Resistance exercise with whey protein ingestion affects

mTOR signaling pathway and myostatin in men

Juha J. Hulmi, Jrgen Tannerstedt, Harri Selnne, Heikki Kainulainen, Vuokko
Kovanen and Antti A. Mero
J Appl Physiol 106:1720-1729, 2009. First published 19 March 2009;
doi:10.1152/japplphysiol.00087.2009
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J Appl Physiol 106: 17201729, 2009.

First published March 19, 2009; doi:10.1152/japplphysiol.00087.2009.

HIGHLIGHTED TOPIC

Regulation of Protein Metabolism in Exercise and Recovery

Resistance exercise with whey protein ingestion affects mTOR signaling

pathway and myostatin in men
Juha J. Hulmi,1 Jorgen Tannerstedt,2 Harri Selanne,3 Heikki Kainulainen,1 Vuokko Kovanen,4
and Antti A. Mero1
1

Department of Biology of Physical Activity and Neuromuscular Research Center, University of Jyvaskyla, Jyvaskyla,
Finland; 2Astrand Laboratory, Swedish School of Sport and Health Sciences and Department of Physiology
and Pharmacology, Karolinska Institutet, Stockholm, Sweden; 3LIKES Research Center, Jyvaskyla, Finland; and 4Department
of Health Sciences, University of Jyvaskyla, Jyvaskyla, Finland
Submitted 28 January 2009; accepted in final form 17 March 2009

hypertrophy; training; nutrition; S6K1; skeletal muscle

ADEQUATE MUSCLE MASS is crucial for human well-being. It is,
therefore, important to identify the mechanisms that stimulate
muscle hypertrophy or prevent atrophy. The most efficient way
to increase the size of a skeletal muscle is by resistance training
(RT) in combination with protein-containing nutrition. Muscle
hypertrophy due to RT and protein nutrition seems largely to
result from cumulative acute increases in muscle protein syn-

Address for reprint requests and other correspondence: J. Hulmi, Dept. of

Biology of Physical Activity, Univ. of Jyvaskyla, P.O. Box 35, 40014 Jyvaskyla, Finland (e-mail: [email protected]).
1720

thesis. One resistance exercise (RE) bout can within 1 h

increase muscle protein synthesis (9), which can last up to 72 h
after exercise (39). Protein ingestion before or after a bout of
RE has been shown to significantly enhance this response (52)
and be possibly more beneficial in terms of muscle hypertrophy
than nutrient ingestion at other times of day (7, 11). It would
thus be important to understand how protein ingestion affects
pathways regulating intracellular hypertrophy of muscle in the
context of a bout of RE and long-term RT.
Muscle protein synthesis and hypertrophy are stimulated by
the mammalian target of rapamycin (mTOR) pathway protein
kinase enzymes that are activated or inactivated by phosphorylation or dephosphorylation at different amino acid sites (13,
28). In this pathway, phosphorylation of eukaryotic initiation
factor 4E (eIF4E) binding protein (4E-BP1) and p70S6K/S6K1
(p70 ribosomal S6 kinase) by mTOR have been shown to be
important in muscle protein synthesis and hypertrophy (2, 12,
28, 33, 35, 40, 43, 51). The importance of 4E-BP1 is due to
the fact that its phosphorylation prevents the interaction and
inhibition of 4E-BP1 with eIF4E and therefore increases
translation of the protein synthesis (24, 42). On the other
hand, p70S6K affects muscle hypertrophy at least through
ribosomal protein S6 (rpS6) as well as possibly through
some other proteins such as eukaryotic elongation factor 2
(eEF2) (44, 45).
Ingestion of protein with carbohydrate or only branchedchain amino acids in the context of a bout of RE has been
shown to increase the phosphorylation of mTOR (3), p70S6K
(26, 30), and rpS6 (26, 30) at 0 4 h post-RE in humans. The
effect of an intact protein source alone, such as whey, on this
pathway in humans, and especially in the longer term after a
bout of RE, i.e., from 12 to 72 h or after months of resistance
training, is unknown.
The mTOR pathway is opposed by myostatin signaling,
which inhibits muscle growth (37, 46), partially possibly
through inhibiting mTOR signaling (1). The only published
studies so far on the myostatin response in humans to a bout of
RE or long-term RT combined with protein ingestion are based
on studies carried out recently in our laboratory (21, 22). These
studies suggested that protein ingestion may acutely hinder the
RE-induced decrease in myostatin mRNA expression in both
young and old men; however, it remains unknown whether that
would also lead to a change in a protein level of myostatin.

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Hulmi JJ, Tannerstedt J, Selanne H, Kainulainen H, Kovanen V, Mero AA. Resistance exercise with whey protein ingestion affects mTOR signaling pathway and myostatin in men. J Appl
Physiol 106: 1720 1729, 2009. First published March 19, 2009;
doi:10.1152/japplphysiol.00087.2009.Signaling pathways sense local and systemic signals and regulate muscle hypertrophy. The effects
of whey protein ingestion on acute and long-term signaling responses
of resistance exercise are not well known. Previously untrained young
men were randomized into protein (n 9), placebo (n 9), and
control (n 11) groups. Vastus lateralis (VL) muscle biopsies were
taken before and 1 h and 48 h after a leg press of 5 10 repetitions
[resistance exercise (RE)] and after 21 wk (2 times per week) of
resistance training (RT). Protein (15 g of whey) or nonenergetic
placebo was ingested before and after a single RE bout and each RE
workout throughout the RT. The protein group increased its body
mass and VL muscle thickness (measured by ultrasonography) already at week 10.5 (P 0.05). At week 21, the protein and placebo
groups had similarly increased their myofiber size. No changes were
observed in the nonexercised controls. However, the phosphorylation
of p70S6K and ribosomal protein S6 (rpS6) were increased at 1 h
post-RE measured by Western blotting, the former being the greatest
with protein ingestion. Mammalian target of rapamycin (mTOR)
phosphorylation was increased after the RE bout and RT only in the
protein group, whereas the protein ingestion prevented the post-RE
decrease in phosphorylated eukaryotic initiation factor 4E binding
protein 1 (p-4E-BP1). Akt phosphorylation decreased after RT,
whereas no change was observed in phosphorylated eukaryotic elongation factor 2. A post-RE decrease in muscle myostatin protein
occurred only in the placebo group. The results indicate that resistance
exercise rapidly increases mTOR signaling and may decrease myostatin protein expression in muscle and that whey protein increases
and prolongs the mTOR signaling response.

1721

mTOR AND MYOSTATIN AFTER EXERCISE WITH PROTEIN

The purpose of this randomized controlled and doubleblinded trial was to examine acute and long-term/chronic
responses to resistance training in terms of protein signaling
known to be related to muscle hypertrophy. Specifically, our
main focus was to investigate these pathways when a highquality milk protein fraction, whey (15), is supplemented to a
normal diet both immediately before and after a resistance
exercise workout. We hypothesized that ingestion of whey
proteins immediately before and after a resistance exercise
bout would have fast acute but not long-lasting effects on the
phosphorylation levels of the mTOR signaling pathway and on
myostatin protein expression.
MATERIALS AND METHODS

and discomforts. Each then signed an informed consent to participate

in the study, which was approved by the local Ethics Committee of the
University of Jyvaskyla and was conducted in accordance with the
Declaration of Helsinki.
Design
This investigation examined acute and long-term responses of
adding high-quality protein to a normal diet (with no other nutritional
supplements). The experimental design involved both a single bout of
RE and 0- to 48-h postexercise responses to it, and following that, 21
wk of RT consisting of 42 RE workouts (Fig. 1). A control group was
included, and all the measurements were always carried out at the
same time of day to exclude the effects of biopsy sampling or effects
of time of year or daily variations (47, 57). All the measurements were
preceded by at least 2 days of rest from physical activity.

Subjects
Experimental RT
Whole body heavy RE workouts were carried out twice a week. A
minimum of 2 days of rest was required between workouts. All
training sessions were supervised by experienced trainers who ensured that proper techniques and progression were used in each
exercise (32). The leg exercises included two exercises for the leg
extensor muscles, the bilateral leg press and bilateral knee extension,
and one exercise for the leg flexors, bilateral knee flexion. The RT
program also included exercises for the other main muscle groups:
chest and shoulders (bench press), upper back, trunk extensors and
flexors, upper arms, ankle extensors, and hip abductors and adductors.
RT was performed with progressive training loads of 40 85% of the
subjects one-repetition maximum (1RM) in a periodized training
program. For each exercise in a workout the number of sets increased
(from 23 to 35) and the number of repetitions in each set decreased
(from 1520 to 5 6) during the 21-wk RT period. The loads were
individually determined throughout the RT period. Recovery between

Table 1. Anthropometry: height, body mass, fat percent, muscle fiber cross-sectional area, and muscle thickness
of the protein, placebo, and control groups
Variable/Group

Height, cm
Protein
Placebo
Control
Body mass, kg
Protein
Placebo
Control
Fat, %
Protein
Placebo
Control
Fiber size: type I, m2
Protein
Placebo
Control
Fiber size: type II, m2
Protein
Placebo
Control
Muscle thickness, cm
Protein
Placebo
Control

Baseline

10.5 wk

21 wk

76.78.1
75.98.0
74.57.8

80.19.5*
77.68.5
75.68.4*

80.19.6*
78.28.9*
74.58.1

16.84.0
17.33.9
16.63.5

17.74.3
17.24.5
17.54.5

17.54.6
17.13.9
16.94.6

P Valuepre vs. 21

P Value%group21

181.86.9
181.06.2
181.94.7
0.009
0.03
0.93
0.35
0.99
0.64

0.001
0.02
0.50
0.46

4,650178
4,198185
4,940411

6,582511*
5,910288*
5,099391

0.009
0.003
0.21

0.03
0.04

5,021402
4,617336
5,501407

7,599576*
6,951484*
5,629380

0.002
0.003
0.72

0.01
0.04

2.930.19*
2.740.25*
2.780.13

0.003
0.046
0.10

0.03
0.12

2.610.14
2.470.22
2.680.15

2.890.18*
2.720.32
2.650.17

Values are means SD, except muscle size variables, which are means SE. P valuepre vs.21 designates Holm-Bonferroni corrected P values compared with
baseline. P value%group21 designates the difference between the training group and the control group in percent change between the baseline and post-21 wk
values. *Significant (P 0.05) P value vs. pre. Significant value compared with percentage change vs. the control group. See text for further description of
groups and time points.
J Appl Physiol VOL

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The subjects were randomly assigned to either a whey protein

group (n 9, age 24.7 5.0 yr), placebo group (n 9, 27.4 3.1
yr), or control group (n 11, 25.2 2.7 yr). Anthropometric details
of the subjects are presented in Table 1. The subjects were recruited
for the study by advertising in newspapers and through e-mail lists. A
subgroup from a previous study (22) was used in the present study.
All the subjects were examined by a physician, and none had
medical problems that might confound the results of this investigation.
None of the subjects had any regular RT experience, but they were
moderately active. Their normal habitual activities included walking,
jogging, swimming, or ball games, and they were urged to continue
the normal activities and daily living exactly the same during the
experimental period. The subjects had a typical Finnish diet containing a rather large amount of protein and moderate amounts of fat and
carbohydrates. The subjects were urged to continue their normal diet
throughout the project. Before the investigation, each subject was
informed about the experimental design and possible associated risks

1722

mTOR AND MYOSTATIN AFTER EXERCISE WITH PROTEIN

Fig. 1. Experimental design. B, vastus lateralis muscle biopsy; T1, testing at

baseline; T2, testing at post 21 wk; 2, venous blood sampling; RE, resistance
exercise bout [5 10-repetition maximum (RM) leg press]; RT, heavy and
progressive resistance training; D, protein (15 g of whey protein) or placebo
(no energy) drink.

the sets was 23 min. No RT was done in the control group; instead
they continued their habitual activity such as jogging, swimming, or
ball games.
Nutritional Supplementation During RT

Heavy RE Protocol and Nutritional Supplementation Before

and After a Bout of RE
A bilateral leg press machine (David 210, David Fitness and
Medical) was used for the single heavy RE bout carried out before the
experimental RT period. The RE bout protocol was same as in earlier
studies (19, 21, 22). The total number of sets was five. Each set
contained 10 repetition maximums. Recovery time between the sets
was 2 min. The first set started with the 75% 1RM load based on the
two earlier strength tests to measure baseline strength of the subjects
(22). The loads were adjusted during the course of the RE bout due to
fatigue so that each subject would be able to perform 10 repetitions at
each set. If the load was too heavy, the subject was assisted slightly
during the last repetitions of the set. Either 15 g of whey protein or the
placebo was ingested immediately before and after the bout of RE.
Anthropometry
After an overnight fasting, body mass (kg) and fat percentage were
measured. Body fat was measured using a skinfold caliper (biceps and
J Appl Physiol VOL

Muscle Biopsies
Muscle biopsies were obtained 0.5 h before (pre) and 1 h (post 1 h)
and 48 h (post 48 h) after the bout of RE, or resting in the control
group, before the RT period (Fig. 1). The post-1 h biopsy time point
represents fast responses of the RE bout and the 48-h time point the
more delayed responses. The biopsy after RT (post 21 wk) was taken
4 5 days after the last exercise workout to minimize the effects of the
last exercise workout on the post-RT biopsy. Biopsies were taken
from the VL muscle with a 5-mm Bergstrom biopsy needle, midway
between the patella and greater trochanter. The pre-RE and the 48-h
as well as post-21 wk biopsies were taken from the right leg. To avoid
any residual effects of the prebiopsy, the 1-h post-RE biopsy was
taken from the left leg and the 48-h biopsy was taken 2 cm above the
previous biopsy location. Before the baseline and the 21-wk biopsy, a
3-h fasting period was required. Of 11 control subjects, for 5 subjects
only pre and post-21 wk biopsies were available.
The muscle sample was cleaned of any visible connective and
adipose tissue as well as blood and frozen immediately in liquid
nitrogen (180C) and stored at 80C. The pre-21 wk and post-21
wk samples for immunohistochemical analysis were obtained with
another needle, and they were immediately mounted on a cork, and
frozen rapidly in isopentane cooled to 160C in liquid nitrogen and
thereafter stored at 80C.
Tissue Processing
Muscle biopsy specimens were hand-homogenized in ice-cold
buffer [20 mM HEPES (pH 7.4), 1 mM EDTA, 5 mM EGTA, 10 mM
MgCl2, 100 mM -glycerophosphate, 1 mM Na3VO4, 2 mM DTT,
1% Triton X-100, 0.2% sodium deoxycholate, 30 g/ml leupeptin, 30
g/ml aprotinin, 60 g/ml PMSF, and 1% phosphatase inhibitor
cocktail (P 2850; Sigma, St. Louis, USA)] at a dilution of 15 l/mg
of wet weight muscle. Homogenates were rotated for 30 min at 4C,
centrifuged at 10,000 g for 10 min at 4C to remove cell debris, and
stored at 80C. Total protein was determined using the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL).
Western Immunoblot Analyses
Aliquots of muscle lysate were solubilized in Laemmli sample
buffer and heated at 95C to denaturate proteins. For 4E-BP1, but not
others, homogenates were first heated 10 min at 95C, centrifuged at
7,000 g for 30 min at 4C, and then continued with the Laemmli
buffer and heating similarly as the other proteins (9).
Samples containing 30 g of total protein were separated by
SDS-PAGE for 60 to 90 min at 200 V using 4 20% gradient gels on
Criterion electrophoresis cell (Bio-Rad Laboratories, Richmond, CA).
All four samples from each subject were run on the same gel. Proteins
were transferred to PVDF membranes at 300-mA constant current for
3 h on ice at 4C. The uniformity of protein loading was checked by
staining the membrane with Ponceau S. Membranes were blocked in
TBS with 0.1% Tween 20 (TBS-T) containing 5% nonfat dry milk for
1 h and then incubated overnight at 4C with commercially available
rabbit polyclonal primary phosphospecific antibodies. Antibodies rec-

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The subjects ingested immediately before and after each RE workout in the gym either 15 g of whey isolate protein with minimal
lactose and fat (Protarmor 907 LSI, Armor Proteins, Brittany, France)
dissolved in 250 ml of water or an equivalent volume of nonenergetic
placebo. The drinks were provided for the subjects in a double-blind
fashion. The essential amino acid composition of the protein drink (15
g) was as follows: histidine (0.2 g), isoleucine (1.0 g), leucine (1.7 g),
lycine (1.4 g), methionine (0.4 g), phenylalanine (0.5 g), threonine
(1.0 g), tryptophan (0.2 g), and valine (0.8 g). Both of the drinks
contained equal amounts of exotic fruit (flavor), acesulfame-K (sweetener), and beta-carotene (color). The drinks were as identical as
possible, differing mainly in the amount of the added viscosity
substance (xanthane gum 3 g/l in the placebo and 1 g/l in the protein)
and obviously in the protein content. Protein drink contained also
trinatriumsitrate [to increase its pH to be equal with placebo (pH 7)].
The reason for the selection of a nonenergetic placebo drink instead of
isocaloric carbohydrate drink was because carbohydrates per se can
have also many effects on many of the studied variables (6).
The dietary intake was recorded in diaries for 3 days before the first
biopsy day at the start of the study, on the biopsy day, and on the day
thereafter (pre; 5 days overall), after 10.5 wk (mid; 4 days), and again
before the 21st-week biopsy (post 21 wk; 3 days before, and on the
biopsy day). The diaries were analyzed using the Micro Nutrica
nutrient-analysis software (version 3.11, Social Insurance Institution
of Finland). The subjects did not eat anything 1 h before and 0.5 h
after the experimental exercise workouts during the RT period. Food
restriction during these time periods was used to ascertain whether the
supplementation of whey, considered a fast-acting and high-quality
protein, has an additive effect where the normal meal ingestion is not
forbidden 23 h before and after each RE bout.

triceps brachii, subscapularis, and iliac crest) (10). Vastus lateralis

(VL) muscle thickness (at the middle of the VL muscle) was measured
by ultrasonography in a standardized supine position (Aloka SSD2000, Tokyo, Japan). The scanning head was coated with transmission
gel to provide acoustic contact without depressing the dermal surface.
The distance between the subcutaneous adipose tissue-muscle interface and intramuscular interface (i.e., aponeurosis) was defined as VL
muscle thickness. The ultrasonography (US) measurement site was
tattooed to ensure that the same site was used both before and after
training. Intraclass correlation coefficient for body weight was r
0.996, for fat percent was r 0.982, and for the VL muscle thickness
in US was r 0.914.

1723

mTOR AND MYOSTATIN AFTER EXERCISE WITH PROTEIN

ognized phosphorylated Akt on Ser473, mTOR on Ser2448, p70S6K on

Thr389, rpS6 on Ser235/236, 4E-BP1 on Thr37/46, and eEF2 on Thr56
(Cell Signaling Technology, Beverly, MA) and COOH-terminal myostatin protein (Chemicon/Millipore AB 3239) (38). The rabbit polyclonal antibody used was raised against a peptide residing in the
COOH terminus of myostatin corresponding to amino acids 349 364
of human myostatin and, therefore, being a similar antibody to those
used previously (14, 50).
All the antibodies were diluted 1:2,000 (except eEF2 on Thr56,
which was 1:3,000) in TBS-T containing 2.5% nonfat dry milk.
Membranes were then washed in TBS-T, incubated with secondary
antibody (horseradish peroxidase-conjugated anti-rabbit IgG; Cell
Signaling Technology) diluted 1:5,000 in TBS-T with 2.5% milk for
1 h followed by washing in TBS-T. Phosphorylated proteins were
visualized by ECL according to the manufacturers protocol (SuperSignal
West femto maximum sensitivity substrate, Pierce Biotechnology)
and quantified using a ChemiDoc XRS in combination with Quantity
One software (version 4.6.3. Bio-Rad Laboratories).
The membranes described above were incubated in Restore Western blot stripping buffer (Pierce Biotechnology) for 30 min and
reprobed with appropriate antibodies for detection of the total expression levels of Akt and rpS6 (rabbit monoclonal) (Cell Signaling
Technology) and p70S6K (Santa Cruz Biotechnology) by immunoblot
analysis as described above.

The blood samples were drawn from the antecubital vein before
and 0, 15, 30, and 60 min after the bout of RE using 21-gauge
disposable needles. Blood was centrifuged at 3,500 rpm in 4C for 10
min to separate serum and stored frozen at 80C until assayed.
Serum testosterone, sex hormone-binding globulin (SHBG), and insulin concentrations were analyzed by an immunometric chemiluminescence method with an Immulite 1000 (DPC, Los Angeles, CA).
The sensitivity of the assay for testosterone and coefficient of variation (CV) are 0.5 nmol/l and 5.7%, for SHBG 0.2 nmol/l and 2.4%,
and for insulin 2 mIU/l and 3.4%, respectively. Free testosterone was
calculated from total testosterone and SHBG concentrations (56). The
results are presented as uncorrected to plasma volume changes as
there were no differences between the protein and placebo group in
the decrease of the plasma volume during and after the bout of RE
(data not shown).
Immunohistochemistry
Muscle fiber cross-sectional area. Serial 8-m-thick transverse
sections were cut on a cryomicrotome (Leica CM 3000) at 24C.
Fiber type was classified by staining using myofibrillar ATPase
method according to the earlier study (31). Fiber sarcolemma was
visualized with an antibody against dystrophin (DYS2, Novocastra
Laboratories) using avidin-biotin peroxidase kit (Vectastain PK-4002,
Vector Laboratories) with diaminobenzidine (Abbott Laboratories) as
a chromogen. The measurements of fiber cross-sectional area (CSA)
comprised an average of 125 57 type I and 129 61 type II muscle
fibers. Stained cross sections were analyzed by Tema Image-Analysis
System (Scan Beam) using a microscope (Olympus BX 50) and color
video camera (Sanyo High Resolution CCD).
Immunohistochemical staining of rpS6 and mTOR. For immunohistochemical staining of rpS6 and mTOR, 8-m longitudinal and
cross sections before the RT period from resting state muscle of the
present subjects were fixed 15 min with 4% PFA-PBS, permeabilized
with 0.2% Triton-X for 10 min, and blocked 30 min with 3%
BSA-PBS and thereafter incubated with primary antibodies overnight
at 4C. Double immunolabeling was performed using a rabbit
monoclonal antibody against rpS6 or rabbit polyclonal antibodies
against phospho-rpS6 on Ser235/236 or phospho-mTOR on Ser2448
(Cell Signaling Technology; 1:40 in 1% BSA-PBS) with either mouse
monoclonal antibody against human slow myosin heavy chain
J Appl Physiol VOL

Statistical Analyses
All data are expressed as means SD, except where designated.
The data were analyzed by a repeated-measures general linear model
ANOVA. Any violations of the assumptions of sphericity were
explored and, if needed, corrected with a Greenhouse-Geisser or
Huynh-Feldt estimator. The Shapiro-Wilk test revealed that Western
blot data were not normally distributed, and therefore for the statistical
tests, all those values were log-transformed. Holm-Bonferroni post
hoc tests were performed to localize the effects. SPSS version 13.0 for
Windows was used for statistical analyses (SPSS, Chicago, IL). The
level of significance was set at P 0.05.
RESULTS

Daily Nutrient Intake

Nutrient intake did not differ between the protein and placebo conditions at weeks 0, 10.5, or 21 or in the averaged
values of those three time points (Table 2). The subjects
habitually consumed 1.48 0.35 g protein/kg body mass in the
protein group and 1.41 0.42 g/kg body mass in the placebo
group.
Anthropometry
Body mass increased significantly in the training groups
after 21 wk compared with the control group (Table 1).
However, at the 10.5-wk time point, the protein group already
showed an increase in body mass compared with the control
group (P 0.01), but the placebo group did not (P 0.56).
There was no change in the fat percent in any group. The
protein group increased VL thickness after both 10.5 wk (P
0.05) and 21 wk (P 0.01) of RT, whereas the placebo group
did so after 21 wk of RT (P 0.05) but not after 10.5 wk (P
0.16) (Table 1). As in the case of body mass, only the protein
group increased its muscle thickness significantly after both

Table 2. Dietary intake: averaged energy and macronutrient

intakes in the protein and placebo groups throughout
the 21-wk training period (week 0, week 10.5, and week 21)
Variable

Protein Group

Placebo Group

P Value

E, 1,000 kJ
E, kJ/kg body mass
Protein, g/kg body mass
CHO, g/kg body mass
Fat, g/kg body mass

10.51.5
14023
1.50.3
3.90.7
1.20.3

10.23.0
13534
1.40.4
3.81.0
1.20.4

0.73
0.57
0.57
0.63
0.91

Values are means SD. E, energy; CHO, carbohydrate. P value is statistical

difference between the protein and placebo groups.

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Blood analysis

(MyHC I) [Developmental Studies Hybridoma Bank (DSHB), Iowa

City, IA: A4.951] diluted to 1:150 (18), or mouse monoclonal antibody against caveolin-3 (1:100) (BD Transduction Laboratories) to
visualize muscle sarcolemma. Nuclei were stained by Hoechst 33258
(Sigma, St. Louis, MO). Secondary antibodies used were goat antirabbit Alexa Fluor 488 or 546 and goat anti-mouse Alexa Fluor 546
or 488 (Molecular Probes, Eugene, OR). Negative controls were done
by omitting the primary or secondary antibody. An Olympus BX-50F
light microscope (Olympus Optical, Tokyo, Japan) with Olympus
color CCD camera (Colorview III, Olympus Optical) and Analysis
software (version 5.0, Soft-Imaging System, Munster, Germany) were
used for the imaging and analysis. Two samples were also further
analyzed using Olympus IX81 confocal microscope with imaging
system and software (Olympus Fluoview 1.6a) (29).

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10.5 and 21 wk compared with the control group (P 0.05),

while the placebo group only approached a trend after 21 wk
(P 0.12).
Acute RE Bout at Week 0 and Training Volumes
The total volume of the work performed in the RE bout
(loads sets repetitions) was similar in the placebo (88.7
15.4 kg/kg body weight) and protein group (91.5 15.7 kg/kg
body wt) at week 0. Average training volumes (loads sets
repetitions) for the leg extensor muscles (leg press and knee
extension) were calculated for weeks 17, 8 14, and 1521.
No significant difference was found between the protein and
placebo groups (data not shown).
Western Blotting Results
ANOVA revealed a time effect in both the protein and
placebo groups for the phosphorylation of p70S6K on Thr389,
rpS6 on Ser235/236 and dephosphorylation of Akt on Ser473
(P 0.05) (Fig. 2). In the protein group only, a significant time

effect was also seen for the phosphorylation of mTOR at

Ser2448 and in the placebo group for the dephosphorylation of
4E-BP1 on Thr37/46 (Fig. 3A). In the control group, no time
effect was seen in any of the studied proteins. The post hoc test
revealed that the phosphorylation of p70S6K and rpS6 was
increased in the protein and placebo groups 1 h after the RE
bout. The change in the phosphorylation of p70S6K was significantly greater with the protein ingestion compared with the
placebo group (P 0.001). The phosphorylation of mTOR
was increased only in the protein group, the increase persisting
at all time points (post 1 h, post 48 h, and post 21 wk) (P
0.05). There was a strong decreasing trend in the phosphorylation of 4E-BP1 in the placebo group at post 1 h (P 0.06).
The decrease was significant compared with the controls (P
0.05). Of the individual changes of phosphorylated 4E-BP1
from pre to post 1 h, seven of nine subjects in the placebo
group showed a decrease (average 43%) and two of nine
subjects showed an increase (8%), whereas six of nine in the
protein group showed an increase (112%) and three of nine

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Fig. 2. Phosphorylation of p70S6K on Thr389 (p-p70; A), mammalian target of rapamycin (mTOR) on Ser2448 (p-mTOR; B), Akt on Ser473 (p-Akt; C), and
ribosomal protein S6 (rpS6) on Ser235/236 (p-rps6; D). Immunoblot of 1 individual is shown on top of AD, as well as total (tot) forms of p70S6K, Akt, and rpS6.
p70S6K blot shows also that phosphorylation of other isoform of S6K1, p85S6K on Thr412 (p-p85), followed the same trend as p70S6K. Values are arbitrary units
(means SE). *P 0.05 vs. pre. # P 0.05, difference between protein and placebo. -TE-, P 0.05 time effect of ANOVA. n 9 for protein and placebo
groups and n 6 for control group. Five control subjects served only as pre and post-21 wk subjects and thus biopsies were obtained from 6/11 control subjects
from all the 4 time points. See text for description of time points (pre, post 1 h, post 48 h, post 21 wk).
J Appl Physiol VOL

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mTOR AND MYOSTATIN AFTER EXERCISE WITH PROTEIN

1725

a decrease (32%) (between-group difference in the change:

P 0.03). The phosphorylation of Akt decreased after 21
wk of RT in both training groups (P 0.05). There was,
however, no change in the phospho-eEF2 (p-eEF2) or total
protein expression of p70S6K, Akt, and rpS6. The myostatin
COOH-terminal protein was decreased at post 1 h in the
placebo group (P 0.02) but not in the protein or control
groups (Fig. 3C).
There were no significant correlations between the REinduced change in the protein kinases or in the myostatin
protein with corresponding changes in VL fiber size or
muscle thickness (by ultrasonography) or VL CSA [by MRI
previously (22)].
Immunohistochemistry
The CSA of type I and II fiber types increased significantly
and similarly after 21 wk of RT in both the protein and placebo
groups (P 0.01) and also significantly (P 0.05) compared
with the control group (Table 1).
Both phosphorylated mTOR at Ser2448 and rpS6 at Ser235/236
as well as total rpS6 were primarily localized close to the
nuclei and sarcolemma, outside the area where contractile
proteins are located (Fig. 4). The signal for these proteins
emanated in large part from inside the muscle fibers but also to
some extent from outside the sarcolemma. No clear cell-type
difference was seen.
J Appl Physiol VOL

Serum Testosterone and Insulin

Compared with the control group, serum total testosterone
concentration elevated significantly during the bout of RE only
in the placebo group (P 0.04). No differences between the
groups were observed in free testosterone or in serum insulin
(not shown).
DISCUSSION

The main findings of the present study were that ingestion of

whey proteins before and after a bout of RE rapidly increased
the phosphorylation of p70S6K and also prevented the decrease
in the phosphorylation of 4E-BP1. Moreover, the RE bout
acutely decreased the active form of myostatin protein, but
only when protein was not supplemented. The phosphorylation of mTOR remained increased after the RE bout from
post 1 h to post 48 h and also after 21 wk of RT when the
protein was ingested. However, RT itself decreased Akt
phosphorylation. The control group results ensured that the
results were not due to repeated biopsy, diurnal rhythm, or
time of year (47, 57).
Whey protein rapidly increases mTOR signaling. In the
present study, whey protein ingestion rapidly increased the
phosphorylation of p70S6K on Thr389 at 1 h post-RE, showing
the activation of the TORC1 complex, including mTOR and its
regulatory proteins (25, 54, 58). Probably also the activation of
p70S6K was increased especially in the protein group because

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Fig. 3. Phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) on Thr37/46 (p-4E-BP1; A), eukaryotic elongation factor 2 (eEF2) on Thr56
(p-eEF2; B), and myostatin 26 kDa (MSTN) COOH-terminal protein (C). Ponceau S (Pon S) staining shows equal protein loading. Immunoblot of 1 individual
is shown at right. See text for further explanation. There was a between-group difference between the protein and placebo group and also between the placebo
and control group in the change from pre to post 1 h (*P 0.05).

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mTOR AND MYOSTATIN AFTER EXERCISE WITH PROTEIN

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Fig. 4. Confocal microscopy images of localization of total (A) and phosphorylated rpS6 (B) and phopshorylated mTOR (C) in muscle cross sections. Nuclei
were stained with Hoechst 33258, sarcolemma with antibody against caveolin-3, and myofibrillar area with an antibody against myosin heavy chain I (MyHC I). All
images are representative of 2 subjects visualized with confocal microscope and of a total of 5 subjects with epifluorescence microscope. Phosphorylated
mTOR at Ser2448, rpS6 at Ser235/236, and total rpS6 were primarily localized close to the nuclei and sarcolemma (caveolin-3), outside the area where
contractile proteins are located (MyHC). The images were taken with the settings in which the secondary antibody (not shown) only gave minimal signal.
Scale bars are 50 m.

phosphorylation from this site is the chief event in the activation of p70S6K (44) and since there was also a tendency for
larger phosphorylation of one of its downstream target rpS6
(protein vs. placebo, P 0.11) and the increase in mTOR
phosphorylation at Ser2448 was observed only in the protein
group. This site of mTOR has been shown to be phosphorylated by p70S6K (5). The present results agree with those of a
J Appl Physiol VOL

recent study showing that protein intake together with carbohydrate before, during, and 1 h after a RE bout increased
phosphorylation of p70S6K at post 0 4 h compared with
carbohydrate only (30). Interestingly, the phosphorylation of
the second isoform of S6K1, p85S6K, clearly followed the same
pattern in the present study as that of p70S6K (see Fig. 2,
protein blot image).

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mTOR AND MYOSTATIN AFTER EXERCISE WITH PROTEIN

J Appl Physiol VOL

ingestion were probably due to 1) a very long-lasting 4- to

5 day effect from the last RE workout, and/or 2) a more
permanent RT-induced change in the basal state of phosphorylation of these proteins. These changes may affect the level of
the protein synthesis of the resting muscle (59) and/or the
amount of the signal needed to activate the cascade through
these pathways. It seems obvious that the change in the state of
phosphorylation of at least some of the mTOR pathway protein
kinases can be rather chronic and less transient due to, e.g.,
aging or exercise (16, 34, 59) and, at least partially, also due to
protein ingestion.
Muscle Hypertrophy after RT, and Myostatin
The earlier MRI results obtained from the present study
design showed a larger increase in VL muscle hypertrophy
with whey protein ingestion (22). The present study also
showed a somewhat faster increase in VL muscle thickness and
body mass with protein ingestion. However, a larger proteininduced increase after the full 21-wk RT in fiber size was not
observed. Recently, in older men, there were no positive
effects of 10 g of casein protein ingested also immediately
before and after a RE workout (55). It is possible that larger
effects on muscle size would have been seen in the present
study using subjects with a higher level of RT background or
whose habitual ingestion of protein is smaller than 1.4 1.5
g/body weight (49). Therefore, while the positive effects of the
protein or amino acid ingestion on muscle hypertrophy signaling can often be clear when studied acutely after each exercise,
especially when the study was performed in a fasting state, the
long-term positive effects may not be as robust with normal
daily high protein consumption.
The present study is the first in humans showing that
myostatin peptide concentration, thought to be the active form
of myostatin, can follow the decreased mRNA transcript of
myostatin after a RE bout. Interestingly, protein ingestion
seemed to prevent the decrease in myostatin after the RE bout.
This may have hindered larger hypertrophy in the protein
group, which could have been predicted from the mTOR
pathway results because myostatin inhibits muscle growth (14,
37, 46). The hindering effect of protein ingestion for downregulating myostatin expression after the bout of RE supports
our earlier mRNA-level findings among younger (22) and older
men (21), suggesting that the change in myostatin was transcriptionally regulated. Indeed, with the present subjects the
myostatin mRNA and protein level changes from pre to post
1 h also correlated positively (r 0.66, P 0.007). It is
assumed that the detected 26-kDa myostatin is a glycosylated
tightly bound dimer of a 110-amino acid COOH-terminal
peptide of myostatin and/or that the monomer of myostatin is
strongly bound by some other protein (14, 50). Recently, a
myostatin propeptide of size 28 kDa and myostatin protein
complexes of size 50 kDa were not changed 24 h after a bout
of RE in humans (27).
Whey protein also seemed to prevent the elevation in serum
total testosterone seen in the placebo group after the bout of
RE, thereby supporting the previous results of protein ingestion
(4, 20, 23). The testosterone response may be due to a decrease
in the synthesis/secretion of testosterone and/or an increase in
metabolic clearance. As was the case with myostatin protein
concentration, the effect of protein ingestion on testosterone

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The most important component responsible for the increased

phosphorylation of p70S6K with the whey protein ingestion is
probably its large content of branched-chain amino acids that
can elicit a similar p70S6K response in the context of a bout of
RE as observed in the present study (3, 26). S6K1/p70S6K has
been shown in animal and cell models to be especially important in muscle hypertrophy (2, 40, 48). Moreover, in humans an
acute increase in the phosphorylation of p70S6K after a bout of
RE has also correlated with a long-term loading-induced
increase in fiber size as well as fat-free mass in trained
humans (51), and with a RE-induced myofibrillar protein
synthesis (33).
Whey protein intake alone prevented a RE-induced decrease
in the phosphorylation of eukaryotic initiation factor 4E
(eIF4E) binding protein (4E-BP1). This supports a recent
finding in humans with a slightly different time scale and
nutrients (30). An RE bout per se has also previously decreased
the phosphorylation of 4E-BP1 shortly after exercise when
protein or amino acids are not supplemented (8, 9, 30, 36).
Prevention of the dephosphorylation of 4E-BP1 after a bout of
RE by ingestion of whey proteins probably prevents association of the 4E-BP1 with eIF4E (43). This allows a larger
increase in protein synthesis (12, 28, 35, 43), a phenomenon
previously observed after the ingestion of whey proteins (52,
53). It can be speculated that whey protein alone can affect
mTOR signaling TORC1 dependently and independently as the
phosphorylation of 4E-BP1 from this site (Thr37/46) may occur
independently of TORC1 unlike the phosphorylation of p70S6K
(25, 54, 58). Our results suggest that these effects occurred
independently of blood insulin or the phosphorylation of Akt at
Ser473.
The phosphorylated mTOR is localized mainly close to the
sarcolemmal membrane as has been shown in rodents (17, 41)
while rpS6 and phospho-rpS6 (p-rpS6) were mostly located
very close to the nuclei as has been found earlier with p-rpS6
(30). Magnification showed that rpS6 usually surrounded the
nuclei, which is theoretically optimal for efficient protein
synthesis.
Protein and training affects the phosphorylation of mTOR
and Akt, respectively. In contrast to the rapid changes, the RE
bout itself did not seem to have a consistent effect on the
phosphorylation of the mTOR pathway proteins at 48 h
post-RE or after a longer term RT, supporting recent human
studies investigating time points 48 h (36) and 24 h post-RE (8,
36), and 4 days after a RT period (34, 59). The only
long-lasting effect of protein ingestion was the increased phosphorylation of mTOR, which remained increased in the protein
group from 1 to 48 h after the RE bout and also after 21 wk of
RT. Surprisingly, the phosphorylation of Akt decreased 0.5fold in both training groups after 21 wk of RT. In contrast, in
previous studies 8 10 wk of RT increased the phopshorylation
of Akt at Ser473 (34, 59). This different response may, owing to
possible complexity of the temporal pattern of the Akt phosphorylation state, depend on the timing of the biopsies or
additionally on the length or type of the training period or
possibly the nutritional state.
The time point 4 5 days post-RE when the biopsy was taken
represents roughly the time point when the next RE workout
would have taken place. Therefore, the pre and post-21 wk
biopsies are comparable. The observed more chronic Akt
and mTOR responses to the RT with or without protein

1727

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mTOR AND MYOSTATIN AFTER EXERCISE WITH PROTEIN

was, however, small. Thus the physiological significance of

these responses warrants future studies.
In conclusion, resistance exercise rapidly increases mTOR
signaling, and whey protein increases and prolongs the mTOR
signaling response to exercise and training. Active form of
myostatin peptide rapidly decreases after a RE bout when
protein nutrition is not supplemented.

15.
16.
17.

ACKNOWLEDGMENTS
We thank Hanna Salmijarvi, Marja Katajavuori, Liisa Kiviluoto, Marko
Haverinen, Paavo Rahkila, Tuovi Nykanen, Risto Puurtinen, and Aila Ollikainen for help in the data collection and analysis. We also thank the very
dedicated group of subjects who made this project possible. The monoclonal
antibody A4.951 for MyHC I, developed by Dr. H. M. Blau (18), was obtained
from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and
maintained by the Univ. of Iowa, Dept. of Biological Science, Iowa City, IA
52242.

18.

19.

20.

GRANTS
The Finnish Ministry of Education and the Ellen and Artturi Nyyssonen
Foundation (Juha Hulmi personal grant) supported this research.

21.

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