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Transcript of Resistance Exercise Induced MTORC1 Signalling is Not Impaired by Subsequent Endurance Exercise in...
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Resistance exercise induced mTORC1 signalling is not impaired by subsequent 1
endurance exercise in human skeletal muscle 2
William Apr1,2*, Li Wang1*, Marjan Pontn1, Eva Blomstrand1 and Kent Sahlin1 3
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1strand Laboratory, Swedish School of Sport and Health Sciences, Stockholm, Sweden, 5
2Department of Clinical Science, Intervention and Technology, Karolinska Institutet, 6
Stockholm, Sweden 7
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* W. Apr and L. Wang contributed equally to this work 11
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Corresponding author:
William Apr
The Swedish School of Sport and Health Sciences
Box 5626, SE-114 86 Stockholm, Sweden
E-mail: [email protected]
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Running head: Concurrent exercise does not inhibit mTORC1 signalling 14
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Key words: concurrent exercise, mTORC1, AMPK, interference 17
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Articles in PresS. Am J Physiol Endocrinol Metab (April 30, 2013). doi:10.1152/ajpendo.00091.2013
Copyright 2013 by the American Physiological Society.
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ABSTRACT 19
The current dogma is that the muscle adaptation to resistance exercise is blunted when 20
combined with endurance exercise. The suggested mechanism (based on rodent experiments) 21
is that activation of adenosine-monophosphate-activated protein-kinase (AMPK) during 22
endurance exercise impairs muscle growth through inhibition of the mechanistic-target-of-23
rapamycin-complex 1 (mTORC1). The purpose of this study was to investigate potential 24
interference of endurance training on the signalling pathways of resistance training (mTORC1 25
- phosphorylation of ribosomal protein S6 kinase 1 (S6K1)) in human muscle. Ten healthy 26
and moderately trained male subjects performed on two separate occasions either acute high 27
intensity and high volume resistance exercise (leg press, R) or R followed by 30 min of 28
cycling (RE). Muscle biopsies were collected before, 1 and 3h post resistance exercise. 29
Phosphorylation of mTOR (Ser2448) increased 2-fold (p
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INTRODUCTION 44
Skeletal muscle possesses remarkable plasticity and as such has the unique ability to adapt to 45
various types of contractile activity which ultimately results in divergent phenotypes in 46
accordance with the specificity of training principle (5). The divergent adaptations following 47
resistance and endurance exercise places these exercise modalities in contrasting ends of the 48
training adaptation continuum. As such, the opposing phenotypes are likely dependent on 49
highly specific adaptations which may be incompatible when different exercise modes are 50
performed simultaneously (5). 51
This notion is supported by several studies showing negative effects on the 52
development of strength and power (23, 31) as well as muscle fibre hypertrophy (34, 40) 53
when both modes of exercise have been performed concurrently over longer periods of time. 54
Collectively, these findings have given rise to the current paradigm of the training 55
interference effect (30), yet not all studies support the existence of such a phenomenon (8, 37, 56
42, 43). The reasons for the discrepancies within the literature are not readily obvious but are 57
likely related to experimental variables such as exercise intensity and volume, exercise 58
sequence or nutritional status. 59
Assessment of physical performance with hard outcome measures offers little 60
insight into the molecular mechanisms regulating long term training adaptations. In recent 61
years, our understanding of the molecular events underlying the various training adaptations 62
has increased considerably and several distinct signalling pathways have been identified. For 63
instance, it is well established that skeletal muscle growth to a large extent is mediated by 64
activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway (12, 28). 65
Similarly, peripheral adaptations responsible for mitochondrial biogenesis include induction 66
of the peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) (7, 50) pathway 67 which in turn mediates the characteristic increase in muscle oxidative capacity seen after 68
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endurance training (38). Mechanistically, crosstalk between these two pathways has been 69
linked to the adenosine monophosphate-activated protein kinase (AMPK), an enzyme 70
activated during energetic stress (53). Activation of AMPK by pharmacological agents has 71
been shown to repress mTORC1 signalling (13, 39, 47) but elevate mRNA expression of 72
PGC-1 (32) in rodent muscle. 73 To date, only a small number of studies have been performed in humans to 74
examine the impact of acute concurrent exercise on the signalling pathways leading to 75
different phenotypes. Some of the investigations found support for an interference effect (15, 76
16), whereas others did not (20, 35). More specifically, when concurrent exercise was 77
performed in the fed state (20, 35) and with several hours of recovery time between exercise 78
modes (35), mTORC1 signalling was similar compared with single mode resistance exercise. 79
However, from these studies and from a mechanistic perspective, it is difficult to evaluate the 80
impact of concurrent exercise per se as feeding in itself may influence signalling though the 81
mTORC1 pathway (33). In contrast, when endurance (16) and sprint (15) exercise preceded 82
resistance exercise in the fasted state, with only 15 minutes of rest in between, mTORC1 83
signalling was attenuated compared to when the exercise order was reversed. However, none 84
of these studies included a single mode exercise for comparison. Thus, to date, no study has 85
investigated the signalling response following concurrent exercise compared with single mode 86
resistance exercise in the fasted state. 87
Therefore, the aim of the present investigation was to examine whether 88
endurance exercise following a heavy resistance exercise protocol would repress molecular 89
signalling through the mTORC1 pathway, compared to single mode resistance exercise. To 90
this end, muscle biopsies from moderately trained men were analyzed with regard to protein 91
signalling and mRNA expression involved in skeletal muscle hypertrophy and mitochondrial 92
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biogenesis. It was hypothesized that concurrent exercise would impair growth related 93
signalling and gene expression. 94
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METHODS 96 97 Subjects 98
Ten healthy moderately trained male subjects were recruited for this study. After being 99
informed of the purpose of the study and of all associated risks, all subjects gave written 100
consent. To be eligible for enrollment in the study, subjects were required to have performed 101
resistance exercise 2-3 times per week and endurance exercise 1-2 times a week during the 102
last 6 months and to have a maximal leg strength equaling four times their body weight or 103
more. Subject characteristics are presented in Table 1. The study was approved by the 104
Regional Ethical Review Board in Stockholm and performed in accordance with the 105
principles outlined in the Declaration of Helsinki. 106
107
Study design 108
The study employed a randomized cross-over design in which each subject performed one 109
session of resistance exercise (R) and another session of resistance exercise followed by 110
endurance exercise (RE). The two sessions were separated by approximately two weeks. A 111
schematic overview of the experimental protocols is provided in Fig.1. All subjects were 112
instructed to maintain their habitual dietary intake and physical activity pattern throughout the 113
entire experimental period. Subjects were instructed to refrain from physical exercise for two 114
days before each trial as well as to record and duplicate their food intake before the first and 115
second trials, respectively. 116
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Pre-tests 119
Before initiation of the actual experiments, each subjects two-legged one repetition 120
maximum (1RM) was determined on a leg press machine (243 Leg press 45, Gymleco, 121
Stockholm, Sweden) after warming up on a cycle ergometer for 10 min. The 1RM was 122
assessed by gradually increasing the load until the subject was unable to perform no more 123
than one single repetition (90-180 knee angle). Maximal and submaximal oxygen uptake was 124
determined on a mechanically braked cycle ergometer (Monark 839E, Vansbro, Sweden) with 125
the work rate gradually increased until volitional exhaustion as described by strand & 126
Rodahl (56). Oxygen uptake was measured continuously utilizing an on-line system (Oxycon 127
Pro, Erich Jaeger GmbH, Hoechberg, Germany) and heart rate (HR) was recorded 128
continuously (Polar Electro Oy, Kempele, Finland). Following initial testing, subjects 129
performed two familiarization sessions in order to minimize any training effects during the 130
live experiments (see below). 131
On the day of each trial, subjects reported to the laboratory at approximately 7.30 am 132
following an overnight fast from 9.00 pm the evening before. Upon arrival, subjects were 133
placed in a supine position and rested for 10 min prior to the collection of a resting blood 134
sample from an arm vein. After subsequent administration of local anaesthesia, a resting 135
biopsy was collected from the middle portion of the vastus lateralis muscle of one leg using a 136
Bergstrm needle (11) with manually applied suction. 137
Following blood and tissue sampling, subjects were seated in the leg press machine 138
and performed 3 warm-up sets of 10 repetitions at 0, 30 and 60% of 1RM with 3 min of rest 139
between each set. Thereafter, the subjects performed 10 sets of heavy resistance exercise (R) 140
or resistance exercise followed by endurance exercise (RE). The resistance exercise protocol 141
consisted of 4 sets of 8-10 repetitions at 85 % of 1 RM, 4 sets of 10-12 repetitions at 75 % of 142
1RM and lastly 2 sets to volitional fatigue at 65 % of 1RM with 3 min of recovery allowed 143
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between each set. The load and number of repetitions was recorded during the first trial and 144
duplicated during the second trial. Time under tension was recorded for each set during the 145
first trial and was used to match, as closely as possible, the repetition speed in each set during 146
the second trial. Following resistance exercise in the RE trial, subjects rested for 15 min after 147
which 30 min of cycling was initiated at an intensity equal to 70 % of the subjects' maximal 148
oxygen consumption (Table 2). 149
In both trials, two additional muscle biopsies were collected in the contra lateral leg 150
at 1 and 3 h following resistance exercise together with blood samples. For each biopsy 151
sampling, a new incision was made approximately 3-4 cm proximal to the previous one and 152
after biopsy collection, samples were immediately blotted free of blood and frozen in liquid 153
nitrogen and stored at -80C for later analysis. Due to technical difficulties, tissue sampling at 154
3 h post resistance exercise was unsuccessful for one subject in the R-trial and for another 155
subject in the RE-trial. 156
157
Plasma analysis 158
Blood samples (4 ml) were centrifuged at 1500 g at 4C for 10 min and the plasma obtained 159
was stored at -20C. Plasma samples were later analyzed for glucose and lactate 160
concentrations as described by Bergmeyer (10). 161
162
Immunoblot analysis 163
Muscle samples were lyophilized, cleaned from blood and connective tissue under a 164
dissection microscope (Carl Zeiss, Germany), and then homogenized in ice-cold buffer (80 165
l/mg dry weight) containing 2 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM EGTA, 10 mM 166
MgCl2, 50 mM -glycerophosphate, 1% TritonX-100, 1 mM Na3VO4, 2 mM dithiothreitol, 20 167 g/ml leupeptin, 50 g/ml aprotinin, 1% phosphatase inhibitor cocktail (Sigma P-2850) and 168
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40 g/l PMSF. Homogenates were then cleared by centrifugation at 10,000 g for 10 min at 169
4C and the resulting supernatant was stored at -80C. 170
Protein concentrations were determined in aliquots of supernatant diluted 1:10 in 171
distilled water using a bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL, USA). 172
Samples were diluted in Laemmli sample buffer (Bio-Rad Laboratories, Richmond, CA, 173
USA) and homogenizing buffer to obtain a final protein concentration of 1.5 g/l. Following 174
dilution, all samples were heated at 95C for 5 min to denature proteins present in the 175
supernatant. Samples were then kept at -20C until further analysis. 176
Details of the western blotting procedures have been described elsewhere (3). Briefly, 177
and with minor modifications, samples containing 30 g total protein were separated by SDS-178
PAGE on Criterion cell gradient gels (4-20% acrylamide; Bio-Rad Laboratories). 179
Electrophoresis was performed on ice at 200 V for 120 min, after which the gels were 180
equilibrated in transfer buffer (25 mM Tris base, 192 mM glycine, and 10% methanol) for 30 181
min. The proteins were then transferred to polyvinylidine fluoride membranes (Bio-Rad 182
Laboratories) at a constant current of 300 mA for 3 h at 4C. Following transfer, membranes 183
were stained with MemCodeTM Reversible Protein Stain Kit (Pierce Biotechnology) (2) to 184
confirm equal loading of the samples. All samples from each subject were run on the same gel. 185
Membranes were blocked for 1 h at room temperature in Tris-buffered saline (TBS; 186
20 mM Tris base, 137 mM NaCl, pH 7.6) containing 5% non-fat dry milk. After blocking, 187
membranes were incubated overnight with commercially available primary antibodies diluted 188
in TBS supplemented with 0.1% Tween-20 containing 2.5% non-fat dry milk (TBS-TM). 189
After incubation with these primary antibodies, the membranes were washed with TBS-TM 190
and incubated for 1 h at room temperature with secondary antibodies conjugated with 191
horseradish peroxidise. Next, the membranes were washed serially (2 x 1 min, 3 x 15 min) 192
with TBS-TM, followed by 4 additional washes with TBS for 5 min each. Finally, membranes 193
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with the antibodies bound to the target proteins were visualized by chemiluminescent 194
detection on a Molecular Imager ChemiDocTM XRS system and the bands were analysed 195
using the contour tool in the Quantity One version 4.6.3 software (Bio-Rad Laboratories). 196
All values are expressed relative to total levels of -tubulin. 197 198
Antibodies 199
Primary antibodies raised in rabbit against phospho-Akt (Ser473; #9271), phospho-mTOR 200
(Ser2448; #2971), phospho-S6K1 (Thr389; #9234), phospho-4E-BP1 (Thr37/46; #2855), phospho-201
eEF2 (Thr56; #2331), phospho-AMPK (Thr172; #4188), phospho-ACC (Ser79; #3661), 202
phospho-p38 (Thr180/Tyr182; #9211), phospho-ERK1/2 (Thr202/Tyr204; #9101) and phospho-203
CaMKII (Thr286; #3361) was purchased from Cell Signaling Technology (Beverly, MA, USA). 204
Primary total antibodies for mouse -tubulin (#T6074) and rabbit REDD1 (#ab63059) were 205 purchased from Sigma-Aldrich (St. Louis, MO, USA) and Abcam (Cambridge, UK), 206
respectively. All primary antibodies were diluted 1:1000 except for eEF2 and -tubulin which 207 were diluted 1:5000. Secondary anti-rabbit (#7074) and anti-mouse (#7076) antibodies 208
(1:10000) were purchased from Cell Signaling Technology. 209
210
RNA extraction and quantitative Real-Time PCR (qRT-PCR) 211
Total RNA was extracted from approximately 2 mg lyophilized and cleaned tissue which was 212
homogenized in PureZOL RNA isolation reagent (Bio-Rad Laboratories) according to the 213
manufacturers instructions. The concentration and purity of the RNA was determined by 214
spectrofotometry and 1 g RNA was used for reverse transcription of 20 l cDNA with 215 iScript cDNA Synthesis Kit (Bio-Rad Laboratories). The primers for the specific genes 216
analyzed here have been presented in a previous publication from this laboratory (50). The 217
concentration of cDNA, annealing temperature and PCR cycle protocol were determined for 218
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each primer pair to ensure optimal conditions for amplification. Samples were run in triplicate 219
and all samples from each subject were run on the same plate to allow direct relative 220
comparisons. qRT-PCR amplification mixtures (25 l) contained 12.5 l 2SYBR Green 221 Supermix (Bio-Rad Laboratoies), 0.5 l 10 M forward and reverse primers, respectively, and 222 11.5 l template cDNA in RNase-free water. qRT-PCR was performed with Bio-Rad iCycler 223 (Bio-Rad Laboratories) and relative changes in mRNA levels were analyzed by the 2-CT 224
method with GAPDH used as the reference gene. 225
226
Statistical analyses 227
All data are expressed as means SE. For protein signalling, gene expression and plasma data, 228
a two-way repeated-measures ANOVA (trial and time) was used for statistical analysis. For 229
missing data points, weighted means were used in the analysis. When a significant main effect 230
or an interaction effect was observed, Fisher LSD post hoc analysis was performed to locate 231
differences. For some positively skewed distributed variables, log-transformation was 232
performed before analyses. For analysis of time under tension and number of repetitions, a 233
paired t-test was used. Statistical significance was set at P < 0.05. 234
235
RESULTS 236
Task performance 237
Eight subjects performed the same number of repetitions in both trials while two subjects 238
performed one and two repetition less, respectively, in the RE-trial. Time under tension was 239
similar between trials (Table 2). Average heart rate during cycling corresponded to 88 1 % 240
of maximal heart rate. 241
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Blood parameters 244
Plasma concentrations of glucose were significantly decreased at both time points during 245
recovery in the R-trial (Table 3). In the RE-trial, plasma levels of glucose remained 246
unchanged until the 3 h time point at which these levels decreased but only compared to 1 h 247
post resistance exercise (Table 3). Plasma levels of lactate were increased in both trials 1 h 248
after resistance exercise, but more so in the RE-trial. By the 3 h time point, lactate levels had 249
dropped back to baseline values in both trials (Table 3). 250
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Protein signalling 252
Phosphorylation of Akt at Ser473 was decreased 1 h after resistance exercise in the R trial 253
compared to resting values as well as being lower compared to the RE trial at the same time 254
point (Fig. 2A). For phosphorylation of mTOR at Ser2448, statistical analysis revealed main 255
effects of time as well as trials (p
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Phosphorylation of AMPK at Thr172 was unchanged 1 h after resistance exercise in 269
both trials. At the 3 h time point, phosphorylation of this protein was decreased 33% (p
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levels, although these levels had returned to baseline values in both trials at the 3 h time point 293
(Table 4). 294
Both exercise protocols induced significant and similar elevations in mRNA 295
expression of REDD1 at the 1 h time point (p
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Mechanistically, activation of mTORC1 results in a coordinated signalling cascade to 317
various components within the translational machinery, which when repeated over time, has 318
been shown to correlate with muscle hypertrophy in both rodent (6) and human muscle (46). 319
The regulatory role of mTORC1 in muscle growth was recently confirmed by Goodman and 320
colleagues (28, 29), who in a series of elegantly designed experiments using various genetic 321
mouse models showed that not only is load induced muscle growth dependent on mTORC1 322
signalling (28), but activation of this complex is sufficient to induce muscle hypertrophy (29). 323
We had initially hypothesized that combining resistance and endurance exercise would impair 324
mTORC1 signalling. However, in contrast to this hypothesis, both modes of exercise induced 325
substantial elevations in mTOR phosphorylation at Ser2448 as well as of its immediate 326
downstream target S6K1 at the Thr389 residue. While mTOR phosphorylation increased 2-fold, 327
the phosphorylation level of S6K1 increased 5-fold at the 1 h time point but continued to 328
increase until 3 h post exercise, reaching 14-fold higher values compared to baseline. 329
In addition to regulating translation initiation, mTORC1 mediated signaling also 330
stimulates translation elongation by repressing phosphorylation of the eukaryotic elongation 331
factor 2 (eEF2) at Thr56 (51). In the present study, both exercise protocols induced dramatic 332
reductions in eEF2 phosphorylation during recovery and in line with the mTOR and S6K1 333
phosphorylation data, we could not detect any difference in eEF2 phosphorylation between 334
trials. Collectively, these findings indicate that the added endurance exercise does not impose 335
an inhibitory effect on growth related signaling, neither at the level of translation initiation nor 336
translation elongation. 337
The reason for the lack of AMPK phosphorylation in this trial is not readily apparent 338
as we (49) and others (14, 26, 48) have shown that endurance exercise with similar intensity 339
and duration as that utilized here induces robust elevations in phosphorylation as well as 340
activity of this kinase. Some studies show that endurance trained subjects with high VO2max 341
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values (i.e. 65 ml min-1 kg-1) may have a diminished AMPK response to endurance 342 exercise (17), especially compared to untrained subjects (55). However, other studies (14, 55) 343
have shown elevated activity and phosphorylation of AMPK in subjects with similar training 344
status (~50 ml min-1 kg-1) as those involved in this study. In addition, sampling was 345
performed 15 min post endurance exercise (1 h after resistance exercise) in the RE-trial. This 346
time point is well within the time frame of expected elevations in AMPK phosphorylation 347
and/or activity as shown previously (21, 49). Thus, the lack of AMPK phosphorylation in the 348
present study is likely related to other factors than those discussed above. 349
Although activation of AMPK following endurance exercise is well recognized (14, 350
26, 36, 48, 54), the molecular interplay with mTORC1 in human muscle is much less 351
established. The complexity of these interactions is demonstrated by several studies showing 352
simultaneous increases in AMPK and mTORC1 signaling following endurance (9, 36, 52) and 353
resistance exercise (21, 52) as well as after concurrent exercise (49). Furthermore, even 354
though AMPK phosphorylation was increased in these trials, protein synthesis was elevated 355
(21, 36, 52), a finding that is contradictory to the suggested mechanism of the interference 356
effect (13, 39, 47). Thus, it is unclear whether or not activation of AMPK impairs mTORC1 357
signalling in human muscle under physiological conditions. 358
Unexpectedly, at the 3 h time point, phosphorylation of AMPK was actually 359
decreased approximately 30% compared to baseline values. This reduction was also seen on 360
the phosphorylation levels of ACC, although to a larger extent (~50%). This finding was 361
present in both trials, suggesting it may be related to activation of mTORC1 induced by the 362
resistance exercise protocol. In support of this idea, Atherton and colleagues (4) demonstrated 363
that high frequency electrical stimulation of rat muscle resulted in pronounced increases in 364
phosphorylation of S6K1 at Thr389 three hours post stimulation and at this time point, AMPK 365
phosphorylation at Thr172 was depressed below resting values. Similarly, in a study by Fujita 366
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et al. (27), resistance exercise combined with nutrient provision dramatically induced 367
mTORC1 signalling while concomitantly reducing AMPK phosphorylation at Thr172. In 368
contrast, some studies have failed to detect a decrease in AMPK phosphorylation despite 369
elevated phosphorylation of S6K1 (17, 22). Our findings may be related, at least in part, to the 370
intensity and volume of the exercise protocol used here, which was much higher than in the 371
previously mentioned studies. 372
In the present study, decreased AMPK phosphorylation coincided with maximal 373
phosphorylation of S6K1 at Thr389 in both trials, an association also observed by others (4, 27). 374
Dagon et al. (19) recently demonstrated a direct inhibitory phosphorylation of AMPK at 375
Ser491 by S6K1 in mouse hypothalamic cells and although not yet confirmed in skeletal 376
muscle, these findings identify a negative feed-back loop in which AMPK is influenced by the 377
mTORC1 pathway. Additional support for a regulatory role of S6K1 on AMPK activity is 378
provided by Aguliar and colleagues (1) who showed increased phosphorylation of the Thr172 379
residue of AMPK in cultured myotubes of S6K1-deficient mice. Therefore, based on these 380
observations, we propose that prior activation of mTORC1 inhibits the activity of AMPK 381
which would suggest bidirectional crosstalk between these pathways. Furthermore, in the 382
studies showing an inhibitory effect of AMPK on mTORC1 (13, 39, 47), pharmacological 383
activation of AMPK was initiated prior to determination of mTORC1 signalling indicating the 384
existence of an order effect. 385
We also measured gene expression of several positive (hVps34, Rheb and cMyc) 386
and negative (TSC1/2, REDD1/2) effectors of mTORC1 signaling, as well as expression of 387
key components (mTOR and S6K1) of this pathway (Table 4). There were only modest 388
fluctuations in mRNA expression of most of these genes but most importantly, we could not 389
detect any differences between trials at any time point, in line with the signaling data 390
presented above. Interestingly, the pattern of mRNA expression of the growth related 391
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transcription factor cMyc mirrored that of S6K1 phosphorylation (Fig. 5D), suggesting that 392
expression of this gene may, at least in part, be regulated by mTORC1 as has been shown in 393
Drosophila (45). These findings show that the lack of inhibition induced by endurance 394
exercise is not only evident at the level of translation initiation and elongation, but also at the 395
level of transcription. 396
In addition to measuring mRNA abundance, we also analyzed total levels of REDD1 397
protein. Both REDD1 and REDD2 have been identified as negative regulators of mTORC1 398
signaling under various conditions of cellular stress (24), including energetic stress (44). 399
Although the precise mechanism by which REDD 1/2 inhibits mTORC1 signaling remains 400
elusive, in vitro studies have shown that induction of REDD1 occurs within hours following 401
hormonal treatment (25, 41). As such, 3 h post resistance exercise could have been an 402
appropriate time point to detect a potential increase in expression of this protein. However, 403
even though the RE-trial arguably induced a greater energetic stress, we could not detect any 404
differences in REDD1 protein levels between trials (Fig. 5A). 405
In contrast to the other genes examined, concurrent exercise resulted in superior 406
mRNA expression of PGC-1 (Fig. 6A), a molecule shown to have a major regulatory role in 407 mitochondrial biogenesis (38). Although an expected finding, the reason for the differential 408
expression of this gene is not readily obvious, at least from a mechanistic perspective. 409
Induction of PGC-1 has been linked to increased signalling by several upstream effectors 410 such as AMPK, calcium/calmodulin-dependent protein kinase (CaMK) and the mitogen-411
activated protein kinase p38 (38), none of which differed between trials in the present study. 412
In addition to these kinases, mTOR has also been implicated in the induction of PGC-1 (18), 413 yet, phosphorylation of mTOR and of all its immediate downstream targets was similar 414
between protocols. Consequently, these data do not allow for any clear conclusions regarding 415
the involvement of these kinases in the PGC-1 response seen during recovery in the RE-trial. 416
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In summary, both single mode resistance exercise and concurrent exercise 417
resulted in robust and positive alterations of regulatory proteins involved in translation 418
initiation (i.e. mTOR and S6K1) and elongation (eEF2), thus promoting skeletal muscle 419
hypertrophy (6, 28, 29, 46). These data demonstrate that moderate intensity endurance 420
exercise performed subsequent to a high intensity high volume resistance exercise protocol 421
does not inhibit mTORC1 signaling during acute recovery in moderately trained men. This 422
conclusion is further supported by the reduction in AMPK phosphorylation following both 423
modes of exercise which we propose is due to prior activation of mTORC1 induced by the 424
resistance exercise protocol. This finding suggests that the interference effect between these 425
pathways is bidirectional. We therefore propose that endurance exercise may be included in 426
training regimes aiming to promote muscle growth. However, further studies are required to 427
assess if the acute changes in the signalling pathways examined here fully reflect long term 428
training adaptations. 429
430
ACKNOWLEDGMENTS 431
We thank all participants for their valuable time and efforts in this study. This study was 432
supported financially by grants from the Swedish National Centre for Research in Sports and 433
the Swedish School of Sport and Health Sciences in Stockholm, Sweden. 434
435
DISCLOSURES 436
No conflicts of interest, financial or otherwise, are declared by the authors.
437 438 439 440 441 442
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47. Thomson DM, Fick CA, and Gordon SE. AMPK activation attenuates S6K1, 589 4E-BP1, and eEF2 signaling responses to high-frequency electrically stimulated skeletal 590 muscle contractions. J Appl Physiol 104: 625-632, 2008. 591 48. Wadley GD, Lee-Young RS, Canny BJ, Wasuntarawat C, Chen ZP, 592 Hargreaves M, Kemp BE, and McConell GK. Effect of exercise intensity and hypoxia on 593 skeletal muscle AMPK signaling and substrate metabolism in humans. American journal of 594 physiology 290: E694-702, 2006. 595 49. Wang L, Mascher H, Psilander N, Blomstrand E, and Sahlin K. Resistance 596 exercise enhances the molecular signaling of mitochondrial biogenesis induced by endurance 597 exercise in human skeletal muscle. J Appl Physiol 111: 1335-1344, 2011. 598 50. Wang L, Psilander N, Tonkonogi M, Ding S, and Sahlin K. Similar 599 expression of oxidative genes after interval and continuous exercise. Med Sci Sports Exerc 41: 600 2136-2144, 2009. 601 51. Wang X and Proud CG. The mTOR pathway in the control of protein 602 synthesis. Physiology (Bethesda, Md 21: 362-369, 2006. 603 52. Wilkinson SB, Phillips SM, Atherton PJ, Patel R, Yarasheski KE, 604 Tarnopolsky MA, and Rennie MJ. Differential effects of resistance and endurance exercise 605 in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle. 606 The Journal of physiology 586: 3701-3717, 2008. 607 53. Winder WW. Energy-sensing and signaling by AMP-activated protein kinase 608 in skeletal muscle. J Appl Physiol 91: 1017-1028, 2001. 609 54. Wojtaszewski JF, Nielsen P, Hansen BF, Richter EA, and Kiens B. Isoform-610 specific and exercise intensity-dependent activation of 5'-AMP-activated protein kinase in 611 human skeletal muscle. The Journal of physiology 528 Pt 1: 221-226, 2000. 612 55. Yu M, Stepto NK, Chibalin AV, Fryer LG, Carling D, Krook A, Hawley JA, 613 and Zierath JR. Metabolic and mitogenic signal transduction in human skeletal muscle after 614 intense cycling exercise. The Journal of physiology 546: 327-335, 2003. 615 56. strand P-O and Rodahl K. Textbook of work physiology. New York: 616 McGraw Hill, 1986. 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638
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23
FIGURE CAPTIONS 639 640 641
FIGURE 1 642
Schematic overview of the experimental trials. R-protocol, resistance exercise only; RE-643
protocol, resistance exercise followed by cycling. Arrows indicate sampling time points for 644
muscle biopsies and blood samples. 645
646
FIGURE 2 647
Phosphorylation levels of Akt (60 kDa) at Ser473 (A), mTOR (289 kDa) at Ser2448 (B), S6K1 648
(70 kDa) at Thr389 (C) and eEF2 (95 kDa) at Thr56 (D) before, 1 and 3 h post resistance 649
exercise in both trials. Representative immunoblots from one subject are shown above each 650
graph. Values are normalized to -tubulin and presented as means SE for 10 subjects (n = 9 651 for 3 h Post). Symbols above lines denote differences revealed by a post-hoc test when a main 652
effect was observed. Symbols without lines denote differences revealed by a post-hoc test 653
when an interaction effect was observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post; P < 0.05 654
vs. R. 655
656
FIGURE 3 657
Phosphorylation levels of AMPK (62 kDa) at Thr172 (A), ACC (280 kDa)at Ser79 (B), p38 658
(~43 kDa) at Thr180/Tyr182 (C) before, 1 and 3 h post resistance exercise in both trials. 659
Representative immunoblots from one subject are shown above each graph. Values are 660
normalized to -tubulin and presented as means SE for 10 subjects (n = 9 for 3 h Post). 661 Symbols above lines denote differences revealed by a post-hoc test when a main effect was 662
observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post. 663
664
-
24
FIGURE 4 665
Phosphorylation levels of 4EBP1 (~15-20 kDa) at Thr37/46 (A), ERK1/2 (42/44 kDa) at 666
Thr202/Tyr204 (B), CaMKII (~50 kDa) at Thr286 (C) before, 1 and 3 h post resistance exercise in 667
both trials. Representative immunoblots from one subject are shown above each graph. 668
Values are normalized to -tubulin and presented as means SE for 10 subjects (n = 9 for 3 h 669 Post). Symbols above lines denote differences revealed by a post-hoc test when a main effect 670
was observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post. 671
672
673
FIGURE 5 674 Total protein levels of REDD1 (~34 kDa) (A) and mRNA expression of REDD1 (B), REDD2 675
(C) and cMyc (D) before, 1 and 3 h post resistance exercise in both trials. Representative 676
immunoblots from one subject are shown above graph A. Protein levels are normalized to -677 tubulin and presented as means SE for 10 subjects (n = 9 for 3 h Post). Symbols above lines 678
denote differences revealed by a post-hoc test when a main effect was observed. *P < 0.05 vs. 679
Rest; #P < 0.05 vs. 1h Post. 680
681 682 FIGURE 6 683
mRNA expression of PGC-1 (A), PRC (B) and PDK4 (C) before, 1 and 3 h post resistance 684 exercise in both trials. Values are presented as means SE for 10 subjects (n = 9 for 3 h Post). 685
Symbols above lines denote differences revealed by a post-hoc test when a main effect was 686
observed. Symbols without lines denote differences revealed by a post-hoc test when an 687
interaction effect was observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post; P < 0.05 vs. R. 688
689 690 691 692 693
-
25
TABLES 694 695 TABLE 1. Subject characteristics 696 697 Variables Mean SEM Age (years) 26 2 Weight (kg) 85 3 Height (cm) 179 2 1 RM (kg) 389 17 VO2max (l min-1) 4.27 0.12 Relative VO2max (ml min-1 kg-1) 50.8 1.6 HRmax (bpm) 192 2 Wattmax (W) 308 15 698
Values are presented as means SE for 10 subjects. 1RM, one repetition maximum; VO2max, 699
maximum oxygen uptake; HRmax, maximum heart rate; Wattmax, maximum cycling intensity 700
expressed in watts. 701
702
TABLE 2. Details of the performed resistance exercise 703
704
705
706
707
708
709
710
711
Values are presented as means SE for 10 subjects. R, resistance exercise only; RE, 712
resistance exercise followed by cycling; 1RM, one repetition maximum. 713
714 715 716 717 718 719 720
Set # % of 1RM Load, kg Repetitions, times Time under tension, sec
Trial R RE R RE 1 0 0 0 10 0 10 0 27.2 0.9 29.1 1.0 2 30 89 5 10 0 10 0 27.2 0.9 26.5 1.2 3 60 217 10 10 0 10 0 27.0 1.1 26.0 1.3 4 85 313 12 9.7 0.2 9.7 0.2 28.9 1.2 28.8 1.5 5 9.3 0.3 9.3 0.3 29.7 0.9 28.8 1.7 6 9.3 0.3 9.3 0.3 30.0 1.1 30.5 1.6 7 8.4 0.5 8.4 0.5 29.8 1.4 29.5 1.9 8 75 271 10 11.4 0.3 11.4 0.3 32.4 1.2 33.7 1.4 9 11.1 0.3 11.1 0.3 33.9 1.7 34.4 1.3 10 10.3 0.5 10.3 0.5 31.5 1.3 33.0 1.9 11 10.0 0.9 9.8 0.9 30.5 2.7 31.7 2.7 12 65 225 9 13.8 0.7 13.8 0.7 38.5 2.1 38.2 1.9 13 12.7 0.9 12.6 0.9 37.2 2.6 37.5 2.3
Total 136.0 135.7 403.8 407.7
-
26
TABLE 3. Plasma concentrations of glucose and lactate. 721
Values are presented as means SE for 10 subjects. Blood was sampled at rest and at 1 and 3 722
h post resistance exercise. R, resistance exercise only; RE, resistance exercise followed by 723
cycling. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post; P < 0.05 vs. R. 724
725 726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
Trial Rest 1 h Post 3 h Post
Glucose, mmol/l R 5.65 0.11 5.03 0.11* 5.29 0.06* RE 5.53 0.10 5.77 0.16 5.42 0.11#
Lactate, mmol/l R 1.43 0.23 3.51 0.28* 1.35 0.09# RE 1.49 0.23 6.13 0.41* 1.42 0.08#
-
27
TABLE 4. Gene expression related to muscle growth. 743
744
745
746
747
748
749
750
751
752
753
754
Values are presented as means SE for 10 subjects (n = 9 for 3 h Post). Muscle was sampled 755
at rest and at 1 and 3 h post resistance exercise. R, resistance exercise only; RE, resistance 756
exercise followed by cycling. Rheb, ras homolog enriched in brain; mTOR, mechanistic target 757
of rapamycin; S6K1, 70 kD ribosomal protein S6 kinase 1; hVps34, human vacuolar protein 758
sorting 34; TSC1/2, tuberous sclerosis complex 1 and 2. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h 759
Post. 760
761
Genes Exercise Rest 1h post 3h post Main effects Int. effect Time Exr Time x Exr
Rheb R 0.64 0.11 0.69 0.09* 0.95 0.15* P
-
1Figure 1.
~45 min
R - protocol
Resistance exercise Rest
Biopsy and blood samples~45 min
Rest 3 h1 h
Resistance exercise RestCyclingRest Rest
30 min15 min 15 min
Biopsy and blood samples
Rest 3 h1 h
RE - protocol
0 h
0 h
~45 min
R - protocol
Resistance exercise Rest
Biopsy and blood samples~45 min
Rest 3 h1 h
Resistance exercise RestCyclingRest Rest
30 min15 min 15 min
Biopsy and blood samples
Rest 3 h1 h
RE - protocol
0 h
0 h
-
2Figure 2.
Rest 1 h Post 3 h Post
Pho
spho
ryla
tion
of A
kt a
t S47
3(a
rbitr
ary
units
)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
*
p-Akt
a-tubulin
Rest 1 h Post 3 h Post
Pho
spho
ryla
tion
of A
kt a
t S47
3(a
rbitr
ary
units
)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
*
p-Akt
a-tubulin
R RERest 1 h 3 h Rest 1 h 3 h
Rest
Phos
phor
ylat
ion
of e
EF2
at T
56(a
rbitr
ary
units
)
*
RRE
0.0
0.5
1.0
1.5
2.0
2.5
*
p-eEF2
a-tubulin
1 h Post 3 h Post
R RERest 1 h 3 h Rest 1 h 3 h
Rest
Pho
spho
ryla
tion
of S
6K1
at T
389
(arb
itrar
y un
its)
*
RRE
0.0
0.5
1.0
1.5
2.0
2.5
* #
p-S6K1
a-tubulin
1 h Post 3 h Post
R RERest 1 h 3 h Rest 1 h 3 h
DC
A B
Rest
Pho
spho
ryla
tion
of m
TOR
at S
2448
(arb
itrar
y un
its)
*
RRE
0.0
0.5
1.0
1.5
2.0
2.5
*
p-mTOR
a-tubulin
1 h Post 3 h Post
R RERest 1 h 3 h Rest 1 h 3 h
-
3Figure 3.
C
A B
Rest
Phos
phor
ylat
ion
of p
38 a
t T18
0/Y
182
(arb
itrar
y un
its)
*RRE
0.0
0.5
1.0
1.5
2.0
2.5
* #
p-p38
a-tubulin
1 h Post 3 h Post
R RERest 1 h 3 h Rest 1 h 3 h
Rest
Pho
spho
ryla
tion
of A
MP
K a
t T17
2(a
rbitr
ary
units
)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
* #
p-AMPK
a-tubulin
1 h Post 3 h Post
R RERest 1 h 3 h Rest 1 h 3 h
Rest
Phos
phor
ylatio
n of
AC
C a
t S79
(arb
itrar
y un
its)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
* #
p-ACC
a-tubulin
1 h Post 3 h Post
R RERest 1 h 3 h Rest 1 h 3 h
-
4Figure 4.
Rest
Phos
phor
ylatio
n of
ER
K1/2
at T
202/
Y20
4(a
rbitr
ary
units
)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
1 h Post 3 h Post
p-ERK1/2
a-tubulin
R RERest 1 h 3 h Rest 1 h 3 h
Rest
Pho
spho
ryla
tion
of 4
EBP
1 at
T37
/46
(arb
itrar
y un
its)
*
RRE
0.0
0.5
1.0
1.5
2.0
2.5
#
1 h Post 3 h PostRest
Pho
spho
ryla
tion
of 4
EBP
1 at
T37
/46
(arb
itrar
y un
its)
*
RRE
0.0
0.5
1.0
1.5
2.0
2.5
#
1 h Post 3 h Post
p-4EBP1
a-tubulin
R RERest 1 h 3 h Rest 1 h 3 h
Rest 1 h 3 h
RRE
0.0
0.5
1.0
1.5
2.0
2.5
Pho
spho
ryla
tion
of C
aMK
II at
T28
6(a
rbitr
ary
units
)
Rest 1 h 3 h
RRE
0.0
0.5
1.0
1.5
2.0
2.5
Pho
spho
ryla
tion
of C
aMK
II at
T28
6(a
rbitr
ary
units
)
p-CaMKII
a-tubulin
R RERest 1 h 3 h Rest 1 h 3 h
C
A B
-
5Figure 5.
Rest 1 h 3 h
RE
DD1
mR
NA
exp
ress
ion
(arb
itrar
y un
its)
* #
RRE
0.0
0.5
1.0
1.5
2.0
2.5 *
Rest 1 h 3 h
RED
D2
mR
NA
exp
ress
ion
(arb
itrar
y un
its)
* #
RRE
0.0
0.5
1.0
1.5
2.0
2.5
DC
A B
Rest
Expr
essi
on o
f RE
DD
1(a
rbitr
ary
units
)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
* #
REDD1
a-tubulin
1 h Post 3 h Post
R RERest 1 h 3 h Rest 1 h 3 h
0.0
0.5
1.0
1.5
2.0
2.5
Rest 1 h 3 h
RRE
cMyc
mR
NA
exp
ress
ion
(arb
itrar
y un
its)
* #
*
-
6Figure 6.
* #
* #
Rest 1 h 3 h
PG
C-1
am
RN
A ex
pres
sion
(arb
itrar
y un
its)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
*
#
Rest 1 h 3 h
PR
C m
RN
A ex
pres
sion
(arb
itrar
y un
its)
RRE
0.0
0.5
1.0
1.5
2.0
2.5
A B
C
Rest 1 h 3 h
PD
K4 m
RN
A ex
pres
sion
(arb
itrar
y un
its)
* *RRE
0.0
0.5
1.0
1.5
2.0
2.5
Article FileFigure 1Figure 2 A-DFigure 3 A-CFigure 4 A-CFigure 5 A-DFigure 6 A-C