Articles in PresS. Am J Physiol Endocrinol Metab (April 30, 2013). doi:10.1152/ajpendo.00091.

2013

Resistance exercise induced mTORC1 signalling is not impaired by subsequent

endurance exercise in human skeletal muscle

William Apr1,2*, Li Wang1*, Marjan Pontn1, Eva Blomstrand1 and Kent Sahlin1

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strand Laboratory, Swedish School of Sport and Health Sciences, Stockholm, Sweden,

Department of Clinical Science, Intervention and Technology, Karolinska Institutet,

Stockholm, Sweden

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* W. Apr and L. Wang contributed equally to this work

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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: william.apro@gih.se
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Running head: Concurrent exercise does not inhibit mTORC1 signalling

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Key words: concurrent exercise, mTORC1, AMPK, interference

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Copyright 2013 by the American Physiological Society.

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ABSTRACT

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The current dogma is that the muscle adaptation to resistance exercise is blunted when

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combined with endurance exercise. The suggested mechanism (based on rodent experiments)

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is that activation of adenosine-monophosphate-activated protein-kinase (AMPK) during

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endurance exercise impairs muscle growth through inhibition of the mechanistic-target-of-

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rapamycin-complex 1 (mTORC1). The purpose of this study was to investigate potential

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interference of endurance training on the signalling pathways of resistance training (mTORC1

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- phosphorylation of ribosomal protein S6 kinase 1 (S6K1)) in human muscle. Ten healthy

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and moderately trained male subjects performed on two separate occasions either acute high

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intensity and high volume resistance exercise (leg press, R) or R followed by 30 min of

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cycling (RE). Muscle biopsies were collected before, 1 and 3h post resistance exercise.

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Phosphorylation of mTOR (Ser2448) increased 2-fold (p<0.05) and that of S6K1 (Thr389) 14-

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fold (p<0.05), with no difference between R and RE. Phosphorylation of eukaryotic

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elongation factor 2 (eEF2, Thr56) was reduced ~70% during recovery in both trials (p<0.05).

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An interesting finding was that phosphorylation of AMPK (Thr172) and acetyl-CoA

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carboxylase (ACC, Ser79) decreased ~30% and ~50%, respectively, 3h post exercise (p<0.05).

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Proliferator-activated receptor--coactivator-1 (PGC-1) mRNA increased more after RE

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(6.5-fold) than after R (4-fold) (RE vs. R: p< 0.01) and was the only gene expressed

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differently between trials. These data show that the signalling of muscle growth through the

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mTORC1-S6K1 axis after heavy resistance exercise is not inhibited by subsequent endurance

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exercise. It is also suggested that prior activation of mTORC1 signalling may repress

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subsequent phosphorylation of AMPK.

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WORDS: 250

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INTRODUCTION

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Skeletal muscle possesses remarkable plasticity and as such has the unique ability to adapt to

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various types of contractile activity which ultimately results in divergent phenotypes in

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accordance with the specificity of training principle (5). The divergent adaptations following

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resistance and endurance exercise places these exercise modalities in contrasting ends of the

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training adaptation continuum. As such, the opposing phenotypes are likely dependent on

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highly specific adaptations which may be incompatible when different exercise modes are

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performed simultaneously (5).

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This notion is supported by several studies showing negative effects on the

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development of strength and power (23, 31) as well as muscle fibre hypertrophy (34, 40)

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when both modes of exercise have been performed concurrently over longer periods of time.

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Collectively, these findings have given rise to the current paradigm of the training

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interference effect (30), yet not all studies support the existence of such a phenomenon (8, 37,

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42, 43). The reasons for the discrepancies within the literature are not readily obvious but are

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likely related to experimental variables such as exercise intensity and volume, exercise

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sequence or nutritional status.

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Assessment of physical performance with hard outcome measures offers little

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insight into the molecular mechanisms regulating long term training adaptations. In recent

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years, our understanding of the molecular events underlying the various training adaptations

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has increased considerably and several distinct signalling pathways have been identified. For

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instance, it is well established that skeletal muscle growth to a large extent is mediated by

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activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway (12, 28).

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Similarly, peripheral adaptations responsible for mitochondrial biogenesis include induction

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of the peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) (7, 50) pathway

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which in turn mediates the characteristic increase in muscle oxidative capacity seen after

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endurance training (38). Mechanistically, crosstalk between these two pathways has been

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linked to the adenosine monophosphate-activated protein kinase (AMPK), an enzyme

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activated during energetic stress (53). Activation of AMPK by pharmacological agents has

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been shown to repress mTORC1 signalling (13, 39, 47) but elevate mRNA expression of

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PGC-1 (32) in rodent muscle.

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To date, only a small number of studies have been performed in humans to

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examine the impact of acute concurrent exercise on the signalling pathways leading to

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different phenotypes. Some of the investigations found support for an interference effect (15,

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16), whereas others did not (20, 35). More specifically, when concurrent exercise was

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performed in the fed state (20, 35) and with several hours of recovery time between exercise

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modes (35), mTORC1 signalling was similar compared with single mode resistance exercise.

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However, from these studies and from a mechanistic perspective, it is difficult to evaluate the

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impact of concurrent exercise per se as feeding in itself may influence signalling though the

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mTORC1 pathway (33). In contrast, when endurance (16) and sprint (15) exercise preceded

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resistance exercise in the fasted state, with only 15 minutes of rest in between, mTORC1

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signalling was attenuated compared to when the exercise order was reversed. However, none

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of these studies included a single mode exercise for comparison. Thus, to date, no study has

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investigated the signalling response following concurrent exercise compared with single mode

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resistance exercise in the fasted state.

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Therefore, the aim of the present investigation was to examine whether

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endurance exercise following a heavy resistance exercise protocol would repress molecular

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signalling through the mTORC1 pathway, compared to single mode resistance exercise. To

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this end, muscle biopsies from moderately trained men were analyzed with regard to protein

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signalling and mRNA expression involved in skeletal muscle hypertrophy and mitochondrial

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biogenesis. It was hypothesized that concurrent exercise would impair growth related

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signalling and gene expression.

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METHODS

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Ten healthy moderately trained male subjects were recruited for this study. After being

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informed of the purpose of the study and of all associated risks, all subjects gave written

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consent. To be eligible for enrollment in the study, subjects were required to have performed

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resistance exercise 2-3 times per week and endurance exercise 1-2 times a week during the

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last 6 months and to have a maximal leg strength equaling four times their body weight or

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more. Subject characteristics are presented in Table 1. The study was approved by the

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Regional Ethical Review Board in Stockholm and performed in accordance with the

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principles outlined in the Declaration of Helsinki.

Subjects

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Study design

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The study employed a randomized cross-over design in which each subject performed one

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session of resistance exercise (R) and another session of resistance exercise followed by

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endurance exercise (RE). The two sessions were separated by approximately two weeks. A

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schematic overview of the experimental protocols is provided in Fig.1. All subjects were

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instructed to maintain their habitual dietary intake and physical activity pattern throughout the

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entire experimental period. Subjects were instructed to refrain from physical exercise for two

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days before each trial as well as to record and duplicate their food intake before the first and

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second trials, respectively.

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Pre-tests

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Before initiation of the actual experiments, each subjects two-legged one repetition

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maximum (1RM) was determined on a leg press machine (243 Leg press 45, Gymleco,

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Stockholm, Sweden) after warming up on a cycle ergometer for 10 min. The 1RM was

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assessed by gradually increasing the load until the subject was unable to perform no more

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than one single repetition (90-180 knee angle). Maximal and submaximal oxygen uptake was

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determined on a mechanically braked cycle ergometer (Monark 839E, Vansbro, Sweden) with

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the work rate gradually increased until volitional exhaustion as described by strand &

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Rodahl (56). Oxygen uptake was measured continuously utilizing an on-line system (Oxycon

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Pro, Erich Jaeger GmbH, Hoechberg, Germany) and heart rate (HR) was recorded

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continuously (Polar Electro Oy, Kempele, Finland). Following initial testing, subjects

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performed two familiarization sessions in order to minimize any training effects during the

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live experiments (see below).

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On the day of each trial, subjects reported to the laboratory at approximately 7.30 am

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following an overnight fast from 9.00 pm the evening before. Upon arrival, subjects were

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placed in a supine position and rested for 10 min prior to the collection of a resting blood

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sample from an arm vein. After subsequent administration of local anaesthesia, a resting

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biopsy was collected from the middle portion of the vastus lateralis muscle of one leg using a

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Bergstrm needle (11) with manually applied suction.

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Following blood and tissue sampling, subjects were seated in the leg press machine

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and performed 3 warm-up sets of 10 repetitions at 0, 30 and 60% of 1RM with 3 min of rest

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between each set. Thereafter, the subjects performed 10 sets of heavy resistance exercise (R)

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or resistance exercise followed by endurance exercise (RE). The resistance exercise protocol

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consisted of 4 sets of 8-10 repetitions at 85 % of 1 RM, 4 sets of 10-12 repetitions at 75 % of

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1RM and lastly 2 sets to volitional fatigue at 65 % of 1RM with 3 min of recovery allowed

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between each set. The load and number of repetitions was recorded during the first trial and

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duplicated during the second trial. Time under tension was recorded for each set during the

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first trial and was used to match, as closely as possible, the repetition speed in each set during

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the second trial. Following resistance exercise in the RE trial, subjects rested for 15 min after

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which 30 min of cycling was initiated at an intensity equal to 70 % of the subjects' maximal

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oxygen consumption (Table 2).

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In both trials, two additional muscle biopsies were collected in the contra lateral leg

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at 1 and 3 h following resistance exercise together with blood samples. For each biopsy

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sampling, a new incision was made approximately 3-4 cm proximal to the previous one and

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after biopsy collection, samples were immediately blotted free of blood and frozen in liquid

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nitrogen and stored at -80C for later analysis. Due to technical difficulties, tissue sampling at

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3 h post resistance exercise was unsuccessful for one subject in the R-trial and for another

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subject in the RE-trial.

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Plasma analysis

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Blood samples (4 ml) were centrifuged at 1500 g at 4C for 10 min and the plasma obtained

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was stored at -20C. Plasma samples were later analyzed for glucose and lactate

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concentrations as described by Bergmeyer (10).

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Immunoblot analysis

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Muscle samples were lyophilized, cleaned from blood and connective tissue under a

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dissection microscope (Carl Zeiss, Germany), and then homogenized in ice-cold buffer (80

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l/mg dry weight) containing 2 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM EGTA, 10 mM

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MgCl2, 50 mM -glycerophosphate, 1% TritonX-100, 1 mM Na3VO4, 2 mM dithiothreitol, 20

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g/ml leupeptin, 50 g/ml aprotinin, 1% phosphatase inhibitor cocktail (Sigma P-2850) and

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40 g/l PMSF. Homogenates were then cleared by centrifugation at 10,000 g for 10 min at

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4C and the resulting supernatant was stored at -80C.

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Protein concentrations were determined in aliquots of supernatant diluted 1:10 in

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distilled water using a bicinchoninic acid assay (Pierce Biotechnology, Rockford, IL, USA).

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Samples were diluted in Laemmli sample buffer (Bio-Rad Laboratories, Richmond, CA,

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USA) and homogenizing buffer to obtain a final protein concentration of 1.5 g/l. Following

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dilution, all samples were heated at 95C for 5 min to denature proteins present in the

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supernatant. Samples were then kept at -20C until further analysis.

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Details of the western blotting procedures have been described elsewhere (3). Briefly,

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and with minor modifications, samples containing 30 g total protein were separated by SDS-

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PAGE on Criterion cell gradient gels (4-20% acrylamide; Bio-Rad Laboratories).

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Electrophoresis was performed on ice at 200 V for 120 min, after which the gels were

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equilibrated in transfer buffer (25 mM Tris base, 192 mM glycine, and 10% methanol) for 30

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min. The proteins were then transferred to polyvinylidine fluoride membranes (Bio-Rad

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Laboratories) at a constant current of 300 mA for 3 h at 4C. Following transfer, membranes

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were stained with MemCodeTM Reversible Protein Stain Kit (Pierce Biotechnology) (2) to

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confirm equal loading of the samples. All samples from each subject were run on the same gel.

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Membranes were blocked for 1 h at room temperature in Tris-buffered saline (TBS;

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20 mM Tris base, 137 mM NaCl, pH 7.6) containing 5% non-fat dry milk. After blocking,

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membranes were incubated overnight with commercially available primary antibodies diluted

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in TBS supplemented with 0.1% Tween-20 containing 2.5% non-fat dry milk (TBS-TM).

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After incubation with these primary antibodies, the membranes were washed with TBS-TM

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and incubated for 1 h at room temperature with secondary antibodies conjugated with

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horseradish peroxidise. Next, the membranes were washed serially (2 x 1 min, 3 x 15 min)

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with TBS-TM, followed by 4 additional washes with TBS for 5 min each. Finally, membranes

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with the antibodies bound to the target proteins were visualized by chemiluminescent

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detection on a Molecular Imager ChemiDocTM XRS system and the bands were analysed

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using the contour tool in the Quantity One version 4.6.3 software (Bio-Rad Laboratories).

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All values are expressed relative to total levels of -tubulin.

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Antibodies

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Primary antibodies raised in rabbit against phospho-Akt (Ser473; #9271), phospho-mTOR

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(Ser2448; #2971), phospho-S6K1 (Thr389; #9234), phospho-4E-BP1 (Thr37/46; #2855), phospho-

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eEF2 (Thr56; #2331), phospho-AMPK (Thr172; #4188), phospho-ACC (Ser79; #3661),

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phospho-p38 (Thr180/Tyr182; #9211), phospho-ERK1/2 (Thr202/Tyr204; #9101) and phospho-

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CaMKII (Thr286; #3361) was purchased from Cell Signaling Technology (Beverly, MA, USA).

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Primary total antibodies for mouse -tubulin (#T6074) and rabbit REDD1 (#ab63059) were

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purchased from Sigma-Aldrich (St. Louis, MO, USA) and Abcam (Cambridge, UK),

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respectively. All primary antibodies were diluted 1:1000 except for eEF2 and -tubulin which

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were diluted 1:5000. Secondary anti-rabbit (#7074) and anti-mouse (#7076) antibodies

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(1:10000) were purchased from Cell Signaling Technology.

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RNA extraction and quantitative Real-Time PCR (qRT-PCR)

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Total RNA was extracted from approximately 2 mg lyophilized and cleaned tissue which was

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homogenized in PureZOL RNA isolation reagent (Bio-Rad Laboratories) according to the

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manufacturers instructions. The concentration and purity of the RNA was determined by

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spectrofotometry and 1 g RNA was used for reverse transcription of 20 l cDNA with

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iScript cDNA Synthesis Kit (Bio-Rad Laboratories). The primers for the specific genes

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analyzed here have been presented in a previous publication from this laboratory (50). The

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concentration of cDNA, annealing temperature and PCR cycle protocol were determined for

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each primer pair to ensure optimal conditions for amplification. Samples were run in triplicate

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and all samples from each subject were run on the same plate to allow direct relative

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comparisons. qRT-PCR amplification mixtures (25 l) contained 12.5 l 2SYBR Green

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Supermix (Bio-Rad Laboratoies), 0.5 l 10 M forward and reverse primers, respectively, and

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11.5 l template cDNA in RNase-free water. qRT-PCR was performed with Bio-Rad iCycler

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(Bio-Rad Laboratories) and relative changes in mRNA levels were analyzed by the 2-CT

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method with GAPDH used as the reference gene.

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Statistical analyses

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All data are expressed as means SE. For protein signalling, gene expression and plasma data,

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a two-way repeated-measures ANOVA (trial and time) was used for statistical analysis. For

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missing data points, weighted means were used in the analysis. When a significant main effect

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or an interaction effect was observed, Fisher LSD post hoc analysis was performed to locate

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differences. For some positively skewed distributed variables, log-transformation was

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performed before analyses. For analysis of time under tension and number of repetitions, a

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paired t-test was used. Statistical significance was set at P < 0.05.

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RESULTS

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Task performance

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Eight subjects performed the same number of repetitions in both trials while two subjects

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performed one and two repetition less, respectively, in the RE-trial. Time under tension was

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similar between trials (Table 2). Average heart rate during cycling corresponded to 88 1 %

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of maximal heart rate.

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Blood parameters

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Plasma concentrations of glucose were significantly decreased at both time points during

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recovery in the R-trial (Table 3). In the RE-trial, plasma levels of glucose remained

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unchanged until the 3 h time point at which these levels decreased but only compared to 1 h

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post resistance exercise (Table 3). Plasma levels of lactate were increased in both trials 1 h

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after resistance exercise, but more so in the RE-trial. By the 3 h time point, lactate levels had

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dropped back to baseline values in both trials (Table 3).

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Protein signalling

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Phosphorylation of Akt at Ser473 was decreased 1 h after resistance exercise in the R trial

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compared to resting values as well as being lower compared to the RE trial at the same time

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point (Fig. 2A). For phosphorylation of mTOR at Ser2448, statistical analysis revealed main

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effects of time as well as trials (p<0.05) but no interaction effect. Further analysis of the data

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with paired t-tests showed no difference between R and RE trials in the fold change of mTOR

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phosphorylation. The significant main effect of trial was therefore likely due to the difference

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in pre exercise values. Post hoc analysis showed statistically significant elevations in mTOR

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phosphorylation at both post exercise time points (p<0.01), with a trend (p=0.057) for a

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further increase at 3 h after resistance exercise (Fig. 2B).

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Phosphorylation of S6K1 at residue Thr389 increased over time in both trials,

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exhibiting 5- (p<0.01) and 14-fold (p<0.01) increases at 1 and 3 h after resistance exercise,

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respectively (Fig. 2C). Phosphorylation of eEF2 at Thr56 decreased at the 1 h time point to

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about 30% of pre exercise values (p<0.01) with no difference between trials (Fig. 2D). At 3 h

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after resistance exercise, phosphorylation of this residue remained significantly depressed in

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both trials (p<0.01) and tended to decrease further compared to the 1 h time point (p=0.084)

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(Fig. 2D).

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Phosphorylation of AMPK at Thr172 was unchanged 1 h after resistance exercise in

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both trials. At the 3 h time point, phosphorylation of this protein was decreased 33% (p<0.01)

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in both trials compared to pre-exercise as well as 1 h values (Fig. 3A). Similarly,

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phosphorylation of ACC at residue Ser79 was unaffected 1 h after resistance exercise but

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decreased 47% (p<0.01) at the 3 h time point in both trials (Fig. 3B). The degree of p38

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phosphorylation at Thr180/Tyr182 increased approximately two-fold in both trials at the 1 h

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time point (p<0.01) and remained elevated at 3 h (p<0.01) after resistance exercise (Fig. 3C).

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Phosphorylation of 4E-BP1 at residues Thr37/46 showed a small but significant

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decrease (p<0.05) 1 h after resistance exercise but at the 3 h time point phosphorylation of

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4E-BP1 had returned to baseline values (Fig. 4A). Phosphorylation of ERK 1/2 at residues

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Thr202/Tyr204 remained unchanged in both trials at all time points (Fig. 4B). Similarly,

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phosphorylation of CamKII at Thr286 was unaltered by exercise in both trials (Fig. 4C). Totals

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levels of REDD1 protein was unchanged 1 h post resistance exercise but increased modestly

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by the 3 h time point in both trials (Fig. 5A).

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Gene expression

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There were minor but significant increases in mRNA expression of Rheb at both the 1

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(p<0.05) and 3 h (p<0.01) time points in both trials (Table 4). Expression of mTOR, hVps34

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and TSC1 was unaffected by either protocol at the 1 h time point, but decreased at the 3 h

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time point compared to before exercise (p<0.05) as well as to 1 h post resistance exercise

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values (p<0.01; Table 4). On the contrary, both protocols resulted in elevated mRNA levels of

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TSC2 1h (p<0.01) after resistance exercise, but these levels had returned to baseline values by

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the 3 h time point (Table 4). Both exercise protocols resulted in minor but significant

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elevations in S6K1 mRNA expression at the 1 h time point (p<0.05) compared to pre exercise

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levels, although these levels had returned to baseline values in both trials at the 3 h time point

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(Table 4).

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Both exercise protocols induced significant and similar elevations in mRNA

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expression of REDD1 at the 1 h time point (p<0.01), although, at 3 h after exercise, mRNA

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levels of REDD1 had dropped below baseline values (p<0.01; Fig. 5B). Expression of

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REDD2 was unchanged after both protocols at the 1 h time point but was significantly

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reduced in both trials 3 h post resistance exercise (p<0.01; Fig. 5C). mRNA abundance of

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cMyc increased continuously and similarly during recovery in both trials (7-fold at 1 h Post vs.

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26-fold at 3 h Post; p<0.01) (Fig. 5D).

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Expression of PGC-1 increased in both trials at 3 h post resistance exercise but

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significantly more so in the RE-trial (6.5-fold vs. 4.1-fold in R; p<0.01) (Fig. 6A). mRNA

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expression of PRC was similar in both trials but more pronounced 3 h after resistance exercise

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compared to the 1 h time point (p<0.01; Fig. 6B). Both trials resulted in pronounced and

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similar increases in PDK4 mRNA expression at the 1 h time point (~7-fold; p<0.01) as well as

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the 3 h time point (~8-fold; p<0.01) after resistance exercise (Fig. 6C).

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DISCUSSION

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The main finding of the present investigation is that endurance exercise performed subsequent

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to resistance exercise does not blunt growth related signaling through the mTORC1 pathway

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in human skeletal muscle. This conclusion is supported by similar and positive alterations of

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mTOR as well as several downstream targets of mTORC1 (i.e. S6K1 and eEF2) following

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both modes of exercise. In addition, both modes of exercise induced similar responses at the

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transcriptional level with the exception of PGC-1, showing superior expression of this gene

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with concurrent exercise.

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Mechanistically, activation of mTORC1 results in a coordinated signalling cascade to

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various components within the translational machinery, which when repeated over time, has

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been shown to correlate with muscle hypertrophy in both rodent (6) and human muscle (46).

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The regulatory role of mTORC1 in muscle growth was recently confirmed by Goodman and

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colleagues (28, 29), who in a series of elegantly designed experiments using various genetic

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mouse models showed that not only is load induced muscle growth dependent on mTORC1

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signalling (28), but activation of this complex is sufficient to induce muscle hypertrophy (29).

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We had initially hypothesized that combining resistance and endurance exercise would impair

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mTORC1 signalling. However, in contrast to this hypothesis, both modes of exercise induced

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substantial elevations in mTOR phosphorylation at Ser2448 as well as of its immediate

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downstream target S6K1 at the Thr389 residue. While mTOR phosphorylation increased 2-fold,

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the phosphorylation level of S6K1 increased 5-fold at the 1 h time point but continued to

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increase until 3 h post exercise, reaching 14-fold higher values compared to baseline.

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In addition to regulating translation initiation, mTORC1 mediated signaling also

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stimulates translation elongation by repressing phosphorylation of the eukaryotic elongation

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factor 2 (eEF2) at Thr56 (51). In the present study, both exercise protocols induced dramatic

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reductions in eEF2 phosphorylation during recovery and in line with the mTOR and S6K1

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phosphorylation data, we could not detect any difference in eEF2 phosphorylation between

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trials. Collectively, these findings indicate that the added endurance exercise does not impose

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an inhibitory effect on growth related signaling, neither at the level of translation initiation nor

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translation elongation.

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The reason for the lack of AMPK phosphorylation in this trial is not readily apparent

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as we (49) and others (14, 26, 48) have shown that endurance exercise with similar intensity

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and duration as that utilized here induces robust elevations in phosphorylation as well as

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activity of this kinase. Some studies show that endurance trained subjects with high VO2max

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values (i.e. 65 ml min-1 kg-1) may have a diminished AMPK response to endurance

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exercise (17), especially compared to untrained subjects (55). However, other studies (14, 55)

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have shown elevated activity and phosphorylation of AMPK in subjects with similar training

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status (~50 ml min-1 kg-1) as those involved in this study. In addition, sampling was

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performed 15 min post endurance exercise (1 h after resistance exercise) in the RE-trial. This

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time point is well within the time frame of expected elevations in AMPK phosphorylation

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and/or activity as shown previously (21, 49). Thus, the lack of AMPK phosphorylation in the

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present study is likely related to other factors than those discussed above.

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Although activation of AMPK following endurance exercise is well recognized (14,

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26, 36, 48, 54), the molecular interplay with mTORC1 in human muscle is much less

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established. The complexity of these interactions is demonstrated by several studies showing

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simultaneous increases in AMPK and mTORC1 signaling following endurance (9, 36, 52) and

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resistance exercise (21, 52) as well as after concurrent exercise (49). Furthermore, even

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though AMPK phosphorylation was increased in these trials, protein synthesis was elevated

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(21, 36, 52), a finding that is contradictory to the suggested mechanism of the interference

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effect (13, 39, 47). Thus, it is unclear whether or not activation of AMPK impairs mTORC1

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signalling in human muscle under physiological conditions.

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Unexpectedly, at the 3 h time point, phosphorylation of AMPK was actually

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decreased approximately 30% compared to baseline values. This reduction was also seen on

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the phosphorylation levels of ACC, although to a larger extent (~50%). This finding was

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present in both trials, suggesting it may be related to activation of mTORC1 induced by the

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resistance exercise protocol. In support of this idea, Atherton and colleagues (4) demonstrated

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that high frequency electrical stimulation of rat muscle resulted in pronounced increases in

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phosphorylation of S6K1 at Thr389 three hours post stimulation and at this time point, AMPK

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phosphorylation at Thr172 was depressed below resting values. Similarly, in a study by Fujita

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et al. (27), resistance exercise combined with nutrient provision dramatically induced

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mTORC1 signalling while concomitantly reducing AMPK phosphorylation at Thr172. In

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contrast, some studies have failed to detect a decrease in AMPK phosphorylation despite

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elevated phosphorylation of S6K1 (17, 22). Our findings may be related, at least in part, to the

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intensity and volume of the exercise protocol used here, which was much higher than in the

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previously mentioned studies.

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In the present study, decreased AMPK phosphorylation coincided with maximal

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phosphorylation of S6K1 at Thr389 in both trials, an association also observed by others (4, 27).

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Dagon et al. (19) recently demonstrated a direct inhibitory phosphorylation of AMPK at

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Ser491 by S6K1 in mouse hypothalamic cells and although not yet confirmed in skeletal

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muscle, these findings identify a negative feed-back loop in which AMPK is influenced by the

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mTORC1 pathway. Additional support for a regulatory role of S6K1 on AMPK activity is

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provided by Aguliar and colleagues (1) who showed increased phosphorylation of the Thr172

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residue of AMPK in cultured myotubes of S6K1-deficient mice. Therefore, based on these

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observations, we propose that prior activation of mTORC1 inhibits the activity of AMPK

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which would suggest bidirectional crosstalk between these pathways. Furthermore, in the

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studies showing an inhibitory effect of AMPK on mTORC1 (13, 39, 47), pharmacological

384

activation of AMPK was initiated prior to determination of mTORC1 signalling indicating the

385

existence of an order effect.

386

We also measured gene expression of several positive (hVps34, Rheb and cMyc)

387

and negative (TSC1/2, REDD1/2) effectors of mTORC1 signaling, as well as expression of

388

key components (mTOR and S6K1) of this pathway (Table 4). There were only modest

389

fluctuations in mRNA expression of most of these genes but most importantly, we could not

390

detect any differences between trials at any time point, in line with the signaling data

391

presented above. Interestingly, the pattern of mRNA expression of the growth related

16

392

transcription factor cMyc mirrored that of S6K1 phosphorylation (Fig. 5D), suggesting that

393

expression of this gene may, at least in part, be regulated by mTORC1 as has been shown in

394

Drosophila (45). These findings show that the lack of inhibition induced by endurance

395

exercise is not only evident at the level of translation initiation and elongation, but also at the

396

level of transcription.

397

In addition to measuring mRNA abundance, we also analyzed total levels of REDD1

398

protein. Both REDD1 and REDD2 have been identified as negative regulators of mTORC1

399

signaling under various conditions of cellular stress (24), including energetic stress (44).

400

Although the precise mechanism by which REDD 1/2 inhibits mTORC1 signaling remains

401

elusive, in vitro studies have shown that induction of REDD1 occurs within hours following

402

hormonal treatment (25, 41). As such, 3 h post resistance exercise could have been an

403

appropriate time point to detect a potential increase in expression of this protein. However,

404

even though the RE-trial arguably induced a greater energetic stress, we could not detect any

405

differences in REDD1 protein levels between trials (Fig. 5A).

406

In contrast to the other genes examined, concurrent exercise resulted in superior

407

mRNA expression of PGC-1 (Fig. 6A), a molecule shown to have a major regulatory role in

408

mitochondrial biogenesis (38). Although an expected finding, the reason for the differential

409

expression of this gene is not readily obvious, at least from a mechanistic perspective.

410

Induction of PGC-1 has been linked to increased signalling by several upstream effectors

411

such as AMPK, calcium/calmodulin-dependent protein kinase (CaMK) and the mitogen-

412

activated protein kinase p38 (38), none of which differed between trials in the present study.

413

In addition to these kinases, mTOR has also been implicated in the induction of PGC-1 (18),

414

yet, phosphorylation of mTOR and of all its immediate downstream targets was similar

415

between protocols. Consequently, these data do not allow for any clear conclusions regarding

416

the involvement of these kinases in the PGC-1 response seen during recovery in the RE-trial.

17

417

In summary, both single mode resistance exercise and concurrent exercise

418

resulted in robust and positive alterations of regulatory proteins involved in translation

419

initiation (i.e. mTOR and S6K1) and elongation (eEF2), thus promoting skeletal muscle

420

hypertrophy (6, 28, 29, 46). These data demonstrate that moderate intensity endurance

421

exercise performed subsequent to a high intensity high volume resistance exercise protocol

422

does not inhibit mTORC1 signaling during acute recovery in moderately trained men. This

423

conclusion is further supported by the reduction in AMPK phosphorylation following both

424

modes of exercise which we propose is due to prior activation of mTORC1 induced by the

425

resistance exercise protocol. This finding suggests that the interference effect between these

426

pathways is bidirectional. We therefore propose that endurance exercise may be included in

427

training regimes aiming to promote muscle growth. However, further studies are required to

428

assess if the acute changes in the signalling pathways examined here fully reflect long term

429

training adaptations.

430
431

ACKNOWLEDGMENTS

432

We thank all participants for their valuable time and efforts in this study. This study was

433

supported financially by grants from the Swedish National Centre for Research in Sports and

434

the Swedish School of Sport and Health Sciences in Stockholm, Sweden.

435
436

DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.

437
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639
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641

FIGURE CAPTIONS

642

FIGURE 1

643

Schematic overview of the experimental trials. R-protocol, resistance exercise only; RE-

644

protocol, resistance exercise followed by cycling. Arrows indicate sampling time points for

645

muscle biopsies and blood samples.

646
647

FIGURE 2

648

Phosphorylation levels of Akt (60 kDa) at Ser473 (A), mTOR (289 kDa) at Ser2448 (B), S6K1

649

(70 kDa) at Thr389 (C) and eEF2 (95 kDa) at Thr56 (D) before, 1 and 3 h post resistance

650

exercise in both trials. Representative immunoblots from one subject are shown above each

651

graph. Values are normalized to -tubulin and presented as means SE for 10 subjects (n = 9

652

for 3 h Post). Symbols above lines denote differences revealed by a post-hoc test when a main

653

effect was observed. Symbols without lines denote differences revealed by a post-hoc test

654

when an interaction effect was observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post; P < 0.05

655

vs. R.

656
657

FIGURE 3

658

Phosphorylation levels of AMPK (62 kDa) at Thr172 (A), ACC (280 kDa)at Ser79 (B), p38

659

(~43 kDa) at Thr180/Tyr182 (C) before, 1 and 3 h post resistance exercise in both trials.

660

Representative immunoblots from one subject are shown above each graph. Values are

661

normalized to -tubulin and presented as means SE for 10 subjects (n = 9 for 3 h Post).

662

Symbols above lines denote differences revealed by a post-hoc test when a main effect was

663

observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post.

664

23

665

FIGURE 4

666

Phosphorylation levels of 4EBP1 (~15-20 kDa) at Thr37/46 (A), ERK1/2 (42/44 kDa) at

667

Thr202/Tyr204 (B), CaMKII (~50 kDa) at Thr286 (C) before, 1 and 3 h post resistance exercise in

668

both trials. Representative immunoblots from one subject are shown above each graph.

669

Values are normalized to -tubulin and presented as means SE for 10 subjects (n = 9 for 3 h

670

Post). Symbols above lines denote differences revealed by a post-hoc test when a main effect

671

was observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post.

672
673
674
675

FIGURE 5
Total protein levels of REDD1 (~34 kDa) (A) and mRNA expression of REDD1 (B), REDD2

676

(C) and cMyc (D) before, 1 and 3 h post resistance exercise in both trials. Representative

677

immunoblots from one subject are shown above graph A. Protein levels are normalized to -

678

tubulin and presented as means SE for 10 subjects (n = 9 for 3 h Post). Symbols above lines

679

denote differences revealed by a post-hoc test when a main effect was observed. *P < 0.05 vs.

680

Rest; #P < 0.05 vs. 1h Post.

681
682
683

FIGURE 6

684

mRNA expression of PGC-1 (A), PRC (B) and PDK4 (C) before, 1 and 3 h post resistance

685

exercise in both trials. Values are presented as means SE for 10 subjects (n = 9 for 3 h Post).

686

Symbols above lines denote differences revealed by a post-hoc test when a main effect was

687

observed. Symbols without lines denote differences revealed by a post-hoc test when an

688

interaction effect was observed. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post; P < 0.05 vs. R.

689
690
691
692
693
24

694
695
696
697

TABLES
TABLE 1. Subject characteristics
Variables
Age (years)
Weight (kg)
Height (cm)
1 RM (kg)
VO2max (l min-1)
Relative VO2max (ml min-1 kg-1)
HRmax (bpm)
Wattmax (W)

Mean SEM
26 2
85 3
179 2
389 17
4.27 0.12
50.8 1.6
192 2
308 15

698
699

Values are presented as means SE for 10 subjects. 1RM, one repetition maximum; VO2max,

700

maximum oxygen uptake; HRmax, maximum heart rate; Wattmax, maximum cycling intensity

701

expressed in watts.

702
703

TABLE 2. Details of the performed resistance exercise

Set #

% of 1RM

Load, kg

1
2
3

0
30
60

00
89 5
217 10

4
5
6
7

85

8
9
10
11
12
13

Trial

Total

Repetitions, times

Time under tension, sec

704

R
10 0
10 0
10 0

RE
10 0
10 0
10 0

R
27.2 0.9
27.2 0.9
27.0 1.1

RE
705
29.1 1.0
26.5 1.2
26.0 1.3

313 12

9.7 0.2
9.3 0.3
9.3 0.3
8.4 0.5

9.7 0.2
9.3 0.3
9.3 0.3
8.4 0.5

28.9 1.2
29.7 0.9
30.0 1.1
29.8 1.4

28.8 1.5
28.8 1.7
707
30.5 1.6
29.5 1.9

75

271 10

11.4 0.3
11.1 0.3
10.3 0.5
10.0 0.9

11.4 0.3
11.1 0.3
10.3 0.5
9.8 0.9

32.4 1.2
33.9 1.7
31.5 1.3
30.5 2.7

708
33.7 1.4
34.4 1.3
33.0 1.9
709
31.7 2.7

65

225 9

13.8 0.7
12.7 0.9

13.8 0.7
12.6 0.9

38.5 2.1
37.2 2.6

38.2 1.9
710
37.5 2.3

136.0

135.7

403.8

706

407.7

711

712

Values are presented as means SE for 10 subjects. R, resistance exercise only; RE,

713

resistance exercise followed by cycling; 1RM, one repetition maximum.

714
715
716
717
718
719
720
25

721

TABLE 3. Plasma concentrations of glucose and lactate.

Trial

Rest

1 h Post

3 h Post

Glucose, mmol/l

R
RE

5.65 0.11
5.53 0.10

5.03 0.11*
5.77 0.16

5.29 0.06*
5.42 0.11#

Lactate, mmol/l

R
RE

1.43 0.23
1.49 0.23

3.51 0.28*
6.13 0.41*

1.35 0.09#
1.42 0.08#

722

Values are presented as means SE for 10 subjects. Blood was sampled at rest and at 1 and 3

723

h post resistance exercise. R, resistance exercise only; RE, resistance exercise followed by

724

cycling. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h Post; P < 0.05 vs. R.

725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742

26

743

TABLE 4. Gene expression related to muscle growth.

744
Genes

Rheb
mTOR
S6K1
hVps34
TSC1
TSC2

Exercise

Rest

1h post

3h post

Main effects

0.64 0.11

0.69 0.09*

0.95 0.15*

RE

0.53 0.06

0.82 0.14*

0.76 0.10*

2.28 0.50

2.39 0.53

2.10 0.34*

RE

1.90 0.34

2.26 0.40

1.40 0.31*

1.03 0.17

1.10 0.13*

1.07 0.14

RE

0.99 0.08

1.23 0.11*

0.83 0.11

1.13 0.23

0.97 0.09

0.94 0.10*

RE

1.00 0.10

1.12 0.14

0.82 0.10*

1.55 0.29

1.49 0.19

1.46 0.17*

RE

1.43 0.15

1.55 0.15

0.99 0.18*

1.72 0.34

2.97 1.14*

2.37 0.68

RE

1.36 0.24

2.25 0.33*

1.54 0.37

Time

Exr

P<0.05

n.s

n.s747

P<0.05

n.s

n.s748

P<0.05

n.s

n.s749

P<0.05

n.s

n.s750

P<0.05

n.s

n.s751

P<0.05

n.s

n.s752

745

Int. effect

Time x Exr

746

753
754
755

Values are presented as means SE for 10 subjects (n = 9 for 3 h Post). Muscle was sampled

756

at rest and at 1 and 3 h post resistance exercise. R, resistance exercise only; RE, resistance

757

exercise followed by cycling. Rheb, ras homolog enriched in brain; mTOR, mechanistic target

758

of rapamycin; S6K1, 70 kD ribosomal protein S6 kinase 1; hVps34, human vacuolar protein

759

sorting 34; TSC1/2, tuberous sclerosis complex 1 and 2. *P < 0.05 vs. Rest; #P < 0.05 vs. 1h

760

Post.

761

27

Figure 1.

R - protocol
Resistance exercise

Rest

~45 min
Biopsy and blood samples
0h

Rest

RE - protocol
Resistance exercise

3h

1h

15 min

30 min

15 min

Rest

Cycling

Rest

Rest

~45 min
Biopsy and blood samples
Rest

0h

1h

3h

Figure 2.

R
Rest

1h

RE
3h

Rest

1h

3h

Rest

p-Akt

p-mTOR

a-tubulin

a-tubulin
2.5

R
RE

2.0

Phosphorylation of mTOR at S2448

(arbitrary units)

Phosphorylation of Akt at S473

(arbitrary units)

2.5

1.5

1.0
0.5
0.0

1 h Post

R
Rest

Rest

1h

3h

R
RE

2.0
1.5

1.0
0.5

1h

3 h Post

RE
3h

Rest

1h

R
Rest

3h

p-S6K1

p-eEF2
a-tubulin

2.0

1.5

1.0
0.5

Phosphorylation of eEF2 at T56

(arbitrary units)

2.5

R
RE

1 h Post

Rest

a-tubulin

Phosphorylation of S6K1 at T389

(arbitrary units)

RE
3h

0.0
Rest

2.5

1h

1h

3 h Post

RE
3h

Rest

1h

3h

R
RE

2.0
1.5
1.0

0.5
0.0

0.0
Rest

1 h Post

3 h Post

Rest

1 h Post

3 h Post

Figure 3.

R
Rest

1h

RE
3h

Rest

1h

R
Rest

3h

p-AMPK

p-ACC

a-tubulin

a-tubulin
2.5

R
RE

2.0

1.5

1.0
0.5

1 h Post

Rest

R
Rest

1h

3 h Post

RE
3h

Rest

1h

3h

p-p38
a-tubulin

Phosphorylation of p38 at T180/Y182

(arbitrary units)

RE
3h

Rest

1h

3h

R
RE

2.0
1.5

1.0
0.5
0.0

0.0

2.5

Phosphorylation of ACC at S79

(arbitrary units)

Phosphorylation of AMPK at T172

(arbitrary units)

2.5

1h

R
RE

2.0

1.5
1.0
0.5
0.0
Rest

1 h Post

3 h Post

Rest

1 h Post

3 h Post

Figure 4.

R
Rest

1h

RE
3h

Rest

1h

3h

Rest
p-ERK1/2

a-tubulin

a-tubulin

Phosphorylation of 4EBP1 at T37/46

(arbitrary units)

2.5

R
RE

2.0

1.5

1.0
0.5
0.0

1 h Post

Rest

R
Rest

1h

3 h Post

RE
3h

Rest

1h

3h

p-CaMKII
a-tubulin

Phosphorylation of CaMKII at T286

(arbitrary units)

2.5

R
RE

2.0
1.5
1.0
0.5
0.0
Rest

1h

3h

Phosphorylation of ERK1/2 at T202/Y204

(arbitrary units)

p-4EBP1

2.5

1h

RE
3h

Rest

1h

3h

R
RE

2.0
1.5
1.0
0.5
0.0
Rest

1 h Post

3 h Post

Figure 5.

R
Rest

1h

RE
3h

Rest

1h

3h

REDD1
a-tubulin

2.5

R
RE

2.0

REDD1 mRNA expression

(arbitrary units)

Expression of REDD1
(arbitrary units)

2.5

1.5
1.0
0.5
0.0

2.0
1.5
1.0
#

0.5
0.0

Rest

1 h Post

3 h Post

Rest

1h

3h

2.5

R
RE

2.0
1.5

1.0
0.5
0.0

cMyc mRNA expression

(arbitrary units)

2.5

REDD2 mRNA expression

(arbitrary units)

R
RE

R
RE

2.0
1.5
1.0

0.5
0.0

Rest

1h

3h

Rest

1h

3h

PDK4 mRNA expression

(arbitrary units)

PGC-1a mRNA expression

(arbitrary units)

2.5

2.5

R
RE

2.0

1.5

*#

1.0

0.5

Rest

R
RE

2.0

Rest

PRC mRNA expression

(arbitrary units)

Figure 6.
6

A
*

0.0

1.5

1.0

0.5

0.0
1h
3h

1h
3h

B
2.5
R
RE

2.0
#

1.5

1.0

0.5

0.0
Rest
1h
3h