Amino Acids (2009) 37:297–308
DOI 10.1007/s00726-008-0150-6
ORIGINAL ARTICLE
Acute and long-term effects of resistance exercise with or without
protein ingestion on muscle hypertrophy and gene expression
Juha J. Hulmi Æ Vuokko Kovanen Æ Harri Selänne Æ William J. Kraemer Æ
Keijo Häkkinen Æ Antti A. Mero
Received: 12 March 2008 / Accepted: 2 June 2008 / Published online: 27 July 2008
Ó Springer-Verlag 2008
Abstract The effects of timed ingestion of high-quality
protein before and after resistance exercise are not well
known. In this study, young men were randomized to
protein (n = 11), placebo (n = 10) and control (n = 10)
groups. Muscle cross-sectional area by MRI and muscle
forces were analyzed before and after 21 weeks of either
heavy resistance training (RT) or control period. Muscle
biopsies were taken before, and 1 and 48 h after 5 9 10
repetition leg press exercise (RE) as well as 21 weeks after
RT. Protein (15 g of whey both before and after exercise)
or non-energetic placebo were provided to subjects in the
context of both single RE bout (acute responses) as well as
each RE workout twice a week throughout the 21-weekRT. Protein intake increased (P B 0.05) RT-induced
muscle cross-sectional area enlargement and cell-cycle
kinase cdk2 mRNA expression in the vastus lateralis
muscle suggesting higher proliferating cell activation
J. J. Hulmi (&) K. Häkkinen A. A. Mero
Department of Biology of Physical Activity,
University of Jyväskylä, P.O. Box 35,
40014 Jyväskylä, Finland
e-mail: juha.hulmi@sport.jyu.fi
V. Kovanen
Department of Health Sciences, University of Jyväskylä,
Jyväskylä, Finland
V. Kovanen
Finnish Centre for Interdisciplinary Gerontology (FCIG),
University of Jyväskylä, Jyväskylä, Finland
H. Selänne
LIKES Research Center, Jyväskylä, Finland
W. J. Kraemer
Human Performance Laboratory, Department of Kinesiology,
University of Connecticut, Storrs, CT, USA
response with protein supplementation. Moreover, protein
intake seemed to prevent 1 h post-RE decrease in myostatin and myogenin mRNA expression but did not affect
activin receptor IIb, p21, FLRG, MAFbx or MyoD
expression. In conclusion, protein intake close to resistance
exercise workout may alter mRNA expression in a manner
advantageous for muscle hypertrophy.
Keywords cdk2 Myostatin Activin receptor IIb
Skeletal muscle Whey
Introduction
One of the hallmarks of resistance training is an increase in
muscle cross-sectional area and improved maximal force
production, especially in previously untrained subjects (for a
comprehensive review, see Wernbom et al. 2007). In addition to resistance training, protein ingestion may play an
important role as a regulator of muscle mass and recovery
from exercise. The timing of the nutrient intake seems to be
also of importance. Nutrient intake before and/or immediately after a resistance exercise (RE) session may be more
beneficial in terms of muscle protein anabolism than nutrient
ingestion at other times such as in the morning and late
evening at least 5 h before or after the workout (Cribb and
Hayes 2006) or 2 h after the workout (Esmarck et al. 2001).
Especially whey/milk protein supplementation may be
advantageous for gaining muscle size (Andersen et al. 2005;
Hartman et al. 2007) and improving muscle protein balance
after a RE bout (Tipton et al. 2007; Wilkinson et al. 2007).
Fast recovery from RE-induced myofibrillar disruption
(Gibala et al. 1995) is also important. Many molecular
factors are important in the recovery process from exercise
as well as in the regulation of muscle hypertrophy per se.
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Of these, myostatin, a well-known negative regulator of
muscle size (McPherron et al. 1997) and proteins downstream to myostatin such as myogenic regulatory factors
and cell-cycle kinases as well as their inhibitors all have
been shown to be crucial (Charge and Rudnicki 2004;
Kuang et al. 2006; McCroskery et al. 2003; Rios et al.
2002; Wagner 2005). A single heavy RE bout provides a
high loading stimulus to skeletal muscle, from which
complete recovery takes usually at least 2–4 days, while
also affecting myostatin, myogenic regulatory factors, and
other cell-cycle related factors (Hulmi et al. 2007; Kim
et al. 2007; Mascher et al. 2008). However, it is not known
whether high-quality protein such as whey (Ha and Zemel
2003) intake close to a resistance exercise modifies exercise-induced gene expression responses both acutely and
after some months of systematic training.
In the present study we investigated whether supplementation of high-quality whey protein has additive effects
compared to normal dietary intakes only, when ingested in
conjunction with RE. The rational for the timed addition of
a high-quality protein such as whey, is the possibility that it
could improve the muscle protein synthesis response to
exercise without interfering with the response to normal
food. Indeed, recent results in an acute design by PaddonJones et al. (2005) suggest that an essential amino acid and
carbohydrate supplement does not acutely interfere with
the normal muscle protein synthesis response to a mixed
meal. Whey is considered to be a high-quality protein
source containing large amounts of essential amino acids,
important in the protein synthesis (Borsheim et al. 2002),
and also fast acting compared to many other protein
sources such as casein (Boirie et al. 1997). It is thus possible that addition of whey, when used chronically in
conjunction with RE may be more anabolic for skeletal
muscle than ingesting only normal mixed meals throughout
the day. Therefore, the purpose of this investigation was to
examine long-term adaptations from resistance training in
terms of whole-muscle size, force production, and muscle
hypertrophy related gene expression that may occur when
high-quality protein is added to a ‘‘normal diet’’ both
immediately before and after each resistance exercise
session. To the best of our knowledge, this is also the first
study combining both acute and long-term gene expression
responses with nutrition and cross-sectional area and
maximal force of trained muscles. We hypothesized that
whey protein intake immediately before and after a resistance exercise bout has both acute and long-term effects on
possible resistance exercise-induced myostatin and cellcycle related gene expression responses and this timed
protein ingestion also increases whole-muscle hypertrophy
response from 21 weeks of resistance training.
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Materials and methods
Subjects
The subjects were randomly assigned after control testing
sessions to either the whey protein group (n = 13), placebo group (n = 14) or to control group (n = 11). There
was no RT in the controls but they continued their
habitual activity such as jogging, swimming or ballgames. The number of subjects who completed the study
was 31. The average age in the three groups were as
follows: protein: 25.2 ± 5.2 years (n = 11), placebo:
27.2 ± 3.0 years (n = 10) and control: 24.9 ± 2.7 years
(n = 10) (Table 1).
All the subjects were examined by a physician and none of
them had medical problems that would confound the results
of this investigation. All subjects were also free from neuromuscular dysfunction and thus were cleared to perform
heavy RT. None of the subjects had prior heavy RT experience. Prior to the investigation, each subject was informed
about the experimental design and the associated risks and
discomforts that may occur. Each then signed an informed
consent document to participate in the study, which was
approved by the local Ethics Committee of the University
and was done in accordance with the Declaration of
Helsinki.
Design
This investigation examined long-term adaptations of
adding high-quality protein to a ‘‘normal diet’’ (including
no nutritional supplements) to increase its bioactivity.
Because both acute and long-term molecular responses of
resistance exercise without and especially with protein
have not been carefully studied, several different muscle
hypertrophy related gene transcript levels were examined
both acutely after a single RE bout but also after longterm RT consisting of more than 40 RE workouts with
either protein or placebo supplementation. The study
design included a control group and all measurements
were performed always at the same time to exclude the
effects of biopsy sampling or effects of time of a year or
daily variations (Sedliak et al. 2007; Vissing et al. 2005).
The total duration of the present study was 23 weeks from
which the first 2 weeks was a control period in which no
experimental RT was carried out but the subjects maintained their normal recreational activities. All of the
measurements (muscle force, muscle cross-sectional area,
anthropometry and muscle biopsies) were preceded by at
least 3 days of rest from physical activity. The experimental design is depicted in Fig. 1.
Short and long-term effects of exercise and protein
299
Table 1 Anthropometry. P valuepre designates Holm–Bonferroni corrected P values compared to baseline
Variable
Height (cm)
Body mass (kg)
Fat (%)
Group
2-week-control period
21-week-resistance training
Control
Mean ± SD
10.5 weeks
Mean ± SD
Baseline
Mean ± SD
21 weeks
Mean ± SD
P valuep
P valueDgroup
DChange
Mean ± SD
PROT
182.2 ± 6.2
PLAC
181.0 ± 5.8
CONT
PROT
182.6 ± 4.8
76.1 ± 7.6
76.5 ± 7.3
79.5 ± 8.7
79.7 ± 8.7
3.2 ± 2.0
0.003*
0.001*
PLAC
74.7 ± 8.5
74.8 ± 8.4
76.5 ± 8.7
77.3 ± 8.9
2.6 ± 2.1
0.008*
0.01*
CONT
75.5 ± 8.1
75.7 ± 8.3
76.7 ± 8.6
75.9 ± 8.8
0.2 ± 1.5
0.65
PROT
17.0 ± 3.7
17.1 ± 3.8
17.5 ± 4.0
17.4 ± 4.2
0.3 ± 1.5
1
0.85
PLAC
16.9 ± 4.4
16.6 ± 4.4
16.5 ± 4.7
16.6 ± 4.0
0.1 ± 0.9
1
0.55
CONT
17.3 ± 3.8
16.7 ± 3.4
17.7 ± 4.3
17.1 ± 4.5
0.4 ± 1.5
0.86
P valueDgroup designates the difference between the training groups and control group in the change between baseline and post 21-weeks values.
DChange is the absolute difference from baseline to 21 weeks
* Significant (P B 0.05) P value
Fig. 1 Experimental design. B Vastus lateralis muscle biopsy, T
testing, RE resistance exercise bout (5 9 10 RM leg press), RT heavy
and progressive resistance training and D protein (15 g of whey
protein) or placebo (no energy) drink. MRI was measured in T2 and
T4 and muscle forces and anthropometry in T1–4
Experimental resistance training
During the 21-week-RT period, total-body heavy RE
workouts were carried out twice a week. A minimum of
2 days of rest was required between the two sessions each
week. All training sessions were supervised by experienced
trainers making sure that proper techniques and progression
was used in each exercise (Kraemer et al. 2002). The
training program was especially focused on knee extensors
since the analysis of muscle cross-sectional area and
muscle biopsies were obtained from the knee extensor
muscle (i.e., vastus lateralis). The following exercises were
used in each training session: two exercises for the leg
extensor muscles, 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 of the body: chest and
shoulders, upper back, trunk extensors and flexors, upper
arms, ankle extensors, and hip abductors and adductors.
Both leg press and knee extension exercises were thought
to activate especially the VL muscle and it is the muscle in
which the biopsy was taken. These exercises, previously
utilized by our laboratory, produce somewhat larger
hypertrophy responses in the VL and VM muscles compared to the other two quadriceps muscles during a
comparable 21-week-RT program (Häkkinen et al. 2001).
The first two exercises in each workout were always the
leg press and bench press. Recovery between the sets was
2–3 min. RT was performed with progressive training
loads of 40–85% of the subject’s 1 RM in a periodized
training program. The number of sets of each exercise
during RE workout increased (from 2–3 to 3–5) and the
number of repetitions in each set decreased (15–20 to 5–6)
during the 21-week-RT period. The loads were individually
determined throughout the RT period.
Nutritional provision during resistance training
Either 15 g of whey isolate protein (Protarmor 907 LSI,
Armor Proteins, Brittany, France, with minimal lactose and
fat) dissolved in 250 ml of water or an equivalent amount
of non-energetic placebo was ingested immediately before
and after each bout of RE in the gym (Fig. 1). Whey is the
most popular protein supplement by those resistance
training and it effectively increases net muscle total protein
synthesis and balance when consumed before or after RE
bout at about similar doses used in this study (Tipton et al.
2007). The essential amino-acid composition of the protein
drink (15 g) was as follows: histidine (0.2 g), isoleucine
(1.0), leucine (1.7), lycine (1.4), methionine (0.4), phenylalanine (0.5), threonine (1.0), tryptophan (0.2) and valine
(0.8). The drinks were provided for the subjects in a double-blind fashion. The drinks were made in our own
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laboratory by the personnel who coded the drinks for the
training supervisors. Drinks contained exotic fruit, trinatriumsitrate, acesulfame-K, xanthane gum and betacarotene
for flavor, viscosity and color. The protein and placebo
supplements looked and tasted as identical as possible.
Dietary intake of the subjects was registered with dietary
diaries for 3 days before the first biopsy day at the start of
the study, on the biopsy day, and the day thereafter (pre,
5 days overall), after 10.5 weeks (mid, 4 days) and again
before the 21st week biopsy (post 21 weeks, 3 days before,
and on the biopsy day). All of the diaries were analyzed
using the Micro Nutrica nutrient-analysis software version
3.11 (The Social Insurance Institution of Finland). The
subjects in either protein or placebo group did not eat
anything 60 min before and 30 min after experimental
exercise workouts during RT period. Food restriction during only these time periods was utilized to ascertain
whether addition of a whey, considered fast acting and
high-quality protein, has an additive effect even if the
normal meal ingestion is not forbidden *2–3 h before and
after each RE bout.
Muscle cross-sectional area and anthropometry
The muscle cross-sectional area (CSA) of the right quadriceps femoris muscle was determined before and after the
21-week-period from both RT and control subjects using
magnetic resonance imaging (MRI) (GE Signa Exite HD
1.5 T) at a local MRI center (Keski-Suomen Magneettikuvaus). During the measurement, the subjects’ legs
were kept parallel and strapped with a belt and a special cast
designed to standardize the measurement as well as possible.
Four axial-plane MRI scans were taken. The first image was
taken 4 cm above the midway between the patella and
greater trochanter (image1) and thereafter the next three
scans were taken at 2, 4 and 6 cm towards patella (images2–4). All the MRI images were analyzed by the same
experienced researcher with OsiriX (version 2.7.5) software.
After an overnight fasting, body mass (kg) and fat percentage were measured. Body fat was measured with
skinfolds (biceps and triceps brachii, subscapular and iliac
crest) (Durnin and Womersley 1974) by the same research
assistant each time. Pearson Product correlations from
control measurements spaced 2 weeks apart (n = 8 for
MRI and n = 38 for skinfolds) showed high reproducibility for the measurement of quadriceps femoris CSA
(r [ 0.96) and for body fat percentage (r = 0.97).
Maximal force
Maximal isometric force of the bilateral leg extensor
muscles was measured on an electromechanical dynamometer with knee angle of 107° and hip angle of 110°
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(Häkkinen et al. 2001; Hulmi et al. 2007). Unilateral isometric knee extension and flexion as well as bilateral bench
press tests were performed with a David 200 system (David
Fitness and Medical, Finland) (Häkkinen et al. 1998). The
knee and elbow angles were 90°. A minimum of three trials
were completed for each subject and the best performance
trial was used in the subsequent statistical analysis. The
force signal of the isometric measurements was recorded
and analyzed with a Signal software version 2.15 (Cambridge Electronic Design Ltd., Cambridge, UK). A David
210 dynamometer (David Fitness and Medical, Finland)
was used to measure maximal bilateral concentric force
production for leg extensors (hip and knee extensors).
Separate trials were performed for concentric 1 repetition
maximum (RM) testing. After each repetition, the load was
increased until the subject was unable to extend his legs to
the full-extended *180° knee angle position. The highest
acceptable load was determined as the 1 RM. The subjects
were carefully familiarized with the test procedures and
had several warm-up contractions in all devices. Intra-class
correlation coefficients with the present subjects (n = 31)
from the control to baseline measures was 0.97 for bilateral
isometric leg extension force, 0.98 for both 1 RM leg
extension and isometric bench press and 0.90 for both knee
extension and flexion.
Heavy RE protocol and nutritional supplementation
when studying the acute effects of protein and RE bout
The heavy RE bout was carried out using the bilateral leg
press machine (David 210) with similar protocols as
described in earlier studies (Häkkinen et al. 2001; Hulmi
et al. 2007, 2008) and the total number of sets for the leg
press was five (each with 10 repetition maximums).
Recovery time between sets was 2 min. The loads were
adjusted during the course of the RE bout due to fatigue so
that each subject would be able to perform ten repetitions
at each set. If the load was too heavy, the subject was
assisted slightly during the last repetitions of the set.
Maximal isometric force was measured bilaterally before
and after each set with an electromechanical dynamometer
with a knee angle of 107° (Hulmi et al. 2007, 2008).
The details of the nutrition for this acute part of the
study were exactly the same as in our earlier study with
older men investigating acute gene expression responses to
a single RE bout (Hulmi et al. 2008). The subjects fasted
for 3 h before the first biopsy. Either a 250 ml of whey
protein isolate or an equivalent amount of placebo was
ingested immediately before and after the bout of RE (15 g
of protein before and after the RE bout). The drinks were
provided to the subjects in a double-blind fashion. Details
of the drinks and dietary diaries were explained above in
this article.
Short and long-term effects of exercise and protein
Muscle biopsies
Muscle biopsies were obtained 0.5 h before and 1 and 48 h
after the RE session before RT as well as 4–5 days after the
last RE workout from 21 weeks of RT (Fig. 1). The 1 h
post-biopsy time point was selected to represent fast
responses of RE bout and the 48 h post-time point the more
delayed responses. We wanted to minimize the effects of
the last exercise workout and the protein ingestion on the
post-training biopsy. Therefore, the biopsy after RT was
taken 4–5 days after the last exercise workout. Biopsies
were taken from the VL muscle with a 5-mm Bergström
biopsy needle, midway between the patella and greater
trochanter. The pre-RE biopsy and the 48 h post-RE
biopsies were taken from the right leg. Avoiding any
residual effects of the pre-biopsy, 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. The 21-weekbiopsy was taken from the same leg as the baseline biopsy
(right). The muscle sample was cleaned of any visible
connective and adipose tissue as well as blood. It was then
immediately frozen in liquid nitrogen and stored at -80°C
for future mRNA analysis.
Analysis of muscle messenger RNA
Total RNA isolation, reverse transcription and cDNA
synthesis. Homogenization of the muscle samples were
done with FastPrep (Bio101 Systems, USA) tubes and total
RNA was extracted using the Trizol-reagent (Invitrogen,
Carlsbad, CA, USA). An OD260/OD280 ratio of 1.8–2.0 and
gel electrophoresis showed that our extraction yielded
DNA-free and un-degraded RNA, respectively. A total of
3 lg of total RNA was reverse transcribed to synthesize
cDNA according to the manufacturer’s instructions using
High Capacity cDNA Archive Kit (Applied Biosystems,
Foster City, CA, USA).
Real-time RT-PCR. The mRNA expression levels were
quantified with a real-time reverse transcriptase-PCR
(RT-PCR) assay using an Abi 7300 Real-Time PCR
System (Applied Biosystems, Foster City, CA, USA).
The probes and primers used were pre-designed transcripts validated by Applied Biosystems bioinformatics
design pipelines. The gene bank accession numbers and
Applied Biosystems assay IDs, respectively were: NM
005259 and Hs00193363_m1 (myostatin), NM 001106
and Hs00609603_m1 (activin receptor IIb), NM 005860
and Hs00610505_m1 (follistatin related gene protein:
FLRG), NM 002478 and Hs00159528_m1 (MyoD), NM
002479 and Hs00231167_m1 (myogenin), NM_078467.1
and Hs00355782_m1 (p21), NM_052827.1 and
Hs00608082_m1 (cdk2), NM002046, Hs99999905_m1
301
(GAPDH), NM_148177.1 and NM_058229.2 and
Hs00369714_m1 (Muscle Atrophy F-Box: MAFbx/atrogin-1) (Hulmi et al. 2007, 2008; Mascher et al. 2008).
Each sample was analyzed in triplicates. PCR cycle
parameters used were for all genes: 50°C for 2 min, +95°C
for 10 min, 37–45 (depending on the mRNA analyzed)
cycles at 95°C for 15 s, and 60°C for 1 min. GAPDH
mRNA was used as an endogenous control because it was
shown to be rather stable and better as a housekeeping gene
than 18sRNA in our previous study (Hulmi et al. 2007,
2008). Moreover, in the present study, GAPDH mRNA and
total RNA (lg/mg wet muscle) were stable across all data
points in both protein and placebo groups (P [ 0.17). Gene
transcript results were calculated according to the Liu and
Saint (2001) mathematical model (Liu and Saint 2002).
SigmaPlot (version 9.0, Systat Software inc., Richmond,
CA, USA) was used as a curve fitting software needed in
the method. The intra-assay CV%s for the triplicate-samples in the PCR runs were as follows: GAPDH (5.8%),
myostatin (9.3%), FLRG (14.3%), activin receptor IIb
(7.9%), p21 (9.1%), cdk2 (8.9%), myogenin (7.6%), MyoD
(10.6%), MAFbx (9.3%).
Statistical analyses
All data are expressed as mean ± SD, except where designated. The data were analyzed by a two-factor repeated
measures General Linear Model (GLM) ANOVA. Any
violations of the assumptions of sphericity were explored
and, if needed, corrected with a Greenhouse-Geisser or
Huynh-Feldt estimator. In muscle cross-sectional area
measurements there were only two levels in the sample
time factor and therefore dependent t tests were used for
their analyses. Shapiro–Wilk test revealed that mRNA data
were not normally distributed and therefore for the statistical tests, all the mRNA values were log-transformed.
Holm–Bonferroni post hoc tests were performed to localize
the effects. All the analyses were performed by means of
SPSS 14.0. The level of significance was set at P B 0.05.
Results
Daily nutrient intake
There were no statistically significant differences in the
total absolute or body weight adjusted energy consumption
or any macronutrient (protein, carbohydrate or fat) intake
between the protein and placebo conditions at weeks 0,
10.5 or 21 (P [ 0.23) (Table 2). The subjects habitually
consumed protein 1.5 ± 0.4 g/kg in the protein group and
1.4 ± 0.4 g/kg in the placebo group (assessed via an
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Table 2 Daily dietary intake: at the beginning of the study, at week 10.5, and during the last week (week 21)
Variable
Week 0
Protein
Week 10.5
Placebo
P value
Week 21
Protein
Placebo
P value
Protein
Placebo
P value
E (1,000 kJ)
10.4 ± 1.6
9.6 ± 2.0
0.37
10.5 ± 2.1
9.7 ± 3.2
0.60
10.2 ± 0.3
11.6 ± 3.2
0.48
E (kJ/kg bw)
136.6 ± 20.3
131.2 ± 20.9
0.57
139.3 ± 34.3
125.4 ± 37.1
0.50
145.7 ± 11.9
154.8 ± 36.7
0.70
1.4 ± 0.3
1.3 ± 0.3
0.67
1.5 ± 0.4
1.5 ± 0.5
0.91
1.7 ± 0.4
1.5 ± 0.4
0.59
Prot (g/kg bw)
CHO (g/kg bw)
3.9 ± 0.6
3.7 ± 0.7
0.35
4.0 ± 0.8
3.5 ± 1.0
0.34
3.4 ± 0.7
4.4 ± 1.1
0.23
Fat (g/kg bw)
1.2 ± 0.3
1.2 ± 0.3
0.47
1.1 ± 0.4
1.1 ± 0.4
0.99
1.5 ± 0.3
1.3 ± 0.4
0.54
Dietary diaries were kept at week 0 (3 days before the biopsy), on the biopsy day, and the day thereafter. At week 21 the dietary diary was
recorded during the 3 days before the biopsy and the biopsy day. Week 10.5 also included a 4-day diary. E energy, g/kg bw g per kg body mass,
Prot protein, CHO carbohydrates. P value is statistical difference between the protein and placebo groups. There were no differences between
weeks 0, 10.5 and 21 in the macronutrient consumption in either protein or placebo groups (P [ 0.05)
average of all food diaries: week 0, week 10.5 and week
21) (P = 0.71).
Anthropometric measurements
Body mass
protein and
no change
Body fat%
(P [ 0.86).
increased significantly during RT in both
placebo groups (P \ 0.01) while there was
in the control group (P = 0.65) (Table 1).
did not change significantly in any group
Muscle CSA
The cross-sectional area (CSA) of the quadriceps femoris
(QF) increased significantly after 21 weeks of RT in both
protein and placebo groups (P \ 0.01) but not in the control group (P [ 0.05) (Fig. 2a). The change of the average
QF CSA was higher in the protein group (9.9 ± 7.4%)
compared to placebo (7.5 ± 4.8%) but the difference did
not reach statistical significance (P [ 0.05). CSA of the
VL muscle increased in all four axial-plane images in both
protein and placebo groups (P \ 0.001) but not in the
control group (P [ 0.25) (Fig. 2b). The average increase in
the VL muscle (VL1–4) was significantly higher in the
protein group (relative increase: 14.8 ± 6.8%) compared to
the placebo (11.2 ± 5.6%) (P \ 0.05). In VI, VM and RF
there were no significant differences in the CSA change
between the protein and placebo groups in any of the CSA
images (P [ 0.05).
Muscle force
Maximal bilateral 1 RM leg extension, bilateral isometric
bench press and unilateral isometric knee extension and
flexion increased significantly and similarly during the
21-week-training period in both the protein and placebo
groups (P B 0.05) (Table 3). However, compared to the
control group (8.0 ± 9.5%, P [ 0.05), isometric leg
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Fig. 2 The average CSA of a quadriceps femoris and b vastus
lateralis from all four MRI images. Significantly (P B 0.05) different
compared to the Pre (asterisks), to the Control (daggers) or to the
Placebo (hash). All the values are mean ± SD
extension increased significantly only in the protein
group (a relative increase of 24.3 ± 12.3%, difference
between the groups P = 0.02), whereas the increase was
not significant in the placebo group (19.3 ± 15.5%,
P = 0.23).
Short and long-term effects of exercise and protein
303
Table 3 Muscle strength
Variable
Group 2-week-control period
Control
Mean ± SD
Baseline
Mean ± SD
P valuepre P valueDgroup
21-week-resistance training
10.5 weeks
Mean ± SD
21 weeks
Mean ± SD
%Change
Mean ± SD
Dynamic (kg)
PROT 163.2 ± 31.8
168.6 ± 28.4
191.4 ± 33.0
200.9 ± 32.5
19.3 ± 4.7
\0.001*
\0.001*
1RM leg press
PLAC 161.0 ± 28.5
164.0 ± 29.9
184.5 ± 26.5
194.8 ± 26.3
19.8 ± 9.5
\0.001*
\0.001*
Isometric
CONT 165.5 ± 22.2 167.5 ± 24.4 171.5 ± 20.4
PROT 4017 ± 1533 3961 ± 1241 4485 ± 1548
173.0 ± 21.6
3.7 ± 6.1
4957 ± 1796 24.3 ± 12.2
0.28
0.002*
0.02*
Leg press (N)
PLAC
4163 ± 1369 19.3 ± 15.5
0.007*
0.23
3647 ± 1333
3624 ± 1344
3823 ± 1350
3901 ± 815
CONT 3812 ± 925
3882 ± 978
Isometric
PROT 853.3 ± 97.6
862.0 ± 104.5 981.1 ± 96.1
Knee extension (N)
PLAC 813.5 ± 124.7 817.3 ± 96.6
4202 ± 1152
8.0 ± 9.5
1029.1 ± 112.7 19.8 ± 8.6
956.1 ± 233.6
CONT 841.0 ± 166.4 862.5 ± 194.1 860.0 ± 190.8
0.06
\0.001*
936.7 ± 117.5 14.9 ± 10.1
0.004*
899.7 ± 209.6
4.4 ± 6.7
0.29
\0.001*
0.05*
Isometric knee flexion (N) PROT 388.2 ± 54.2
399.5 ± 55.0
436.4 ± 56.9
461.9 ± 55.8
16.5 ± 12.7
0.004*
0.007*
PLAC 422.3 ± 52.9
396.2 ± 66.2
441.4 ± 65.2
452.8 ± 73.9
15.3 ± 13.5
0.009*
0.01*
CONT 394.3 ± 78.7
405.4 ± 95.9
397.6 ± 100.1
408.7 ± 95.6
1.3 ± 7.5
1.00
Isometric bench press (N) PROT 648.0 ± 141.5 655.0 ± 128.9 747.1 ± 154.9
803.9 ± 169.5 22.5 ± 8.3
\0.001*
\0.001*
PLAC 628.6 ± 139.7 620.6 ± 149.1 716.0 ± 162.6
782.5 ± 174.0 25.2 ± 8.3
\0.001*
\0.001*
CONT 613.2 ± 106.5 608.2 ± 118.3 633.5 ± 126.8
623.2 ± 130.1
2.4 ± 5.9
0.60
P valuepre designates Holm–Bonferroni corrected P values compared to baseline. P valueDgroup designates difference between the training groups
and control group in the change between baseline and post 21 week values. DChange is the percentage difference from the baseline to 21 weeks
* Significant (P B 0.05) P value
Acute resistance exercise bout at the week 0
The total volume of the work in the RE bout
(loads 9 sets 9 repetitions) was 6,722 ± 1,210 kg in the
placebo and 6,753 ± 1,189 kg in protein group (P = 0.96)
at week 0 of the study. Maximal isometric leg extension
force was significantly decreased (P \ 0.01) immediately
following the RE session but there were no differences
between the groups in this decrease after the RE
bout (P = 0.70) (placebo: from 3,449 ± 1,118 N to
2,041 ± 369 N and protein: from 3,568 ± 1,184 N to
2,186 ± 431 N).
Muscle mRNA levels
There was no change in any measured mRNA values in the
control group at any time point (P [ 0.05) (Figs. 3, 4). A
significant 31% decrease in myostatin mRNA was
observed 1 h after the RE bout but only in the placebo
condition (P = 0.02), not in the protein group (P [ 0.69)
(Fig. 3). The receptor of myostatin, activin receptor IIb
mRNA, decreased in both protein and placebo groups after
the RE bout being significant at 48 h after RE in both
placebo and (P = 0.04) protein groups (P = 0.01).
A significant 340% increase in cdk2 mRNA was
observed at 1 h after RE in the protein condition
(P = 0.01) and there was also a trend for an increase both
at 48 h post-RE (320%) and after 21 weeks of RT (120%)
(P = 0.08) (Fig. 4). By contrast, there was a significant
decrease after 21 weeks of RT in the placebo group
(P = 0.04). Thus, the 21-week-responses in the cdk2
mRNA between the protein and placebo group were significantly different (P = 0.04).
p21 mRNA increased in both protein and placebo
groups. The increase was significant in the placebo group
both at post 1 h (679%, P = 0.05) and at post 48 h (976%,
P = 0.05) after RE bout whereas significant increase was
seen in the protein group at 1 h after RE (466%,
P = 0.003). A significant decrease in myogenin mRNA
was observed at 1 h after the RE bout (37%, P = 0.005)
and after 21 weeks of RT (43%, P = 0.02) in the placebo
group but not in the protein group (P [ 0.34).
No significant change due to either protein, RE bout or
21 weeks of RT were observed in MAFbx (P [ 0.43)
(Fig. 3), MyoD (P [ 0.26) and FLRG (P [ 0.10) mRNA
(MyoD and FLRG data not shown).
Discussion
The major findings of the present study investigating both
acute and long-term effects in previously untrained young
men were as follows: timed intake of 15 g of whey protein
both immediately before and after each exercise session (1)
further increased resistance training-induced vastus lateralis muscle hypertrophy measured by MRI without
123
304
Fig. 3 Real-time RT-PCR results for myostatin, activin receptor IIb
and MAFbx mRNA expressions before and after single RE bout
(black bar: 1 h and white bar: 48 h) as well as after 21 weeks of RT
(gray bar: 21 weeks) from vastus lateralis muscle in protein, placebo
and control conditions. Results are normalized to GAPDH mRNA
expression and changes are presented in relation to pre-RE levels. All
J. J. Hulmi et al.
the values are mean ± SE. Asterisks statistical (P B 0.05) difference
compared to the pre-value in either protein or placebo condition and
hash difference in the change between protein and placebo group. The
results are shown as untransformed, whereas statistics were done with
log-transformed values because the mRNA data were not normally
distributed
Fig. 4 Real-time RT-PCR
results for cdk2, p21 and
myogenin mRNA expressions
before and after a single RE
bout as well as after 21 weeks
of RT. See further explanations
in text for previous Fig. 3
significantly increasing cross-sectional areas of other
quadriceps femoris muscles and also (2) increased cellcycle related kinase cdk2 mRNA expression. Moreover,
protein intake (3) seemed to prevent post-exercise decrease
in myostatin and myogenin mRNA expression. The inclusion of the control group in the study assured that the
results were not due to repeated biopsy effect, diurnal
effect, or time of the year (Sedliak et al. 2007; Vissing et al.
2005).
Protein ingestion has been shown also previously to
increase muscle myofiber CSA (Andersen et al. 2005; Cribb
et al. 2007; Hartman et al. 2007) as well as lean or fat-free
body mass (Burke et al. 2001; Candow et al. 2006a; Cribb
et al. 2007; Hartman et al. 2007; Kerksick et al. 2006) during
RT. This study was the first one to investigate the effects of
timed protein nutrition close to a resistance exercise bout on
training-induced whole-muscle hypertrophy by magnetic
123
resonance imaging (MRI). MRI is considered a ‘‘gold
standard’’ for cross-sectional area measurements of muscle
size due to the high quality of the images and high reproducibility (Reeves et al. 2004). The current results
demonstrate that subjects ingesting 15 g of whey both
immediately before and after each RE workout, two times a
week for 21 weeks, had larger quadriceps femoris (QF)
muscle hypertrophy (*10%) than the placebo group
(*7.5%) (ns). Of the individual QF muscles, protein
ingestion significantly increased resistance training-induced
muscle hypertrophy in vastus lateralis, one of the largest
muscles in the body and largest of the four QF muscles
(Häkkinen et al. 2001). The MRI results of the present study,
therefore, further suggests the importance of high-quality
protein consumption soon before and after each heavy RE
workout (Andersen et al. 2005; Cribb and Hayes 2006).
However, there were no statistically significant effects of
Short and long-term effects of exercise and protein
protein intake for other QF muscles although the most distal
vastus medialis MRI-image showed signs for greater
hypertrophy response in the protein group (16.9–19.9 cm2
compared to 16.2–17.5 cm2 for the placebo, P = 0.059,
data not shown). The reason for observing the significant
difference only in the VL muscle may be due to the fact that
our exercise selection was designed to specifically load the
VL muscle, the muscle from which muscle biopsies were
taken. Indeed, compared to the VI and RF, the VL (and with
a smaller extent also VM) exhibited the greatest hypertrophy, both absolutely and relatively, during the 21 weeks of
RT (data not shown) supporting earlier results from our
laboratory (Häkkinen et al. 2001).
Of the muscle strength variables, protein intake had a
positive effect only in isometric leg force production in the
leg press (the increase was significant compared to the
controls only in the protein group). The finding that protein
did not have consistent effect on maximal muscle force is
in agreement with some (Andersen et al. 2005; Burke et al.
2001; Candow et al. 2006b; Kerksick et al. 2006), but not
all previous studies (Candow et al. 2006a; Cribb et al.
2007). Accordingly, the effect of protein intake on
improved muscle force in previously untrained subjects
was only minor. This may be due to neural mechanisms,
which may explain most of the force production enhancement during the first weeks of RT (Häkkinen et al. 2001).
The effects of protein could, therefore, become significant
and more consistent in terms of both muscle hypertrophy
and muscle force production after much longer term
training (e.g., 1–2 years), or possibly even faster with
inclusion of already well-trained subjects or an amount
larger than 2 9 15 g of protein per RE workout. These
possibilities need further investigation.
In addition to muscle phenotype, many different gene
transcript levels from the muscle were examined both
acutely after the single RE bout and also after RT for
21 weeks. We found that cyclin-dependent kinase 2 (cdk2)
mRNA levels increased significantly after the RE bout only
in the protein group, the same phenomenon that has been
shown earlier with older men in response to a similar RE
bout and protein protocols (Hulmi et al. 2008). Interestingly, this increase in cdk2 mRNA remained elevated after
21 weeks of RT, but again only in the protein group.
Therefore, it seems evident that protein ingestion close to
the RE bout increases cdk2 mRNA expression both in
young and old men.
Cyclin-dependent kinases are probably the most
important regulators of cell proliferation (for review, see
Malumbres et al. 2000). Cdk2 is especially important in the
G1/S progression of the cell cycle (Berthet et al. 2003).
Whereas cdk2 is a protein of which expression in both
mRNA and protein levels as well as activity are increased
in proliferating myoblasts (Hlaing et al. 2002; McCroskery
305
et al. 2003; Ohkubo et al. 1994), it is possible that protein
ingestion before and after a RE workout may increase
satellite cell proliferation. This may lead to increased
satellite cell count which is important in muscle recovery
from micro-damage (Charge and Rudnicki 2004) and
possibly also to myonuclear addition, a phenomenon
important in muscle growth (Adams et al. 2002). Indeed, a
recent study by Olsen et al. (2006) suggests that protein
ingestion may have positive effects on the muscle satellite
cell number during RT in humans. Moreover, Halevy et al.
(2003) found that feeding increased DNA synthesis and
satellite cell number in the culture of turkey breast muscle
satellite cells when compared to a food-deprived state.
Additional proof that protein possibly affects gene
expression in human satellite cells in vivo comes from the
present finding that protein ingestion seemed to prevent a
small but rapid decrease in myogenin mRNA after the
single RE bout and also a decrease after 21 weeks of RT,
both observed only in the placebo group. This may be
explained by an in vitro finding showing that feeding
increases myogenin levels in satellite cell culture when
compared to a food-deprived state (Halevy et al. 2003).
Myogenin is a transcription factor expressed in myogenic
cells, and like cdk2, is also downstream to myostatin (Rios
et al. 2002). Myogenin is an important regulator for muscle
satellite cell differentiation (Charge and Rudnicki 2004;
Rios et al. 2002).
It is also possible that at least part of the observed higher
cdk2 mRNA response with protein intake comes from
proliferating cells other than satellite cells (or other muscle
myogenic cells), such as fibroblasts and endothelial cells.
Previously, one week of low protein intake decreased
transcript levels in muscle positively relating to cell proliferation and increased transcript levels that negatively
regulate cell proliferation (Thalacker-Mercer et al. 2007).
The possibly larger cell proliferating capacity response
could enhance muscle recovery after exercise workouts.
The upstream mechanisms for the increased cdk2 gene
expression response with whey protein are, however,
unknown. It is possible that either (1) whey proteins in
general or particular amino acids, (2) some bioactive
peptides or other related functional components in it (Ha
and Zemel 2003) or (3) energy in itself could affect cellcycle regulators in skeletal muscle when the muscle
metabolism is most active (i.e., during and after a RE bout
when protein was provided). Whey proteins have a large
amount of leucine, which was recently shown to activate
myogenic satellite cells in pigs through the mTOR pathway
(Han et al. 2008). Interestingly, cdk2 knockout mice are
slightly smaller than wild-type mice (Berthet et al. 2003)
and this difference could be related to possible positive
effects of cdk2, a protein downstream of myostatin
(McCroskery et al. 2003), on muscle mass.
123
306
In addition to cdk2, we also found effects of protein
ingestion on myostatin mRNA itself. More specifically, the
decrease in myostatin mRNA, occurring 1 h after the RE
bout in the placebo group, was prevented with whey protein ingestion. We have observed that in older men, protein
ingestion prevented the delayed post 48 h decrease in
myostatin mRNA (Hulmi et al. 2008). Therefore, protein
intake seems to affect muscle myostatin gene expression in
healthy men, but possibly with a different time-scale in the
young versus old. However, the results from various animal
species and study settings on the effects of different
nutritional protocols for the expression of myostatin are
contradictory (Guernec et al. 2004; Jeanplong et al. 2003;
Nakazato et al. 2006) and, therefore, more research is
warranted. The positive effect of timed protein intake on
vastus lateralis muscle growth was observed in the present
study. Therefore, it is possible that the acute myostatin
mRNA decrease in vastus lateralis muscle and protein
ingestion effect on preventing this decrease may not have
an especially important effect on muscle hypertrophy. This
agrees with a recent study utilizing cluster analysis, which
showed that subjects who had largest increase in muscle
fiber CSA during a RT period did not have different postRE myostatin mRNA response compared to individuals
who experienced low to no increases in muscular hypertrophy (Kim et al. 2007).
We did not observe any effect of protein ingestion on
myostatin binding protein FLRG mRNA response to the
RE bout in contradiction with our earlier results with older
men (Hulmi et al. 2008). Protein intake did not have a
significant effect on the RE-induced response of cdk
inhibitor p21 and activin receptor IIb mRNA, which supports our earlier results with older men (Hulmi et al. 2008).
The significant down-regulation of activin receptor IIb
48 h after the RE bout confirms our previous findings with
both untrained and trained older men (Hulmi et al. 2007).
This RE-induced response is interesting since myostatin
mediates its signals mainly through activin receptor IIb
(Lee and McPherron 2001). Therefore, the decrease in
activin receptor IIb mRNA gene expression after a RE bout
may lead to lower myostatin signalling in muscle fibres, a
response being theoretically advantageous for muscle
growth. This possibility, however, needs further investigation. Since protein ingestion seems to have at least a
minor acute effect on decreasing endogenous muscle protein degradation (Nagasawa et al. 1998; Tipton and Wolfe
2001), we were also interested in studying whether protein
intake could affect transcription of enzymes regulating
proteolysis. The results suggest that protein ingestion close
to the RE bout does not have an effect on ubiquitin-ligase
MAFbx mRNA expression (also called atrogin-1), a factor
important in muscle proteolysis and atrophy (Bodine et al.
2001). This suggests that if protein affects RE-induced
123
J. J. Hulmi et al.
proteolysis it is not through transcriptional regulation of
MAFbx.
In conclusion, high-quality whey protein intake before
and after resistance exercise appears to further augment
resistance training-induced muscle hypertrophy in previously untrained subjects. It also increased cyclin-dependent
kinase 2 (cdk2) gene expression and may prevent an exercise-induced decrease in myostatin and myogenin mRNA.
The increase in cdk2 gene expression suggests a higher
proliferating cell activation response with protein supplementation that can be advantageous for muscle hypertrophy.
Acknowledgments The authors thank Hanna Salmijärvi, Marja
Katajavuori, Liisa Kiviluoto, Marko Haverinen, Mikko Pietikäinen,
Hermanni Oksanen, Tuomas Kaasalainen, Tuovi Nykänen, Risto
Puurtinen, and Aila Ollikainen for their help in data collection and
analysis. We also thank the very dedicated group of subjects who
made this project possible. The Finnish Ministry of Education and the
Ellen and Artturi Nyyssönen Foundation (Juha Hulmi personal grant)
supported this research.
References
Adams GR, Caiozzo VJ, Haddad F, Baldwin KM (2002) Cellular and
molecular responses to increased skeletal muscle loading after
irradiation. Am J Physiol Cell Physiol 283:C1182–C1195
Andersen LL, Tufekovic G, Zebis MK, Crameri RM, Verlaan G,
Kjaer M, Suetta C, Magnusson P, Aagaard P (2005) The effect of
resistance training combined with timed ingestion of protein on
muscle fiber size and muscle strength. Metabolism 54:151–156
Berthet C, Aleem E, Coppola V, Tessarollo L, Kaldis P (2003) Cdk2
knockout mice are viable. Curr Biol 13:1775–1785
Bodine SC, Latres E, Baumhueter S, Lai VK, Nunez L, Clarke BA,
Poueymirou WT, Panaro FJ, Na E, Dharmarajan K, Pan ZQ,
Valenzuela DM, DeChiara TM, Stitt TN, Yancopoulos GD,
Glass DJ (2001) Identification of ubiquitin ligases required for
skeletal muscle atrophy. Science 294:1704–1708
Boirie Y, Dangin M, Gachon P, Vasson MP, Maubois JL, Beaufrere B
(1997) Slow and fast dietary proteins differently modulate
postprandial protein accretion. Proc Natl Acad Sci USA
94:14930–14935
Borsheim E, Tipton KD, Wolf SE, Wolfe RR (2002) Essential amino
acids and muscle protein recovery from resistance exercise. Am
J Physiol Endocrinol Metab 283:E648–E657
Burke DG, Chilibeck PD, Davidson KS, Candow DG, Farthing J,
Smith-Palmer T (2001) The effect of whey protein supplementation with and without creatine monohydrate combined with
resistance training on lean tissue mass and muscle strength. Int J
Sport Nutr Exerc Metab 11:349–364
Candow DG, Burke NC, Smith-Palmer T, Burke DG (2006a) Effect
of whey and soy protein supplementation combined with
resistance training in young adults. Int J Sport Nutr Exerc
Metab 16:233–244
Candow DG, Chilibeck PD, Facci M, Abeysekara S, Zello GA
(2006b) Protein supplementation before and after resistance
training in older men. Eur J Appl Physiol 97:548–556
Charge SB, Rudnicki MA (2004) Cellular and molecular regulation of
muscle regeneration. Physiol Rev 84:209–238
Cribb PJ, Hayes A (2006) Effects of supplement timing and resistance
exercise on skeletal muscle hypertrophy. Med Sci Sports Exerc
38:1918–1925
Short and long-term effects of exercise and protein
Cribb PJ, Williams AD, Stathis CG, Carey MF, Hayes A (2007)
Effects of whey isolate, creatine, and resistance training on
muscle hypertrophy. Med Sci Sports Exerc 39:298–307
Durnin JV, Womersley J (1974) Body fat assessed from total body
density and its estimation from skinfold thickness: measurements
on 481 men and women aged from 16 to 72 years. Br J Nutr
32:77–97
Esmarck B, Andersen JL, Olsen S, Richter EA, Mizuno M, Kjaer M
(2001) Timing of postexercise protein intake is important for
muscle hypertrophy with resistance training in elderly humans. J
Physiol 535:301–311
Gibala MJ, MacDougall JD, Tarnopolsky MA, Stauber WT, Elorriaga
A (1995) Changes in human skeletal muscle ultrastructure and
force production after acute resistance exercise. J Appl Physiol
78:702–708
Guernec A, Chevalier B, Duclos MJ (2004) Nutrient supply enhances
both IGF-I and MSTN mRNA levels in chicken skeletal muscle.
Domest Anim Endocrinol 26:143–154
Ha E, Zemel MB (2003) Functional properties of whey, whey
components, and essential amino acids: mechanisms underlying
health benefits for active people (review). J Nutr Biochem
14:251–258
Halevy O, Nadel Y, Barak M, Rozenboim I, Sklan D (2003) Early
posthatch feeding stimulates satellite cell proliferation and
skeletal muscle growth in turkey poults. J Nutr 133:1376–1382
Han B, Tong J, Zhu MJ, Ma C, Du M (2008) Insulin-like growth
factor-1 (IGF-1) and leucine activate pig myogenic satellite cells
through mammalian target of rapamycin (mTOR) pathway. Mol
Reprod Dev 75:810–817
Hartman JW, Tang JE, Wilkinson SB, Tarnopolsky MA, Lawrence
RL, Fullerton AV, Phillips SM (2007) Consumption of fat-free
fluid milk after resistance exercise promotes greater lean mass
accretion than does consumption of soy or carbohydrate in
young, novice, male weightlifters. Am J Clin Nutr 86:373–381
Hlaing M, Shen X, Dazin P, Bernstein HS (2002) The hypertrophic
response in C2C12 myoblasts recruits the G1 cell cycle
machinery. J Biol Chem 277:23794–23799
Hulmi JJ, Ahtiainen JP, Kaasalainen T, Pollanen E, Häkkinen K, Alen
M, Selanne H, Kovanen V, Mero AA (2007) Postexercise
myostatin and activin IIb mRNA levels: effects of strength
training. Med Sci Sports Exerc 39:289–297
Hulmi JJ, Kovanen V, Lisko I, Selanne H, Mero AA (2008) The
effects of whey protein on myostatin and cell cycle-related gene
expression responses to a single heavy resistance exercise bout in
trained older men. Eur J Appl Physiol 102:205–213
Häkkinen K, Pakarinen A, Newton RU, Kraemer WJ (1998) Acute
hormone responses to heavy resistance lower and upper
extremity exercise in young versus old men. Eur J Appl Physiol
Occup Physiol 77:312–319
Häkkinen K, Pakarinen A, Kraemer WJ, Häkkinen A, Valkeinen H,
Alen M (2001) Selective muscle hypertrophy, changes in EMG
and force, and serum hormones during strength training in older
women. J Appl Physiol 91:569–580
Jeanplong F, Bass JJ, Smith HK, Kirk SP, Kambadur R, Sharma M,
Oldham JM (2003) Prolonged underfeeding of sheep increases
myostatin and myogenic regulatory factor myf-5 in skeletal
muscle while IGF-I and myogenin are repressed. J Endocrinol
176:425–437
Kerksick CM, Rasmussen CJ, Lancaster SL, Magu B, Smith P,
Melton C, Greenwood M, Almada AL, Earnest CP, Kreider RB
(2006) The effects of protein and amino acid supplementation on
performance and training adaptations during ten weeks of
resistance training. J Strength Cond Res 20:643–653
Kim JS, Petrella JK, Cross JM, Bamman MM (2007) Load-mediated
downregulation of myostatin mRNA is not sufficient to promote
307
myofiber hypertrophy in humans: a cluster analysis. J Appl
Physiol 103:1488–1495
Kraemer WJ, Adams K, Cafarelli E, Dudley GA, Dooly C,
Feigenbaum MS, Fleck SJ, Franklin B, Fry AC, Hoffman JR,
Newton RU, Potteiger J, Stone MH, Ratamess NA, TriplettMcBride T, American College of Sports Medicine (2002)
American college of sports medicine position stand. Med Sci
Sports Exerc 34:364–380
Kuang S, Charge SB, Seale P, Huh M, Rudnicki MA (2006) Distinct
roles for Pax7 and Pax3 in adult regenerative myogenesis. J Cell
Biol 172:103–113
Lee SJ, McPherron AC (2001) Regulation of myostatin activity and
muscle growth. Proc Natl Acad Sci USA 98:9306–9311
Liu W, Saint DA (2002) Validation of a quantitative method for real
time PCR kinetics. Biochem Biophys Res Commun 294:347–
353
Malumbres M, Ortega S, Barbacid M (2000) Genetic analysis of
mammalian cyclin-dependent kinases and their inhibitors. Biol
Chem 381:827–838
Mascher H, Tannerstedt J, Brink-Elfegoun T, Ekblom B, Gustafsson T,
Blomstrand E (2008) Repeated resistance exercise training
induces different changes in mRNA expression of MAFbx and
MuRF-1 in human skeletal muscle. Am J Physiol Endocrinol
Metab 294:E43–E51
McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R
(2003) Myostatin negatively regulates satellite cell activation
and self-renewal. J Cell Biol 162:1135–1147
McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal
muscle mass in mice by a new TGF-beta superfamily member.
Nature 387:83–90
Nagasawa T, Hirano J, Yoshizawa F, Nishizawa N (1998) Myofibrillar protein catabolism is rapidly suppressed following protein
feeding. Biosci Biotechnol Biochem 62:1932–1937
Nakazato K, Hirose T, Song H (2006) Increased myostatin synthesis
in rat gastrocnemius muscles under high-protein diet. Int J Sport
Nutr Exerc Metab 16:153–165
Ohkubo Y, Kishimoto T, Nakata T, Yasuda H, Endo T (1994) SV40
large T antigen reinduces the cell cycle in terminally differentiated myotubes through inducing Cdk2, Cdc2, and their partner
cyclins. Exp Cell Res 214:270–278
Olsen S, Aagaard P, Kadi F, Tufekovic G, Verney J, Olesen JL, Suetta
C, Kjaer M (2006) Creatine supplementation augments the
increase in satellite cell and myonuclei number in human skeletal
muscle induced by strength training. J Physiol 573:525–534
Paddon-Jones D, Sheffield-Moore M, Aarsland A, Wolfe RR,
Ferrando AA (2005) Exogenous amino acids stimulate human
muscle anabolism without interfering with the response to mixed
meal ingestion. Am J Physiol Endocrinol Metab 288:E761–E767
Reeves ND, Maganaris CN, Narici MV (2004) Ultrasonographic
assessment of human skeletal muscle size. Eur J Appl Physiol
91:116–118
Rios R, Carneiro I, Arce VM, Devesa J (2002) Myostatin is an
inhibitor of myogenic differentiation. Am J Physiol Cell Physiol
282:C993–C999
Sedliak M, Finni T, Cheng S, Haikarainen T, Häkkinen K (2007)
Diurnal variation in maximal and submaximal strength, power
and neural activation of leg extensors in men: multiple sampling
across two consecutive days. Int J Sports Med 29(3):217–224
Thalacker-Mercer AE, Fleet JC, Craig BA, Carnell NS, Campbell WW
(2007) Inadequate protein intake affects skeletal muscle transcript
profiles in older humans. Am J Clin Nutr 85:1344–1352
Tipton KD, Wolfe RR (2001) Exercise, protein metabolism, and
muscle growth. Int J Sport Nutr Exerc Metab 11:109–132
Tipton KD, Elliott TA, Cree MG, Aarsland AA, Sanford AP, Wolfe
RR (2007) Stimulation of net muscle protein synthesis by whey
123
308
protein ingestion before and after exercise. Am J Physiol
Endocrinol Metab 292:E71–E76
Vissing K, Andersen JL, Schjerling P (2005) Are exercise-induced
genes induced by exercise? FASEB J 19:94–96
Wagner KR (2005) Muscle regeneration through myostatin inhibition.
Curr Opin Rheumatol 17:720–724
Wernbom M, Augustsson J, Thomee R (2007) The influence of
frequency, intensity, volume and mode of strength training on
123
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J. J. Hulmi et al.
whole muscle cross-sectional area in humans. Sports Med
37:225–264
Wilkinson SB, Tarnopolsky MA, Macdonald MJ, Macdonald JR,
Armstrong D, Phillips SM (2007) Consumption of fluid skim
milk promotes greater muscle protein accretion after resistance
exercise than does consumption of an isonitrogenous and
isoenergetic soy-protein beverage. Am J Clin Nutr 85:1031–
1040