody. By immunoprecipitation of AS160 protein from paired human lysate samples (basal-insulin and basal-exercise) using the TBC1D4 antibody, it was shown that the 160-kDa band recognized by both the TBC1D4 and the PAS antibody by Western blotting was completely removed in the postimmunoprecipitate samples and present in the immunoprecipitate samples. In addition, the same band is recognized by the AS160 antibody (catalog no. 07–741) made by Upstate Biotechnologies (Waltham, MA; data not shown). This demonstrates that the band detected with these antibodies is likely to be AS160 (Fig. 1B).

AS160 phosphorylation during exercise. There were no significant changes in total AS160 protein expression in any of the exercise trials. During exercise at 67% VO2 peak, AS160 phosphorylation increased after 60 min (P = 0.06) and 90 min (P < 0.05) of exercise only. In addition, the increase in AS160 phosphorylation after 90 min was higher (P = 0.06) than after 60 min of exercise (Fig. 2A). In contrast, AS160 phosphorylation was not changed by exercise in the two short-term high-intensity exercise trials (30 s or 2 min), and a small but significant decrease in AS160 phosphorylation was seen after 20 min (80% VO2 peak) of exercise (Fig. 2B).


Figure 2
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  		Fig. 2. AS160 phosphorylation in response to exercise. AS160 phosphorylation was determined at rest (open bars) and during exercise (filled bars) using the PAS antibody in the four different exercise trials and related to total AS160 protein. A: AS160 phosphorylation increased significantly after 90 min and tended to be increased after 60 min of exercise (P = 0.06). Also, phosphorylation after 90 min tended (P = 0.06) to higher than after 60 min. B: AS160 phosphorylation was unchanged after 30 s and 2 min and decreased after 20 min of exercise (P < 0.05). Results are means ± SE; n = 8–11 experiments. Effect of exercise compared with rest: *P < 0.05 and (*)P = 0.06. Different from preceding time point: ({dagger})P = 0.06.

 
AMPK activity during exercise. In a recent paper (1), AMPK activity during the high-intensity exercise trials (30 s, 2 min, and 20 min) was reported. Because these data are important in the present context, they are also shown in Table 1. Thus the activity of the {alpha}1beta2{gamma}1 and the {alpha}2beta2{gamma}1 complexes were either unchanged or decreased with exercise, whereas the activity of the {alpha}2beta2{gamma}3 complex increased dramatically in all three exercise trials. In response to exercise performed at 67% VO2 peak, {alpha}2beta2{gamma}3-AMPK activity increased significantly after 30 min, but there was no further increase after this time point (Fig. 3B). The pattern of {alpha}2beta2{gamma}1-AMPK activation in response to exercise was distinct from that of {alpha}2beta2{gamma}3-AMPK in that the activity was not increased significantly until 60 min of exercise and increased further after 90 min (Fig. 3C). {alpha}1beta2{gamma}1-AMPK activity was not regulated to any major extent, but a small significant increase was observed at 60 min of exercise (Fig. 3A). AS160 may be a substrate for AMPK in rodent muscle. Thus we tested whether any association existed between AMPK signaling and AS160 phosphorylation, and, for several reasons, we looked for correlations specifically between {alpha}2beta2{gamma}1-AMPK activity and AS160 phosphorylation. First of all, AS160 was only phosphorylated in exercise trials in which {alpha}2beta2{gamma}1-AMPK was activated. Second, the time course of AS160 phosphorylation and activation of {alpha}2beta2{gamma}1-AMPK was quite similar. Third, in exercise trials in which {alpha}2beta2{gamma}3-AMPK was activated without any activation of {alpha}2beta2{gamma}1-AMPK, no AS160 phosphorylation was observed. Fourth, the {gamma}3-isoform in rodent muscle is dispensable for contraction to regulate AS160 phosphorylation. Correlation analysis on the 60- and 90-min data from the time course study revealed a fairly strong positive correlation between AS160 phosphorylation and {alpha}2beta2{gamma}1-AMPK activity (r2 = 0.55, P < 0.001; Fig. 4). Because interpretation of such analysis may be biased by the multiple measures from the same individual, correlation analyses at the individual time points at which significant changes had been observed were also performed. In line with the former analysis, positive correlations were observed at time 60 (r2 = 0.73, P = 0.007) and 90 (r2 = 0.42, P = 0.08) min of exercise (Fig. 4).


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  		Table 1. AMPK trimer activity during short-term high-intensity exercise

 

Figure 3
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  		Fig. 3. AMP-activated protein kinase (AMPK) activity in response to exercise in the time course study. AMPK activities of {alpha}1beta2{gamma}1 (A), {alpha}2beta2{gamma}1 (B), and {alpha}2beta2{gamma}3 (C) were determined at rest (open bars) and during/after exercise (filled bars) on the three AMPK complexes in human skeletal muscle purified by immunoprecipitation using AMPK subunit-specific antibodies. Results are means ± SE; n = 8. Effect of exercise compared with rest: *P < 0.05. Different from preceding time point: {dagger}P < 0.05.

 

Figure 4
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  		Fig. 4. Correlation between AS160 phosphorylation and AMPK activity. Pearson's product-moment correlation coefficient for {alpha}2beta2{gamma}1-AMPK activity and AS160 phosphorylation at 60 min ({circ}) and 90 min ({blacktriangleup}) were calculated in the time course study. The short dashed line indicates line of best fit for the 60-min data (n = 8; r2 = 0.73; P < 0.007). The solid line indicates line of best fit for the 90-min data (n = 8; r2 = 0.42; P < 0.084). The long dashed line indicates line of best fit for both data sets (n = 16; r2 = 0.55; P < 0.001).

 
Akt phosphorylation during exercise. AS160 contains at least six known phosphorylation sites of which five lie in ideal positions for Akt recognition, and recombinant Akt can phosphorylate recombinant AS160 (18). We investigated whether Akt was phosphorylated during the different exercise interventions to evaluate the potential role of Akt in exercise-induced AS160 phosphorylation. Akt protein expression did not change by exercise in any of the exercise trials (data not shown). In the short-term high-intensity exercise trials (30 s and 2 min), Akt phosphorylation on site Thr308, but not on site Ser473, was increased (Fig. 5, A and B). In contrast, no phosphorylation of Akt was seen during exercise performed at 80 or 67% VO2 peak (Figs. 5B and 6, A and B). Small oscillations in Akt phosphorylation status in the time course study did occur. However, none of these reached the level of significance (Fig. 6, A and B). Although Akt activation during exercise may have many metabolic consequences, the degree of Akt phosphorylation observed in response to the short-term high-intensity exercise is minor (~90 and ~250% for 30 s and 2 min, respectively) compared with the phosphorylation response (i.e., ~1,100%) on Akt Thr308 observed in human skeletal muscle after 2 h of physiological hyperinsulinemia (100 µU/ml; data not shown).


Figure 5
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  		Fig. 5. Akt phosphorylation after high-intensity exercise. Akt phosphorylation was determined at rest (open bars) and after exercise (filled bars) using phosphospecific antibodies recognizing either Thr308 (A) or Ser473 (B), and data were related to total Akt protein in the same samples. Results are means ± SE; n = 9–11. Effect of exercise compared with rest: *P < 0.05.

 

Figure 6
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  		Fig. 6. Akt phosphorylation during and after exercise of moderate intensity. Akt phosphorylation was determined at rest (open bars) and during/after exercise (filled bars) using phosphospecific antibodies recognizing either Thr308 (A) or Ser473 (B), and data were related to total Akt in the same samples. Results are means ± SE; n = 8.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of this study were that AS160 was not phosphorylated until 60 min of moderate-intensity exercise and increased further after 90 min of exercise and that more intensive exercise of shorter duration did not lead to AS160 phosphorylation. Only one other study has reported increases in AS160 phosphorylation in response to exercise in human skeletal muscle (6). After 60 min of cycle ergometer exercise at 70% VO2 peak, they found that AS160 phosphorylation was increased by ~80% compared with rest. The present study confirms this finding and adds the new information that AS160 phosphorylation increases in a time-dependent manner. However, it cannot be ruled out that exercise performed at an intensity higher or lower than 67% of VO2 peak for 90 min would result in a higher or lower phosphorylation of AS160, respectively. This would indicate that AS160 could also be regulated in an intensity-dependent manner. The question remains as to what the signaling events behind this rather late response in AS160 phosphorylation/deactivation are, and whether this may relate to exercise-regulated GLUT4 recruitment and glucose transport.

It has recently been published that AS160 is phosphorylated during contractions in vitro in mouse skeletal muscle and that this phosphorylation is dependent on {alpha}2-AMPK activity (23, 40). We have also shown that contraction-induced AS160 phosphorylation is not impaired in mice lacking the {gamma}3-AMPK subunit and that a constitutively active recombinant {alpha}2beta2{gamma}1-AMPK trimer is capable of increasing AS160 phosphorylation in crude muscle lysate from mice (40). Total AMPK activity in human skeletal muscle derives primarily from three different complexes, namely {alpha}1beta2{gamma}1, {alpha}2beta2{gamma}1, and {alpha}2beta2{gamma}3 (1, 44). Therefore, based on these observations, we have investigated the pattern of AMPK activation in the different exercise trials and hypothesized that the {alpha}2beta2{gamma}1-AMPK complex in human skeletal muscle may act in the pathway leading to exercise-induced AS160 phosphorylation. In agreement, no AS160 phosphorylation was evident during exercise at which no {alpha}2beta2{gamma}1-activation occurred despite marked activation of the {alpha}2beta2{gamma}3 complex. Furthermore, during exercise at 67% VO2 peak, all AMPK trimers were activated, but with completely different time courses. Although the {alpha}1beta2{gamma}1-AMPK complex showed little regulation, the {alpha}2-AMPK complexes were regulated to a higher extent. This has been observed before using similar exercise protocols (8, 38, 46). However, the pattern of activation of the two {alpha}2-AMPK complexes differed considerably, with the {alpha}2beta2{gamma}1 complex being activated in a time course resembling that of AS160 phosphorylation. In line with this observation, correlation between {alpha}2beta2{gamma}1-AMPK activity and AS160 phosphorylation was rather strong. Thus the available data suggest, but do not directly prove, a connection between {alpha}2beta2{gamma}1 activation and AS160 phosphorylation in skeletal muscle during exercise in humans.

The fact that AS160 phosphorylation was not increased after 30 min of exercise in the time course study, although {alpha}2beta2{gamma}1-AMPK activity tended to be higher compared with rest at this time point, could be related to a reduced level of circulating insulin (9, 26). Although speculative, the tendency for a reduced Akt phosphorylation of both Thr308 and Ser473 at this point may support such a view, and this may have counteracted any effect of {alpha}2beta2{gamma}1-AMPK on AS160 phosphorylation. Similarly, the reason for the decreased AS160 phosphorylation following 20 min of exercise at 80% of VO2 peak could be because of lower levels of plasma insulin, reducing Akt-induced AS160 phosphorylation. Supporting this idea, Akt phosphorylation on Thr308 and Ser473 tended to be lower in response to this exercise protocol. Akt is known to mediate insulin-induced effects on AS160 (36). However, it is not clear whether Akt plays a role for AS160 phosphorylation during exercise in humans. Akt has two phosphorylation sites, both of which are important for activation of the kinase. From in vitro experiments, it appears that phosphorylation of Ser473 precedes phosphorylation of Thr308 and that phosphorylation of the former residue is important for full activation of the kinase (37). Supporting the assumption that Akt was not activated in response to the high-intensity exercise trials was the finding that AS160 phosphorylation was not increased in these trials. Although cellular mechanisms derived from animal studies cannot always be applied to humans, a study published recently showed convincingly that Akt2, the isoform of Akt involved in maintenance of glucose homeostasis (4, 10, 28), is not involved in contraction-induced AS160 phosphorylation in mice (23). Thus, based on the points made in the above discussion, it seems unlikely that AS160 is phosphorylated by Akt during exercise of moderate to high intensity in human skeletal muscle.

The existing data regarding Akt phosphorylation/activation in human skeletal muscle in response to exercise are not clear. Examining the available studies in which Akt activity and/or phosphorylation have been measured under similar conditions as in the present time course study (~67% VO2 peak), four studies report increased Akt activity/phosphorylation (6, 14, 35, 42), and four studies, the present study included, report no significant changes in Akt activity and/or phosphorylation (39, 41, 47). Akt activity/phosphorylation can be increased in certain animal models where muscles are stimulated either in vivo, ex vivo, or in situ. The discrepancy between results from human and animal studies could be because of species differences or because of recruitment of more muscle fibers in these models. In fact, when we measured Akt phosphorylation in the 30-s and 2-min high-intensity studies, we found a ~90 and a 250% increase in Akt Thr308 phosphorylation, respectively, perhaps suggesting that very high muscle tension is the triggering factor for Thr308 phosphorylation. However, phosphorylation of Ser473 on Akt did not change with exercise, indicating that full Akt activation was likely not achieved under these conditions.

We recently reported that AMPK is important for contraction-induced AS160 phosphorylation in skeletal muscle of mice (40). However, because AMPK does not seem to be mandatory for contraction-induced glucose transport in muscles from these same mice (16, 30), contraction-induced glucose uptake may be mediated by AS160-independent mechanisms. It has been known for many years that glucose uptake increases within minutes in response to exercise at moderate intensities (11, 22) and that this increased glucose uptake is related to increased GLUT4 content in the plasma membrane (25). Therefore, because the time course of glucose uptake and GLUT4 translocation and AS160 phosphorylation do not correspond, this would imply that exercise-induced AS160 phosphorylation and glucose uptake in human skeletal muscle may not always be closely related. However, future studies using antibodies targeting specific phosphorylation sites on AS160 may reveal association between glucose uptake and AS160 phosphorylation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Financial support was given from the Copenhagen Muscle Research Centre, the Danish Natural Science Research Council, the Danish Medical Research Council, the Novo Nordisk Foundation, the Danish Diabetes Association, the Lundbeck Foundation, and from an integrated project (contract no. LSHM-CT-2004–005272) from the European Union. A. J. Rose was supported by a postdoctoral fellowship from the Carlsberg Foundation and from the European Union. J. F. P. Wojtaszewski was supported by a Hallas Møller fellowship from the Novo Nordisk Foundation.


   ACKNOWLEDGMENTS
 
We acknowledge the help from Camilla Aunsholm Madsen, Dyval Steinman, Richard Evering, Kim Sjøberg, Bruno Bisiani, Christa Broholm, and Kristian Kiillerich for recruiting and testing the subjects and from Irene Beck Nielsen and Betina Bolmgren for technical assistance during the experiments. We are grateful for the kind donation of the {alpha}1- and {alpha}2-AMPK antibodies from D. G. Hardie.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. F. P. Wojtaszewski, Copenhagen Muscle Research Centre, Dept. of Human Physiology, Institute of Exercise and Sport Sciences, Univ. of Copenhagen, DK-2100, Copenhagen, Denmark (e-mail: jwojtaszewski{at}aki.ku.dk)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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