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Am J Physiol Regul Integr Comp Physiol 296: R326-R333, 2009. First published November 26, 2008; doi:10.1152/ajpregu.90806.2008
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EXERCISE AND RESPIRATORY PHYSIOLOGY

Skeletal muscle eEF2 and 4EBP1 phosphorylation during endurance exercise is dependent on intensity and muscle fiber type

Adam J. Rose,* Bruno Bisiani,* Bodil Vistisen, Bente Kiens, and Erik A. Richter

Molecular Physiology Group, Copenhagen Muscle Research Centre, Dept. of Exercise and Sport Sciences, Section of Human Physiology, University of Copenhagen, Copenhagen, Denmark

Submitted 1 October 2008 ; accepted in final form 6 November 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Protein synthesis in skeletal muscle is known to decrease during exercise, and it has been suggested that this may depend on the magnitude of the relative metabolic stress within the contracting muscle. To examine the mechanisms behind this, the effect of exercise intensity on skeletal muscle eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) phosphorylation, key components in the mRNA translation machinery, were examined together with AMP-activated protein kinase (AMPK) in healthy young men. Skeletal muscle eEF2 phosphorylation at Thr56 increased during exercise but was not influenced by exercise intensity, and was lower than rest 30 min after exercise. On the other hand, 4EBP1 phosphorylation at Thr37/46 decreased during exercise, and this decrease was greater at higher exercise intensities and was similar to rest 30 min after exercise. AMPK activity, as indexed by AMPK {alpha}-subunit phosphorylation at Thr172 and phosphorylation of the AMPK substrate ACCβ at Ser221, was higher with higher exercise intensities, and these indices were higher than rest after high-intensity exercise only. Using immunohistochemistry, it was shown that the increase in skeletal muscle eEF2 Thr56 phosphorylation was restricted to type I myofibers. Taken together, these data suggest that the depression of skeletal muscle protein synthesis with endurance-type exercise may be regulated at both initiation (i.e., 4EBP1) and elongation (i.e., eEF2) steps, with eEF2 phosphorylation contributing at all exercise intensities but 4EBP1 dephosphorylation contributing to a greater extent at high vs. low exercise intensities.

protein synthesis; translation; elongation; initiation; AMPK

IT IS RECOGNIZED THAT THE understanding of the mechanisms controlling skeletal muscle protein turnover may aid in the development of novel therapies to combat skeletal muscle diseases (30). The control of protein turnover in skeletal muscle during exercise is poorly understood (30). It is known that skeletal muscle protein synthesis is blunted during exercise (2, 13). A recent study (24) demonstrated that contractile activity was a potent stimulus to blunt net protein synthesis rate of rat skeletal muscle ex vivo and can even override the anabolic effect of insulin. Studies by Bylund-Fellenius et al. (7) in perfused rat hindquarters demonstrated that protein synthesis rates were lower in contracting compared with resting skeletal muscle, particularly in fast-twitch muscles. In that study (7), it was shown that the magnitude of the blunting of protein synthesis was related to the magnitude of changes in high-energy phosphogens, indicating that signaling downstream of metabolic stress is a likely mechanism behind the fall in protein synthesis in working muscle. Despite this, the signaling mechanisms responsible for the decrease in skeletal muscle protein synthesis during exercise are not well understood.

While the synthesis of individual proteins is likely to be under control of specific gene transcription, global protein synthesis is determined by the rate of messenger RNA translation. Translation is conventionally divided into three phases: initiation, elongation, and termination controlled by proteins called eukaryotic initiation, elongation, and release factors, respectively (28). An initial step involves recruitment of the initiator methionyl-tRNA to the 40S ribosomal subunit that is catalyzed by GTP bound eukaryotic initiation factor 2{alpha} (eIF2) (28). Since phosphorylation of eIF2{alpha} inhibits its activity and can occur as a response to cellular stress (28), it could be that this may be a regulatory mechanism for the blunting of skeletal muscle protein synthesis with exercise. However, studies show no effect of acute resistance exercise (6) or ex vivo contraction (24) on eIF2{alpha} phosphorylation and eIF2B activity in skeletal muscle of rats. Another important step of initiation is 40S ribosomal subunit binding to the mRNA, which is catalyzed by a complex of eIF4A-eIF4E-eIF4G (28). A major regulator of the activity of this complex is eIF4E binding proteins (4EBP), which bind eIF4E when dephosphorylated, thereby preventing this initiation complex formation (28). There are studies that show that deletion of 4EBPs show only a mildly different growth phenotype compared with corresponding wild-type mice (4, 21, 40). However, as these mice were not studied under stress conditions, such as exercise or starvation, no clear evidence was presented that 4EBPs are not involved in the blunting of protein synthesis during such conditions. Indeed, studies of Drosophila show that 4EBP is not essential for growth but is involved in the ability to coordinate metabolic homeostasis during environmental stress (37, 38). In support of a role for of 4EBP in regulation of protein synthesis during stress conditions, Williamson et al. (43) demonstrated that there was polyribosome disaggregation in working murine skeletal muscle during treadmill running and provided evidence for a blunting of mRNA translation initiation. In particular, a dephosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4EBP1) at Thr37/46, which would bind eIF4E and inhibit initiation by preventing its association with eIF4G (28), was observed in skeletal muscle during exercise (43). Indeed, cell culture studies consistently showed dephosphorylation of 4EBP1 and blunted protein synthesis by several different cellular stressors (27). Other studies in rats showed that there is increased formation of 4EBP1-eIF4E complexes and decreased eIF4E-eIF4G complexes, which corresponded to blunted protein synthesis in skeletal muscle with running exercise (16), indicating a functional significance of 4EBP1 dephosphorylation. Concerning upstream signaling, the dephosphorylation of 4EBP1 was accompanied by changes in mTORC1 complex formation and AMPK phosphorylation in skeletal muscle during exercise, suggesting that the depression of initiation is mediated, at least in part, by an AMPK-mTORC1–4EBP1 signaling cascade (43).

In mammalian cells, peptide chain elongation requires two eukaryotic elongation factors (eEF), namely eEF1 and eEF2 (28). eEF2 mediates the translocation of the ribosome relative to the mRNA after addition of each amino acid to the nascent chain (28). Recently, we (32) and others (24) showed that the phosphorylation of eEF2 at Thr56, which decreases its activity (8, 29, 34), increased rapidly in working skeletal muscle during exercise, and it was hypothesized that it is a mechanism by which exercise may blunt skeletal muscle protein synthesis. Hence, since it was shown earlier that the blunting of protein synthesis rate and AMPK activation were related to the degree of metabolic stress of working skeletal muscle (7, 45), we sought to investigate the effect of aerobic exercise intensity on the regulation of AMPK and two key phosphoproteins involved in blunting of mRNA translation, namely 4EBP1 and eEF2. We hypothesized that both increases in eEF2 and decreases in 4EBP1 phosphorylation in skeletal muscle during exercise would be greater at high vs. low aerobic exercise intensities. Furthermore, since increasing exercise intensity in vivo requires progressive recruitment of different motor units (17, 35), and as it has been shown that these signaling events may be related to muscle fiber type and respective stimulation pattern (1), we sought to investigate the potential for muscle fiber-type differences in these signaling cascades.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human Subjects and Interventions

Two separate exercise studies were conducted to investigate the effects of exercise on skeletal muscle eEF2 and 4EBP1 phosphorylation. The subject characteristics, experimental protocols, and basic physiological responses for these studies have been reported in detail previously (3, 31). The subjects were regularly active, but not specifically trained. The present data are published in this separate publication for reason of clarity of discussion, and because the methods to examine eEF2 phosphorylation using immunohistochemistry were not established in the present laboratory at the time of publication of the other studies.

In one study (31), 10 healthy men [26 ± 1 yr, 187 ± 2 cm, 85 ± 2 kg, VO2peak = 53 ± 1 ml·(kg·min)–1] performed bicycling exercise for 30 min, with the exercise intensity being ~35%, 60%, and 85% VO2peak for three consecutive 10-min bouts with biopsies from the vastus lateralis muscle taken before exercise, after each 10-min bout, and 30 min postexercise. In addition, on another day, a control study was performed where the same subjects exercised for 30 min at 35% VO2peak with biopsies taken before, immediately after exercise, and 30 min postexercise.

In the other study (3), nine men [24 ± 1 yr, 183 ± 3 cm, 78 ± 3 kg, VO2peak = 54 ± 2 ml·(kg·min)–1] performed a 120-s bicycle test at a work rate corresponding to 110% of peak work rate, which was defined as the highest work intensity maintained for a whole minute during the incremental VO2peak test. Biopsies of the vastus lateralis muscle were taken before and immediately after the cessation of exercise. Importantly, this cycling exercise intensity is expected to recruit all types of motor units of the vastus lateralis (41), and thereby all muscle fibers should be active.

For both studies, subjects fasted overnight prior to experimentation, and resting biopsies were obtained after 1 h of inactivity in the supine position and rapidly frozen and stored. Muscle samples taken during exercise were frozen in liquid nitrogen between 15 and 30 s after cessation of activity, and the tissue was stored at –80°C until required. For the 120-s study, an aliquot of each muscle biopsy was embedded in Tissue-Tek (Sakura Finetek, NL) mounting medium, frozen in isopentane cooled to the temperature of liquid nitrogen and stored at –80°C for subsequent histochemical analysis. Subjects gave informed consent prior to participation and the studies were carried out in accordance with the Declaration of Helsinki II and studies were reviewed and approved by the Copenhagen Municipal Ethics Committee.

Tissue Preparation

All materials were from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. For skeletal muscle sample protein extraction, samples (15–20 mg) were freeze dried and then homogenized while in an ice slurry (i.e., 0°C) in a buffer (15 µl/mg tissue, original weight) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM Na fluoride, 5 mM Na pyrophosphate, 2 mM Na orthovanadate, 1 mM PMSF, 1 mM dithiothreitol, 1 mM benzamidine, 0.5% (vol/vol) protease inhibitor cocktail, and 1% (vol/vol) Nonidet P-40 using a polytron homogenizer (model PT 1200; Kinematica, Bohemia, NY) until no visible particles remained. The homogenates were mixed thoroughly by end-over-end rotation at 4°C for 30 min, and then spun at 6,000 g for 10 min at 4°C. The clarified supernatant was taken and stored at –80°C until required. A small aliquot of each lysate was taken prior to storage for total protein concentration analysis.

Protein concentration of tissue extracts was determined in triplicate using the bicinchoninic acid method using bovine serum albumin standards (Pierce Biotech) and bicinchoninic acid assay reagents (Pierce Biotech). A maximal coefficient of variance of 5% was accepted between replicates.

Analytical Techniques

Western blotting. Samples were immunoblotted for protein expression and phosphorylation according to Rose et al. (32). The primary antibodies used were anti-pThr56eEF2 (cat. no. 2331; Cell Signaling Technology), anti-pThr37/464EBP1 (cat. no. 9459; Signaling Technology), anti-pThr172AMPK{alpha} (cat no. 2531; Cell Signaling Technology) and anti-pSer221ACCβ (cat. no. 07–303; Upstate Biotechnology). Secondary antibodies were from Dako (Glostrup, Denmark). Phosphorylation of AMPK {alpha}-subunit and the AMPK substrate ACCβ (33) were measured as indices of AMPK activity. Band intensity was quantified by Kodak imaging software (Kodak 1D version 3.5). Preliminary experiments demonstrated that the amounts of protein loaded were within the dynamic range for the conditions used and the results obtained (data not shown) and that the phosphospecific antibodies were indeed phosphospecific (33).

Double immunofluorescence. Transverse cryosections (10 µm) were transferred to uncoated glass slides precleaned with ethanol, and dried for 30 min at room temperature. Sections were fixed for 15 min in 4% paraformaldehyde, washed in PBS and then incubated with 0.1% Triton in PBS for 10 min at room temperature. After several washes with PBS, slides were blocked in 1% BSA in PBS for 30 min at room temperature and then incubated overnight at 4°C with a blocking solution containing anti-pThr56eEF2 (1:250, cat. no. 2331; Cell Signaling Technology) and monoclonal anti-MHC-1 (1:100, clone A4.840 obtained from the Developmental Hybridoma Bank, University of Iowa) primary antibodies. Sections were rinsed in PBS, incubated with secondary fluorescent antibodies (Alexa Fluor 568 goat anti-rabbit IgG 1:400 for ant-pT56eEF2 and Alexa Fluor 488 goat anti-mouse IgM 1:400 for anti-MHC-I) in blocking solution for 60 min at room temperature and mounted with Vetcashield mounting medium (Vector Laboratories, Burlingame, CA). Negative control slides were performed with the primary antibodies omitted.

After 24 h, images were captured at x40 magnification with a Axiophot 2 fluorescence microscope (Zeiss, Jena, Germany) coupled to a Photometrics CoolSNAP cf digital camera (Roper Scientific, Germany). A Texas red excitation filter (540–580 nm) for pThr56eEF2 and an FITC excitation filter (465–495 nm) for MHC-I were utilized for recording the fluorescence signal. Images were analyzed with the public domain ImageJ 1.39u software (National Institutes for Health, Bethesda, MD). A total of 83 ± 6 muscle fibers for each muscle biopsy (46 ± 3, type I and 37 ± 3, type II) were analyzed. pThr56eEF2 fluorescent signal for each subject was quantified as the intracellular mean staining intensity minus the background mean intensity obtained from the corresponding negative control slide. 4EBP1 phosphorylation was not examined with immunoflourescence of muscle sections as preliminary immunoblots showed that it gave additional immunoreactive bands outside of the expected relative mobility of 4EBP1 and thus lacked the necessary specificity required for this technique.

Calculations and Statistics

Statistical analyses were performed using SigmaStat version 3.5 with t-tests, one-way, or two-way ANOVA used where appropriate. With the exercise intensity experiment data two separate two-way ANOVA for repeated measures were done. One was done where the effect of exercise (i.e., time) and trial type were tested. The other was done to compare the effects postexercise vs. basal between the two trial types. This separation was done as we felt that the exercise and postexercise conditions should be handled differently as they are metabolically different conditions (i.e., catabolic vs. anabolic). Student-Newman-Keuls multiple comparison post hoc tests were used when the ANOVA revealed significant interactions between variables. Correlations and regression analyses were performed on delta data (i.e., intervention point minus basal within each trial). To examine differences in eEF2 phosphorylation with the 2-min study, a paired t-test was performed. To examine differences between muscle fiber types, a two-way ANOVA for repeated measures was performed with Student-Newman-Keuls multiple comparison. Post hoc tests were used when the ANOVA revealed significant interactions between variables. Graphs were constructed using SigmaPlot version 10.0. Differences were considered to be significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Exercise Intensity on Skeletal Muscle Translation Factor Signaling

In response to exercise, there was an increase in eEF2 phosphorylation at Thr56 regardless of time or exercise intensity (Fig. 1). In contrast, there was a lower eEF2 phosphorylation 30 min postexercise (Fig. 1). Importantly, there were no differences in eEF2 expression between trials or at any time point (P = 0.81; data not shown). There was a lower 4EBP1 phosphorylation at Thr37/46 at 65% and 85% VO2peak compared with time 0 in the incremental exercise trial (Fig. 1). Furthermore, there was a decrease in 4EBP1 phosphorylation at 30 min of constant load exercise at 35% VO2peak compared with time 0. Given that there was no difference with 10 min of exercise at 35% VO2peak this suggests that there is a time effect of exercise at this intensity. In addition, there was a greater magnitude of 4EBP1 dephosphorylation at 30 min in the incremental exercise trial vs. the constant load trial demonstrating that 4EBP1 dephosphorylation is greater at high vs. low exercise intensities.


Figure 1
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  		Fig. 1. Effects of exercise time and intensity on skeletal muscle translation signaling. Skeletal muscle samples were taken before and after exercise, and extracted proteins from these samples were immunoblotted to detect eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1) phosphorylation. Representative immunoblots are shown. AU, arbitrary units. Data are means ± SE, n = 7–10. *P < 0.05 vs. time 0; #different from time 0, P < 0.05; {dagger}different from incremental, P < 0.05. The dashed line indicates a main effect for time.

 
The phosphorylation of AMPK {alpha}-subunit and the AMPK substrate (44) ACCβ were measured as indices of AMPK activity. The phosphorylation of AMPK{alpha} was higher during exercise only at 85% VO2peak during the incremental trial change with 35% VO2peak in the constant load trial (Fig. 2). Similar results were found for ACCβ phosphorylation at Ser221 except that there was also an increase at 65% VO2peak during the incremental trial (Fig. 2). In addition, there were increases in AMPK{alpha} and ACCβ phosphorylation 30 min postexercise only in the incremental trial. Correlation and regression analyses between changes in eEF2/4EBP1 and AMPK/ACC phosphorylation were performed. There was a positive-linear correlation between changes in pT-AMPK{alpha} and pS-ACCβ (R2 = 0.38; P < 0.001; data not shown). There was no significant correlation between changes in pT-eEF2 and changes in pT-AMPK{alpha} (R2 = 0.002; DF = 59; P = 0.73) or pS-ACCβ (R2 = 0.05; DF = 59; P = 0.09). There were significant, albeit weak, negative-linear correlations between changes in p-4EBP1 and changes in pT-AMPK{alpha} (R2 = 0.09; DF = 51; P < 0.05) as well as pS-ACCβ (R2 = 0.10; DF = 51; P < 0.05). However, it should be noted that 4EBP1 phosphorylation was back to basal levels 30 min postexercise in both trials (Fig. 1), despite significantly higher AMPK and ACC phosphorylation 30 min postexercise compared with basal in the incremental trial (Fig. 2).


Figure 2
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  		Fig. 2. Effects of exercise time and intensity on skeletal muscle AMP activated protein kinase (AMPK) signaling. Skeletal muscle samples were taken before and after exercise, and extracted proteins from these were immunoblotted to detect AMPK {alpha}-subunit and acetyl-CoA carboxylase-β (ACCβ) phosphorylation. Representative immunoblots are shown. Data are means ± SE, n = 10. *P < 0.05 vs. time 0; different from time 0, P < 0.05; {dagger}different from constant load, P < 0.05.

 
Effect of Exercise on eEF2 Phosphorylation in Skeletal Muscle Fibers

In response to an exhaustive 120-s exercise, phosphorylation at Thr56 of human skeletal muscle eEF2 (expressed as eEF2 phosphorylation at Thr56 divided by eEF2 protein expression) increased by 2.3-fold when compared with resting values (Fig. 3, top). There was no difference in eEF2 expression between rest and exercise (data not shown). In contrast, phosphorylation of skeletal muscle 4E-BP1 at Thr37/46 after the 120-s exercise was not different compared with rest samples (data not shown). Furthermore, we sought to investigate whether the increase in eEF2 phosphorylation was fiber-type specific. To do so, we were able to use the immunofluorescence technique, since preliminary Western blotting experiments demonstrated that the pThr56eEF2 antibody detected a single immunoreactive band of the predicted molecular weight (data not shown), demonstrating the high specificity of this antibody. In addition, we performed negative controls slides (primary antibodies omitted) for each biopsy when running the histochemical analyses, and the results showed absence of fluorescent signal (representative negative image in Fig. 3). As shown in Fig. 3, the increase (~55%) in eEF2 phosphorylation after the 120-s exercise was restricted to type I fibers. In type II fibers there was a small, albeit significant, decrease in eEF2 phosphorylation after exercise. Moreover, in the basal state there was a clear difference in eEF2 phosphorylation between type I and type II fibers, with values 55% lower in type I compared with type II fibers.


Figure 3
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  		Fig. 3. Effect of exercise on muscle fiber-type changes in eEF2 phosphorylation at Thr56. Skeletal muscle samples were taken before and after exercise and were immunoblotted for eEF2 phosphorylation (top; n = 9). *Different from rest, P < 0.001. In addition, double immunoflourescence histochemistry was performed on sections of a subset of the same skeletal muscle samples (n = 5) with myosin heavy chain I (MHC1) and pThr56- eEF2 antibodies used. *Fiber-type difference at rest, P < 0.05; #different from rest, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main novel findings of this study were that the increase in skeletal muscle eEF2 phosphorylation with exercise was insensitive to exercise intensity, whereas 4EBP1 dephosphorylation was greater with high vs. low exercise intensities (Fig. 1). In addition, immunohistochemistry showed that the increase in eEF2 phosphorylation was restricted to type I fibers (Fig. 3). Of note, while it has been shown that resistance-type exercise in the postabsorptive state decreases skeletal muscle protein synthesis (13), no study has systematically investigated the effect of exercise intensity on protein turnover during endurance-type exercise in humans. Hence, it is clear that further studies are warranted to investigate the possible functional role of differences in eEF2 and 4EBP1 phosphorylation during endurance-type exercise.

The increase in eEF2 phosphorylation was not surprising, as it has been reported that there are rapid and sustained increases in eEF2 phosphorylation in working skeletal muscle in humans during exercise (32) and rodent skeletal muscle ex vivo (1, 24). Indeed, studies show that phosphorylation of eEF2 at this site reduces its activity and protein translation in cells (8, 29, 34). Further work is required to demonstrate a functional role for eEF2 phosphorylation in working muscle. However, inexplicably, others have failed to observe changes in skeletal muscle eEF2 phosphorylation directly after endurance exercise (22) and resistance exercise (13). As the increase in eEF2 phosphorylation is thought to be downstream of a Ca2+-calmodulin-eEF2 kinase signaling cascade (32) and there would be greater muscle fiber recruitment with higher exercise intensities (17, 35), it was predicted that there would be a higher level of eEF2 phosphorylation with higher exercise intensities, similar to what has been shown for CaMKII (33). In contrast, there was no further increase in eEF2 phosphorylation between exercise intensities requiring 35% to 85% VO2peak (Fig. 1). Of note, similar to other studies (24, 32) there was no correlation between changes in eEF2 phosphorylation and indices of AMPK activity during exercise.

One possible explanation of the lack of the effect of exercise intensity on eEF2 phosphorylation is that there may be muscle fiber-type specific differences in increases in eEF2 phosphorylation with exercise, as type I myofibers are recruited at low exercise intensities, whereas type I and type II myofibers are recruited at high exercise intensities (17, 35). Indeed, Atherton et al. (1) demonstrated that when rat muscles (both soleus and extensor digitorum longus) were isolated and electrically stimulated to contract with a pattern simulating slow motor neuron activity continuously for 3 h there was an increase in eEF2 phosphorylation, whereas there was a decrease in eEF2 phosphorylation when muscles were stimulated with an intermittent fast motor neuron-like stimulation intermittently for 12 min. While the differences between these two may relate to the differences of pattern and duration of stimulation, they may also relate to the pulse frequency applied (i.e., slow: 10 Hz vs. fast: 100 Hz).Therefore we hypothesized that there may be differences in eEF2 phosphorylation in muscle fibers innervated by different motor nerves during exercise in vivo. To investigate this, we performed immunofluorescence analyses on human skeletal muscle biopsies taken at rest and immediately after a 120-s exhaustive exercise bout. This kind of exercise was chosen because it is known to result in recruitment of all muscle fiber types, both type I and type II (41). Western blot analyses on the same samples revealed a marked (2.3-fold) increase in phosphorylated eEF2 after exercise. This approximately twofold increase as measure with Western blot was supported by the immunohistochemical measure where there was an ~30% net increase in eEF2 phosphorylation with exercise compared with rest. When looking at the individual muscle fiber types, the phosphorylation of eEF2 after exercise was found to increase in type I fibers only, whereas a small but significant decrease was observed in type II fibers, when compared with rest (Fig. 3). A few considerations can be drawn from these results. First, the magnitude of increase in phosphorylation of eEF2 was markedly higher when detected by Western blotting compared with its detection by immunohistochemistry, which is not entirely surprising, given that these two methods have different sensitivities and that there are known limitations with the quantification of immunohistochemical analyses (42). Secondly, the increase in eEF2 phosphorylation observed only in type I fibers is in line with what has been found by Atherthon et al. (1), where the authors found an increase in eEF2 phosphorylation immediately after low-frequency stimulation of rat skeletal muscle, but not after high-frequency stimulation.

Interestingly, we observed a 55% higher phosphorylation of eEF2 in type II fibers compared with type I in the resting state, which suggests that there may be fiber-type differences in the elongation rates of preexisting mRNA in human skeletal muscle at rest. Since eEF2 phosphorylation is thought to be the key mechanism responsible for inhibition of the elongation step (28), it can be hypothesized that elongation rates, and possibly protein synthesis rates, are higher in type I fibers vs. type II fibers in human skeletal muscle at rest. In support of this, several studies have shown that, in animals, muscle composed of mainly type I fibers present much higher basal protein turnover than muscles expressing mainly type II fibers (15, 20). However, this conclusion should be taken with caution, since it has been shown in humans that protein synthesis rates at rest in triceps brachii vs. soleus and vastus lateralis, three muscles known to markedly differ in fiber-type composition, were only slightly (i.e., within 15%) different (25).

There was a lower eEF2 phosphorylation 30 min postexercise (Fig. 1), which is similar to prior observations (14, 22). This may be a potential mechanism behind higher skeletal muscle protein synthesis rates after endurance exercise, as has been observed previously (9, 23, 36).

In contrast to eEF2 phosphorylation, the phosphorylation of 4EBP1 at Thr37/46 decreased during exercise and the magnitude of this decrease was greater with high vs. low exercise intensities (Fig. 1). It would be expected that this dephosphorylation would result in a greater binding to eukaryotic initiation factor 4E (eIF4E), subsequently lowering the formation of the eIF4E-eIF4G complex and mRNA translation initiation (28). In particular, phosphorylation of 4EBP1 at Thr37/46 is mediated by mTORC1 and is a priming step for subsequent phosphorylations at other distal sites (28), and it has been shown that there are lower amounts of eIF4E-eIF4G complexes and higher amounts of 4EBP1-eEF4E complexes, which corresponded to blunted protein synthesis in skeletal muscle with running exercise (16), indicating a functional significance of 4EBP1 dephosphorylation. Thus, it may be that there is a greater suppression of skeletal muscle protein synthesis during high vs. low-intensity exercise and that this may be mediated by a greater blunting of translation initiation. This decrease in 4EBP1 phosphorylation in working skeletal muscle was expected, as other studies have shown similar findings in skeletal muscle of humans in response to resistance exercise (13) and mice in response to running exercise (43). As others have provided evidence that the decrease in 4EBP1 phosphorylation is downstream of an AMPK-mTORC1 signaling cascade (5, 12, 39, 43), indices of AMPK activity were also measured. As shown in Fig. 2, AMPK signaling was clearly sensitive to exercise intensity as has been shown before (10, 45). In addition, there were significant, albeit weak, negative linear correlations between changes in indices of AMPK activity and 4EBP1 phosphorylation. However, it should be noted that there was a clear dissociation between postexercise changes in AMPK activity and 4EBP1 phosphorylation, with 4EBP1 phosphorylation back to basal levels 30 min postexercise in both trials (Fig. 1), despite significantly higher AMPK and ACC phosphorylation 30 min postexercise compared with basal in the incremental trial (Fig. 2). However, this could be simply due to differential upstream regulation of phosphorylation of 4EBP1 during vs. after exercise. Indeed, Dreyer et al. (13) have observed higher 4EBP1 phosphorylation despite continued activation of AMPK in skeletal muscle after resistance exercise. Further work is required to determine whether AMPK signaling is required for depressed skeletal muscle protein synthesis as well as mTORC1 activity and 4EBP1 phosphorylation during exercise.

Similar to the other signaling events, the higher changes in AMPK activity and 4EBP1 dephosphorylation with increasing exercise intensity may be explained by muscle fiber recruitment, with AMPK activation and 4EBP1 dephosphorylation mainly occurring in type II fibers. This is plausible, as there was no change in indices of AMPK activity after 30 min at 35% VO2peak despite a probable activation of type I myofibers (17, 35). Clearly further work is required to examine the potential for muscle fiber or stimulation type differences in AMPK activation by exercise.

A study by Atherton et al. (1) demonstrated that when rat muscles (both soleus and extensor digitorum longus) were isolated and electrically stimulated to contract with a pattern simulating slow motor neuron activity continuously for 3 h vs. when stimulated with an intermittent fast motor neuron like stimulation intermittently for 12 min, there was a disparate signaling pattern with AMPK signaling activated during the endurance-like pattern and PKB-mTOR-4EBP1 pathway activated during the resistance-type pattern. However, studies of humans have failed to verify these findings. Indeed, both resistance-type and endurance-type exercise increase AMPK activity (Fig. 2) (13, 19, 32, 45) and decrease 4EBP1 phosphorylation (Fig. 1) (11, 13, 19), and resistance-exercise may actually decrease PKB activity (11) in skeletal muscle of humans. Furthermore, skeletal muscle protein synthesis is blunted during both resistance- (13) and endurance-type (30) exercise. Thus, there are probably similar mechanisms behind the fall in protein synthesis in skeletal muscle during resistance- and endurance-type exercise, the nature of which requires further investigation.

Perspectives and Significance

In summary, these data show that during endurance-type exercise, 4EBP1 dephosphorylation occurs to a greater extent at high vs. low exercise intensities, whereas eEF2 phosphorylation increases similarly at all exercise intensities, which may be explained by a selective increase in type I myofibers. Others have surmised that the suppression of skeletal muscle protein synthesis during exercise may be part of the stimulus by which protein synthesis is enhanced after exercise (7, 26). Given that the intensity of exercise training is important for subsequent adaptive responses of skeletal muscle (18), the putative higher blunting of protein synthesis by greater 4EBP1 dephosphorylation during high-intensity exercise may be part of the molecular mechanism by which high-intensity exercise training promotes greater adaptations than low-intensity training.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Financial support from the Copenhagen Muscle Research Centre, the Danish Medical and Natural Science Research Councils, the Danish Diabetes Association, an Integrated Project (contract no. LSHM-CT-2004–005272) from the European Union, as well as the Novo-Nordisk Research and Lundbeck Foundations, is acknowledged. B. Kiens acknowledges the financial support of the Danish Ministry of Food, Agriculture and Fisheries, as well as the Danish Ministry of Family and Consumer Affairs. A. J. Rose was supported by a postdoctoral fellowship from the Carlsberg Foundation and from the European Union.


   ACKNOWLEDGMENTS
 
Betina Bolmgren and Irene Bech Nielsen are acknowledged for skilled technical assistance. Jesper Birk, Camilla Madsen, Kim Sjøberg, and Jørgen Wojtaszewski are acknowledged for collaborating on the 120-s exercise study. Louise Høeg and Camilla Brolin are acknowledged for assistance with the histochemical analyses.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Adam J. Rose, Molecular Physiology Group, Copenhagen Muscle Research Centre, Dept. of Exercise and Sport Sciences, Sect. of Human Physiology, Univ. of Copenhagen, Universitetsparken 13, Copenhagen, Denmark, 2100 (e-mail: arose{at}ifi.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.

* Adam J. Rose and Bruno Bisiani contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Atherton PJ, Babraj JA, Smith K, Singh J, Rennie MJ, Wackerhage H. Selective activation of AMPK-PGC-1{alpha}; or PKB-TSC2-mTOR signaling can explain specific adaptive responses to endurance or resistance training-like electrical muscle stimulation. FASEB J 19: 786–788, 2005.[Abstract/Free Full Text]
  2. Atherton PJ, Rennie MJ. Protein synthesis a low priority for exercising muscle. J Physiol 573: 288–289, 2006.[Free Full Text]
  3. Birk JB, Wojtaszewski JFP. Predominant {alpha}2/β2/{gamma}3 AMPK activation during exercise in human skeletal muscle. J Physiol 577: 1021–1032, 2006.[Abstract/Free Full Text]
  4. Blackshear PJ, Stumpo DJ, Carballo E, Lawrence J. Disruption of the gene encoding the mitogen-regulated translational modulator PHAS-I in mice. J Biol Chem 272: 31510–31514, 1997.[Abstract/Free Full Text]
  5. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS. AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 277: 23977–23980, 2002.[Abstract/Free Full Text]
  6. Bolster DR, Kubica N, Crozier SJ, Williamson DL, Farrell PA, Kimball SR, Jefferson LS. Immediate response of mammalian target of rapamycin (mTOR)-mediated signalling following acute resistance exercise in rat skeletal muscle. J Physiol 553: 213–220, 2003.[Abstract/Free Full Text]
  7. Bylund-Fellenius AC, Ojamaa KM, Flaim KE, Li JB, Wassner SJ, Jefferson LS. Protein synthesis versus energy state in contracting muscles of perfused rat hindlimb. Am J Physiol Endocrinol Metab 246: E297–E305, 1984.[Abstract/Free Full Text]
  8. Carlberg U, Nilsson A, Nygard O. Functional properties of phosphorylated elongation factor 2. Eur J Biochem 191: 639–645, 1990.[Web of Science][Medline]
  9. Carraro F, Stuart CA, Hartl WH, Rosenblatt J, Wolfe RR. Effect of exercise and recovery on muscle protein synthesis in human subjects. Am J Physiol Endocrinol Metab 259: E470–E476, 1990.[Abstract/Free Full Text]
  10. Chen ZP, Stephens TJ, Murthy S, Canny BJ, Hargreaves M, Witters LA, Kemp BE, McConell GK. Effect of exercise intensity on skeletal muscle AMPK signaling in humans. Diabetes 52: 2205–2012, 2003.[Abstract/Free Full Text]
  11. Deldicque L, Atherton P, Patel R, Theisen D, Nielens H, Rennie M, Francaux M. Decrease in Akt/PKB signalling in human skeletal muscle by resistance exercise. Eur J Appl Physiol 104: 57–65, 2008.[CrossRef][Web of Science][Medline]
  12. Deshmukh AS, Treebak JT, Long YC, Viollet B, Wojtaszewski JFP, Zierath JR. Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling. Mol Endocrinol 22: 1105–1112, 2008.[Abstract/Free Full Text]
  13. Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E, Rasmussen BB. Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and protein synthesis in human skeletal muscle. J Physiol 576: 613–624, 2006.[Abstract/Free Full Text]
  14. Frøsig C, Sajan MP, Maarbjerg SJ, Brandt N, Roepstorff C, Wojtaszewski JFP, Kiens B, Farese RV, Richter EA. Exercise improves phosphatidylinositol-3,4,5-trisphosphate responsiveness of atypical protein kinase C and interacts with insulin signalling to peptide elongation in human skeletal muscle. J Physiol 582: 1289–1301, 2007.[Abstract/Free Full Text]
  15. Garlick PJ, Maltin CA, Baillie AG, Delday MI, Grubb DA. Fiber-type composition of nine rat muscles. II. relationship to protein turnover. Am J Physiol Endocrinol Metab 257: E828–E832, 1989.[Abstract/Free Full Text]
  16. Gautsch TA, Anthony JC, Kimball SR, Paul GL, Layman DK, Jefferson LS. Availability of eIF4E regulates skeletal muscle protein synthesis during recovery from exercise. Am J Physiol Cell Physiol 274: C406–C414, 1998.[Abstract/Free Full Text]
  17. Gollnick PD, Piehl K, Saltin B. Selective glycogen depletion pattern in human muscle fibres after exercise of varying intensity and at varying pedalling rates. J Physiol 241: 45–57, 1974.[Abstract/Free Full Text]
  18. Holm L, Reitelseder S, Pedersen TG, Doessing S, Petersen SG, Flyvbjerg A, Andersen JL, Aagaard P, Kjaer M. Changes in muscle size and MHC composition in response to resistance exercise with heavy and light loading intensity. J Appl Physiol 105: 1454–1461, 2008.[Abstract/Free Full Text]
  19. Koopman R, Zorenc AHG, Gransier RJJ, Cameron-Smith D, van Loon LJC. Increase in S6K1 phosphorylation in human skeletal muscle following resistance exercise occurs mainly in type II muscle fibers. Am J Physiol Endocrinol Metab 290: E1245–E1252, 2006.[Abstract/Free Full Text]
  20. Lang CH, Wu D, Frost RA, Jefferson LS, Kimball SR, Vary TC. Inhibition of muscle protein synthesis by alcohol is associated with modulation of eIF2B and eIF4E. Am J Physiol Endocrinol Metab 277: E268–E276, 1999.[Abstract/Free Full Text]
  21. Le Bacquer O, Petroulakis E, Paglialunga S, Poulin F, Richard D, Cianflone K, Sonenberg N. Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2. J Clin Invest 117: 387–396, 2007.[CrossRef][Web of Science][Medline]
  22. Mascher H, Andersson H, Nilsson PA, Ekblom B, Blomstrand E. Changes in signalling pathways regulating protein synthesis in human muscle in the recovery period after endurance exercise. Acta Physiol (Oxf) 191: 67–75, 2007.[CrossRef][Medline]
  23. Miller BF, Olesen JL, Hansen M, Dossing S, Crameri RM, Welling RJ, Langberg H, Flyvbjerg A, Kjaer M, Babraj JA, Smith K, Rennie MJ. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise. J Physiol 567: 1021–1033, 2005.[Abstract/Free Full Text]
  24. Miranda L, Horman S, De Potter I, Hue L, Jensen J, Rider M. Effects of contraction and insulin on protein synthesis, AMP-activated protein kinase and phosphorylation state of translation factors in rat skeletal muscle. Pflügers Arch 455: 1129–1140, 2008.[CrossRef][Web of Science][Medline]
  25. Mittendorfer B, Andersen JL, Plomgaard P, Saltin B, Babraj JA, Smith K, Rennie MJ. Protein synthesis rates in human muscles: neither anatomical location nor fibre-type composition are major determinants. J Physiol 563: 203–211, 2005.[Abstract/Free Full Text]
  26. Pain V, Manchester K. The influence of electrical stimulation in vitro on protein synthesis and other metabolic parameters of rat extensor digitorum longus muscle. Biochem J 118: 209–220, 1970.[Web of Science][Medline]
  27. Patel J, McLeod LE, Vries RG, Flynn A, Wang X, Proud CG. Cellular stresses profoundly inhibit protein synthesis and modulate the states of phosphorylation of multiple translation factors. Eur J Biochem 269: 3076–3085, 2002.[Web of Science][Medline]
  28. Proud CG. Signalling to translation: how signal transduction pathways control the protein synthetic machinery. Biochem J 403: 217–234, 2007.[CrossRef][Web of Science][Medline]
  29. Redpath NT, Price NT, Severinov KV, Proud CG. Regulation of elongation factor-2 by multisite phosphorylation. Eur J Biochem 213: 689–699, 1993.[Web of Science][Medline]
  30. Rennie MJ. Why muscle stops building when it's working. J Physiol 569: 3, 2005.[Free Full Text]
  31. Richter EA, Vistisen B, Maarbjerg SJ, Sajan M, Farese RV, Kiens B. Differential effect of bicycling exercise intensity on activity and phosphorylation of atypical protein kinase C and extracellular signal-regulated protein kinase in skeletal muscle. J Physiol 560: 909–918, 2004.[Abstract/Free Full Text]
  32. Rose AJ, Broholm C, Kiillerich K, Finn SG, Proud CG, Rider MH, Richter EA, Kiens B. Exercise rapidly increases eukaryotic elongation factor 2 phosphorylation in skeletal muscle of men. J Physiol 569: 223–228, 2005.[Abstract/Free Full Text]
  33. Rose AJ, Kiens B, Richter EA. Ca2+-calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise. J Physiol 574: 889–903, 2006.[Abstract/Free Full Text]
  34. Ryazanov AG, Davydova EK. Mechanism of elongation factor 2 (EF-2) inactivation upon phosphorylation. Phosphorylated EF-2 is unable to catalyze translocation. FEBS Lett 251: 187–190, 1989.[CrossRef][Web of Science][Medline]
  35. Sale DG. Influence of exercise and training on motor unit activation. Exerc Sport Sci Rev 15: 95–151, 1987.[Web of Science][Medline]
  36. Sheffield-Moore M, Yeckel CW, Volpi E, Wolf SE, Morio B, Chinkes DL, Paddon-Jones D, Wolfe RR. Postexercise protein metabolism in older and younger men following moderate-intensity aerobic exercise. Am J Physiol Endocrinol Metab 287: E513–E522, 2004.[Abstract/Free Full Text]
  37. Teleman AA, Chen YW, Cohen SM. 4E-BP functions as a metabolic brake used under stress conditions but not during normal growth. Genes Dev 19: 1844–1848, 2005.[Abstract/Free Full Text]
  38. Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF. Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev 19: 1840–1843, 2005.[Abstract/Free Full Text]
  39. Thomson DM, Fick CA, Gordon SE. AMPK activation attenuates S6K1, 4E-BP1, and eEF2 signaling responses to high-frequency electrically stimulated skeletal muscle contractions. J Appl Physiol 104: 625–632, 2008.[Abstract/Free Full Text]
  40. Tsukiyama-Kohara K, Poulin F, Kohara M, DeMaria CT, Cheng A, Wu Z, Gingras AC, Katsume A, Elchebly M, Spiegelman BM, Harper ME, Tremblay ML, Sonenberg N. Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1. Nat Med 7: 1128–1132, 2001.[CrossRef][Web of Science][Medline]
  41. Van Hall G, González-Alonso J, Sacchetti M, Saltin B. Skeletal muscle substrate metabolism during exercise: methodological considerations. Proc Nutr Soc 58: 899–912, 1999.[Web of Science][Medline]
  42. Walker RA. Quantification of immunohistochemistry—issues concerning methods, utility and semiquantitative assessment I. Histopathology 49: 406–410, 2006.[CrossRef][Web of Science][Medline]
  43. Williamson DL, Kubica N, Kimball SR, Jefferson LS. Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle. J Physiol 573: 497–510, 2006.[Abstract/Free Full Text]
  44. Winder WW, Hardie DG. Inactivation of acetyl-CoA carboxylase and activation of AMP-activated protein kinase in muscle during exercise. Am J Physiol Endocrinol Metab 270: E299–E304, 1996.[Abstract/Free Full Text]
  45. Wojtaszewski JFP, Nielsen P, Hansen BF, Richter EA, Kiens B. Isoform-specific and exercise intensity-dependent activation of 5'-AMP-activated protein kinase in human skeletal muscle. J Physiol 528: 221–226, 2000.[Abstract/Free Full Text]



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