Mechanotransduction is an intricate series of events that converts contraction-mediated stimuli into biological responses in skeletal muscle (7). In this regard, cell signaling networks are a complex yet integral part of the adaptation process responding to numerous inputs and generating a multiplicity of outputs resulting in altered biological function (7). Contractile activity associated with exercise training represents a stimulus capable of inducing transient alterations in cell signal transduction and metabolism that, when repeated over time, acts to promote the selective attainment of specific biochemical and morphological adaptations in skeletal muscle (7). Specifically, high-intensity, short-duration contraction promotes muscle hypertrophy and strength, whereas prolonged, low-intensity contractile activity is associated with increased mitochondrial density and enhanced resistance to fatigue (7).
Many signaling pathways are exercise responsive and have been implicated in adaptation in human skeletal muscle. Indeed, signaling mechanisms in muscle are modulated with the onset of contraction and during the initial minutes or hours after cessation of exercise (8-10,25,30). However, our current understanding of the various signaling responses and interactions that may be involved in orchestrating exercise-induced adaptation is far from complete. For example, the insulin-insulin-like growth factor (IGF) signaling pathway has been implicated in regulating exercise-induced adaptation in skeletal muscle given its putative capacity to direct diverse cell processes such as glucose transport, glycogen resynthesis, hypertrophy, translation, and ubiquitin-mediated protein degradation (7). An important focal point in the insulin-IGF pathway is the serine-threonine kinase Akt (also known as protein kinase B/PKB), which is proposed to regulate many cell signaling responses and adaptation machinery stimulated by vigorous exercise (7).
Previous investigations examining Akt-mediated signaling in skeletal muscle after exercise have shown disparate responses (2,10,13,25), possibly reflecting differences in research design such as exercise mode, intensity, and/or duration. Regardless, the apparent lack of clarity makes it difficult to ascertain the precise role of Akt-mediated signaling in promoting or inhibiting activity of important exercise-induced mechanisms that may contribute to the specificity of training adaptation in skeletal muscle. Accordingly, the primary aim of the present study was to determine the early time course of phosphorylation for cell signaling proteins after resistance and endurance exercise and to provide new information regarding the optimal timing for postexercise muscle biopsy sampling. We hypothesized that the prolonged, moderate-intensity contraction, and greater glycogen depletion associated with endurance cycling would initiate early Akt-mediated signaling for glucose uptake compared with the anabolic Akt-mTOR-S6K phosphorylation response to resistance exercise occurring later for translation and subsequent protein synthesis. Thus, the divergent exercise modes would generate a disparity in the timing and/or magnitude of phosphorylation events in human skeletal muscle.
Sixteen healthy male subjects with a training history in recreational fitness or team sports volunteered for this study. Subjects were randomly assigned to either a cycling (n = 8; mean ± SE: age = 29.0 ± 2.3 yr, body mass = 77.1 ± 5.2 kg, peak oxygen uptake (V˙O2peak) = 54.3 ± 1.3 mL·kg−1·min−1) or a resistance (n = 8; mean ± SE: age = 28.4 ± 1.6 yr, body mass = 81.8 ± 5.6 kg, one-repetition maximum (1RM) leg extension = 120.0 ± 10.4 kg) exercise group. The diverse exercise modes used in the present study would be expected to generate a significant disparity in total work (cycling ∼660 kJ vs resistance exercise <130 kJ) (4). In addition, the study was restricted to between-group comparisons because of the prohibitive number of muscle biopsies (i.e., 10) with a cross-over design. The experimental procedures and the possible risks associated with the study were explained to each subject, who all gave written informed consent before participation. The study was approved by the Human Research Ethics Committee of the RMIT University (Melbourne, Australia) and the Massey University (Palmerston North, New Zealand).
V˙O2peak was determined during an incremental test to volitional fatigue on a Lode cycle ergometer (Groningen, The Netherlands). The protocol has been described in detail previously (17). In brief, subjects commenced cycling at a workload equivalent to 1.5 W·kg−1 for 150 s. Thereafter, the workload was increased by 25 W every 150 s until volitional fatigue, defined as the inability to maintain a cadence >70 rpm. Throughout the test, which typically lasted 10-12 min, subjects breathed through a mouthpiece attached to a metabolic cart (Vista TurboFit; VacuMed, Ventura, CA) to record oxygen consumption.
Quadriceps strength was determined during a series of single repetitions on a standard pulley leg extension machine (Fitness Works, Auckland, New Zealand) until the maximum load lifted was established (1RM). Repetitions were separated by a 3-min recovery and were used to establish the maximum load or weight that could be moved through the full range of motion once, but not a second time. Exercise range of motion was 85°, with leg extension end point set at −5° from full extension.
Diet or exercise controls.
Before both experimental trials (described subsequently), subjects were instructed to refrain from alcohol consumption and vigorous physical activity for a minimum of 48 h. Subjects were provided with standardized prepacked meals that consisted of 3 g of carbohydrate per kilogram of body mass, 0.5 g of protein per kilogram of body mass, and 0.3 g of fat per kilogram of body mass consumed as the final caloric intake the evening before reporting for an experimental trial.
On the morning of an experimental trial, subjects reported to the laboratory after an approximately 10-h overnight fast. After subjects rested in the supine position for approximately 15 min, local anesthesia (2-3 mL of 2% Xylocaine (lignocaine)) was administered to the skin, subcutaneous tissue, and fascia of the vastus lateralis muscle (∼15 cm above the patella) in preparation for the series of muscle biopsies. A resting (basal) biopsy was taken using a 5-mm Bergstrom needle modified with suction, and approximately 150 mg of muscle was removed, blotted to remove excess blood, and immediately frozen in liquid nitrogen. Four additional incisions were made in preparation for subsequent postexercise biopsies. Subjects then completed a bout of either cycling or resistance exercise (described in detail subsequently), and a second biopsy was taken immediately after the cessation of exercise. Subjects then rested in the supine position for 60 min, and further biopsies were taken after 15, 30, and 60 min postexercise recovery. Muscle biopsies were taken from a separate site (distal to proximal) from the same leg. Samples were stored at −80°C until subsequent analysis. In addition, a catheter was inserted into the antecubital vein for blood sampling, and blood samples (∼2 mL) were taken at equivalent time points with muscle biopsies.
Subjects performed 60 min of continuous cycling at a power output that elicited approximately 70% of individual V˙O2peak. Subjects were fan cooled and allowed ad libitum access to water throughout the ride. Visual feedback for pedal frequency, power output, and elapsed time was provided to subjects.
After a standardized warm-up (1 × 5 repetition at 50% and 60% 1RM), subjects performed eight sets of five repetitions at approximately 80% 1RM. Each set was separated by a 3-min recovery period during which the subject remained seated on the leg extension machine. Contractions were performed at a set metronome cadence, and strong verbal encouragement was provided during each set.
Blood glucose, lactate, and insulin.
Whole blood samples were collected in EDTA-containing tubes and immediately analyzed for glucose and lactate concentration using an automated glucose/lactate analyzer (YSI 2300, Yellow Springs, OH). Blood samples were then centrifuged at 1000g (4°C) for 15 min, and aliquots of plasma were stored at −80°C until analysis. Plasma insulin concentration was determined using an immunoassay (EIA) kit (ALPCO Diagnostics, Salem, NH).
A small piece of frozen muscle (∼20 mg) was freeze dried and powdered to determine muscle glycogen concentration. Freeze-dried muscle was extracted with 500 μL of 2 M hydrochloric acid, incubated at 100°C for 2 h, and then neutralized with 1.5 mL of 0.67 M sodium hydroxide for subsequent determination of glycogen concentration via enzymatic analysis with fluorometric detection (Jasco FP-750 spectrofluorometer, Easton, MD) at excitation 365 nm/emission 455 nm. Glycogen concentration was expressed as millimoles of glycogen per kilogram of dry weight.
Muscle samples were homogenized in ice-cold buffer (1:8 mg muscle:mL buffer) containing 50 mM of Tris-HCl, pH 7.5, 1 mM of EDTA, 1 mM of EGTA, 10% glycerol, 1% Triton X-100, 50 mM of NaF, 5 mM of sodium pyrophosphate, 1 mM of DTT, 10 μg·mL−1 of trypsin inhibitor, 2 μg·mL−1 of aprotinin, 1 mM of benzamidine, and 1 mM PMSF using a motorized pellet pestle (Sigma-Aldrich, St. Louis, MO) with 5-s pulses. The lysate was kept on ice at all times and was then centrifuged at 12,000g for 20 min at 4°C. The supernatant was transferred to a sterile tube and was subsequently aliquoted for determination of protein concentration using a BCA protein assay (Pierce, Rockford, IL). Lysate was then resuspended in Laemmli sample buffer, with 50 μg of protein loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes blocked with 5% nonfat milk, washed with 10 mM of Tris-HCl, 100 mM of NaCl, and 0.02% Tween 20, and incubated with primary antibody (1:1000) overnight at 4°C. Membranes were incubated with secondary antibody (1:2000), and proteins were detected via enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK; Pierce Biotechnology, Rockford, IL) and quantified by densitometry. All sample time points for each subject were run on the same gel, and data are expressed relative to α-tubulin. Polyclonal anti-phospho-AktSer473 (cat no.9271), anti-phospho-mTORSer2448 (2971), anti-phospho-glycogen synthase (GS) Ser641 (3891), anti-phospho-4E-BP1Thr70 (9455), and anti-phospho-Ser/Thr Akt substrate (9611) and monoclonal anti-phospho-AktThr308 (4056), anti-phospho-tuberin (TSC2) Thr1462 (3617), anti-phospho-GSK-3βSer9 (9323), and anti-phospho-4E-BP1Thr37/46 (2855) were from Cell Signaling Technology (Danvers, MA). Polyclonal anti-phospho-eIF2BϵSer539 (44-530G) was from Biosource (Carlsbad, CA), and p70S6KThr389 (04-392) was from Millipore (Temecula, CA). Anti-phospho-AMPKαThr172 was raised against AMPK-α peptide (KDGEFLRpTSCGAPNY) as described previously (6). Monoclonal anti-α-tubulin control protein antibody was from Sigma-Aldrich (T6074).
All data were analyzed by two-way ANOVA (two factor: time × exercise) with Student-Newman-Keuls post hoc analysis. Statistical significance was established when P < 0.05 (SigmaStat for Windows, Version 3.11, Chicago, IL). Phosphorylation at rest was not different between exercise groups for any of the proteins of interest. Consequently, data are expressed relative to rest in arbitrary units ± SEM.
Blood Lactate, Insulin, and Glucose
Peak blood lactate concentration after cycling (CYC) occurred immediately postexercise (P < 0.05; Table 1). Blood lactate remained elevated above rest after 15 min (146%, P < 0.001) and 30 min (79%, P < 0.01) of recovery from CYC. After resistance exercise (REX), blood lactate concentration was significantly elevated above rest immediately postexercise (106%, P < 0.05). Blood lactate concentrations after CYC were higher compared with REX at each corresponding time point during the 60-min recovery period (P < 0.01; Table 1). There were no differences in plasma insulin and blood glucose concentration (Table 1).
Muscle glycogen concentration decreased approximately 56% after 60 min of CYC (214 mmol·kg·−1 of dry weight, P < 0.001; Fig. 1), whereas REX reduced muscle glycogen approximately 26% (103 mmol·kg−1 of dry weight, P < 0.01). Of note, preexercise muscle glycogen concentration was not different, but greater glycogen utilization during 60 min CYC compared with REX resulted in significantly lower postexercise glycogen concentration (REX vs CYC ∼93%, P < 0.01; Fig. 1).
Akt/tuberous sclerosis complex (TSC)2/mammalian target of rapamycin (mTOR).
There were comparable changes in AktThr308 and AktSer473 phosphorylation during the 60-min postexercise time course after both CYC and REX (Figs. 2A and B). Moreover, despite only minor changes during the initial recovery period (0-15 min), there were significant differences for 30 and 60 min postexercise compared with rest. Specifically, CYC increased AktThr308 phosphorylation approximately 250%-300% and AktSer473 phosphorylation approximately 130% after 30 and 60 min of recovery, respectively (P < 0.05). Similarly, REX induced a significant increase in AktThr308/Ser473 phosphorylation above rest (∼100%-200%) at equivalent time points (P < 0.05; Figs. 2A and B).
Changes in TSC2Thr1462 phosphorylation after CYC were increased above rest at all time points peaking 30 min postexercise (∼130%, P < 0.001), whereas REX did not alter TSC2 phosphorylation (Fig. 2C). Consequently, phosphorylation of TSC2Thr1462 during recovery from CYC was higher compared with corresponding time points after REX (P < 0.01; Fig. 2C). There were variable responses in mTORSer2448 phosphorylation that resulted in significant effects for time and exercise mode (P < 0.05; Fig. 2D). CYC generated a significant increase in mTORSer2448 phosphorylation above rest immediately postexercise (∼100%, P < 0.05) then rapidly abated during the initial 15-min recovery but was elevated 60 min postexercise (∼90%, P = 0.051). In contrast, mTORSer2448 phosphorylation after REX was largely unaffected during the initial recovery period (0-15 min) but increased 30 min postexercise (∼100%, P < 0.05). The divergent mTORSer2448 phosphorylation immediately postexercise resulted in a significant difference between exercise modes (P < 0.05; Fig. 2D).
p70 S6 kinase (S6K)/eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1).
There were similar increases in S6KThr389 phosphorylation for each exercise mode during the acute postexercise recovery period (Fig. 3A). However, the increase in S6K phosphorylation above rest failed to reach significance throughout the 60-min recovery period after CYC (62%-140%), whereas phosphorylation of S6KThr389 was only different from rest 60 min postexercise after REX (∼176%, P = 0.050; Fig. 3A).
There were comparable postexercise responses between exercise modes for phosphorylation of 4E-BP1Thr37/46 but a greater magnitude of response during recovery from CYC (Fig. 3B). There was a significant dephosphorylation at 4E-BP1Thr37/46 compared with rest immediately postexercise after CYC (−92%, P < 0.01). During the subsequent 30-min recovery period, 4E-BP1Thr37/46 phosphorylation rapidly increased and was elevated at 30 min (95%, P < 0.05) and 60 min of recovery from CYC (77%, P < 0.05). In contrast, REX failed to generate a significant change in 4E-BP1Thr37/46 phosphorylation during the recovery period, resulting in a difference from CYC at 30 and 60 min postexercise (∼60%, P < 0.05; Fig. 3B). Changes in 4E-BP1Thr70 phosphorylation were discordant compared with 4E-BP1Thr37/46 (Fig. 3C). Specifically, 4E-BP1Thr70 phosphorylation decreased after CYC and remained suppressed during the 60-min recovery period (52%-58%), approaching significance 15 min postexercise (−72%, P = 0.056; Fig. 3C). Conversely, there was a modest increase in phosphorylation at 4E-BP1Thr70 that was sustained throughout the recovery period after REX, but these increases failed to reach statistical significance (40%-52%). The divergent 4E-BP1Thr70 phosphorylation responses resulted in a significant exercise mode effect at all postexercise time points (P < 0.01; Fig. 3C).
Glycogen synthase kinase (GSK) 3β/glycogen synthase (GS)/eukaryotic translation initiation factor (eIF) 2Bϵ.
GSK-3βSer9 phosphorylation increased above rest immediately postexercise and 30-60 min after CYC (∼80%-100%, P < 0.05; Fig. 4A). The increase (51%) in GSK-3βSer9 phosphorylation immediately after REX failed to reach significance and was not different from rest throughout recovery. Consequently, phosphorylation of GSK-3β during recovery from CYC was higher 30-60 min postexercise compared with REX (∼60%, P < 0.05; Fig. 4A).
CYC induced a significant dephosphorylation of GSSer641 immediately postexercise (−59%, P < 0.05), which was sustained throughout the 60-min recovery period (∼−74%, P < 0.05; Fig. 4B). Conversely, GSSer641 phosphorylation after REX was largely unchanged during the postexercise period (30%-45%). As a result, phosphorylation of GSSer641 during recovery from REX was higher compared with equivalent time points after CYC (P < 0.01; Fig. 4B).
CYC generated a significant increase in eIF2BϵSer539 phosphorylation above rest immediately postexercise (55%, P < 0.05), and this increase was also different from the corresponding REX time point (P < 0.05; Fig. 4C). Phosphorylation at eIF2BϵSer539 after CYC then rapidly abated during the initial 15-min recovery (−59%, P < 0.05) and remained suppressed for the remainder of the recovery period. Despite a modest attenuation in eIF2BϵSer539 phosphorylation throughout recovery from REX, these changes were not significantly different from rest.
AMP-activated protein kinase (AMPK)/Akt substrate (AS) 160 kDa.
AMPKThr172 phosphorylation after CYC was elevated above rest throughout the 60-min recovery period and was highest 30 min postexercise (∼195%, P < 0.001), whereas REX failed to induce any significant changes in postexercise AMPKThr172 phosphorylation (Fig. 5A). Phosphorylation of AMPKThr172 during recovery from CYC was higher 0 and 30 min postexercise compared with equivalent time points after REX (P < 0.05; Fig. 5A).
After modest increases in phosphorylation of AS160 during the initial postexercise period (0-15 min), there was a rapid elevation 30 and 60 min after CYC (240%-270%, P < 0.001; Fig. 5B). REX failed to induce a change in AS160 phosphorylation during the postexercise recovery period. Phosphorylation of AS160 after CYC was also higher compared with REX 15-60 min after exercise (104%-140%, P < 0.01; Fig. 5B).
Cell signaling transduction networks are essential pathways for converting contraction-mediated signals to physiological responses. The aim of the present study was to establish the early time course of putative exercise-mediated phosphorylation responses after endurance and resistance exercise. Our results show a similar time course for Akt-mTOR-S6K phosphorylation during the initial 60-min postexercise recovery period despite differences in contractile activity with diverse exercise modes. Conversely, we observed distinct phosphorylation status to promote glucose transport and glycogen synthesis after endurance but not resistance exercise. Our results indicate that peak phosphorylation for many of the proteins of the insulin-insulin-like growth factor pathway occurs 30-60 min after exercise in the fasted state.
The first finding of the present study was the highly coordinated changes in the AktThr308 and AktSer473 phosphorylation time course after divergent exercise (Figs. 2A and B), which occurred independent of blood glucose or insulin concentration (Table 1). Previous studies in humans have reported increased (10,11,15,19,25,32), decreased (13,36), or unchanged (2,10,11) Akt phosphorylation after exercise in the fasted state. Accordingly, although the possibility exists that the apparent lack of agreement in exercise-induced Akt phosphorylation responses in previous studies may be attributed in part to differences in exercise intensity, duration, and/or subject muscle glycogen and training status, our results indicate that the timing of muscle biopsy sampling is an essential consideration. Moreover, exercise mode appears to have little impact in generating divergence in the phosphorylation status of Akt, at least during the first hour postexercise. Work in rodents has shown an earlier peak activation of Akt in skeletal muscle (∼5-10 min) after cessation of contractile activity (5,33). However, our findings are similar to those of Mascher et al. (25) and Dreyer et al. (15), showing elevated AktSer473 phosphorylation 60 min postexercise after endurance and resistance exercise in humans, respectively.
Akt may directly activate mTOR and subsequent downstream targets through phosphorylation at the mTORSer2448 residue (27) or indirectly by phosphorylation and subsequent inhibition of tuberous sclerosis complex 2 (TSC2) at Thr1642 (21). There is a paucity of information relating to the exercise-induced TSC2 phosphorylation response in vivo skeletal muscle, and we can only speculate that the low- to moderate-intensity and prolonged duration of contraction associated with endurance but not resistance exercise initiates a yet to be defined mechanism for the TSC2Thr1462 phosphorylation we observed. In contrast, exercise-induced mTORSer2448 phosphorylation corresponded more closely with that of Akt (Fig. 2D). Thus, endurance and resistance exercise appear capable of enhancing mTOR phosphorylation, and the diverse contractile stimuli both appear to initiate pathways to promote translation in skeletal muscle. There are numerous cell functions regulated by mTOR, and the immediate postexercise increase in phosphorylation after cycling may reflect the putative role of mTOR in control of mitochondrial oxidative function (12). Moreover, Mascher et al. (25) and Benziane et al. (2) have also shown an increase in mTOR phosphorylation immediately after 60 min of cycling at approximately 70%-75% V˙O2peak.
Regulation of translation for protein synthesis via Akt-mTOR includes two parallel effectors: S6K and 4E-BP1 (3,31). S6K phosphorylation is proposed to enhance translation of mRNAs encoding ribosomal proteins and elongation factors, whereas 4E-BP1 can bind to eIF4E and prevent cap-dependent mRNA translation (38). Our data provide further support for the contraction-mediated increase in S6K phosphorylation during the early recovery phase after exercise in human skeletal muscle (10,15,34,36). Although there is evidence that elevated S6K phosphorylation may persist 2-6 h after resistance exercise in the fasted state (10,15,16,34), the increase in translational activity appears to be initiated within the first 60 min of recovery.
4E-BP1 inhibits translation initiation by binding to eIF4E, preventing formation of the multiprotein eIF4E-G scaffolding complex (eIF4F), which binds the 40S ribosomal subunit to mRNA (18). There were comparable patterns of 4E-BP1Thr37/46 phosphorylation throughout the 60-min recovery period in the present study but a greater magnitude of effect after cycling (Fig. 3B). The decrease in 4E-BP1Thr37/46 phosphorylation inhibiting translation immediately postexercise is not surprising, given the results of Rose et al. (30) showing depression of translation signaling during endurance exercise, indicative of the suppression of the energy consuming process of protein synthesis while other cellular demands are met. The divergent phosphorylation status of 4E-BP1Thr70 compared with 4E-BP1Thr37/46 during the 60-min postexercise period was unexpected. To the best of our knowledge, this is the first study to investigate 4E-BP1Thr70 phosphorylation and to compare 4E-BP1 phosphorylation sites after exercise in humans. Undoubtedly, the regulation of 4E-BP1 activity is complex, and phosphorylation has been proposed to occur in a hierarchical manner with 4E-BP1Thr37/46 phosphorylation required for subsequent 4E-BP1Thr70 modification (38). However, it is unclear how the changes in phosphorylation correspond with in vivo activity in humans after exercise. Moreover, whether the discordance between phosphorylation sites reflects differences in eIF4E binding in skeletal muscle and/or characterizes alternate mechanisms through which each exercise mode modulates translation requires further investigation.
Collectively, the data regarding phosphorylation of putative Akt-mTOR-regulated signaling proteins proximal to translation initiation or elongation emphasize the complexity of translational machinery for exercise-induced protein synthesis in skeletal muscle. How the adaptation response of these key signaling components is modified by various volumes and intensity of contractile activity remains equivocal. The early signaling responses may represent the cumulative effect of recovery from contraction-induced disruption to muscle homeostasis and translation initiation for early response genes. Regardless, our findings highlight differences in site-specific phosphorylation and divergence between exercise modes for 4E-BP1Thr70. In addition, despite differences in the magnitude of effect, the exercise-induced increase in 4E-BP1Thr37/46 and S6KThr389 phosphorylation is inhibited or delayed during the early recovery period (0-30 min) but is eventually enhanced after endurance and resistance exercise, respectively.
Phosphorylation of GSK-3 is reported to result in hypophosphorylation (activation) of glycogen synthase (GS) and/or eIF2Bϵ (22,26). In the present study, we observed a lack of coordination in exercise-specific GSK-3βSer9 phosphorylation when compared against phosphorylation of Akt. This disparity may be due to GSK3 being part of the wingless-type MMTV integration site family (Wnt) signaling pathway. The Wnt pathway is exercise responsive and inhibits GSK3 activity (26), and Wnt signaling may have been differentially modulated by the divergent contractile stimuli. The changes in GSSer641 phosphorylation largely corresponded with GSK-3βSer9 with sustained dephosphorylation of GSSer641 during the 60-min postexercise period after cycling but not resistance exercise (Fig. 4B). As a critical regulator of glycogen resynthesis increased GS activity (dephosphorylation) following a bout of endurance cycling would be expected given the prolonged, constant load contractile activity and greater glycogen depletion (Fig. 1). Taken together, the changes in GSK-3 and GS phosphorylation provide a time course for the molecular signaling mechanisms regulating resynthesis of muscle glycogen during the early recovery period from endurance exercise (25,28,32). Conversely, glycogen resynthesis during initial recovery from heavy resistance exercise may represent a lesser priority.
Phosphorylation of GSK-3βSer9 may also enhance translation by derepressing its inhibition of eIF2Bϵ (22,39). The increase in eIF2BϵSer539 phosphorylation (inactivation) immediately after cycling was analogous with changes in 4E-BP1Thr37/46, indicating the suppression of global translational signaling during endurance exercise (Fig. 4C). Similarly, eIF2BϵSer539 phosphorylation rapidly diminished thereafter (15 min), characterizing the enhanced translation status during recovery from endurance exercise also seen with 4E-BP1Thr37/46 (Fig. 3C). To the best of our knowledge, this is the first study to investigate the eIF2BϵSer539 response to an endurance exercise bout, although our results for resistance exercise are similar to that of Glover et al. (16), showing decreased eIF2B phosphorylation after an acute bout of resistance exercise in humans.
As a metabolic regulator and energy-sensing protein, it is not surprising that AMPK phosphorylation was more pronounced after prolonged exercise duration (Fig. 5A). The effect of resistance exercise on AMPK has received less scrutiny, but Dreyer et al. (15) and Koopman et al. (23) have shown increased AMPK activity in the early recovery period from resistance exercise in the fasted state. AMPK has been implicated in repressing anabolic processes in skeletal muscle via inhibition of mTOR-mediated signaling to initiate translation (1,15). Intriguingly, the time-course data in the present study show that the increase in AMPKThr172 occurred concomitantly with phosphorylation events for enhancing translation. Benziane et al. (2) have also shown increased AMPKThr172 coinciding with elevated mTORSer2448 after 60 min cycling. Likewise, Mascher et al. (25) examined Akt-mTOR signaling after prolonged cycling and also observed increased mTOR phosphorylation when AMPK activity would be expected to be elevated. As such, there is accumulating evidence that AMPKThr172 phosphorylation may have limited capacity to subdue translational signaling, at least in the early postexercise period in humans (29).
Finally, divergent responses between exercise modes were evident for AS160 phosphorylation likely because of exacerbated glycogen depletion and subsequent stimulation of glucose transport machinery with endurance exercise (19). AS160 phosphorylation may be regulated by Akt- and AMPK-mediated signaling pathways to release inhibition on the glucose transporter Glut4 (24,37). Our results are similar to those of Sriwijitkamol et al. (35) and Howlett et al. (20), showing increased Akt and AMPK phosphorylation associated with elevated AS160 phosphorylation after prolonged (40-60 min) moderate-intensity cycling but not resistance exercise, respectively. Dreyer et al. (14) have previously shown an increase in AS160 phosphorylation and glucose uptake 60 min after resistance exercise in humans. However, an important distinction between the study of Dreyer et al. (14) and the present investigation was significantly greater volume of exercise. Our results showing equivalent Akt responses yet divergence in the AMPK-AS160 time course with resistance exercise versus cycling indicate that regulation of AS160 phosphorylation may have been an AMPK- rather than an Akt-mediated response. Nonetheless, the relative role of AMPK and Akt in regulation of AS160 remains contentious, and further research is required to determine the contribution of each signaling pathway in modulating exercise-induced AS160 activation.
In conclusion, our findings offer insight of the IGF pathway and AMPK phosphorylation time course during the early recovery period after diverse exercise in skeletal muscle and extend our current knowledge regarding the exercise-specific signaling responses in humans (40). Specifically, we provide new information showing similar Akt-mTOR-S6K phosphorylation concomitant with divergent AMPK-AS160 and GS phosphorylation that was only enhanced after cycling. We propose that some of the uncertainty in establishing the contraction-induced signaling responses in human skeletal muscle has been due to differences in the timing of postexercise muscle biopsies. Moreover, our results indicate that endurance and resistance exercise are equally capable of "switching on" translational signaling. Consequently, it appears unlikely that alterations in translation significantly contribute to the specificity of training adaptation in skeletal muscle. Indeed, to better understand acute exercise-specific adaptive profiles and any incompatibility of adaptation machinery when comparing divergent exercise, elucidating changes at the transcriptional level may be more prudent. Regardless, given the limitations in the maximum number of muscle biopsies from any single individual when undertaking in vivo human research, our findings provide new information for determining the optimal postexercise time point when investigating cell signaling after exercise in the fasted state.
Donny M. Camera and Johann Edge contributed equally to this study.
This study was funded by the Emerging Researcher Grant Scheme at RMIT University (awarded to VGC). The authors thank Andrea Short, Jessica Dent, and Chelsea Aim for technical assistance with experimental trials.
No funding for this work was received from the National Institute of Health, the Wellcome Trust, the Howard Hughes Medical Institute, or others.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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