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

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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

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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

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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

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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

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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

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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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REFERENCES 443 444 1. Aguilar V, Alliouachene S, Sotiropoulos A, Sobering A, Athea Y, Djouadi F, 445 Miraux S, Thiaudiere E, Foretz M, Viollet B, Diolez P, Bastin J, Benit P, Rustin P, 446 Carling D, Sandri M, Ventura-Clapier R, and Pende M. S6 kinase deletion suppresses 447 muscle growth adaptations to nutrient availability by activating AMP kinase. Cell metabolism 448 5: 476-487, 2007. 449 2. Antharavally BS, Carter B, Bell PA, and Krishna Mallia A. A high-affinity 450 reversible protein stain for Western blots. Analytical biochemistry 329: 276-280, 2004. 451 3. Apro W and Blomstrand E. Influence of supplementation with branched-chain 452 amino acids in combination with resistance exercise on p70S6 kinase phosphorylation in 453 resting and exercising human skeletal muscle. Acta physiologica (Oxford, England) 200: 237-454 248, 2010. 455 4. Atherton PJ, Babraj J, Smith K, Singh J, Rennie MJ, and Wackerhage H. 456 Selective activation of AMPK-PGC-1alpha or PKB-TSC2-mTOR signaling can explain 457 specific adaptive responses to endurance or resistance training-like electrical muscle 458 stimulation. Faseb J 19: 786-788, 2005. 459 5. Baar K. Training for endurance and strength: lessons from cell signaling. 460 Medicine and science in sports and exercise 38: 1939-1944, 2006. 461 6. Baar K and Esser K. Phosphorylation of p70(S6k) correlates with increased 462 skeletal muscle mass following resistance exercise. The American journal of physiology 276: 463 C120-127, 1999. 464 7. Baar K, Wende AR, Jones TE, Marison M, Nolte LA, Chen M, Kelly DP, 465 and Holloszy JO. Adaptations of skeletal muscle to exercise: rapid increase in the 466 transcriptional coactivator PGC-1. Faseb J 16: 1879-1886, 2002. 467 8. Balabinis CP, Psarakis CH, Moukas M, Vassiliou MP, and Behrakis PK. 468 Early phase changes by concurrent endurance and strength training. Journal of strength and 469 conditioning research / National Strength & Conditioning Association 17: 393-401, 2003. 470 9. Benziane B, Burton TJ, Scanlan B, Galuska D, Canny BJ, Chibalin AV, 471 Zierath JR, and Stepto NK. Divergent cell signaling after short-term intensified endurance 472 training in human skeletal muscle. American journal of physiology 295: E1427-1438, 2008. 473 10. Bergmeyer HU. Methods of enzymatic analysis. New York: Academic Press, 474 1974. 475 11. Bergstrom J. Muscle electrolytes in man determined by neutron activation 476 analysis on needle biopsy specimens. Scand J Clin Lab Invest Suppl 68: 1-110, 1962. 477 12. Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, 478 Zlotchenko E, Scrimgeour A, Lawrence JC, Glass DJ, and Yancopoulos GD. Akt/mTOR 479 pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy 480 in vivo. Nat Cell Biol 3: 1014-1019, 2001. 481 13. Bolster DR, Crozier SJ, Kimball SR, and Jefferson LS. AMP-activated 482 protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated 483 mammalian target of rapamycin (mTOR) signaling. The Journal of biological chemistry 277: 484 23977-23980, 2002. 485 14. Chen ZP, Stephens TJ, Murthy S, Canny BJ, Hargreaves M, Witters LA, 486 Kemp BE, and McConell GK. Effect of exercise intensity on skeletal muscle AMPK 487 signaling in humans. Diabetes 52: 2205-2212, 2003. 488 15. Coffey VG, Jemiolo B, Edge J, Garnham AP, Trappe SW, and Hawley JA. 489 Effect of consecutive repeated sprint and resistance exercise bouts on acute adaptive 490 responses in human skeletal muscle. Am J Physiol Regul Integr Comp Physiol 297: R1441-491 1451, 2009. 492

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16. Coffey VG, Pilegaard H, Garnham AP, O'Brien BJ, and Hawley JA. 493 Consecutive bouts of diverse contractile activity alter acute responses in human skeletal 494 muscle. J Appl Physiol 106: 1187-1197, 2009. 495 17. Coffey VG, Zhong Z, Shield A, Canny BJ, Chibalin AV, Zierath JR, and 496 Hawley JA. Early signaling responses to divergent exercise stimuli in skeletal muscle from 497 well-trained humans. Faseb J 20: 190-192, 2006. 498 18. Cunningham JT, Rodgers JT, Arlow DH, Vazquez F, Mootha VK, and 499 Puigserver P. mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha 500 transcriptional complex. Nature 450: 736-740, 2007. 501 19. Dagon Y, Hur E, Zheng B, Wellenstein K, Cantley LC, and Kahn BB. 502 p70S6 kinase phosphorylates AMPK on serine 491 to mediate leptin's effect on food intake. 503 Cell metabolism 16: 104-112, 2012. 504 20. Donges CE, Burd NA, Duffield R, Smith GC, West DW, Short MJ, 505 Mackenzie R, Plank LD, Shepherd PR, Phillips SM, and Edge JA. Concurrent resistance 506 and aerobic exercise stimulates both myofibrillar and mitochondrial protein synthesis in 507 sedentary middle-aged men. J Appl Physiol 112: 1992-2001, 2012. 508 21. Dreyer HC, Fujita S, Cadenas JG, Chinkes DL, Volpi E, and Rasmussen 509 BB. Resistance exercise increases AMPK activity and reduces 4E-BP1 phosphorylation and 510 protein synthesis in human skeletal muscle. The Journal of physiology 576: 613-624, 2006. 511 22. Drummond MJ, Dreyer HC, Pennings B, Fry CS, Dhanani S, Dillon EL, 512 Sheffield-Moore M, Volpi E, and Rasmussen BB. Skeletal muscle protein anabolic 513 response to resistance exercise and essential amino acids is delayed with aging. J Appl Physiol 514 104: 1452-1461, 2008. 515 23. Dudley GA and Djamil R. Incompatibility of endurance- and strength-training 516 modes of exercise. J Appl Physiol 59: 1446-1451, 1985. 517 24. Ellisen LW. Growth control under stress: mTOR regulation through the 518 REDD1-TSC pathway. Cell cycle (Georgetown, Tex 4: 1500-1502, 2005. 519 25. Frost RA, Huber D, Pruznak A, and Lang CH. Regulation of REDD1 by 520 insulin-like growth factor-I in skeletal muscle and myotubes. Journal of cellular biochemistry 521 108: 1192-1202, 2009. 522 26. Fujii N, Hayashi T, Hirshman MF, Smith JT, Habinowski SA, Kaijser L, 523 Mu J, Ljungqvist O, Birnbaum MJ, Witters LA, Thorell A, and Goodyear LJ. Exercise 524 induces isoform-specific increase in 5'AMP-activated protein kinase activity in human 525 skeletal muscle. Biochemical and biophysical research communications 273: 1150-1155, 526 2000. 527 27. Fujita S, Dreyer HC, Drummond MJ, Glynn EL, Cadenas JG, Yoshizawa F, 528 Volpi E, and Rasmussen BB. Nutrient signalling in the regulation of human muscle protein 529 synthesis. The Journal of physiology 582: 813-823, 2007. 530 28. Goodman CA, Frey JW, Mabrey DM, Jacobs BL, Lincoln HC, You JS, and 531 Hornberger TA. The role of skeletal muscle mTOR in the regulation of mechanical load-532 induced growth. The Journal of physiology 589: 5485-5501, 2011. 533 29. Goodman CA, Miu MH, Frey JW, Mabrey DM, Lincoln HC, Ge Y, Chen J, 534 and Hornberger TA. A phosphatidylinositol 3-kinase/protein kinase B-independent 535 activation of mammalian target of rapamycin signaling is sufficient to induce skeletal muscle 536 hypertrophy. Molecular biology of the cell 21: 3258-3268, 2010. 537 30. Hawley JA. Molecular responses to strength and endurance training: are they 538 incompatible? Applied physiology, nutrition, and metabolism = Physiologie appliquee, 539 nutrition et metabolisme 34: 355-361, 2009. 540

21

31. Hickson RC. Interference of strength development by simultaneously training 541 for strength and endurance. European journal of applied physiology and occupational 542 physiology 45: 255-263, 1980. 543 32. Jorgensen SB, Wojtaszewski JF, Viollet B, Andreelli F, Birk JB, Hellsten Y, 544 Schjerling P, Vaulont S, Neufer PD, Richter EA, and Pilegaard H. Effects of alpha-545 AMPK knockout on exercise-induced gene activation in mouse skeletal muscle. Faseb J 19: 546 1146-1148, 2005. 547 33. Kimball SR and Jefferson LS. New functions for amino acids: effects on gene 548 transcription and translation. The American journal of clinical nutrition 83: 500S-507S, 2006. 549 34. Kraemer WJ, Patton JF, Gordon SE, Harman EA, Deschenes MR, 550 Reynolds K, Newton RU, Triplett NT, and Dziados JE. Compatibility of high-intensity 551 strength and endurance training on hormonal and skeletal muscle adaptations. J Appl Physiol 552 78: 976-989, 1995. 553 35. Lundberg TR, Fernandez-Gonzalo R, Gustafsson T, and Tesch PA. Aerobic 554 exercise alters skeletal muscle molecular responses to resistance exercise. Medicine and 555 science in sports and exercise 44: 1680-1688, 2012. 556 36. Mascher H, Ekblom B, Rooyackers O, and Blomstrand E. Enhanced rates of 557 muscle protein synthesis and elevated mTOR signalling following endurance exercise in 558 human subjects. Acta physiologica (Oxford, England) 202: 175-184, 2011. 559 37. Nelson AG, Arnall DA, Loy SF, Silvester LJ, and Conlee RK. Consequences 560 of combining strength and endurance training regimens. Physical therapy 70: 287-294, 1990. 561 38. Olesen J, Kiilerich K, and Pilegaard H. PGC-1alpha-mediated adaptations in 562 skeletal muscle. Pflugers Arch 460: 153-162, 2010. 563 39. Pruznak AM, Kazi AA, Frost RA, Vary TC, and Lang CH. Activation of 564 AMP-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside 565 prevents leucine-stimulated protein synthesis in rat skeletal muscle. The Journal of nutrition 566 138: 1887-1894, 2008. 567 40. Putman CT, Xu X, Gillies E, MacLean IM, and Bell GJ. Effects of strength, 568 endurance and combined training on myosin heavy chain content and fibre-type distribution 569 in humans. European journal of applied physiology 92: 376-384, 2004. 570 41. Regazzetti C, Bost F, Le Marchand-Brustel Y, Tanti JF, and Giorgetti-571 Peraldi S. Insulin induces REDD1 expression through hypoxia-inducible factor 1 activation 572 in adipocytes. The Journal of biological chemistry 285: 5157-5164, 2009. 573 42. Sale DG, Jacobs I, MacDougall JD, and Garner S. Comparison of two 574 regimens of concurrent strength and endurance training. Medicine and science in sports and 575 exercise 22: 348-356, 1990. 576 43. Sale DG, MacDougall JD, Jacobs I, and Garner S. Interaction between 577 concurrent strength and endurance training. J Appl Physiol 68: 260-270, 1990. 578 44. Sofer A, Lei K, Johannessen CM, and Ellisen LW. Regulation of mTOR and 579 cell growth in response to energy stress by REDD1. Molecular and cellular biology 25: 5834-580 5845, 2005. 581 45. Teleman AA, Hietakangas V, Sayadian AC, and Cohen SM. Nutritional 582 control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell metabolism 7: 583 21-32, 2008. 584 46. Terzis G, Georgiadis G, Stratakos G, Vogiatzis I, Kavouras S, Manta P, 585 Mascher H, and Blomstrand E. Resistance exercise-induced increase in muscle mass 586 correlates with p70S6 kinase phosphorylation in human subjects. European journal of applied 587 physiology 102: 145-152, 2008. 588

22

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

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

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Biopsy and blood samples~45 min

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Biopsy and blood samples~45 min

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Resistance exercise RestCyclingRest Rest

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Article FileFigure 1Figure 2 A-DFigure 3 A-CFigure 4 A-CFigure 5 A-DFigure 6 A-C