effect of branched-chain amino acid supplementation during...

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,


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)


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


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


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


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)



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)



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


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


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


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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