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Exercise-induced AMPK activation does not interfere with muscle
hypertrophy in response to resistance training in men
Tommy R. Lundberg,
1
Rodrigo Fernandez-Gonzalo,
2,3
and Per A. Tesch
2
1
Department of Health Sciences, Mid Sweden University, Östersund, Sweden;
2
Department of Physiology and Pharmacology,
Karolinska Institutet, Stockholm, Sweden; and
3
Department of Laboratory Medicine, Section for Clinical Physiology,
Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden
Submitted 26 September 2013; accepted in final form 8 January 2014
Lundberg TR, Fernandez-Gonzalo R, Tesch PA. Exercise-induced
AMPK activation does not interfere with muscle hypertrophy in response
to resistance training in men. J Appl Physiol 116: 611–620, 2014. First
published January 9, 2014; doi:10.1152/japplphysiol.01082.2013.—As
aerobic exercise (AE) may interfere with adaptations to resistance
exercise (RE), this study explored acute and chronic responses to
consecutive AE (⬃45 min cycling) and RE (4 ⫻7 maximal knee
extensions) vs. RE only. Ten men performed acute unilateral AE ⫹
RE interspersed by 15 min recovery. The contralateral leg was
subjected to RE. This exercise paradigm was then implemented in a
5-wk training program. Protein phosphorylation, gene expression, and
glycogen content were assessed in biopsies obtained from the vastus
lateralis muscle of both legs immediately before and 3 h after acute
RE. Quadriceps muscle size and in vivo torque were measured, and
muscle samples were analyzed for citrate synthase activity and gly-
cogen concentration, before and after training. Acute AE reduced
glycogen content (32%; P⬍0.05) and increased (P⬍0.05) phos-
phorylation of AMPK (1.5-fold) and rpS6 (1.3-fold). Phosphorylation
of p70S6K and 4E-BP1 remained unchanged. Myostatin gene expres-
sion was downregulated after acute AE ⫹RE but not RE. Muscle size
showed greater (P⬍0.05) increase after AE ⫹RE (6%) than RE
(3%) training. Citrate synthase activity (18%) and endurance perfor-
mance (22%) increased (P⬍0.05) after AE ⫹RE but not RE. While
training increased (P⬍0.05) in vivo muscle strength in both legs,
normalized and concentric torque increased after RE only. Thus AE
activates AMPK, reduces glycogen stores, and impairs the progres-
sion of concentric force, yet muscle hypertrophic responses to chronic
RE training appear not to be compromised.
aerobic exercise; gene expression; human skeletal muscle signaling;
muscle strength and power
IF PRECEDED BY AEROBIC EXERCISE (AE), typical outcomes of
resistance exercise (RE) training, e.g., increased strength,
power, and muscle size, may be compromised (30, 37). While
the requisites and associated mechanisms of such interference
have not been elucidated, residual fatigue and antagonistic
molecular responses derived from previous exercise have been
put forth as tentative causes to such an effect (27, 40). How-
ever, when allowing for 6 h recovery between bouts, we
recently reported that 5 wk concurrent AE ⫹RE training
produced similar improvements in muscle strength and power,
and even greater increases in muscle size, as RE alone (42).
Thus time allowed for recovery between bouts may be vital in
optimizing muscle adaptations to AE ⫹RE training.
It is well founded that acute AE reduces muscle glycogen
content (13, 29) and may compromise neuromuscular function
(5, 36). Thus undertaking RE immediately after AE may impair
muscle strength and power (39). Apart from interfering with in
vivo function, decreased glycogen availability may also di-
rectly or indirectly impact aspects of muscle signaling follow-
ing RE (17). Although this notion has been challenged (12),
collectively, it appears that acute AE induces fatigue that could
blunt the desired response to subsequent RE and hence atten-
uate muscle adaptations to chronic RE training.
At the cellular level, AE upregulates adenosine monophos-
phate-activated protein kinase (AMPK) to restore AMP/ATP
balance and to trigger transcriptional activators, regulating
mitochondrial biogenesis (14). RE increases protein synthesis
through the mammalian target of rapamycin (mTOR) signaling
pathway, and hence, cumulative RE favors contractile protein
accretion (45). In support of this stereotypic mode-specific
response, rat muscles subjected to either high- or low-fre-
quency electrical stimulation purported to mimic RE and AE,
respectively, showed increased mTOR phosphorylation after
RE but not AE (3). Conversely, AE hyperphosphorylated
AMPK and increased protein levels of the peroxisome prolif-
erator-activated receptor-␥coactivator-1 (PGC-1␣) (3). While
those findings imply that mode-specific signaling through
AMPK and mTOR pathways dictates classical end-point ad-
aptations to chronic AE and RE training, results of human
studies are equivocal. For example, when strength- and endur-
ance-trained athletes performed their habitual or nonhabitual
exercises, robust anabolic muscle signaling was noted after
unaccustomed exercise only (16). As reports also suggest
AMPK increases in response to RE (20) and mTOR to be
activated by AE (43), it appears important signaling routes
dictating human skeletal muscle adaptations are shared for
different exercise modes.
In the rat, contraction-induced mTOR signaling is inhibited
by prior AMPK activation caused by endurance exercise (60).
More specifically, translational signaling is compromised
through attenuation of key downstream regulators such as
eukaryotic initiation factor 4E-binding protein (4E-BP1) and
p70S6 kinase (p70S6K) (60). This is in concert with the
marked suppression of protein synthesis following AMPK
activation (8). Collectively, this scenario offers reasonable
mechanistic support for myofiber protein accretion to be
blunted, as noted after concurrent AE and RE training (27).
In human subjects who performed AE prior to RE (15), the
muscle anabolic response was somewhat attenuated compared
with exercise performed in the reversed order. Indeed, per-
forming RE followed by AE shows neither molecular interfer-
ence (2) nor altered rate of protein synthesis (19) compared
with RE alone. Our recent studies, examining responses to AE
followed by RE, showed uncompromised translational signal-
Address for reprint requests and other correspondence: T. R. Lundberg,
Dept. of Health Sciences, Mid Sweden Univ., 831 25 Östersund, Sweden
(e-mail: tommy.lundberg@miun.se).
J Appl Physiol 116: 611–620, 2014.
First published January 9, 2014; doi:10.1152/japplphysiol.01082.2013.
8750-7587/14 Copyright ©2014 the American Physiological Societyhttp://www.jappl.org 611
ing and muscle hypertrophy when recovery to restore muscle
function between bouts was allowed (41, 42). Given that acute
AE transiently impairs muscle function (5), reduces glycogen
stores (36), and activates AMPK (11), it may be that back-to-
back AE ⫹RE interferes with translational signaling and
hence attenuates muscle hypertrophy following chronic
training.
Therefore, the aim of the current study was to investigate
acute and chronic outcomes of consecutive AE ⫹RE and
training compared with RE alone. It was hypothesized that 1)
acute AE would reduce muscle glycogen stores and activate
AMPK signaling, 2) AE would compromise RE-induced
mTOR signaling, and 3) acute and chronic AE ⫹RE would
impair in vivo contractile function and blunt muscle hypertro-
phy compared with RE alone.
METHODS
General design. Ten men performed unilateral consecutive bouts of
knee extensor AE (⬃45 min) and RE (4 sets of 7 reps) interspersed by
15 min recovery. The contralateral limb was subjected to RE only.
Analysis of vastus lateralis muscle biopsies, obtained in both legs
before (PRE) and 3 h after (POST) RE (Fig. 1), measured glycogen
concentration, gene expression, and protein phosphorylation. These
two exercise regimens were subsequently implemented into a 5-wk
training program. Before and after training, muscle strength and
power were assessed and quadriceps femoris (QF) muscle volume,
cross-sectional area (CSA), and signal intensity (SI) determined by
means of magnetic resonance imaging (MRI). Resting muscle biop-
sies assessed training-induced changes in citrate synthase (CS) activ-
ity and glycogen content.
Subjects. Ten healthy male volunteers (26 ⫾5 yr, 183 ⫾7 cm, and
77 ⫾9 kg) completed the study protocol. Subjects were moderately
trained college students performing recreational exercise ⬃3 days/wk.
While most subjects had practiced RE training at some time, none had
performed structured weight training in the past year prior to the
study. The study experiments and procedures were explained before
subjects gave their written informed consent to participate. The study
was approved by the Regional Ethical Review Board in Stockholm.
Exercise equipment. AE was performed using a modified one-
legged cycle ergometer (model 828E, Monark Exercise AB, Varberg,
Sweden) as previously described (1, 42). Open-chain concentric
(CON) knee extensions were performed at a target cadence of 60 rpm.
Power and cadence were sampled at 2 Hz (SRM GmbH, Jülich,
Germany). RE was performed using a seated knee extension ergom-
eter (YoYo Technology, Stockholm, Sweden) equipped with a fly-
wheel offering inertial resistance (0.11 kg/m
2
) during coupled CON
and eccentric (ECC) muscle actions (59). Peak torque and power were
calculated from force (MuscleLab, Langesund, Norway) and speed
(SmartCoach, Stockholm, Sweden) measurements sampled at 100 Hz.
The coefficient of variation (CV%) for assessing flywheel peak power
across two different sessions within 1 wk was 5%. Maximal isometric
and isokinetic torque were assessed using a Cybex II (Lumex, New
York) dynamometer employing protocols described elsewhere (42).
For all apparatuses used, individual settings were maintained through-
out the study. Subjects completed three familiarization sessions in the
course of 2 wk prior to the study. Standardized warm-ups preceded
any exercise test or training session.
Acute exercise bouts. The acute exercise experiment was performed
1 wk prior to commencing 5 wk training. First, one randomly chosen
limb completed 40-min one-legged AE at ⬃70% of maximal work-
load (W
max
) using a cadence of 60 rpm. To ensure strenuous efforts,
ratings of perceived exertion (RPE; central and local) were obtained
using the 6–20 Borg scale (9). At 40 min, the workload was increased
by ⬃20 W and subjects were requested to continue until failure. Heart
rate was recorded continuously (Polar Electro, Kempele, Finland).
Fifteen minutes after completing AE, unilateral RE (4 ⫻7 maximal
repetitions; 2 min rest between sets) was executed for each leg in a
random order on the knee extension ergometer. Subjects were verbally
encouraged and requested to perform each repetition at maximal
effort. Peak power was measured in each repetition.
Training protocols. The AE ⫹RE training regimen was dedicated
to the leg performing AE ⫹RE during the acute experiments. The
other limb served as control and performed RE only. Subjects com-
pleted 15 AE sessions (3 nonconsecutive days/wk) and 12 unilateral
RE sessions for both limbs (2 days/wk during weeks 1, 3, and 5and
3 days/wk during weeks 2 and 4). Thus any RE session (i.e., 4 ⫻7
knee extensions for each leg) was preceded by AE for the intervention
leg, and allowed for 15 min recovery between bouts. All AE and RE
training sessions were supervised.
Muscle biopsies and diet control. Muscle biopsies were obtained
from the mid portion of vastus lateralis under local anesthesia imme-
diately before (PRE) and 3 h after (POST) the acute RE bout of either
leg (Fig. 1). The 3 h time point was chosen to accommodate for
changes in both protein phosphorylation and gene expression. In
addition, resting muscle samples from either leg were collected 72 h
after the last training session. Five millimeter Bergström-needles (7)
with suction applied were used to obtain ⬃150 mg tissue samples that
were cleansed of excess blood, fat, and connective tissue before being
frozen in liquid nitrogen-precooled isopentane and stored at ⫺80°C.
Subsequent biopsies were obtained from separate incisions, moving in
direction distal to proximal. A standardized dinner (pasta, tomato
sauce, and juice) consisting of 2.21 g carbohydrates/kg body wt (bw),
0.22 g protein/kg bw, and 0.04 g fat/kg bw was provided the night
before experiments. On days of scheduled biopsies, a liquid formula
(1.01 g carbohydrates/kg bw, 0.31 g protein/kg bw, and 0.24 g fat/kg
bw) was provided as breakfast 2 h prior to the first biopsy. The liquid
formulas contained 6.3 g protein (0.55 g leucine), 20.2 g carbohy-
drates, and 4.9 g fat per 100 ml (Ensure Plus, Abbott Laboratories,
Maidenhead, UK).
Pre- and posttesting. MRI scans (see below) were scheduled prior
to any test or biopsy. Three or four days after acute experiments,
maximal isometric and isokinetic strength, knee extension torque and
power, and one-legged endurance performance were assessed over 2
days. Muscle strength and power for the right leg and endurance for
the left leg were measured on day 1. The order of tests was reversed
on day 2. Measures of peak torque were obtained at constant velocities
of 0.52, 1.05, 2.09, 3.14, 3.67, and 4.19 rad/s using the Cybex II
dynamometer. Subjects performed 2–3 attempts (30 s rest) at each
velocity and the best result represented peak torque. Maximal isomet-
ric torque was measured at knee angle 120°. Subjects were instructed
to push with maximal effort for ⬃5 s. The best score (two trials) in a
1 s window defined peak isometric torque. Subsequently, peak torque
and power were assessed on the flywheel knee extension ergometer.
Subjects performed 2 ⫻7 repetitions (2 min rest between sets) under
strong verbal encouragement. Peak values were averaged across sets
and repetitions, and normalized torque (N·m/cm
2
) was calculated as
the ratio between peak knee extension torque and average muscle
CSA. Finally, the one-legged ergometer incremental test assessed
5 wk AE+RE
training
5 wk RE
training
RE
AE
B B
RE
AE+RE
15 min RE + 3 h
RE
0 h
Basal
B
Basal
Fig. 1. Schematic overview of the study protocol. AE, aerobic exercise; RE,
resistance exercise; B, muscle biopsy.
612 Compatibility of Aerobic and Resistance Training •Lundberg TR et al.
J Appl Physiol •doi:10.1152/japplphysiol.01082.2013 •www.jappl.org
W
max
and endurance performance. Resistance was increased by 2.5 N
every 2nd minute until failure to maintain cadence. W
max
was defined
as the last successfully completed workload. Heart rate was recorded
continuously throughout the test. RPE was obtained every 2nd minute
and at exhaustion. Postperformance tests were completed identical to
the pretests at the same time of the day (⫾2 h). Subjects were blind
to any test result to ensure nonbiased efforts. The post-MRI scans
were obtained 48–72 h after the last training session. Resting muscle
biopsies were obtained within 2 days after the MRI scans. Throughout
the study, subjects were instructed to maintain ordinary daily routines,
yet to refrain from strenuous activities involving the lower limbs.
Magnetic resonance imaging. Subjects rested in the supine position
for 1 h prior to any MRI scan (6). Cross-sectional T2 weighted images
were obtained using a 1.5-Tesla Philips MR Systems Intera (Best, The
Netherlands) unit as previously described (42). During each scan, 50
images with 10-mm slice thickness were obtained. Anatomical land-
marks and standardized procedures ensured that the same segment
was scanned before and after training. CSA and SI (mean gray value;
MGV) of each individual QF muscle [vastus lateralis (VL), vastus
intermedius (VI), vastus medialis (VM), and rectus femoris (RF)]
were analyzed from the image where gluteus maximus was no longer
visible, to the last image in which RF appeared. Every third image was
analyzed to quantify CSA and SI using ImageJ software (National
Institutes of Health, Bethesda, MD). As an additional control, SI of
the biceps femoris (BF) muscle was analyzed in the third image of
each subject. Mean CSA was multiplied by slice thickness to obtain
muscle volume. In our laboratory, the CV for interindividual assess-
ment of muscle volume amounts to 1%.
CS activity and glycogen content. Freeze-dried muscle samples
(⬃3 mg) were homogenized in phosphate buffer with 0.5% BSA. CS
activity and glycogen content were subsequently determined in du-
plicates through fluorometric assays as described elsewhere (42).
RNA isolation, reverse transcription, and real-time PCR. Gene
expression of established markers of muscle adaptations to AE and
RE were analyzed. Twenty-milligram muscle samples were homog-
enized using TRIzol and total RNA was extracted. One microgram of
total RNA from each sample was used for reverse transcription into
cDNA for a final volume of 20 l (High Capacity Reverse Transcrip-
tion Kit, Applied Biosystems, Foster City, CA). Real-time PCR
(ABI-PRISMA 7700 Sequence Detector, Perkin-Elmer Applied Bio-
systems) procedures were employed to determine mRNA expression.
Probes and primers (TaqMan) for atrogin-1 (Hs00369714_m1), Mus-
cle RING-finger protein-1 (MuRF-1; Hs00822397_m1), myostatin
(Hs00193363_m1), PGC-1␣(Hs01016724_m1), and vascular endothe-
lial growth factor (VEGF; Hs99999070_m1) were purchased from Ap-
plied Biosystems. GAPDH (Hs99999905_m1) and 18S (Hs01375212_
g1) were used as reference genes. The expression of reference genes
did not differ across time points and the GAPDH/18S ratio did not
change. Reaction and amplification mixes (10 l) consisted of the
diluted (1:100) cDNA (4.5 l), TaqMan Fast Universal PCR Master
Mix (5.0 l), and specific primers (0.5 l). Subsequent cycling
protocols were 2 min at 50°C and 10 min at 90°C followed by 40
cycles at 95°C for 15 s and 60°C for 1 min. Target gene expression
was reported as a ratio to the average of the two reference genes using
the 2
⫺⌬CT
formula.
Protein extraction and western blotting. About 30 mg muscle tissue
was manually homogenized in RIPA buffer, and proteins were recov-
ered as previously described (41). Protein concentrations were subse-
quently determined using the Bradford technique. Thirty micrograms
of protein per sample were loaded on 10% SDS precast gels (Bio-Rad)
and separated through electrophoresis together with a protein ladder.
Gels were transferred to PVDF-membranes using the Trans-Blot
Turbo Transfer System from Bio-Rad. Blocking was completed using
fluorescent blocking buffer (Millipore, Billerica, MA) during 60 min
at room temperature (RT). Membranes were incubated overnight at
4°C with primary antibodies (1:1,000) for phospho-P70S6K (Thr389),
phospho-rpS6 (Ser235/236), phospho-4E-BP1 (Thr37/46), and phos-
pho-AMPK␣(Thr172). All antibodies were from Cell Signaling
Technology (Beverly, MA), except for the antibody against p70S6K
which was from Santa Cruz Biotechnology (Santa Cruz, CA). After
the overnight incubation, membranes were washed (4 ⫻5 min) in
PBST (0.1%) and incubated with IRDye secondary antibody (LI-COR
Biosciences, Cambridge, UK) for 60 min at RT. A final series of
washes were then performed before scanning the membranes (Odys-
sey SA Infrared Imaging System, LI-COR Bioscience). The blots
were subsequently quantified using ImageJ. To control for loading
error, phosphorylated proteins were expressed relative to total ␣-tu-
bulin abundance (1:20,000; Sigma-Aldrich, St. Louis, MO).
Data analysis. Dependent variables were analyzed by two-way
repeated-measures ANOVA (factors: time and leg). When CON and
ECC muscle actions were analyzed separately, three-way ANOVAs
were employed. CS activity was analyzed by one-way ANOVA over
time. Data skewness was assessed through histograms and the Sha-
piro-Wilk test. Positively skewed variables (PGC-1␣and VEGF) were
log-transformed. When two-way interaction was found significant, a
priori planned simple effect comparisons within each level were
performed. The false discovery rate (FDR) procedure was employed
to adjust for these comparisons (18). Significance was accepted at the
5% level (P⬍0.05). Data are presented as means ⫾SD.
RESULTS
Acute exercise experiment. Average power during 40 min
AE was 37 ⫾6 W. Power increased to 60 ⫾10 W during the
final increment to exhaustion, which lasted 2 min ⫾12 s. RPE
rose in a linear fashion, to attain 13 (central) and 15 (local)
during the final 10 min of exercise, and 14 and 18 at exercise
completion. Average HR during AE was 118 ⫾18 beats/min.
HR amounted to 148 ⫾16 beats/min during the final exercise
stage, and peaked at 161 ⫾14 beats/min. In the subsequent
RE, the leg that had completed AE showed 10% lower (351 ⫾
97 W; P⫽0.020) peak power than the leg subjected to RE
only (387 ⫾88 W).
Aerobic exercise training. Average power across the 15 AE
sessions was 46 ⫾13 W. Average power amounted to 39 ⫾7
W in the first and increased (P⫽0.004) to 53 ⫾15 W during
the last session. At the final incremental step to exhaustion (2
min ⫾25 s), power averaged 66 ⫾18 W. RPE (local)
increased over the course of exercise to attain near maximal
values (19 ⫾1) at exercise completion. HR averaged 120 ⫾17
beats/min during the 40-min sessions and increased to 146 ⫾
21 beats/min during the final exercise stage. Average peak HR
across training sessions was 160 ⫾18 beats/min.
Resistance exercise training. Changes in peak power for
CON and ECC muscle actions were similar over the 5-wk train-
ing; hence CON-ECC data were merged. There was no leg ⫻
time interaction for peak power. Thus the progression in power
across RE training sessions (main effect of time, P⬍0.0005;
Fig. 2) was comparable for the different legs. However, the leg
subjected to AE ⫹RE performed ⬃20% lower peak power
across the 5-wk training (main effect of leg, P⫽0.001).
Endurance performance. Time to exhaustion in the one-
legged incremental test increased 22% (P⫽0.001) after AE ⫹
RE and was unchanged after RE (interaction: P⫽0.002, Table 1).
Peak HR was similar across time and leg (Table 1). At failure,
RPE (local) amounted to 19 ⫾1 for both legs at pre- and
posttests.
Strength performance. Flywheel knee extension peak torque
increased after training (Table 1). However, while peak ECC
torque increased equally across legs, peak CON torque in-
613Compatibility of Aerobic and Resistance Training •Lundberg TR et al.
J Appl Physiol •doi:10.1152/japplphysiol.01082.2013 •www.jappl.org
creased after RE (10%, P⫽0.004), yet remained unchanged
after AE ⫹RE (P⫽0.237). Consequently, CON torque
normalized to muscle CSA was compromised after AE ⫹RE
training (interaction P⫽0.010). Maximal isometric strength
was unaltered by training (Table 1). The isokinetic tests
showed large variations in individual responses, resulting in no
differences across time or legs (all P⬎0.05, Table 1).
Muscle volume, CSA, and signal intensity. Total QF volume
and CSA increased after either intervention (Table 2). How-
ever, the increase was greater after AE ⫹RE (6%) than RE
(3%, Fig. 3; interaction QF volume: F⫽38.5, P⬍0.0005).
Likewise, the increased volume of the four individual QF
muscles was greater after AE ⫹RE than RE (Table 2). SI
increased 7% after AE ⫹RE (P⫽0.009), yet was unaltered by
RE. This effect was consistent across the four individual QF
muscles (Table 2). BF of either leg showed no change in SI.
Glycogen content. There was a leg ⫻time interaction for
glycogen content (F⫽19.5, P⫽0.001). Thus at PRE, the leg
that had completed AE showed 32% lower (P⬍0.0005)
glycogen concentration than the rested leg (Fig. 4). This effect
was still evident 3 h after the acute RE bout (P⬍0.0005). In
the rested state after training, the leg subjected to AE ⫹RE
showed greater glycogen content than the RE leg (P⫽0.003)
and compared with basal values (P⫽0.004).
CS activity. CS activity increased (18%; P⫽0.001) in AE ⫹
RE (from 44.8 to 53.1 mmol·kg
⫺1
·min
⫺1
), but not in RE (43.3
mmol·kg
⫺1
·min
⫺1
).
Gene expression. There was a leg ⫻time interaction for
PGC-1␣expression (F⫽116.6, P⬍0.0005; Fig. 5). Thus the
increased expression from pre to post was greater after AE ⫹
RE (10.3-fold, P⬍0.0005), than RE (2.0-fold, P⫽0.001).
Likewise, there was an interaction effect for VEGF expression
(F⫽28.4, P⬍0.0005), due to a greater increase after AE ⫹
RE (2.5-fold, P⬍0.0005) vs. RE (1.2-fold, P⬍0.042).
Myostatin expression showed a tendency for interaction (P⫽
0.086) because the downregulation was greater after AE ⫹RE
(65%; P⬍0.0005) than RE (31%; not significant after FDR
procedures). MuRF-1 expression increased after AE ⫹RE
(2.9-fold, P⫽0.003) and was unchanged after RE (interaction:
F⫽20.4, P⫽0.001). Atrogin-1 showed interaction (F⫽42.0,
P⬍0.0005), as mRNA levels decreased after RE (43%, P⫽
0.004) and tended to increase after AE ⫹RE (1.3-fold, P⫽
0.101).
Protein phosphorylation. AMPK phosphorylation showed a
leg ⫻time interaction (F⫽11.4, P⫽0.008); values at PRE
were greater (1.5-fold, P⫽0.034) in the leg that had per-
formed AE, compared with the “rested” (RE) leg (Fig. 6).
Similarly, rpS6 signaling showed interaction (F⫽14.4, P⫽
0.004) such that phosphorylation was elevated (1.3-fold, P⫽
0
100
200
300
400
500
600
700
123456789101112
AE+RE
RE
Power (W)
No. of resistance exercise session
b, c
Fig. 2. Peak power measured in the flywheel ergometer during 12 resistance
exercise sessions with (AE ⫹RE) or without (RE) preceding aerobic exercise.
Means ⫾SD. Significant effect (P⬍0.05): b ⫽leg; c ⫽time.
Table 1. Selected performance measures pre and post resistance training with (AE
⫹
RE) or without (RE) preceding
aerobic exercise
AE ⫹RE RE
PRE POST ⌬% PRE POST ⌬%
Endurance performance
a,b,c
,s 632 ⫾105 770 ⫾145*# 22 624 ⫾151 654 ⫾142 5
W
maxa,b,c
,W 57 ⫾15 77 ⫾19*# 35 56 ⫾21 62 ⫾18 11
Peak heart rate at W
maxb
, beats/min 163 ⫾7 168 ⫾16 3 160 ⫾10 159 ⫾15 ⫺1
Flywheel merged peak power
c
,W 428 ⫾110 496 ⫾129* 16 435 ⫾80 515 ⫾109* 18
Flywheel peak CON power
c
,W 425 ⫾105 480 ⫾126* 13 427 ⫾76 501 ⫾104* 17
Flywheel peak ECC power
c
,W 432 ⫾116 512 ⫾135* 19 443 ⫾88 529 ⫾118* 19
Flywheel merged peak torque
c
, N·m 249 ⫾37 274 ⫾50* 10 251 ⫾34 280 ⫾39* 12
Normalized merged torque, N·m/cm
2
3.12 ⫾0.40 3.23 ⫾0.47 4 3.08 ⫾0.37 3.36 ⫾0.51 9
Flywheel peak CON torque
a,c
, N·m 236 ⫾33 247 ⫾43 5 234 ⫾26 258 ⫾35* 10
Normalized CON torque
a
, N·m/cm
2
2.95 ⫾0.28 2.90 ⫾0.34# ⫺2 2.87 ⫾0.26 3.08 ⫾0.39* 7
Flywheel peak ECC torque
c
, N·m 263 ⫾44 301 ⫾63* 14 268 ⫾46 303 ⫾51* 13
Normalized ECC torque, N·m/cm
2
3.29 ⫾0.55 3.56 ⫾0.66* 8 3.29 ⫾0.54 3.64 ⫾0.71* 11
Maximal isometric torque, N·m 323 ⫾84 305 ⫾52 ⫺6 297 ⫾52 314 ⫾68 6
Isometric torque, N·m
at 0.52 rad/s 300 ⫾58 293 ⫾55 ⫺2 291 ⫾46 296 ⫾56 2
at 1.05 rad/s 265 ⫾57 254 ⫾46 ⫺4 250 ⫾45 254 ⫾54 2
at 2.09 rad/s 218 ⫾51 201 ⫾44 ⫺8 204 ⫾35 206 ⫾49 1
at 3.14 rad/s 178 ⫾35 168 ⫾37 ⫺6 173 ⫾29 173 ⫾41 0
at 3.67 rad/s 164 ⫾39 148 ⫾28 ⫺10 156 ⫾30 156 ⫾40 0
at 4.19 rad/s 140 ⫾35 128 ⫾31 ⫺9 140 ⫾26 141 ⫾37 1
Values are means ⫾SD. W
max
, maximal workload; CON, concentric; ECC, eccentric. Significant main effects (P⬍0.05): a ⫽interaction, b ⫽leg, c ⫽time.
Significant simple effects (P⬍0.05): *time (POST vs. PRE); #leg (AE ⫹RE vs. RE).
614 Compatibility of Aerobic and Resistance Training •Lundberg TR et al.
J Appl Physiol •doi:10.1152/japplphysiol.01082.2013 •www.jappl.org
0.021) in AE ⫹RE at PRE. Although 4E-BP1 signaling was
similar across legs, there was a trend toward a main effect of
time (P⫽0.070) due to a slight increase from PRE to POST.
There were no changes across time or legs for p70S6K phos-
phorylation. Representative blots are shown in Fig. 7.
DISCUSSION
The current study scrutinized the proposed negative effect of
AE-induced AMPK activation on subsequent muscle signaling
and hypertrophic responses to RE training. Our novel results
show that AMPK activation prompted by AE does not com-
promise hypertrophy in human muscle subjected to RE. In-
deed, concurrent AE ⫹RE rather produced greater increase in
muscle size than RE. Thus, while we refute the purported
incompatibility between AMPK signaling and muscle hyper-
trophic responses, the results reinforce that AE could obstruct
the progression of in vivo muscle function to subsequent
cumulative RE. If employed in competitive athletes or exe-
cuted over extended time, there are obvious reasons to suspect
this effect would have adverse impact on performance.
AMPK activation induced by AE has been held responsible
for interfering with muscle growth to subsequent RE training
(4, 27). Indeed, animal and in vitro studies suggest AMPK
signaling blocks mTOR activity (34), impairs protein synthesis
(8, 60), and provokes myofibrillar protein degradation (51). In
stark contrast, and somewhat as a surprise, we recently re-
ported robust muscle hypertrophy after RE preceded by AE,
and allowing for 6 h recovery, that overshadowed that pro-
duced by RE only (42). Collectively these findings spurred us
to hypothesize that recovery between exercise bouts is a
prerequisite to optimize functional and cellular responses to
concurrent AE ⫹RE training.
In the current investigation, and similar to the findings of our
previous study (42), AE ⫹RE produced more substantial
increases in muscle size than RE. Thus inevitably, back-to-
back AE ⫹RE did not induce AMPK-mediated blunting of
muscle hypertrophy. This may be because suppressed protein
synthesis is associated with increased AMPK signaling only
during and immediately after acute exercise (20). In fact, there
0
200
400
600
800
1000
PRE POST BASAL
AE+RE RE
5 wk training
a, b, c
##
# ‡
Glycogen (mmol/kg dry wt)
Fig. 4. Muscle glycogen concentration before (PRE) and 3 h after (POST)
acute resistance exercise with (AE ⫹RE) or without (RE) preceding aerobic
exercise and in the rested state (BASAL) after 5 wk training. Significant effect
(P⬍0.05): a ⫽interaction, b ⫽leg, c ⫽time. Significant differences (P⬍
0.05) vs. #opposite leg.
Table 2. Selected MRI measures pre and post resistance training with (AE
⫹
RE) or without (RE) preceding aerobic
exercise
AE ⫹RE RE
PRE POST ⌬% PRE POST ⌬%
QF muscle volume
a,c
,cm
3
1,217 ⫾246 1,286 ⫾247* 6 1,239 ⫾231 1,273 ⫾237* 3
VL muscle volume
a,c
,cm
3
403 ⫾95 425 ⫾95* 6 411 ⫾96 421 ⫾96* 2
VI muscle volume
a,c
,cm
3
376 ⫾77 389 ⫾79* 3 390 ⫾67 395 ⫾71 1
VM muscle volume
a,c
,cm
3
322 ⫾70 336 ⫾69*# 4 318 ⫾64 325 ⫾64* 2
RF muscle volume
a,c
,cm
3
117 ⫾26 135 ⫾28* 15 120 ⫾33 131 ⫾34* 9
QF mean CSA
a,c
,cm
2
80.7 ⫾13.2 85.2 ⫾12.9* 6 82.2 ⫾12.5 84.4 ⫾12.5* 3
QF signal intensity
a
, MGV 48.0 ⫾5.9 51.5 ⫾7.6* 7 47.3 ⫾5.8 46.8 ⫾6.1 ⫺1
VL signal intensity
a,b,c
, MGV 47.3 ⫾4.5 51.1 ⫾5.7*# 8 46.6 ⫾4.2 46.6 ⫾4.6 0
VI signal intensity
a
, MGV 47.4 ⫾4.3 49.8 ⫾4.8*# 5 46.5 ⫾4.6 45.5 ⫾4.4 ⫺2
VM signal intensity
a
, MGV 50.4 ⫾5.9 51.9 ⫾7.0 3 49.6 ⫾7.4 48.2 ⫾7.3 ⫺3
RF signal intensity
a,c
, MGV 47.0 ⫾11.9 53.2 ⫾15.4* 13 46.6 ⫾9.5 46.8 ⫾10.5 0
BF signal intensity, MGV 41.2 ⫾5.7 41.6 ⫾5.8 1 39.7 ⫾4.2 39.9 ⫾5.1 1
Values are means ⫾SD. CSA, cross-sectional area; MGV, mean gray value; QF, quadriceps femoris; VL, vastus lateralis; VI, vastus intermedius; VM, vastus
medialis; RF, rectus femoris; BF, biceps femoris. Significant main effects (P⬍0.05): a ⫽interaction, b ⫽leg, c ⫽time. Significant simple effects (P⬍0.05):
*time (PRE vs. POST); #leg (AE ⫹RE vs. RE).
0
25
50
75
100
125
150 AE+RE
RE
*
Muscle volume (Δ cm3)
Fig. 3. Individual and mean increase in quadriceps muscle volume after
resistance training with (AE ⫹RE) or without (RE) concurrent aerobic
exercise. *Greater increase after AE ⫹RE.
615Compatibility of Aerobic and Resistance Training •Lundberg TR et al.
J Appl Physiol •doi:10.1152/japplphysiol.01082.2013 •www.jappl.org
is sound support for this presumption. First, increased AMPK
activity is typically evident ⱕ1 h postexercise (11, 20, 46)
returning to baseline shortly thereafter (20, 62), and cumulative
exercise may even offset this response (46). Second, when RE
was executed subsequent to AE (15), the only inhibitory effect
occurred at the IGF-1 mRNA level while mTOR signaling was
not impacted. Third, muscle protein fractional synthetic rate,
assessed 1–4 h postexercise, was comparable across AE ⫹RE
and RE (13). Taken together, and given that protein synthesis
may be elevated 48–72 h after acute RE (38), the very
short-lived AMPK activation induced by AE most likely
evokes minute, if any, impact on the net protein balance
accumulated between exercise sessions.
While the lack of enhanced mTOR signaling after RE was
unexpected, rpS6 phosphorylation was elevated after AE. This
increase occurred in parallel with increased AMPK phosphor-
ylation following AE, yet was unaccompanied by increases in
other mTOR effectors. This response seems at odds with the
current understanding of enhanced translation initiation and
protein synthesis after acute exercise. However, increases in
muscle protein synthesis and rpS6 phosphorylation may occur
in the absence of increased mTOR signaling (19). Further, our
gene expression records support that AE ⫹RE served a more
potent stimulus for tissue remodeling than RE. More specifi-
cally, while RE alone failed to downregulate myostatin and
increase MuRF-1 expression, these genes showed robust re-
sponses after AE ⫹RE. Myostatin suppression has been
associated with muscle hypertrophy and decreased specific
force (47), and MuRF-1 expression appears crucial for loading-
induced growth to occur (33). These findings largely also
corroborate with changes in expression of other genes (i.e.,
PGC-1␣, VEGF and atrogin-1), which showed augmented
mRNA response in the leg that was subjected to concurrent
exercise. These results are intriguing, because apart from
controlling mitochondrial biogenesis induced by AE, PGC-1␣
induction may also protect against muscle protein breakdown
(10) and elicit hypertrophy in response to loading (57). Further,
it is noteworthy our results accord with the exaggerated gene
expression response to AE ⫹RE with 6 h between bouts (22,
41). Thus, at least during the initial training phase and regard-
PRE POST
AE+RE RE
PGC-1α mRNA (a.u.)
0
1
2
3
4
5
A
a, b, c
* #
*
PRE POST
AE+RE RE
0
1
2
3
4
5
VEGF mRNA (a.u.)
Ba, b, c
* #
*
PRE POST
AE+RE RE
* #
0
1
2
3
4
5
MuRF-1 mRNA (a.u.)
Ca, b, c
PRE POST
AE+RE RE
0
1
2
3
4
5
Atrogin-1 mRNA (a.u.)
Da, b
*
#
PRE POST
AE+RE RE
* #
0
1
2
3
4
5
Myostatin mRNA (a.u.)
E
b, c
Fig. 5. A–E: PGC-1␣, VEGF, MuRF-1,
atrogin-1, and myostatin mRNA levels be-
fore (PRE) and 3 h after (POST) resistance
exercise with (AE ⫹RE) or without (RE)
preceding aerobic exercise. Significant ef-
fects (P⬍0.05): a ⫽interaction, b ⫽leg,
c⫽time. Significant differences (P⬍0.05)
vs. #opposite leg, *same leg at PRE.
616 Compatibility of Aerobic and Resistance Training •Lundberg TR et al.
J Appl Physiol •doi:10.1152/japplphysiol.01082.2013 •www.jappl.org
less of recovery, it appears the more voluminous AE ⫹RE
paradigm induced a more favorable cellular environment for
muscular, vascular, and mitochondrial protein turnover.
It is well established that AE reduces glycogen stores, and
commencing exercise in a low-glycogen state may evoke
different metabolic and molecular responses compared with
muscles displaying normal or supranormal glycogen levels (17,
28). It has been postulated that glycogen depletion, and/or
associated changes in muscle energy balance, could contribute
to the incompatibility of mixed-mode training (40, 50). In the
current study, however, glycogen utilization induced by AE
was not accompanied by altered mTOR signaling 3 h post RE.
Similarly, translational signaling and protein synthetic re-
sponses to RE were not compromised in glycogen-voided
skeletal muscle at onset of exercise (12). Taken together, it
appears human muscle commencing RE at ⬃30% reduced
glycogen stores and ⬃20% compromised function possesses
undiminished ability to undergo hypertrophy.
Evidently AE ⫹RE exaggerated the increased muscle size
shown with RE only. These findings were very consistent and
the hierarchical response across individual QF muscles was
identical to what we have reported earlier (42, 52). Moreover,
SI of the RE leg and the nonused BF muscle serving as control
was unaltered, providing further evidence that reported differ-
ences across legs were specific to the QF muscle group and the
intervention imposed. However, given the established relation-
ship between muscle anatomical CSA and in vivo force (44),
the finding of greater muscle hypertrophy, but not force, after
AE ⫹RE than RE suggests that the increase in size was not
entirely “functional.” At first, and consistent with the notion of
increased SI of MRIs (26, 42), it would be tempting to attribute
the reduced specific force to edema or swelling due to the
preceding exercise. However, it should be recalled both legs
executed the high-load ECC component of RE, and none of the
subjects reported delayed onset of muscle soreness, associated
with ECC exercise (23). It also remains that the acute molec-
ular response implied greater anabolic environment after AE ⫹
RE compared with RE. Likewise, muscle samples obtained
before and after 5 wk AE ⫹RE or RE (42) showed unaltered
protein concentrations despite the robust hypertrophy (unpub-
lished observation). Altogether, these findings suggest that the
increased SI was caused by events unrelated to cell swelling.
As mitochondria constitute 4– 6% of muscle tissue (32) and
our proxy markers (CS activity; endurance performance)
showed substantial increases after AE ⫹RE, it may be that
A
PRE POST
AE+RE RE
0
1
2
3
4
5
p-AMPK at Thr 172 (a.u.)
a
#
PRE POST
AE+RE RE
0
1
2
3
4
5
p-p70S6K at Thr 389 (a.u.)
B
C
PRE POST
AE+RE RE
0
1
2
3
4
5
p-rpS6 at Ser 235/236 (a.u.)
a
#
PRE POST
AE+RE RE
0
1
2
3
4
5
p-4E-BP1 at Thr 37/46 (a.u.)
D
Fig. 6. A–D: phosphorylation of phospho-
P70S6K (Thr389), phospho-rpS6 (Ser235/
236), phospho-4E-BP1 (Thr37/46), and phospho-
AMPK␣(Thr172) relative to total ␣-tubulin
before (PRE) and 3 h after (POST) resis-
tance exercise with (AE ⫹RE) or without
(RE) preceding aerobic exercise. Significant
effects (P⬍0.05): a ⫽interaction. Signifi-
cant differences (P⬍0.05) vs. #opposite leg.
AE+RE RE
p-AMPK
p-p70S6K
p-rpS6
p-4E-BP1
α-Tubulin
α-Tubulin
α-Tubulin
α-Tubulin
Pre Pre Post Post
Pre Pre Post Post
RE AE+RE
Fig. 7. Representative blots of phospho-P70S6K (Thr389), phospho-rpS6
(Ser235/236), phospho-4E-BP1 (Thr37/46) and phospho-AMPK␣(Thr172)
with corresponding ␣-tubulin bands.
617Compatibility of Aerobic and Resistance Training •Lundberg TR et al.
J Appl Physiol •doi:10.1152/japplphysiol.01082.2013 •www.jappl.org
noncontractile protein constituents had subtle, yet significant
impact on size measures. In fact, increases in mitochondrial
volume and glycogen stores after short-term endurance training
have been estimated to account for 3–4% of the increase in
muscle CSA (53, 54). While it remains to establish a credible
explanation for the more ample increase in muscle volume
after AE ⫹RE than RE, evidently increases in both myofi-
brillar and mitochondrial/sarcoplasmic protein pools occur in
parallel.
Albeit the AE insult impaired succeeding RE performance
throughout the training period (Fig. 2), force and power in-
creased after both AE ⫹RE and RE in the current investiga-
tion. However, RE-induced improvements in normalized and
absolute CON torque were blunted by AE ⫹RE. Previous
research also suggests that concurrent training may interfere
with the progression of in vivo muscle function (40). Such
interference appears most evident for explosive strength and
power (21, 24, 48, 55) and is not necessarily accompanied by
compromised muscle hypertrophy (25, 35, 48). Regardless of
mechanism(s) involved, it is imperative that certain unidenti-
fied events responsible for functional shortcomings brought
about by the previous AE, must be normalized prior to RE. In
support, high- but not low-intensity RE produced strength
gains, whereas muscle size increased regardless of intensity
(31). Further, back-to-back AE ⫹RE impeded progression of
strength, but not muscle size (58). Concurrent exercise allow-
ing for 6 h recovery between exercise modes did not (42).
Altogether, it is apparent that restored muscle function between
exercise bouts is a prerequisite for attaining optimal gains in
muscle function in response to AE ⫹RE training. This con-
trasts the muscle hypertrophic response, which seems to occur
independent of recovery.
The one-legged AE model employed here promotes early
endurance adaptations (42, 49, 56). This was manifested in
marked increases in PGC-1␣and VEGF expression after acute
AE, and enhanced CS activity and endurance performance
after AE ⫹RE training. The current RE protocol increased
muscle size and strength at rates comparable to what has been
shown elsewhere (52, 61). While we have no apparent expla-
nation for the somewhat less pronounced RE-induced increases
in muscle size and strength than reported by us recently using
the same RE paradigm (42), this observation should not dis-
tract from the mutual finding of significantly different response
across AE ⫹RE and RE in the two studies.
It should be acknowledged that in the current study, total
work performed differed across legs. Such a response is inher-
ent in any experimental design aimed at examining the effect of
AE on responses prompted by RE. Given the particular ques-
tion posted here, this effect presents no drawback in interpret-
ing our results. The unique loading feature of the RE method-
ology used here allows for execution of maximal voluntary
force or power through the entire range of motion in each CON
action of each set, as well as ECC overload (59). Inevitably, the
preceding AE, which consisted of CON knee extensions only,
compromised peak power during this task (Fig. 2). In this
context it should be appreciated that employing an AE mode
other than the current cycling model, e.g., running comprising
ECC actions and characterized by different loading history,
may have produced different results. While being outside the
scope of the present study, a recent meta-analysis, quantifying
a total of 422 effect sizes, reported that the interference effect
is exacerbated with running compared with cycling (63).
In conclusion, consecutive bouts of AE ⫹RE performed
over 5 wk exaggerated the increase in muscle size shown with
RE alone. This occurred despite increased AMPK phosphory-
lation, reduced glycogen content, and attenuated muscle func-
tion elicited by AE. Thus we demonstrate that AMPK activa-
tion induced by AE does not interfere with muscle growth
produced by concurrent RE training. However, AE ⫹RE blunt
the progression of important aspects of in vivo muscle func-
tion. Employing this approach may therefore be counterpro-
ductive for athletes and individuals aiming at developing max-
imal strength and power.
ACKNOWLEDGMENTS
We thank D. Carlsson, R. Vargas Paris, and M. Pettersson for technical
support.
GRANTS
This study was supported by grants from the Swedish National Centre for
Research in Sports (P. A. Tesch; CIF), the European Space Agency (P. A.
Tesch; ESA), and the Swedish National Space Board (P. A. Tesch; SNSB).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.R.L., R.F.-G., and P.A.T. conception and design of
research; T.R.L. and R.F.-G. performed experiments; T.R.L. and R.F.-G.
analyzed data; T.R.L., R.F.-G., and P.A.T. interpreted results of experiments;
T.R.L. and P.A.T. drafted manuscript; T.R.L., R.F.-G., and P.A.T. edited and
revised manuscript; T.R.L., R.F.-G., and P.A.T. approved final version of
manuscript; R.F.-G. prepared figures.
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