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ORIGINAL ARTICLE
Effect of branched-chain amino acid supplementationduring unloading on regulatory components of protein synthesisin atrophied soleus muscles
Gustavo Bajotto • Yuzo Sato • Yasuyuki Kitaura •
Yoshiharu Shimomura
Received: 3 September 2010 / Accepted: 29 December 2010 / Published online: 11 January 2011
� Springer-Verlag 2011
Abstract Maintenance of skeletal muscle mass depends
on the equilibrium between protein synthesis and protein
breakdown; diminished functional demand during unload-
ing breaks this balance and leads to muscle atrophy. The
current study analyzed time-course alterations in regulatory
genes and proteins in the unloaded soleus muscle and the
effects of branched-chain amino acid (BCAA) supple-
mentation on muscle atrophy and abundance of molecules
that regulate protein turnover. Short-term (6 days) hind-
limb suspension of rats resulted in significant losses of
myofibrillar proteins, total RNA, and rRNAs and pro-
nounced atrophy of the soleus muscle. Muscle disuse
induced upregulation and increases in the abundance of the
eukaryotic translation initiation factor 4E-binding protein 1
(4E-BP1), increases in gene and protein amounts of two
ubiquitin ligases (muscle RING-finger protein 1 and mus-
cle atrophy F-box protein), and decreases in the expression
of cyclin D1, the ribosomal protein S6 kinase 1, the
mammalian target of rapamycin (mTOR), and ERK1/2.
BCAA addition to the diet did not prevent muscle atrophy
and had no apparent effect on regulators of proteasomal
protein degradation. However, BCAA supplementation
reduced the loss of myofibrillar proteins and RNA, atten-
uated the increases in 4E-BP1, and partially preserved
cyclin D1, mTOR and ERK1 proteins. These results indi-
cate that BCAA supplementation alone does not oppose
protein degradation but partly preserves specific signal
transduction proteins that act as regulators of protein syn-
thesis and cell growth in the non-weight-bearing soleus
muscle.
Keywords Rat hindlimb suspension � Disuse-induced
skeletal muscle atrophy � Nutritional countermeasure �Mammalian target of rapamycin � mRNA translation
Introduction
Unload- or disuse-induced atrophy of skeletal muscles is
one of the most challenging problems encountered by
astronauts when exposed to microgravity for prolonged
periods of time. Marked protein loss characterizes skeletal
muscle atrophy (Jackman and Kandarian 2004), and this
phenomenon is closely connected with alterations in
intracellular protein kinetics. In fact, several studies using
ground-based models of microgravity have shown that
unloading of skeletal muscles leads to significant increases
in protein degradation rates and significant decreases in
protein synthesis rates, as measured in vivo or in vitro (see
review, Bajotto and Shimomura 2006). In addition,
diminished amounts of total RNA in rat (Haddad et al.
2003) and human (Gamrin et al. 1998) atrophied muscles
indicates reduced capacity for protein synthesis. These
findings suggest, therefore, that regulation of protein
turnover (i.e., synthesis and degradation) plays a central
Communicated by Jacques R. Poortmans.
G. Bajotto and Y. Shimomura were affiliated with the Nagoya
Institute of Technology (Nagoya, Japan) until August 2008. Part of
this work was performed at that institution.
G. Bajotto � Y. Kitaura � Y. Shimomura (&)
Department of Applied Molecular Biosciences, Graduate School
of Bioagricultural Sciences, Nagoya University,
Nagoya 464-8601, Japan
e-mail: [email protected]
Y. Sato
Department of Health Science, Faculty of Psychological
and Physical Science, Aichi Gakuin University,
Nisshin 470-0195, Japan
123
Eur J Appl Physiol (2011) 111:1815–1828
DOI 10.1007/s00421-010-1825-8
role in disuse-induced skeletal muscle wasting. However,
the molecular regulatory mechanisms that respond to
weightlessness or reduced mechanical tension are still
poorly understood.
On account of its direct or indirect action towards
activation of molecules that control translation initiation
events, the serine/threonine protein kinase mammalian
target of rapamycin (mTOR) has been recognized as a very
important component for the regulation of protein synthesis
in muscles (Proud 2007; Wullschleger et al. 2006).
Accordingly, downstream effectors of mTOR-induced
translational control such as the ribosomal protein S6
kinase 1 (S6K1) and the eukaryotic translation initiation
factor 4E-binding protein 1 (4E-BP1) modulate skeletal
muscle growth. Conversely, the ATP-dependent ubiquitin–
proteasome pathway is constitutively active in muscle
fibers and, during muscle inactivity, predominantly deals
with degradation of cleaved myofibrillar proteins (Jackman
and Kandarian 2004; Taillandier et al. 1996). Enzymatic
polyubiquitination of protein substrates involves the regu-
latory action of two striated muscle-specific ubiquitin
ligases—muscle RING-finger protein 1 (MuRF1) and
muscle atrophy F-box protein (MAFbx)—which have been
identified responsive to changes in mechanical loading and,
to a certain extent, rate-limiting of muscle atrophy (Bodine
et al. 2001a).
The branched-chain amino acid (BCAA) leucine is
widely known as the nutrient with the strongest protein
anabolic effect in mammals (Kimball and Jefferson 2004).
Several studies have reported that administration of
BCAAs or leucine alone induces significant increases in
skeletal muscle protein synthesis rates (Crozier et al. 2005)
with concomitant enhancement in the phosphorylation of
downstream targets of mTOR signaling (Crozier et al.
2005; Anthony et al. 2000), especially after exercise
(Dreyer et al. 2008; Karlsson et al. 2004). In addition,
leucine can mimic postprandial hyperaminoacidemia and
act as a nutrient signal to stimulate protein synthesis in
cardiac and skeletal muscles by increasing eukaryotic
initiation factor (eIF)4E availability for eIF4F complex
assembly (Escobar et al. 2006). Furthermore, other studies
have shown that administration of leucine to chickens
suppresses myofibrillar proteolysis by downregulating the
ubiquitin–proteasome pathway (Nakashima et al. 2005)
and feeding aged rats with a leucine-supplemented diet for
10 days restores the defective postprandial inhibition of
proteasome-dependent proteolysis in skeletal muscle
(Combaret et al. 2005). Hence, the abovementioned find-
ings suggest that dietary supplementation with BCAA is
potentially useful to maintain protein synthetic capacity
and to inhibit protein breakdown in unloaded muscles,
preventing protein loss and, ultimately, attenuating muscle
atrophy.
Rodent hindlimb suspension (HS) is a well-established
ground-based model of microgravity (Morey-Holton and
Globus 2002) which induces pronounced atrophy in anti-
gravitational muscles such as the soleus. A small number of
studies have publicized that short-term HS leads to inac-
tivation of protein synthesis regulators such as mTOR
(Reynolds et al. 2002) and S6K1 (Hornberger et al. 2001)
and significant increases in the expression of genes that
regulate protein degradation (Taillandier et al. 1996) in
hindlimb muscles. However, time-course changes in the
abundance of mRNAs and proteins that regulate protein
turnover in atrophying muscles are unknown and, as
mentioned above, the molecular aspects of the potential
preventive effect of BCAA supplementation on disuse-
induced muscle wasting have not yet been investigated.
Therefore, this study was performed to analyze 1) time-
course alterations in regulatory genes and proteins in
unloaded muscles and 2) the effects of dietary supple-
mentation with BCAA during short-term HS on muscle
atrophy and abundance of regulatory molecules.
Experimental procedures
Materials and diets
Oligonucleotide primers were synthesized by the STAR
Oligo Service of Rikaken Co. Ltd. (Nagoya, Japan). Dye
reagent concentrate for protein determination, bovine
c-globulin standard, dual color prestained Precision Plus
Protein standards, goat antirabbit secondary antibody
(H ? L), and protein G-HRP conjugate were obtained from
Bio-Rad Laboratories Inc. (Hercules, CA). Goat polyclonal
antibody against GAPDH, rabbit polyclonal antibodies
against 4E-BP1, MuRF1 and MAFbx, monoclonal antibody
against ubiquitin, and rabbit antigoat secondary antibody
were purchased from Santa Cruz Biotechnology Inc. (Santa
Cruz, CA). Antimouse secondary antibody (H ? L) was
from Promega Co. (Madison, WI). Rabbit antibodies
against mTOR, phospho-mTOR (Ser2448), S6K1, phos-
pho-S6K1 (Thr389), cyclin D1, and extracellular signal-
regulated kinases (ERK1/2) were purchased from Cell
Signaling Technology Inc. (Danvers, MA). The S6K1
antibody also recognized the 85-kDa isoform of the ribo-
somal protein S6 kinase (p85-S6K). Enhanced chemilumi-
nescence (ECL) Western blotting detection reagents were
from GE Healthcare UK Ltd. (Little Chalfont, Bucking-
hamshire, England). All other reagents were of analytical
grade and were bought from Wako Pure Chemical Indus-
tries Ltd. (Osaka, Japan), Nacalai Tesque Inc. (Kyoto,
Japan), or Sigma–Aldrich Co. (Tokyo, Japan).
The AIN-93G diet was based on the original formula
(Reeves et al. 1993), with the difference that dextrinized
1816 Eur J Appl Physiol (2011) 111:1815–1828
123
cornstarch was substituted for 10% pure dextrin and the
final relative amount of cornstarch was 42.9486%. For the
BCAA-supplemented AIN-93G diet, 5% cornstarch was
replaced with BCAA, in which the ratio among the 3 amino
acids—leucine/isoleucine/valine—was 2:1:1.2; this ratio
was based on the amino acid composition of rat milk
protein (Davis et al. 1993). BCAA was added to the diet at
the concentration of 5% (w/w) to provide the rats with
about twofold the BCAA amount of the control diet.
Dietary formulations were produced by Nippon Formula
Feed Manufacturing Co. Ltd. (Yokohama, Japan).
Animals
In the total, 56 male Sprague–Dawley rats were used in this
study. Rats were specific pathogen-free animals purchased
from Japan SLC Inc. (Hamamatsu, Japan). Rats aged 6
weeks (152 ± 1 g body weight (BW), n = 22), 7 weeks
(230 ± 1 g BW, n = 10) and 5 weeks (140 ± 2 g BW,
n = 24) were used in Experiments 1, 2 and 3, respectively.
Rats were housed in wire-mesh cages, one animal per cage,
room temperature was set at 23 ± 1�C, and lighting was
from 7:00 to 19:00 h. Rats were provided tap water and
pellet-type diets ad libitum. Animal procedures were in
accordance with guidelines set out by the Guide for Care
and Use of Laboratory Animals of Nagoya University
(2000) and with the Guidelines for Proper Conduct of
Animal Experiments (Science Council of Japan, 2006).
The study protocol was approved by the Animal Care
Committee of the Nagoya Institute of Technology.
Experimental design
Experiment 1
The objective of this pilot experiment was to investigate
the time-course variation in the amounts of specific skeletal
muscle mRNAs and proteins during unloading. Rats were
given the standard rodent diet CE-2 (CLEA Japan Inc.,
Tokyo, Japan). After 1 week acclimatization, which
included two sessions of 1 h HS per day for all rats,
animals were divided into the following seven groups: fed
control (n = 3), 0.5-day control (n = 3), 0.5-day HS
(n = 4), 1.5-day HS (n = 3), 3.5-day HS (n = 3), 5.5-day
HS (n = 3), and 5.5-day control (n = 3). Excluding the fed
control group (dissected in the morning at 7:00 h), all rats
were dissected at 19:00 h, after a 12-h starvation period.
Rats were anesthetized by intraperitoneal injection of
pentobarbital sodium (50 mg/kg BW), without allowing
the hindlimbs of HS rats to become weight bearing (they
were anesthetized while suspended), and the bilateral
soleus muscles were excised, promptly freeze-clamped at
liquid nitrogen temperature, weighed, and stored at -80�C
until analyses. Special care was taken to remove connec-
tive tissue and tendons and to wipe up blood from the
specimens before freeze-clamping.
Experiment 2
This experiment was performed to study the short-term
influence of BCAA addition to the diet on food intake, BW,
and plasma BCAA concentration. Animals were divided
into two groups with approximately the same average BW:
AIN-93G diet group (n = 5) and AIN-93G ? 5% BCAA
diet group (n = 5). Daily food intake and BW were
recorded for 9 days and, on the fourth day, food intake in
six equal periods of the day was measured and six blood
samples were collected in 4-h intervals by tail snipping. On
the final day, all rats were anesthetized as described above
and organs were removed and weighed.
Experiment 3
This experiment aimed to analyze the effect of BCAA
supplementation on disuse-induced skeletal muscle atrophy
and its molecular aspects. Rats were randomly divided into
two groups and provided either the AIN-93G (n = 12) or
the AIN-93G ? 5% BCAA diet (n = 12) from the first day
of the experiment. After a 4-day acclimatization period that
included two sessions of 1 h HS per day for all rats
(without using anesthesia to apply the tail harness), animals
were further divided into the following four groups: con-
trol/AIN93 (n = 6), control/BCAA (n = 6), HS/AIN93
(n = 6), and HS/BCAA (n = 6). Rats of the HS groups
were unloaded for 6 days. Food intake was recorded daily
and BW on days 0, 4, and 10 (just prior to dissection). On
the last day of the experiment, food was removed from
cages at the end of the dark phase and, 2–4 h later, all rats
were anesthetized as described under Experiment 1 and
dissected. Blood (2.5–3.0 ml) was collected from the
inferior vena cava using a syringe containing 50 ll of
200 mmol/l EDTA (pH 7.5) and then bilateral muscles
(soleus, gastrocnemius, plantaris, extensor digitorum lon-
gus, tibialis anterior, and triceps brachii) were excised and
handled as described under Experiment 1. As the soleus
was the muscle with the most pronounced degree of atro-
phy after HS, it was selected for further analysis. Ice-cold
blood samples were centrifuged at 30009g for 10 min and
plasma was obtained and stored at -40�C until analyses.
Hindlimb suspension
The HS (also called ‘hindlimb unloading’) rodent model has
been reviewed by Morey-Holton and Globus (2002). For the
present study, we developed original cages measuring
28 9 26 9 40 cm in size and an innovative method to
Eur J Appl Physiol (2011) 111:1815–1828 1817
123
harness the tail of the rat in an attempt to reduce discomfort
as much as possible. Suspension cages had wire-mesh floor
and ceiling, and the internal side of their walls was covered
with 2-mm thick transparent acrylic panels (to a height of
16 cm from the floor) so as to prevent rats from climbing
the walls with their forelimbs. Steel curtain tracks con-
taining carrier rollers (with 2 nylon wheels each) with 360�swivels and small drop chains were affixed to the ceiling of
the cages in the longitudinal direction. The suspension
system was designed so that rats could freely ambulate
around the cages using their forelimbs, but without allowing
their hindlimbs to rest against any supportive surface. The
rat was anesthetized briefly with pentobarbital sodium, the
tail was cleaned thoroughly with 70% ethyl alcohol and air
dried, and 2 narrow strips of Battlewin non-elastic, adhesive
sports tape (Nichiban Co. Ltd., Tokyo, Japan) were applied
longitudinally along the dorsal and ventral sides of the 4-cm
proximal portion of the tail. Using washable glue, a concave
copper sheet and a specially shaped copper hook containing
a small nickel-plated brass snap link were attached to the
ventral and dorsal strips of tape, respectively. Pieces of
sports tape (19 mm wide) were wrapped circumferentially
around the proximal and distal segments of the eye of the
hook and these two parts were attached together with
the same tape applied longitudinally along the laterals of the
tail, forming a stable cuff-like structure that surrounded
approximately 4.2 cm of the proximal portion of the tail.
Shortly after the animal had recovered from the anesthetic,
the snap link was connected to the drop chain of the sus-
pension apparatus, unloading the hindlimbs. The height of
suspension was adjusted so that, when the hindlimbs of the
rat were fully extended, the fingers just cleared the floor of
the cage. Rats were closely monitored during the HS period,
and no obstruction of blood flow to the distal portion of the
tail was observed.
Myofibrillar protein fractionation
Essentially, soleus muscle proteins were fractioned as
described by Garma et al. (2007), with a few modifications.
Briefly, around 20 mg of finely powdered muscle sample
was homogenized in 20 volumes of ice-cold buffer con-
taining 10 mmol/l Tris (pH 6.8), 250 mmol/l sucrose,
100 mmol/l potassium chloride (KCl), 5 mmol/l EDTA,
and a cocktail of protease inhibitors (Roche Diagnostics
GmbH, Mannheim, Germany). Homogenization was per-
formed at high speed for 30 s using a Polytron PT 1200
handheld homogenizer (Kinematica AG, Littau-Lucerne,
Switzerland). Homogenates were centrifuged at 10009g
for 10 min at 4�C and the supernatants were collected for
protein determination (soluble protein fraction). Pellets
were resuspended by vortexing in 20 volumes of a buffer
containing 10 mmol/l Tris (pH 6.8), 175 mmol/l KCl,
2 mmol/l EDTA, and 0.5% (w/v) Triton X-100, suspen-
sions were centrifuged as above, pellets were resuspended
with the same buffer, and suspensions were centrifuged
once again. Obtained pellets were resuspended in 20 vol-
umes of cold washing buffer (10 mmol/l Tris (pH 7.0) and
150 mmol/l KCl), resultant suspensions were centrifuged
as above, and the final myofibrillar pellets were resus-
pended in an appropriate volume (approximately 30
volumes of the initial muscle mass) of 10 mmol/l Tris (pH
7.4), 100 mmol/l KCl, and 1 mmol/l EDTA (myofibrillar
protein fraction). Soluble and myofibrillar fractions were
diluted to 50% with 1.5 mol/l sodium hydroxide and pro-
tein concentrations were determined in double by the
method of Bradford using bovine c-globulin as standard.
Total RNA isolation, quantification and RT-PCR
Skeletal muscle total RNA was isolated from powdered
tissue using the ISOGEN reagent (Nippon Gene Co. Ltd.,
Tokyo, Japan) (Bajotto et al. 2004) for the samples of
Experiment 1 and using the SV Total RNA Isolation System
(Promega Co., Madison, WI) for the samples of Experiment
3, following instructions of the manufacturer. The yield of
total RNA obtained was determined spectrophotometrically
at 260 nm and the integrity of the purified RNA was
determined by formaldehyde denaturing 1% (w/v) agarose
gel electrophoresis and ethidium bromide staining. The
commercial kit SuperScript First-Strand Synthesis System
for RT-PCR (Invitrogen Co., Carlsbad, CA) was used for
the reverse transcription of poly(A)? RNA templates and
subsequent digestion of remaining RNA into the first-strand
cDNA preparations. PCR reaction cocktails containing
40 U/ml of recombinant Taq DNA polymerase and 0.3
lmol/l of each specific oligonucleotide primer (Table 1)
were prepared using the TaKaRa Taq reagents (Takara Bio
Inc., Otsu, Japan). Cycle-course experiments were carried
out using control and atrophied muscles to determine the
optimal number of cycles for amplification of each gene that
fit within the linear range. PCR-amplified fragments were
resolved by electrophoresis on 1.5 or 2% (w/v) agarose gels
containing 0.25 lg/ml ethidium bromide, the gels were then
exposed to ultraviolet light, Polaroid photographs were
taken, and the signals were analyzed using the Scion Image
Beta 4.0.2 software (Scion Corporation, Frederick, MD) for
the semiquantitative determination of the abundance of the
target mRNA molecules.
Protein extraction, electrophoresis and immunoblotting
Muscle powder was homogenized in 7 volumes of ice-cold
homogenization buffer (20 mmol/l HEPES (pH 7.4),
2 mmol/l EGTA, 50 mmol/l sodium fluoride, 100 mmol/l
KCl, 0.2 mmol/l EDTA, 50 mmol/l b-glycerophosphate,
1818 Eur J Appl Physiol (2011) 111:1815–1828
123
1 mmol/l DTT, 0.1 mmol/l PMSF, 1 mmol/l benzamidine,
and 0.5 mmol/l sodium vanadate) using the same homog-
enizer described above. Homogenates were centrifuged at
100009g for 10 min at 4�C and the protein concentration
of the supernatants was determined as above. Proteins were
resolved by one-dimensional SDS-PAGE, using 6%
(for mTOR), 12.5% (for proteins other than mTOR and
4E-BP1) or 15% (for 4E-BP1) gels, and transferred to
polyvinylidene fluoride (PVDF) membranes. Membranes
were incubated overnight at 4�C with primary antibodies
diluted 1:800 to 1:1000 and for 60–90 min at room tem-
perature with secondary antibodies diluted 1:3000. Bound
antibodies were detected and signals on the X-ray films
were quantified as described previously (Bajotto et al.
2004). In addition to the detection of the housekeeping
protein GAPDH, proteins immobilized on PVDF mem-
branes were stained with Coomassie Brilliant Blue G-250
after immunoblot analysis was completed, to make sure
that samples have been evenly loaded.
Plasma biochemical analyses
Plasma glucose, free fatty acids (FFA), and triglycerides
(TG) concentrations were assayed enzymatically using the
Glucose C-II, the NEFA C, and the Triglyceride E com-
mercial kits, respectively, purchased from Wako Pure
Chemical Industries Ltd. (Osaka, Japan). Plasma insulin
concentration was determined by chemiluminescence
enzyme immunoassay (Morgan and Lazarow 1963) by the
SRL Inc. (Tokyo, Japan). Plasma BCAA concentration was
assayed spectrophotometrically by recording end-point
NADH production from the oxidative deamination of
BCAAs catalyzed by leucine dehydrogenase (Beckett 2000).
Statistical analysis
Data are presented as means ± SE. Data were analyzed by
one-way (Experiments 1 and 2), two-way (Experiment 3)
or two-way repeated measures (Experiments 2 and 3)
ANOVA followed by either the Tukey–Kramer (Experi-
ment 1) or the Fisher protected least significant difference
(Experiments 2 and 3) test when the ANOVA demonstrated
significant difference. P \ 0.05 was considered to be sta-
tistically significant. However, following recommendations
of the ‘‘guidelines for reporting statistics in journals pub-
lished by the American Physiological Society’’ (Curran-
Everett et al. 2004), P \ 0.1 will be indicated herein as a
‘tendency’ to statistical significance. The StatView 5.0
software (SAS Institute Inc., Cary, NC) was used for the
statistical analysis of the data.
Results
Experiment 1
Time-course HS resulted in gradual decreases in soleus
muscle mass and in its protein and total RNA contents
(Fig. 1a–c). One-way ANOVA was significant for muscle
mass (P = 0.002), protein (P = 0.047) and total RNA
(P \ 0.001), and post-hoc analysis revealed that the
observed decreases in these three parameters were signifi-
cant after 5.5-day HS (Fig. 1a–c). Expression of genes
encoding ubiquitin C, the C2 subunit of the proteasome,
and the 14-kDa ubiquitin-conjugating enzyme (E2) did not
change significantly in atrophied soleus muscles (Fig. 2a).
On the other hand, expression of MuRF1 and MAFbx
mRNAs increased and cyclin D1 mRNA tended to decrease
in unloaded muscles (Fig. 2a). With regard to changes in
the abundance of regulatory proteins in the atrophying
soleus, MuRF1 and MAFbx appeared to increase, cyclin
D1 was unaltered, and the phosphorylated form of S6K1
(Thr389) and its total protein content decreased (Fig. 2b).
In addition, the c-form of 4E-BP1 disappeared and the
amount of its b-form showed increases in unloaded/atro-
phied soleus muscles (Fig. 2b).
Table 1 Primers used in Experiments 1 and 3
Gene Forward primer Reverse primer Annealing
temperature (�C)
Amplicon
length (bp)
Ubiquitin C 50-GATCCAGGACAAGGAGGGC-30 50-CATCTTCCAGCTGCTTGCCT-30 60 71
C2 subunit 50-GGCTGCTCATTGCTGGTTAG-30 50-CCAACAATCCCAATGGAAAC-30 56 256
14-kDa E2 50-GTGCACCATCTGAAAACAA-30 50-ATCGGTTCTGCAGGATGTCT-30 53 210
MuRF1 50-TACCGAGAGCAGTTGGAAAAGT-30 50-CTCAAGGCCTCTGCTATGTGTT-30 57 215
MAFbx 50-CAGAACAGCAAAACCAAAACTC-30 50-GCGATGCCACTCAGGGATGT-30 56 218
Cyclin D1 50-TCTACACTGACAACTCTATCCG-30 50-TAGCAGGAGAGGAAGTTGTTGG-30 54 304
GAPDH 50-GTGAAGGTCGGTGTGAACG-30 50-GAGATGATGACCCTTTTGG-30 54 356
C2 subunit C2 subunit of the proteasome, 14-kDa E2 14-kDa ubiquitin-conjugating enzyme (E2), MuRF1 muscle RING-finger protein 1, MAFbxmuscle atrophy F-box protein (also called atrogin-1)
Eur J Appl Physiol (2011) 111:1815–1828 1819
123
Experiment 2
Relative food intake throughout the experimental period
did not differ considerably between rats eating the control
and rats eating the BCAA-added diet (77 ± 2 and
74 ± 1 g/kg BW/day, respectively). However, BCAA
intake per day was significantly higher in rats provided
with the test diet (P \ 0.01; 3.1 ± 0.1 and 6.7 ± 0.1 g/kg
BW for control and BCAA groups, respectively). One-day
food intake was similar between both groups of rats, except
that the amount of food eaten by the BCAA group between
06:00 and 10:00 h was markedly less than the amount
eaten by the control group in the same period (Fig. 3a; two-
way repeated measures ANOVA for the main effect of
food and time, and the interaction between food and time
were 0.287, \0.001, and \0.001, respectively). Type of
food and time showed significant interaction concerning
their influence in the growth of the rats during the 9-day
experiment (P = 0.005) and the relative quantity of intra-
abdominal fat in rats supplemented with BCAA tended to
be less than in control animals (P = 0.084; 2.6 ± 0.2 and
2.9 ± 0.1 g/100 g BW, respectively). Two-way repeated
measures ANOVA revealed that the variation in the plasma
BCAA concentration throughout the day was significantly
influenced by the type of food and time, and these two
factors significantly interacted (Fig. 3b; P = 0.002,
\ 0.001, and 0.001, respectively). Plasma BCAA concen-
trations were significantly higher at 14:00, 22:00, 02:00,
and 06:00 h and tended to be higher (P = 0.051) at
10:00 h in rats of the BCAA group than in rats of the
control group (Fig. 3b).
Experiment 3
Daily food intake was almost the same among the four
groups of rats during the 10-day experimental period
Fig. 1 Time-course variation in
muscle mass and amounts of
protein and total RNA in the
unloaded soleus (Experiment 1).
Line graphs show the time-
course variation in soleus
muscle mass (a) and protein
(b) and total RNA (c) contents
after unloading. Data are from
three or four rats in each group
(means ± SE) and represent
values normalized to the actual
body weight of the rats at the
time of dissection. Data of the
0-day time-point, corresponding
to the fed rats, were not
included in the statistical
analysis. *P \ 0.05 vs. 0.5-day
control, 5.5-day control, and
0.5-day HS; #P \ 0.05 vs.
0.5-day control; �P \ 0.05 vs.
0.5-day control, 5.5-day control,
0.5-day HS, and 1.5-day HS
1820 Eur J Appl Physiol (2011) 111:1815–1828
123
(Fig. 4a); however, the main effect of suspension was
significant in the two-way repeated measures ANOVA
(P = 0.025) and the food intake on the second day after
unloading was significantly less in both groups of sus-
pended rats than in control/AIN93 rats. Compared with
controls, the growth rate of hindlimb-suspended rats was
significantly reduced at the time of dissection and no effect
of BCAA supplementation on growth was observed
(Fig. 4b).
Plasma glucose and TG concentrations did not change
among the four groups of rats (Table 2). However, unloading
increased plasma FFA levels and BCAA supplementation
significantly decreased circulating insulin concentrations
and increased BCAA levels (Table 2). Although HS/AIN93
rats had significantly higher plasma levels of FFA than
control/AIN93 rats, no significant difference between HS/
BCAA and control/BCAA groups was observed. In addition,
BCAA supplementation markedly decreased plasma insulin
concentrations in HS rats only (Table 2).
Excepting the extensor digitorum longus, HS, but not
BCAA supplementation, influenced the mass of all muscles
that were collected (Table 3). Compared with control
groups eating the same diet, the mass of the soleus, gas-
trocnemius, and plantaris muscles significantly decreased
Fig. 2 Time-course changes in gene expression and abundance of
specific proteins in the unloaded soleus muscle (Experiment 1).
Representative ethidium bromide signals (a) and immunoblots (b) illus-
trating the time-course alteration in the expression of genes and
abundance of proteins, respectively, in the unloaded soleus muscle. Each
signal represents the average density of signals in each group (n = 3 or 4),
as quantified by scanning densitometry. 0.5C 0.5-day control, 0.5H 0.5-
day HS, 1.5H 1.5-day HS, 3.5H 3.5-day HS, 5.5H 5.5-day HS, 5.5C 5.5-
day control, Ub. C ubiquitin C, C2 C2 subunit of the proteasome, E214-kDa ubiquitin-conjugating enzyme (E2), MuRF1 muscle RING-finger
protein 1, MAFbx muscle atrophy F-box protein (also called atrogin-1),
Cy. D1 cyclin D1 (also known as Ccnd1), S6K1 ribosomal protein S6
kinase 1, p-S6K1 phospho-S6K1 (Thr389), 4E-BP1 eukaryotic translation
initiation factor 4E-binding protein 1 (also known as PHAS-I)
Fig. 3 One-day food intake and alteration in the plasma BCAA
concentration in rats fed the BCAA-supplemented diet (Experiment
2). The bar graph shows the amount of AIN-93G or AIN-93G ? 5%
BCAA diet eaten by the rats in six 4-h periods of the day (a) and the
line graph shows the variation in plasma BCAA concentration in six
time-points during the day (b) for five animals in each group
(means ± SE). CTR control. *P \ 0.05 vs. control; #P \ 0.01 vs.
control; �P = 0.051 vs. control
Eur J Appl Physiol (2011) 111:1815–1828 1821
123
in unloaded rats; however, the same comparison showed
that the mass of the tibialis anterior and triceps brachii
muscles significantly increased in HS rats (Table 3).
Another assessment considering the relative mass of pooled
flexors and extensors also confirmed that HS atrophies
flexors and hypertrophies extensor muscles, with no influ-
ence of BCAA addition to the diet (Table 3).
Soleus soluble protein concentration tended to be higher
in HS/BCAA than in HS/AIN93 rats and, compared with
control rats, the total content of soluble protein was
significantly lower in the two groups of suspended animals
(Table 4). Myofibrillar protein concentration and content in
the soleus were significantly decreased after unloading;
however, HS/BCAA animals tended to have higher myo-
fibrillar protein concentration and total amount than
HS/AIN93 animals (Table 4). Total RNA concentration
and content were also markedly lower in both groups of
suspended rats, and the concentration of total RNA in the
soleus of HS/BCAA rats was significantly higher than in
HS/AIN93 rats (Table 4). Abundances of 18S and 28S
rRNAs were significantly decreased in atrophied soleus
muscles; however, rats of the HS/BCAA group had
markedly higher amounts of 28S rRNA than rats of the HS/
AIN93 group (Table 4).
Compared with controls, the expression of genes encoding
MuRF1 and MAFbx significantly increased and the abun-
dance of cyclin D1 mRNA significantly decreased in unloa-
ded/atrophied soleus muscles (Fig. 5). GAPDH mRNA
expression did not change among the four groups of rats, and
no marked effect of BCAA supplementation on the expression
of the other three genes analyzed was observed (Fig. 5).
The amounts of MuRF1 and MAFbx proteins signifi-
cantly increased and the abundance of phosphorylated
S6K1, total S6K1, and p85-S6K proteins significantly
decreased in unloaded/atrophied soleus muscles, indepen-
dent of the supplementation of BCAA in the diet (Fig. 6).
However, although the amounts of cyclin D1 protein
markedly decreased in the soleus muscles of suspended rats
eating the control diet, cyclin D1 was somewhat preserved in
the soleus of suspended, BCAA-supplemented animals
(Fig. 6). No significant changes in the amounts of GAPDH
protein (Fig. 6) and Coomassie-stained proteins (Fig. 7b)
were observed among the four groups of rats. Marked
increase in the abundance of broad-range ubiquitinated
proteins in the soleus muscle was observed only in the
HS/BCAA group (Fig. 7a). Amounts of phosphorylated and
total mTOR (Fig. 8a), the c-form of 4E-BP1 (Fig. 8b), and
ERK1/2 (Fig. 8c) proteins were significantly decreased in
the soleus muscles of unloaded rats, and no marked effect of
Fig. 4 Daily food intake and growth of hindlimb-suspended rats fed
normal or BCAA-supplemented diet (Experiment 3). Line graphsshow daily food intake (a) and the growth rate (b) of the four groups
of rats during the 10-day experimental period. Data are means ± SE
for six animals in each group. *P \ 0.05 for Ctr/AIN93 vs. HS/
AIN93 and HS/BCAA; #P \ 0.01 for Ctr/AIN93 vs. HS/AIN93 and
for Ctr/BCAA vs. HS/BCAA
Table 2 Plasma biochemistry (Experiment 3)
Control HS ANOVA’s P-values
AIN93 BCAA AIN93 BCAA Food Load F 9 L
Glucose (mmol/l) 9.0 ± 0.3 8.7 ± 0.2 8.6 ± 0.2 8.8 ± 0.4 0.861 0.600 0.413
Free fatty acids (mmol/l) 0.23 ± 0.01 0.22 ± 0.02 0.29 ± 0.03* 0.24 ± 0.02 0.142 0.048 0.336
Triglycerides (mmol/l) 0.99 ± 0.11 0.93 ± 0.12 1.04 ± 0.26 0.83 ± 0.03 0.398 0.869 0.664
Insulin (pmol/l) 367 ± 78 230 ± 45 600 ± 136 261 ± 55# 0.012 0.142 0.259
BCAA (mmol/l) 0.68 ± 0.04 1.13 ± 0.10� 0.75 ± 0.03 1.13 ± 0.10� \0.001 0.633 0.645
Plasma was obtained from blood taken just prior to dissection, 2–4 h after rats were deprived from food. Assays were performed as described
under ‘‘Experimental procedures’’ (except for insulin, each assay was run in duplicate). Values are means ± SE (n = 6)
* P \ 0.05 vs. control eating the same food; # P \ 0.05 vs. HS/AIN93; � P \ 0.01 vs. control/AIN93; � P \ 0.01 vs. HS/AIN93
1822 Eur J Appl Physiol (2011) 111:1815–1828
123
the BCAA supplement on phosphorylated mTOR (Fig. 8a),
the c-form of 4E-BP1 (Fig. 8b), and ERK2 (Fig. 8c) proteins
was observed. However, decreases in total mTOR protein
were less in the soleus muscles of rats eating the BCAA-
supplemented diet, which tended to have higher amounts of
this protein than HS rats eating the control diet (Fig. 8a). In
addition, while the amounts of total 4E-BP1 protein mark-
edly increased in the soleus muscles of HS rats eating the
control diet, these increases were not significant in the soleus
of HS rats eating the diet supplemented with BCAA
(Fig. 8b). Furthermore, while the abundance of ERK1 pro-
tein was markedly decreased in the HS/AIN93 group, it was
preserved in the HS/BCAA group of rats, which had sig-
nificantly higher amounts of ERK1 in their soleus muscles
than HS/AIN93 animals (Fig. 8c).
Discussion
Results of the current study point to the involvement of
cell cycle elements in the progress of muscle atrophy and
indicate that BCAA supplementation alone cannot halt
skeletal muscle wasting. However, BCAA helps in
attenuating decreases in muscle protein and RNA amounts
and partially preserving specific signal transduction pro-
teins that act as regulators of protein synthesis and cell
growth, such as mTOR and ERK1, in the unloaded soleus
muscle.
One of the novel findings of this study was that muscle
disuse suppresses transcription of cyclin D1, a gene with
well-known key regulatory function on cell cycle pro-
gression or cell proliferation (Klein and Assoian 2008).
Cyclin D1 expression has been shown to increase in
overloaded muscles (Adams et al. 2002) and in serum-
stimulated myotubes (Nader et al. 2005), mediating signals
that incite a hypertrophic response in muscles. As tran-
scriptional repression of the cyclin D1 gene is crucial for
maintaining cellular quiescence and preventing unwanted
cell proliferation (Klein and Assoian 2008), our results
suggest that, unless some effective countermeasure is
applied, cell cycle progression is needless or must be
constrained during muscle unloading.
Table 3 Relative mass of muscle pairs and comparison of pooled flexor and extensor muscles (Experiment 3)
Control HS ANOVA’s P-values
AIN93 BCAA AIN93 BCAA Food Load F 9 L
Soleus 77 ± 2 80 ± 2 43 ± 1* 46 ± 2* 0.118 \0.001 0.879
Gastrocnemius 930 ± 21 901 ± 8 815 ± 15* 795 ± 13* 0.121 \0.001 0.766
Plantaris 184 ± 4 182 ± 2 164 ± 3* 165 ± 3* 0.903 \0.001 0.561
Extensor digitorum longus 94 ± 2 94 ± 1 96 ± 2 96 ± 2 0.830 0.262 0.947
Tibialis anterior 327 ± 4 325 ± 6 356 ± 5* 348 ± 8# 0.452 \0.001 0.653
Triceps brachii 779 ± 23 805 ± 15 841 ± 20# 872 ± 19# 0.168 0.003 0.908
Flexors (% control) 100 ± 1 100 ± 1 77 ± 1* 78 ± 2* 0.809 \0.001 0.646
Extensors (% control) 100 ± 2 101 ± 1 106 ± 2* 107 ± 2# 0.584 \0.001 0.931
Values for muscle mass are shown in mg/100 g BW (normalized to the actual body weight of the rats at the time of dissection). Values are
means ± SE (n = 6)
* P \ 0.01 vs. control eating the same food; # P \ 0.05 vs. control eating the same food
Table 4 Soleus muscle soluble and myofibrillar proteins, total RNA, and rRNAs (Experiment 3)
Control HS ANOVA’s P-values
AIN93 BCAA AIN93 BCAA Food Load F 9 L
Soluble protein (mg/g tissue) 82 ± 2 77 ± 3 74 ± 5 82 ± 2* 0.594 0.650 0.034
Soluble protein (mg/100 g BW) 6.3 ± 0.2 6.1 ± 0.3 3.1 ± 0.2# 3. 8 ± 0.2# 0.372 \0.001 0.105
Myofibrillar protein (mg/g tissue) 145 ± 4 131 ± 5 97 ± 7# 111 ± 4*,� 0.952 \0.001 0.016
Myofibrillar protein (mg/100 g BW) 11.2 ± 0.2 10.5 ± 0.5 4.2 ± 0.4# 5.1 ± 0.4*,# 0.710 \0.001 0.036
Total RNA (lg/mg tissue) 1.06 ± 0.10 1.03 ± 0.08 0.44 ± 0.05# 0.68 ± 0.06#,� 0.169 \0.001 0.066
Total RNA (lg/100 g BW) 82 ± 7 82 ± 6 18 ± 2# 31 ± 3# 0.228 \0.001 0.216
18S rRNA (arbitrary unit) 59 ± 5 58 ± 6 30 ± 2# 44 ± 4� 0.175 \0.001 0.122
28S rRNA (arbitrary unit) 57 ± 5 56 ± 6 22 ± 2# 38 ± 3#,� 0.093 \0.001 0.060
Content values were normalized to the actual body weight of the rats at the time of dissection. Values are means ± SE (n = 6)
* P \ 0.1 vs. HS/AIN93; # P \ 0.01 vs. control eating the same food; � P \ 0.05 vs. control eating the same food; � P \ 0.05 vs. HS/AIN93
Eur J Appl Physiol (2011) 111:1815–1828 1823
123
It has been reported that inhibition of the mTOR path-
way obstructs leucine-induced augments in cyclin D1
protein in primary cultured chicken hepatocytes (Lee et al.
2008). In addition, Nader et al. (2005) demonstrated that
rapamycin prevents increases in cyclin D1 protein in
hypertrophying myotubes, suggesting that the role of
mTOR is in part to modulate cyclin D1-dependent cyclin-
dependent kinase-4 activity in the regulation of retino-
blastoma and rRNA synthesis. This proposition is partially
in line with our finding that BCAA-induced recurrent
activation of mTOR (and consequent attenuation in the
disuse-induced loss of total mTOR protein) resulted in
preservation of cyclin D1 protein and rRNAs in the soleus
muscle. In contrast to the effect of resistance exercise
(Adams et al. 2007), however, supplementation with
BCAA had no influence on cyclin D1 mRNA, suggesting
that mTOR-related modulation of cyclin D1 protein in
inactive muscles is likely to be a posttranscriptional event
only.
The total S6K1 protein in the soleus muscle was
decreased with time and, given that others have not iden-
tified significant alterations in this protein after disuse
(Adams et al. 2007), this result was somewhat surprising
and indicates transcriptional regulation of this kinase dur-
ing unloading. In addition to S6K1 cutback, reduced
phosphorylation levels of the translational repressor
4E-BP1 (as evidenced by disappearance of its c-form) and
concurrent increases in the amounts of its b-form
substantiate the conception that protein synthesis is inhib-
ited during muscle inactivity. Conversely, compensatory
hypertrophy of the plantaris muscle has been shown to be
associated with 4E-BP1 downregulation and S6K1 upreg-
ulation in rats (Bodine et al. 2001b), endorsing the
importance of these downstream effectors of mTOR in
skeletal muscle plasticity. Insignificant increases in total
4E-BP1 protein in the soleus of HS/BCAA rats indicate
some positive effect of BCAA supplementation.
Previous time-course experiments have shown that the
activating effect of leucine (Anthony et al. 2002) and food
consumption (Wilson et al. 2009) on several molecules that
regulate translation initiation is transient, ceasing within
2–3 h after amino acid administration or meal feeding. In
our Experiment 3, given that rats were dissected a few
hours after peak food intake, no significant differences on
the phosphorylation levels of proteins such as S6K1 and
mTOR could be observed, and this represents a limitation
of the current study. Nevertheless, considering the well-
documented stimulating effect of BCAAs on components
Fig. 5 Soleus muscle mRNA expression (Experiment 3). Represen-
tative ethidium bromide signals of PCR-amplified fragments demon-
strating the mRNA expression of four genes in normal and atrophied
soleus muscles. Values under the bands indicate the percentage
expressions of mRNAs relative to the Ctr/AIN93 group of rats, for six
animals in each group (means ± SE). *P \ 0.01 vs. control eating
the same food
Fig. 6 Abundance of proteins in the soleus muscle (Experiment 3).
Representative immunoblots showing the abundance of proteins in
normal and atrophied soleus muscles. Values under the blots indicate
the percentage amounts of proteins relative to the Ctr/AIN93 group of
rats, for six animals in each group (means ± SE). *P \ 0.05 vs.
control eating the same food; #P \ 0.01 vs. control eating the same
food
1824 Eur J Appl Physiol (2011) 111:1815–1828
123
of the mTOR signaling pathway (Kimball and Jefferson
2006) and provided that periodic requirement or activation
of signaling proteins may result in preservation of their
intracellular amounts, at this time we may be able to
Fig. 7 Soleus muscle abundance of ubiquitinated and Coomassie-
stained proteins (Experiment 3). Representative immunoblot of
broad-range ubiquitinated proteins (a) and typical distribution of
soleus muscle proteins immobilized on a PVDF membrane (b).
Values under the lanes indicate the percentage amounts of ubiqui-
tinated proteins relative to the Ctr/AIN93 group of rats, for six
animals in each group (means ± SE). *P \ 0.05 vs. control eating
the same food
Fig. 8 Abundance of mTOR, 4E-BP1, and ERK1/2 proteins in the
soleus muscle (Experiment 3). Representative immunoblots illustrat-
ing the abundance of phosphorylated (Ser2448) and total mTOR (a),
4E-BP1 (b), and ERK1/2 (c) proteins in normal and atrophied soleus
muscles. Values under the blots indicate the percentage amounts of
phosphorylated mTOR (a), c-form of 4E-BP1 (b), and ERK2
(c) proteins relative to the Ctr/AIN93 group of rats, for six animals
in each group (means ± SE). Bar graphs give a quantification of the
relative abundance of total mTOR (a), total 4E-BP1 (b), and ERK1
(c) proteins in the soleus muscle of control and hindlimb-suspended
rats eating the AIN-93G (open bars) or the AIN-93G ? 5% BCAA
(closed bars) diet (n = 6, means ± SE). *P \ 0.01 vs. control eating
the same food; #P \ 0.05 vs. control eating the same food
Eur J Appl Physiol (2011) 111:1815–1828 1825
123
interpret our data based chiefly on the total quantity of
proteins, as discussed above. It is indispensable, however,
to confirm these conclusions in future studies, in which
atrophied muscles are excised during the dark period.
Recently, intracellular mitogen-activated protein kinase
(MAPK) signal transduction cascades, especially the
ERK1/2, have emerged as important regulators of numer-
ous functions within mammalian cells (Sturgill 2008). Of
particular interest is the reported involvement of ERK1/2
in the regulation of both growth factor- and contraction-
induced anabolic response in skeletal muscle (Drummond
et al. 2009; Haddad and Adams 2004; Parkington et al.
2004; Tsakiridis et al. 2001; Williamson et al. 2006). In a
recent study, Shi et al. (2009) have demonstrated that
inhibition of MAPK signaling cascades inactivates Akt and
its downstream kinases, upregulates gene transcription of
MuRF1 and MAFbx, and provokes profound muscle atro-
phy in vitro and in vivo. Based on this background, we may
infer that mechanical unloading-induced downregulation of
ERK1/2 functions as a key regulatory mechanism during
disuse-induced muscle wasting, which reduces protein
synthesis at the transcriptional and translational levels,
increases the gene expression of ubiquitin ligases, and also
represses cyclin D1 transcription (Roovers and Assoian
2000). In opposition, BCAA or even leucine alone (Lee
et al. 2008) possibly upregulates ERK1 only (as observed
here), attenuating some of the deleterious effects of muscle
inactivity on protein turnover and myogenesis.
In disagreement with the findings of Taillandier et al.
(1996), time-course changes in the mRNA expression of
ubiquitin C, the C2 subunit of the proteasome, and the
14-kDa ubiquitin-conjugating enzyme (E2) were not
observed in this study and the reason for this divergence is
unclear. However, gradual increases in the mRNA
expression of MuRF1 and MAFbx were in line with pre-
vious reports (Bodine et al. 2001a; Haddad et al. 2006;
Nikawa et al. 2004), and increases in the protein abundance
of these muscle-specific ubiquitin ligases suggested timely
translation of their genes during unloading. Augmented
abundance of ubiquitinated proteins only in the soleus
muscles of suspended rats that were provided the BCAA
diet suggests the presence of plenty amounts of newly
synthesized proteins that, for an unknown reason, were not
promptly used and so ended by being targeted for
degradation.
Comparable to the results of a human study that
examined the effects of a leucine-enriched high protein diet
on long-term bed rest-induced muscle atrophy (Trappe
et al. 2007), in the current study BCAA supplementation
did not prevent decreases in muscles mass. Since admin-
istration of BCAAs could produce a significant increase in
muscle wet weight in mice bearing a cachexia-inducing
tumor (Eley et al. 2007), we hypothesize that the factor
‘muscle load’ is decisive in determining the yield of
diverse results for different models. In this study, however,
the presence of higher amounts of total RNA and rRNAs in
the HS/BCAA group of rats indicates that BCAA addition
to the diet preserved the protein translational capacity in
the soleus muscle (Haddad et al. 2003). Similar to some of
the effects of BCAA observed in this study, others have
shown that heat stress (Naito et al. 2000) and administra-
tion of the b2-adrenergic agonist clenbuterol (Wineski et al.
2002) can also diminish the loss of proteins in inactive
muscles. Further studies are necessary to test whether
BCAA supplementation can boost up the effects of other
countermeasures and, comparable to clenbuterol or
Bowman-Birk inhibitor concentrate (Arbogast et al. 2007),
lead to considerable mitigation of muscle atrophy.
Conclusion
Taken together, the results of this study indicate that
BCAA supplementation alone does not oppose protein
degradation but partly preserves specific signal transduc-
tion proteins that act as regulators of protein synthesis and
cell growth in the non-weight-bearing soleus muscle.
Added to the preservation of muscle proteins and RNA,
these effects of BCAA may, therefore, bring forth faster
recovery of atrophied muscles upon reloading (this
hypothesis will be tested in future experiments).
Acknowledgments This work was partially supported by Grants-in-
Aid for Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (20300216) and from the
Japan Society for the Promotion of Science (17-05171). The authors
sincerely acknowledge Asami Inaguma, Satoko Watanabe, Yosuke
Asai, Yuka Kodera, Hiroki Nagata, Takuma Maekawa, Yuko Yasuda,
and Rie Shikano for their considerate help during dissection of the
animals.
Conflict of interest There are no conflicts of interest.
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