Experimental Gerontology 72 (2015) 269–277

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Experimental Gerontology

journal homepage: www.elsevier.com/locate/expgero

Leucine supplementation improves regeneration of skeletal muscles

from old rats
Marcelo G. Pereira a, Meiricris T. Silva a, Fernanda M. da Cunha b, Anselmo S. Moriscot a,
Marcelo S. Aoki c, Elen H. Miyabara a,⁎
a
Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Prof. Lineu Prestes Av. 2415, São Paulo, SP 05508-000, Brazil
b
Department of Biochemistry, Federal University of São Paulo, 3 de Maio St. 100, São Paulo, SP, Vila Clementino, 04044-020, Brazil
c
School of Arts, Sciences and Humanities, University of São Paulo, Arlindo Bettio Av. 1000, São Paulo, SP 03828-000, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The decreased regenerative capacity of old skeletal muscles involves disrupted turnover of proteins. This study
Received 1 August 2015 investigated whether leucine supplementation in old rats could improve muscle regenerative capacity. Young
Received in revised form 6 October 2015 and old male Wistar rats were supplemented with leucine; then, the muscles were cryolesioned and examined
Accepted 15 October 2015
after 3 and 10 days. Leucine supplementation attenuated the decrease in the expression of eukaryotic translation
Available online 23 October 2015
initiation factor 4E binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4E (eIF4E) in young and
Keywords:
old muscles on day 3 post-injury and promoted an increase in the cross-sectional area of regenerating myofibers
Aging from both young and old soleus muscles on day 10 post-injury. This supplementation decreased the levels of
Skeletal muscle ubiquitinated proteins and increased the proteasome activity in young regenerating muscles, but the opposite ef-
Regeneration fect was observed in old regenerating muscles. Moreover, leucine decreased the inflammation area and induced
Leucine supplementation an increase in the number of proliferating satellite cells in both young and old muscles. Our results suggest that
leucine supplementation improves the regeneration of skeletal muscles from old rats, through the preservation
of certain biological responses upon leucine supplementation. Such responses comprise the decrease in the in-
flammation area, increase in the number of proliferating satellite cells and size of regenerating myofibers, com-
bined with the modulation of components of the phosphoinositide 3-kinase/Akt-protein kinase B/mammalian
target of rapamycin (PI3K/Akt/mTOR) pathway and ubiquitin-proteasome system.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction
Skeletal muscle regeneration is a well-orchestrated process that oc-
curs after muscle injury. This process includes the migration of inflam-
matory cells to the injured area, the proliferation and differentiation of
satellite cells—a muscle-specific stem cell population, the recovery of
myofiber size as well as the re-establishment of neuromuscular tissue,
connective tissue and angiogenesis. Subsequently, the functional recov-
ery of injured muscles occurs (Charge and Rudnicki, 2004; Ciciliot and
Schiaffino, 2010; Ten Broek et al., 2010).
Previous studies have shown that in senescent individuals, the im-
pairment of the regenerative capacity of skeletal muscle is due in part
to impaired satellite cell function (Shavlakadze et al., 2010) and in-
volves the delayed activation, proliferation, and differentiation of satel-
lite cells (Barani et al., 2003; Conboy et al., 2003; Shefer et al., 2006).
Consequently, the number and caliber of nascent myofibers decrease
⁎ Corresponding author.
E-mail addresses: pereiramg@gmail.com (M.G. Pereira), me_tomaz@hotmail.com
(M.T. Silva), fmcunha@unifesp.br (F.M. da Cunha), moriscot@usp.br (A.S. Moriscot),
aoki.ms@usp.br (M.S. Aoki), elenm@usp.br (E.H. Miyabara).
(Charge et al., 2002) and myofibers are gradually replaced by non-
contractile elements, such as connective and adipose tissues (Carosio
et al., 2011). Some studies have reported a remarkable decrease in the
number of satellite cells from old muscles under homeostatic conditions
(Brack et al., 2005; Shefer et al., 2006). Moreover, a recent study has
demonstrated that satellite cells from geriatric rodents show decreased
activation compared with those from old rodents (Sousa-Victor et al.,
2014).
Although the regenerative process of the skeletal muscle during
aging is well known at the structural level, the molecular mechanisms
responsible for the regeneration decline in aged muscles are still not total-
ly understood (Brack et al., 2007; Carlson et al., 2008; Conboy et al., 2003).
In addition, the identification of strategies to improve the regenerative
capacity of aged muscle is on-going. Exposure to factors present in a
young serum or use of growth factors, which stimulate proliferation or
myogenic progression, can partially restore satellite cell proliferation
and differentiation in aged muscles (Brack et al., 2007; Conboy et al.,
2003; Conboy et al., 2005; Shefer et al., 2006).
In addition, we have recently demonstrated that the pharmacologi-
cal treatment with the β2-adrenoceptor agonist formoterol can improve
the recovery of regenerating myofiber size in aged muscles (Conte et al.,

http://dx.doi.org/10.1016/j.exger.2015.10.006
0531-5565/© 2015 Elsevier Inc. All rights reserved.
270 M.G. Pereira et al. / Experimental Gerontology 72 (2015) 269–277
2012). This recovery may be associated with an increase of protein syn-
thesis via mammalian/mechanistic target of rapamycin (mTOR) activa-
tion (Conte et al., 2012). The recovery of myofiber size during muscle
regeneration is controlled by signaling pathways that maintain the bal-
ance between protein synthesis and degradation. Accordingly, the acti-
vation of elements involved in mRNA translation, such as the kinase
mTOR and its downstream targets, the eukaryotic translation initiation
factor 4E-binding protein (4E-BP1) and S6 kinase 1 (S6K1), is an essen-
tial step to accomplish the muscle regenerative response (Ge et al.,
2009; Miyabara et al., 2010).
The ubiquitin proteasome system (UPS), which is the major path-
way of proteolytic activity in muscles, is also important for the muscle
regenerative process (Taillandier et al., 2004; Tintignac et al., 2005).
During degradation of injured muscle tissue, E3 ubiquitin protein li-
gases, such as MuRF1 and MAFbx/atrogin-1, bind ubiquitin conjugates
to target proteins. Then, the ubiquitinated proteins are recognized and
degraded by the 26S proteasome (Taillandier et al., 2004). In fact, a sig-
nificant increase in the levels of ubiquitinated proteins has been shown
at early stages of muscle recovery after injury (Miyabara et al., 2010;
Pereira et al., 2014a).
The deficit in the regenerative capacity of old muscles is associated
with the impaired ability to replace muscle protein (Degens, 2007;
Ryall et al., 2008), in which protein degradation increases and protein
synthesis decreases. In addition, the impairment of signaling pathways
that regulate the mass of aged muscles is due in part to an inadequate
nutrition (Charlton, 2002). Considering these statements, supplementa-
tion with the essential amino acid leucine may be a suitable non-
pharmacological strategy to improve muscle regeneration in senescent
individuals. Leucine has been shown to regulate protein turnover
in skeletal muscle promoting mass gain (Dodd and Tee, 2012; Li et al.,
2011), and decreasing mass loss (Baptista et al., 2010). In addition, it
has been suggested that β-hydroxy-β-methylbutyrate (HMB), a leucine
metabolite, can attenuate muscle mass loss in aged animals, possibly via
a decrease in nuclear apoptosis (Hao et al., 2011) and an increase in the
proliferation of satellite cells (Alway et al., 2014). Moreover, it has been
shown that insulin-like growth factor 1 and leucine can activate myo-
genic satellite cells through the mTOR pathway in pigs (Han et al.,
2008).
We have recently demonstrated that leucine supplementation im-
proves the recovery of myofiber size and contractile function of
regenerating young skeletal muscles after cryolesion, accompanied by
a decrease in the accumulation of ubiquitinated proteins (Pereira
et al., 2014a). In addition, we have shown that leucine accelerates con-
nective tissue repair from regenerating young muscles via attenuation
of the activation of transforming growth factor-β receptor type I (TβR-
I) and Smad2/3 (Pereira et al., 2014b). However, the effect of leucine
supplementation on the regenerative capacity of aged muscles has not
been fully elucidated. Therefore, the aim of the present study was to in-
vestigate the effect of leucine supplementation on the morphological
and molecular recovery of injured skeletal muscles from aged animals.
2. Methods
The protocols used in this study were in strict accordance with the
ethical principles in animal research followed by the Brazilian College
of Animal Experimentation. The study was approved by the Ethics Com-
mittee in Animal Research of the Institute of Biomedical Sciences at the
University of São Paulo (Protocol No. 87/2011).
2.1. Animal rearing and leucine supplementation
Two-month old male Wistar rats (n = 20) and 20–24 month old
male Wistar rats (n = 20) were maintained in standard plastic cages
in an animal facility with controlled environmental conditions (24 °C;
12/12-h light/dark cycle) and reared on standard food and tap water
ad libitum.
Beginning 3 days before muscle injury and continuing until the
end of both experimental period (until post-cryolesion days 3 and 10,
n = 10 for each period), L-leucine (Ajinomoto, Tokyo, Japan) was ad-
ministered once a day by oral gavage at a dose of 1.35 g/kg body mass
(Crozier et al., 2005; Lang et al., 2003; Vary, 2007). Leucine was dis-
solved in water and each animal was gavaged with a 5-mL volume of
the solution (Pereira et al., 2014a; Pereira et al., 2014b). A control
group received saline only. The results of a previous experiment indicat-
ed that muscles from saline-gavaged rats did not show morphological
changes compared with those from non-gavaged animals (data not
shown).
2.2. Experimental design
Rats received leucine supplementation or not and were subjected to
cryolesion of the soleus muscle from the left hind limb. The contralateral
soleus muscle of the right hind limb was used as the intact control
(Conte et al., 2012). Thus, four different groups of muscles were gener-
ated for both ages: control (C, right hind limb from untreated animals at
3 and 10 days, n = 5 for each period); leucine supplementation only
(Leu, right hind limb from supplemented animals at 3 and 10 days,
n = 5 for each period); cryolesion only (Cryo, left hind limb from un-
treated animals at 3 and 10 days, n = 5 for each period) and leucine
supplementation combined with cryolesion (Cryo + Leu, left hind
limb from supplemented animals at 3 and 10 days, n = 5 for each
period).
Prior to cryolesion, animals were anesthetized with ketamine and
xylazine (95 and 12 mg/kg body weight, i.p.) and all efforts were
made to minimize suffering. In each animal, the left soleus muscle was
surgically exposed by a lateral incision between the fascias until the
total surface of the muscle was exposed. The cryolesion procedure
consisted of one freeze–thaw cycle of the muscles in situ. In order to
freeze the entire muscle surface, an iron rod was pre-cooled in liquid ni-
trogen and its flat end (0.4 × 1.0 cm) was brought into contact with the
superior surface of the muscle for 10 s (Miyabara et al., 2010; Pereira
et al., 2014a). This procedure was performed in all groups to avoid vary-
ing the amount of muscle injury in each group. Afterwards, the muscles
were thawed and the wounds were closed with 6–0 silk sutures. There-
after, animals were maintained on a warming plate at 37 °C for a few mi-
nutes to avoid hypothermia. In this procedure the recovery is rapid and
no analgesia is necessary (Conte et al., 2012; Miyabara et al., 2010;
Pereira et al., 2014a; Pereira et al., 2014b; Silva et al., 2014). The contra-
lateral soleus muscle (right hind limb) was used as the intact control
(Conte et al., 2012; Miyabara et al., 2010).
On post-cryolesion days 3 and 10, animals received the last
dose of leucine. One hour later (Pereira et al., 2014a; Pereira et al.,
2014b), each rat was again deeply anesthetized with sodium thiopental
(5 mg/100 g body weight, i.p) and the soleus muscles (left and right
hind limb) were removed, weighed and separated into three parts. All
of them were stored at −80 °C until the moment that they were used
for histological analyses, Western blot procedures and proteasome ac-
tivity assay. Afterwards, the animals were euthanized by cervical
dislocation.
2.3. Muscle morphology and quantitative analyses
Soleus muscle cross-sections from young and old animals on day 10
post-injury were used for the morphometric and quantitative analyses.
Each muscle was cut into 10-μm cross-sections on a cryostat (CM3050;
Leica, Nussloch, Germany). Muscle cross-sections were stained with he-
matoxylin and eosin (H&E) to reveal the overall morphology and were
examined under a light microscope (Nikon, Eclipse E1000, ACT-1 ver-
sion 2.2, Melville, New York, NY, USA). Morphometric and quantitative
analyses were conducted with a digitizing unit linked to computer soft-
ware (Image-Pro Plus; Media Cybernetics, Rockville, MD, USA). To de-
termine the myofiber cross-sectional area (CSA; μm2), approximately
 M.G. Pereira et al. / Experimental Gerontology 72 (2015) 269–277
500 myofibers were measured per muscle (n = 5 per group). In the in-
tact groups, the CSA was measured in randomly selected H&E-stained
muscle cross-sections. In the cryolesion groups, the CSA was measured
only in H&E-stained muscle cross-sections obtained from the
regenerating area, i.e., the area characterized by the presence of
myofibers with centralized nuclei, as previously reported (Miyabara
et al., 2010). Five entire muscle cross-sections per animal were used
for quantification of the area of inflammation, which was characterized
by the presence of clear areas between myofibers with inflammatory
cell infiltrates observed in H&E-stained muscles (Miyabara et al.,
2010). The inflammation area was expressed as a percentage of whole
muscle cross-sections (Silva et al., 2014).
2.4. Immunostaining
Soleus muscle cross-sections from young and old animals on day 3
post-injury were used for immunodetection of MyoD and Pax7. Each
muscle was cut into 10-μm cross-sections on a cryostat (CM3050;
Leica, Nussloch, Germany). Muscle cross-sections were fixed with 4%
paraformaldehyde in 0.2 M phosphate buffer (PB) for 10 min at room
temperature, blocked with 0.1 glycine in phosphate-buffered saline
(PBS) for 5 min, and permeabilized in 0.2% Triton X-100/PBS for
10 min. The slides were incubated overnight in a moisture chamber at
4 °C in a solution containing the primary antibodies, 3% normal goat
serum, and 0.3% Triton X-100/0.1 M PB. After washing (three 10-min
washes with 0.1 M PB), the slides were immersed in a solution contain-
ing the respective secondary antibodies and 0.3% Triton X-100/0.1 M PB
for 2 h in a dark room. The slides were again washed in 0.1 M PB (three
10-min washes) and mounted with Vectashield mounting medium
containing 4′,6-diamidino-2-phenylindole (Vector Laboratories) and
cover-slipped.
The primary antibodies used were mouse monoclonal anti-Pax7
(1:40; Development Studies Hybridoma Bank, Iowa, IA, USA), and rabbit
polyclonal anti-MyoD (1:200; Bioss, Woburn, MA, USA). The secondary
antibodies used were FITC-conjugated goat anti-mouse (1:200; Jackson
ImmunoResearch, West Grove, PA, USA) and rhodamine anti-rabbit
(1:200; Abcam Inc., Cambridge, MA, USA). The percentage of proliferat-
ing satellite cells (MyoD+) was determined in a total of Pax7+ satellite
cells. Three entire cross sections for each group were used to count the
proliferating satellite cells.
The stained sections were analyzed in a fluorescence microscope
(Observer D1, Zeiss, Jena, Germany). Figures were mounted using
Adobe Photoshop v7.0, with image manipulation being restricted to
overall threshold and brightness adjustments.
2.5. Western blot analysis
To detect the expression of components of the PI3K/Akt/mTOR path-
way and E3 ligases, muscle samples were homogenized in an extraction
solubilization buffer composed of 90 mM KCl, 10 mM 4-2-
hydroxyethyl-1-piperazineethanesulfonic acid, 3 mM MgCl2+, 5 mM
ethylenediaminetetraacetic acid (EDTA), 1% glycerol, 1 mM dithiothrei-
tol, 0.04% sodium dodecyl sulfate, and a proteinase and phosphatase in-
hibitor cocktail (1:100; Sigma-Aldrich, St. Louis, MO, USA). To
determine the expression of ubiquitinated proteins, muscles were ho-
mogenized in an extraction solubilization buffer, as described by
Pereira et al. (2014a). Homogenates were centrifuged at 12,000 ×g for
10 min at 4 °C, the supernatant was collected, and the protein was quan-
tified using the Bradford assay (Bio-Rad, Hercules, CA, USA) with bovine
serum albumin as the standard (Bradford, 1976). Equal amounts of pro-
tein (50 μg) were separated by electrophoresis on 8%–12% sodium do-
decyl sulfate polyacrylamide gels and transferred to a nitrocellulose
membrane (Bio-Rad, Hercules, CA, USA). The membranes were stained
with Ponceau S to determine the protein content and rinsed with Tris-
buffered saline/Tween solution (0.5 M NaCl; 50 mM Tris–HCl, pH 7.4;
and 0.1% Tween 20). Membranes were incubated overnight at 4 °C
271
with the primary antibodies. After a 30-min wash in Tris-buffered
saline/Tween solution, the membranes were incubated with the sec-
ondary antibodies for 1 h at room temperature. The membranes were
again washed for 30 min in Tris-buffered saline/Tween solution. The la-
beled proteins were detected using the enhanced chemiluminescence
system (ECL; Amersham, Pittsburg, PA, USA) and autoradiography. Den-
sitometry analysis was performed using ImageJ software (Scion Corp.,
National Institutes of Health, Bethesda, MD, USA).
The primary antibodies used for Western blotting were rabbit poly-
clonal antibodies raised against phospho-p70S6K at the Ser371 and Thr389
residues, 4E-BP1, and eIF4E (1:1000; Cell Signaling Technology, Danvers,
MA, USA). In addition, rabbit polyclonal antibodies against ubiquitin
(1:1500; Boston Biochem, Cambridge, MA, USA), MAFbx/atrogin-1
(1:1000; Abcam, Cambridge, MA, USA), and MuRF1 (1:1000; Cell Signal-
ing Technology, Danvers, MA, USA) were used. The secondary antibodies
used were peroxidase-conjugated goat anti-mouse IgG and goat anti-
rabbit IgG (AffiniPure, 1:10,000; Jackson ImmunoResearch Laboratories
Inc., West Grove, PA, USA). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH, 1:1000; Cell Signaling Technology, Danvers, MA, USA) was
used as standard.
2.6. Proteasome activity
The activity of the 26S proteasome was quantitated in the cytosolic
extract of soleus muscles from young and old rats on day 3 post-injury
in the presence of 1 mM ATP. Muscles were disrupted in lysis buffer
(50 mM Tris, pH 7.5, 1 mM ATP—all from Sigma, St. Louis, MO, USA).
Extracts were centrifuged (10,000 ×g, 10 min, 4 °C), protein concentra-
tion was determined using the Bradford method (Bradford, 1976),
and 25 μg of protein from each sample was incubated with 120 mM of
the fluorogenic proteasome substrate Suc-Leu-Leu-Val-Tyr-AMC
(Calbiochem, Darmstadt, Germany), which is specific for the quantifica-
tion of chymotrypsin-like activity. The increase in fluorescence during
substrate cleavage by the proteasome was monitored for 45 min using
a fluorescence spectrophotometer (Molecular Devices, Sunnyvale, CA,
USA).
2.7. Statistical analysis
Data were presented as mean and standard deviation (SD). A
Kolmogorov–Smirnov test was executed to compare the frequency dis-
tribution of myofiber CSA of the groups. For other analyses, multiple
comparisons of mean values were made using the General Linear
Model (analysis of variance). Whenever a significant F-value was ob-
tained, Tukey's post hoc test was performed for the comparison pur-
poses (SAS 9.2 software; SAS Institute Inc., Cary, NC, USA). Values of
p b 0.05 were considered statistically significant.
3. Results
3.1. Muscle morphological features
Histological cross-sections of soleus muscles were examined micro-
scopically on day 10 post-injury, a time point characterized by intense
protein synthesis in order to recover myofiber size (Ciciliot and
Schiaffino, 2010; Pereira et al., 2014b). The myofibers from intact con-
trol muscles from young animals exhibited peripheral nuclei and a po-
lygonal shape (Fig. 1A), whereas those from old animals exhibited
different sizes and shapes and disorganized fascicles (Fig. 1A).
In the young group analyzed after 10 days of leucine supplementa-
tion, there was a significant increase in the myofiber CSA, compared
with that from the age matched control (Fig. 1B) (3810.1 ± 739.6 vs.
2740.7 ± 641.3; 39%, p b 0.05). Similarly, in the old Leu group the sup-
plementation was able to increase the myofiber CSA when compared
with that from the age matched control (Fig. 1B) (4002.6 ± 1089.2 vs.
2936.9 ± 1118.7; 36%, p b 0.05). Accordingly, the percentage of
272 M.G. Pereira et al. / Experimental Gerontology 72 (2015) 269–277

Fig. 1. Histological features of cross-sections of young and old soleus muscles analyzed on day 10 post-injury (A). Frequency distribution of myofiber CSA of young and old soleus muscles
(B). Area of inflammation in young and old soleus muscles (C). C, intact control muscle; Leu, control muscles supplemented only with leucine; Cryo, injured muscles analyzed on day 10
post-injury; and Cryo + Leu, leucine-supplemented injured muscles analyzed day 10 post-injury. Note that the intact control muscles (C) and the leucine-supplemented muscles (Leu)
have a normal morphology. On day 10 post-cryolesion, the muscles from the Cryo and Cryo + Leu group presented regenerating myofibers with centralized nuclei (arrowheads in
panel A), and there was less inflammatory infiltration in the Cryo + Leu group (asterisks in panel A). b, p b 0.05 vs. Cryo from the age-matched group. c, p b 0.05 vs. Cryo from the
young group. d, p b 0.05 vs. Cryo + Leu from the young group. Data are presented as mean and SD. Scale bars correspond to 50 μm.

myofibers larger than 3200 μm2 in the soleus muscles was 78% and 22%
in the young Leu group and its age-matched intact control group, re-
spectively (Fig. 1B). In addition, we found 77% and 39% in the old Leu
group and its age-matched intact control group, respectively (Fig. 1B).
On day 10 post-injury, the muscles from both young and old animals
presented centrally nucleated myofibers with various sizes and inflam-
matory cells (Fig. 1A). In the young group analyzed on day 10 post-
injury, there was a significant reduction in the myofiber CSA, compared
with that from the age matched control (Fig. 1B) (1342.6 ± 418.1 vs.
2740.7 ± 641.3; 51%, p b 0.05). Leucine supplementation was able to
partially revert it, since it was observed a significant increase in the
myofiber CSA in the Cryo + Leu group, compared with that from the
age matched cryolesioned group (Fig. 1B) (1679.2 ± 168.1 vs.
1342.6 ± 418.1; 25%, p b 0.05). Similarly, there was an increase of
myofiber CSA in the old Cryo + Leu group, compared with that from
the age matched cryolesioned group (Fig. 1B) (1764.7 ± 803.4 vs.
1193.8 ± 360.0; 47%, p b 0.05). Accordingly, the percentage of
myofibers larger than 1600 μm2 was 22% and 44% in young soleus mus-
cles from the Cryo group and Cryo + Leu group, respectively (Fig. 1B);
and 12% and 53% in old soleus muscles from these two groups, respec-
tively (Fig. 1B).
The inflammation area in cryolesioned soleus muscles from old ani-
mals at day 10 post-cryolesion was 44% larger than that from the young
rats (p b 0.05, Fig. 1C). Leucine supplementation reduced the inflamma-
tory area in regenerating old muscles to a level similar to that observed
in regenerating young muscles (reduction of 42%, p b 0.05, Fig. 1C).
3.2. Expression of components of the PI3K/Akt/mTOR signaling pathway
There was a similar increase in the phosphorylation of p70S6K at
Ser371 (89%) and at Thr389 (79%) in soleus muscles of young animals
from the Cryo and Cryo + Leu groups evaluated on day 3 post-injury
compared with their age-matched controls (p b 0.05, Fig. 2A and B). In
muscles from old animals, the phosphorylation of p70S6K increased at
Ser371 (33% and 53%) and at Thr389 (90% and 100%) in the Cryo and
Cryo + Leu groups, respectively, compared with their age-matched
controls (p b 0.05, Fig. 2C and D).
By contrast, the expression of 4E-BP1 and eIF4E decreased 80% and
45%, respectively, in young muscles from the Cryo group compared
with their age-matched intact controls (p b 0.05, Fig. 2A and B). Similar-
ly, the expression of 4E-BP1 and eIF4E decreased 80% and 65%, respec-
tively, in old muscles from the Cryo group compared with their age-
matched controls (p b 0.05, Fig. 2C and D). Leucine promoted a four-
fold increase in the expression of 4E-BP1 in young muscles in the
Cryo + Leu group compared with its age-matched Cryo group
(p b 0.05, Fig. 2A and B) and prevented the decrease in the expression
of eIF4E in young muscles in the Cryo + Leu group compared with its
age-matched Cryo group (p b 0.05, Fig. 2A and B). In old muscles, the ex-
pression of 4E-BP1 and eIF4E increased 72% and 53%, respectively, in the
Cryo + Leu group compared with its age-matched Cryo group (p b 0.05,
Fig. 2C and D). In addition, there were no changes in the expression level
of these proteins in young and old muscles between the Leu group and
the intact control group.
3.3. Expression of E3-ligases
The expression levels of the E3-ligases MAFbx/atrogin-1 and MuRF1
were quantitated in both young and old muscles on day 3 post-injury;
since it was previously demonstrated that, at this time point, FoXO3a ac-
tivation and ubiquitinated protein accumulation started showing signif-
icant changes (Pereira et al., 2014a). In young muscles, no changes were
observed in the expression levels of both MAFbx/atrogin-1 and MuRF1
 M.G. Pereira et al. / Experimental Gerontology 72 (2015) 269–277 273

Fig. 2. Representative Western blots of elements of the PI3K/Akt/mTOR signaling pathway and E3-ligases. Young (A) and old (C) muscle groups are identified at the top. C, intact control
muscle; Leu, control muscles supplemented only with leucine; Cryo, injured muscles analyzed on day 3 post-injury; and Cryo + Leu, leucine-supplemented injured muscles analyzed day 3
post-injury. Densitometry analyses of elements of the PI3K/Akt/mTOR signaling pathway and E3-ligases in young (B) and old soleus muscles (D). Blots were reacted with antibodies spe-
cific for p70S6K phosphorylation at Ser371 and Thr389 residues, 4E-BP1, eIF4E, atrogin-1, MuRF1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as standard. a,
p b 0.05 vs. intact control soleus muscle; b, p b 0.05 vs. Cryo from the age-matched group. Data are presented as mean and SD.

in any groups analyzed (Fig. 2A and B). In old muscles, the expression
level of MAFbx/atrogin-1 did not change in all groups evaluated; how-
ever, MuRF1 expression level decreased 65% in the Cryo group
compared with the level in the age-matched control group (p b 0.05,
Fig. 2C and D). Leucine supplementation alone did not change MuRF1
expression compared with the age-matched control (Fig. 2C and D),
but it increased MuRF1 expression in cryolesioned old muscles to a
level similar to that of the age-matched control (Fig. 2C and D).
3.4. Accumulation of ubiquitinated proteins, and determination of protea-
some activity
The expression of ubiquitin-conjugated proteins in old intact control
muscles showed a tendency of increase (20%) compared with that ob-
served in young intact control muscles (p = 0.72, Fig. 3A). In young
muscles, the ubiquitin-conjugated proteins increased 60% in the Cryo
group compared with those in the control group (p b 0.05, Fig. 3A). In
aged muscles, this increase in the amount of ubiquitinated proteins
was reduced by 20% in the Cryo group compared with the levels in the
age-matched control group (p b 0.05, Fig. 3A). There was no difference
in the ubiquitin-conjugated proteins in young and old muscles from
the Leu groups compared with those in their age-matched controls
(Fig. 3A). The ubiquitin-conjugated proteins decreased 30% in young
muscles from the Cryo + Leu group compared with those in the age-
matched Cryo group (p b 0.05, Fig. 3A). However, the amount of
ubiquitinated proteins increased 30% in aged muscles from the
Cryo + Leu group compared with those in the age-matched Cryo
group (p b 0.05, Fig. 3A).
The proteasome activity levels displayed a trend of decrease in intact
control soleus muscles from old animals compared with those from
young animals (25%, p = 0.79, Fig. 3B). In addition, a trend of reduction
in activity levels was observed in cryolesioned young muscles, but not in
old muscles, compared with those from the age-matched intact control
(20%, p = 0.92, Fig. 3B). In the Leu group, the proteasome activity levels
showed a tendency of increase in young muscles compared with those
in the age-matched intact control (25%, p = 0.73, Fig. 3B); and, this in-
crease was significant in old muscles (44%, p b 0.05, Fig. 3B). The protea-
some activity levels increased 100% in young muscles from the
Cryo + Leu group compared with those in the age-matched Cryo
group (p b 0.05, Fig. 3B). On the other hand, there was only a trend of
increase in these levels in old muscles from the Cryo + Leu group
compared with those in the age-matched Cryo group (23%, p = 0.82,
Fig. 3B).
3.5. Proliferating satellite cell count
Histological cross sections of soleus muscles from Cryo and
Cryo + Leu groups, from both young and old animals, collected on
day 3 post-injury were immunostained against Pax7 and MyoD; in
order to detect the incidence of proliferating MyoD+ satellite cells
from a total of Pax7+ satellite cells. A similar percentage of proliferating
satellite cells was observed in both young and old muscles from Cryo
groups (39% and 38%, respectively, Fig. 4). Leucine supplementation
274 M.G. Pereira et al. / Experimental Gerontology 72 (2015) 269–277

Fig. 3. Representative Western blots and densitometry analyzes of the amount of ubiquitinated proteins in both young and old animal groups (A). Muscle groups are identified at the top.
C, intact control muscles; Leu, control muscles supplemented only with leucine; Cryo, injured muscles analyzed on day 3 post-injury; and Cryo + Leu, leucine-supplemented injured mus-
cles analyzed day 3 post-injury. Blots were reacted with antibodies specific for ubiquitinated proteins. 26S proteasome activity of soleus muscles from both young and old animals analyzed
on day 3 post-injury (B). a, p b 0.05 vs. intact control soleus muscles; b, p b 0.05 vs. Cryo from the age-matched group. Data are presented as mean and SD.

increased the percentage of proliferating satellite cells in both young
and old regenerating muscles (Cryo + Leu groups) compared with
that in age-matched Cryo groups (34% and 39%, respectively, p b 0.05,
Fig. 4).
4. Discussion
The success of the skeletal muscle regenerative process is funda-
mental to maintain skeletal muscle function throughout life. In the pres-
ent study, we aimed to investigate the effect of leucine supplementation
on the regenerative capacity of skeletal muscles from senescent rats.
Based on our previous work (Pereira et al., 2014a), we chose to perform
the first measurements at day 3 post-injury, since at this time point,
we started seeing the effects of leucine supplementation on young
regenerating muscles, i.e., attenuation of inflammatory cell infiltration,
FOXO3 activation and ubiquitinated protein accumulation. In general,
our data showed that leucine supplementation improved the structural
recovery of regenerating soleus muscles from old rats. This recovery in-
volved an increase in the number of proliferating satellite cells, and
modulation of components of the PI3K/AKT/mTOR pathway and the
ubiquitin–proteasome system.
A significant increase in the CSA of regenerating myofibers was ob-
served in soleus muscles from leucine-supplemented old animals ana-
lyzed at day 10 post-injury compared with the only injured control
muscles. A similar result was observed in young animals, which is in
agreement with our previous study, in which leucine supplementation
for 10 days increased the CSA of regenerating myofibers from young so-
leus muscles (Pereira et al., 2014a). The increased myofiber size in
regenerating muscles from old animals supplemented with leucine
led us to investigate the involvement of some components of the
PI3K/Akt/mTOR signaling pathway and ubiquitin–proteasome system.
Previous studies have shown that a single dose of leucine can trigger
protein synthesis through the activation of the PI3K/Akt/mTOR signal-
ing pathway (Crozier et al., 2005; Lang et al., 2003; Murgas Torrazza
et al., 2010; Vary and Lynch, 2007). In our previous work (Pereira
et al., 2014a), we had examined the activation of elements from PI3K/
Akt/mTOR signaling pathway in muscles supplemented with leucine
and analyzed at 10 days post-cryolesion. However, we did not observe
significant effects of leucine supplementation on that. Therefore, in the
present study we decided to evaluate some components of this pathway
in an earlier time point, i.e. three days post-injury.
A decrease in the expression of 4E-BP1 and eIF4E was observed in in-
jured soleus muscle from both young and old rats and herein leucine
was able to revert it. An intense inflammatory process may be responsi-
ble for the decrease in 4E-BP1 and eIF4E expression, considering that
previous studies have shown that the release of pro-inflammatory cyto-
kines and reactive oxygen species can inhibit the formation of the
mTORC1 complex and decrease the phosphorylation of 4E-BP1 (Frost
and Lang, 2011; Lang and Frost, 2004). The decreased muscle inflamma-
tion area observed in this study is a possible mechanism by which pro-
tein expression was restored. In addition, we have previously shown
that leucine does not alter the expression of components of the PI3K/
Akt/mTOR signaling pathway in the tibialis anterior muscle from
young rats analyzed at day 10 post-injury. In this case, an increase in
the rate of protein synthesis was observed in injured muscles from
leucine-supplemented animals (Pereira et al., 2014b). Therefore, it is
possible to hypothesize that the activation of the PI3K/Akt/mTOR signal-
ing pathway during a short period (3 days) stimulated the increase of
protein synthesis and sustained this increase for 10 days after injury.
Moreover, it is possible that leucine may affect other intracellular path-
ways involved in the control of muscle tropism, such as β-adrenergic
and Bone Morphogenetic Protein (BMP), pathways that contribute to
muscle mass gain (Minetti et al., 2011; Sartori et al., 2013).
The attenuation of the inflammation area in old muscles from
leucine-supplemented animals also occurred in young muscles (Pereira
et al., 2014a) and could be associated with the effect of branched-chain
amino acids on inflammation via glutamate transamination. This process
increases the synthesis of glutamine, an amino acid highly metabolized
by inflammatory cells in some diseases or pathological conditions
(Nicastro et al., 2012). Therefore, we can hypothesize that the
 M.G. Pereira et al. / Experimental Gerontology 72 (2015) 269–277 275

Fig. 4. Percentage of proliferating (MyoD+) satellite cells (SC) in a total of Pax7+ SC, analyzed on day 3 post-injury. Cryo, muscles analyzed on day 3 post-injury, and Cryo + Leu, leucine-
supplemented muscles analyzed on day 3 post-injury. Arrowheads indicate Pax7, MyoD, DAPI (nucleus staining), and merged images represent MyoD+/Pax7+ cells. b, p b 0.05 vs. age-
matched Cryo group. Data are presented as mean and SD. Scale bars correspond to 50 μm.

inflammatory process was accelerated in old muscles stimulated by in-
crease in energy substrate.
Age-related muscle adaptations are due, in part, to the disrupted
turnover of muscle proteins, a process mediated by the ubiquitin–pro-
teasome system. In order to evaluate whether leucine modulates pro-
tein degradation during the regenerative process, favoring skeletal
muscle mass integrity, we evaluated some components of the
ubiquitin–proteasome system. Initially, we observed that regenerating
soleus muscles from both young and old animals had high levels of
ubiquitinated proteins at day 3 post-injury, which is in agreement
with the results of a previous study (Miyabara et al., 2010). Leucine sup-
plementation decreased the amount of ubiquitinated proteins in young
regenerating muscles but increased these levels, even further, in old
regenerating muscles. Such effects were not accompanied by changes
in the expression of MAFbx/atrogin-1 and MuRF1 in young muscles.
However, MuRF1 expression decreased in injured soleus muscles in
the old group, and this decrease could be related to the involvement
of MuRF1 in the maintenance of sarcomere structure, being associated
to the M-line (Centner et al., 2001; Franke et al., 2014). In this respect,
leucine may contribute to the recovery of MuRF1 expression in old
regenerating muscles, thereby favoring the reestablishment of muscle
structure.
We also observed that leucine supplementation increased the pro-
teasome activity levels in regenerating muscles from young animals,
but not from old muscles. It suggests that leucine promoted the efficient
recycling of proteins only in young muscles, leading to the decreased ac-
cumulation of ubiquitinated proteins. On the other hand, the unaltered
proteasome activity in leucine-supplemented regenerating muscles
from old animals, evaluated on day 3 post-injury, could be associated
with a delay or impairment in the proteasome activity of injured mus-
cles upon leucine supplementation during aging. These results are cor-
roborated, at least in part, by a recent study (Baehr et al., 2014), which
reported an increase in the proteasome activity in a functional
overload-induced hypertrophy model; suggesting that the activation
of the ubiquitin–proteasome system is essential for skeletal muscle
plasticity. Moreover, it was recently reported that the proteasome activ-
ity is fundamental for muscle growth and development (Kitajima et al.,
2014). In the present study, the increase in proteasome activity was not
accompanied by changes in the expression of E3-ligases MAFbx/
atrogin-1 and MuRF1. Indeed, previous studies have shown an apparent
276 M.G. Pereira et al. / Experimental Gerontology 72 (2015) 269–277
disconnection between the expression of these E3-ligases and muscle
proteolytic activity (Baehr et al., 2011; Baehr et al., 2014; Vary et al.,
2008). However, the possibility that other E3-ligases are involved in
the regulation of muscle proteolysis should be considered (Bodine and
Baehr, 2014).
It was observed that the number of proliferating satellite cells in old
muscles was similar to that of young muscles after injury, which is in
line with the study of Sousa-Victor et al. (2014). In addition, leucine in-
creased the number of proliferating satellite cells to a similar level in
both young and old group muscles. It is possible that the accelerated in-
flammatory process during the regeneration of leucine supplemented
muscles may lead to the early activation and proliferation of satellite
cells, which corroborates their increased number at day 3 post-injury.
There is evidence that supplementation with HMB, a leucine metabolite,
promotes the increase in the proliferation of satellite cells in muscles of
aged rats during recovery from disuse atrophy (Alway et al., 2013).
Moreover, based on our previous studies it is possible to speculate
that leucine can decrease the expression of proteins that form the con-
nective tissue, which may contribute to the remodeling of the extracel-
lular matrix during the muscle regenerative process (Pereira et al.,
2014a; Pereira et al., 2014b). In this respect, studies have suggested
that the adequate structuring of the extracellular matrix is essential
for satellite cell function owing to the preservation of the satellite cell
niche (Bentzinger et al., 2013; Urciuolo et al., 2013).
In summary, our findings suggest that leucine improves the regener-
ation of skeletal muscles from old rats. This beneficial effect involves the
preservation of some biological responses upon leucine supplementa-
tion, including the increase in the number of proliferating satellite
cells and size of regenerating myofibers, combined with the modulation
of components of the PI3K/Akt/mTOR pathway and ubiquitin–protea-
some system. These findings may contribute to future strategies aiming
to accelerate the recovery of old skeletal muscles in clinical cases in
which the appropriate muscle repair can significantly decrease rehabil-
itation time.
Conflicts of interest
The authors declare that there are no conflicts of interest associated
with this manuscript.
Acknowledgments
The authors are grateful to Dr. Alicia J. Kowaltowski from the Chem-
istry Institute at the University of São Paulo for allowing the use of the
fluorimeter from her laboratory, and Rosana Prisco for her assistance
with the statistical analyses.
This work was funded by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP, Grant Nos. 2012/15276-9, 2012/21238-
2, 2011/50688-3, 2012/50500-7 and CEPID Redoxoma 2013/07937-8);
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, Grant No. 471162/2012-4).
M. G. P. and M. T. S. received funding from FAPESP (Grant Nos. 2010/
52520-0 to M.G.P.; 2009/54240-7 and FAPESP/CAPES 2014/13874-1 to
M.T.S.).
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