J Appl Physiol 118: 742749, 2015.

First published January 8, 2015; doi:10.1152/japplphysiol.00054.2014.

Mixed lactate and caffeine compound increases satellite cell activity

and anabolic signals for muscle hypertrophy
Yoshimi Oishi,1 Hayato Tsukamoto,1 Takumi Yokokawa,2 Keisuke Hirotsu,3 Mariko Shimazu,3
Kenji Uchida,3 Hironori Tomi,3 Kazuhiko Higashida,4,5 Nobumasa Iwanaka,5 and Takeshi Hashimoto1,5
1

Graduate school of Sport and Health Science, Ritsumeikan University, Shiga; 2Graduate school of Human and
Environmental Studies, Kyoto; 3Central Research and Development Laboratory, Kobayashi Pharmaceutical, Osaka; 4Faculty
of Sport Science, Waseda University, Saitama; and 5Faculty of Sport and Health Science, Ritsumeikan University, Shiga,
Japan

Submitted 24 January 2014; accepted in final form 15 December 2014

Oishi Y, Tsukamoto H, Yokokawa T, Hirotsu K, Shimazu M,

Uchida K, Tomi H, Higashida K, Iwanaka N, Hashimoto T. Mixed
lactate and caffeine compound increases satellite cell activity and anabolic signals for muscle hypertrophy. J Appl Physiol 118: 742749, 2015.
First published January 8, 2015; doi:10.1152/japplphysiol.00054.2014.
We examined whether a mixed lactate and caffeine compound (LC)
could effectively elicit proliferation and differentiation of satellite
cells or activate anabolic signals in skeletal muscles. We cultured
C2C12 cells with either lactate or LC for 6 h. We found that lactate
significantly increased myogenin and follistatin protein levels and
phosphorylation of P70S6K while decreasing the levels of myostatin
relative to the control. LC significantly increased protein levels of
Pax7, MyoD, and Ki67 in addition to myogenin, relative to control.
LC also significantly increased follistatin expression relative to control and stimulated phosphorylation of mTOR and P70S6K. In an in
vivo study, male F344/DuCrlCrlj rats were assigned to control (Sed,
n 10), exercise (Ex, n 12), and LC supplementation (LCEx, n
13) groups. LC was orally administered daily. The LCEx and Ex
groups were exercised on a treadmill, running for 30 min at low
intensity every other day for 4 wk. The LCEx group experienced a
significant increase in the mass of the gastrocnemius (GA) and tibialis
anterior (TA) relative to both the Sed and Ex groups. Furthermore, the
LCEx group showed a significant increase in the total DNA content of
TA compared with the Sed group. The LCEx group experienced a
significant increase in myogenin and follistatin expression of GA
relative to the Ex group. These results suggest that administration of
LC can effectively increase muscle mass concomitant with elevated
numbers of myonuclei, even with low-intensity exercise training, via
activated satellite cells and anabolic signals.
skeletal muscle cells; low-intensity exercise; sarcopenia; mTOR;
follistatin
SATELLITE CELLS ARE MYOGENIC precursor cells located between
the sarcolemma and the basal lamina of muscle fibers. These
cells regulate myofiber repair, maintenance, and growth (25,
35). Satellite cells are normally maintained in a quiescent state;
however, when the muscle is damaged by exercise, these cells
reenter the cell cycle. Upon reentry, some cells generate new
muscle fibers and provide additional myonuclei to the parent
fiber, while others return to a quiescent status.
Sarcopenia, defined as the gradual decline in skeletal muscle
mass and strength with aging, is a serious problem for the
elderly (39). Age-related loss of muscle mass and strength may
be attributed to a decrease in the number of satellite cells

Address for reprint requests and other correspondence: T. Hashimoto,

Faculty of Sport & Health Science, Ritsumeikan University, 1-1-1 Nojihigashi,
Kusatsu, Shiga 525-8577, Japan (e-mail: thashimo@fc.ritsumei.ac.jp).
742

and/or a decline in their ability to become active and to

proliferate in response to anabolic stimuli (6, 7, 29, 43).
Although resistance training or high-intensity exercise training
can increase the number of satellite cells after myofiber inflammation in both the elderly and the young (28, 48), there is little
evidence showing that low-intensity endurance training increases satellite cell activity.
In addition to satellite cell activity, stimulation of anabolic
signals such as mTOR and P70S6K is important for muscle
hypertrophy. Indeed, Sandri (45) demonstrated that blocking
anabolic signals resulted in muscle atrophy or impaired protein
synthesis. Both resistance training and relatively high-intensity
aerobic exercise could stimulate anabolic signals. Camera et al.
(4) reported that either resistance training for eight sets of five
repetitions at 80% 1 repetition maximum (RM) or cycling
O2 peak could increase Akt,
exercise for 60 min at 70% V
mTOR, and P70S6K phosphorylation. Furthermore, Vissing et
al. (49) reported that 10 wk of resistance training, but not
cycling training, increased Akt, mTOR, and P70S6K phosphorylation. Altogether, both satellite cell activity and anabolic
signals are increased by high-intensity training (16, 41). Because low-intensity exercise is more feasible for the elderly,
developing a method to effectively increase satellite cell activity and anabolic signals in low-intensity exercise would be
protective against sarcopenia.
Myogenin, a myogenic regulatory factor, regulates myogenesis and differentiation of muscle cells (50). When myogenin
expression increases, satellite cells enter the differentiation of
the cell cycle (12). Although myogenin expression is increased
by a single bout of high-intensity exercise (13), our previous
study demonstrated that sodium lactate treatment increases
myogenin mRNA in L6 cells (24). Additionally, the calcium/
calmodulin-dependent serine phosphatase calcineurin regulates
muscle hypertrophy in mature rats (17). Indeed, overexpression
of calcineurin induces slow and fast fiber hypertrophy (46).
Caffeine increases intracellular calcium, which activates calcineurin (1, 30, 33). Based on these data, we hypothesized that
a lactate-based supplement containing caffeine, an activator of
intracellular calcium signals (33), could effectively elicit proliferation and differentiation of satellite cells, activate anabolic
signals in skeletal muscle, and thereby increase muscle mass
when combined with low-intensity exercise training.
To assess this hypothesis, we initially examined whether
lactate and/or lactate-caffeine (LC) treatment could elicit proliferation and differentiation of satellite cells or activate anabolic signals in C2C12 skeletal muscle cells. Furthermore, we
examined whether the administration of a mixed lactate and

8750-7587/15 Copyright 2015 the American Physiological Society

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Lactate-Caffeine Supplement Increases Muscle Mass

caffeine compound (LC compound), concomitant with endurance exercise training, could effectively increase muscle mass
via activated satellite cells and/or anabolic signals in rat skeletal muscle.
MATERIALS AND METHODS

Cell culture protocol. All cell culture reagents were obtained from
Wako (Osaka, Japan) unless otherwise mentioned. Cell culture was
performed as previously described (24). The majority of experiments
aimed at determining the physiology of satellite cells have been
conducted on immortalized myogenic cell lines (10). Among myogenic cell lines, which have been the most appropriate for examining
the biochemical and genetic pathways that direct muscle regeneration,
C2C12 cell transcriptional and cell-signaling responses remain sufficiently similar to those of primary embryonic and adult myoblasts,
such that they have been used broadly and successfully to model both
(10). Thus, we used C2C12 cells to examine the hypertrophic effects
of lactate or LC compound in vitro. Briefly, C2C12 cells were
maintained in DMEM supplemented with 10% FBS. For differentiation, confluent cells (Day 0) were treated with differentiation media
(DMEM supplemented with 2% horse serum). Differentiation media
were replaced every 2 days up to the experimental day.
Differentiated C2C12 cells were incubated in differentiation media
containing either 5 mM caffeine (Wako; 031 06792), 10 mM sodium
lactate (Wako; 195-05965), or 10 mM sodium lactate plus 5 mM
caffeine (LC) for 6 h. Cells were harvested after 6 h. We evaluated
anabolic signals and satellite cell proteins using Western blotting (n
9 in each group).
Animal care. Ethical approval for this study was obtained from the
Committee on Animal Care of Ritsumeikan University. Male F344/
DuCrlCrlj rats (9 wk old) were obtained from Charles River Japan
(Kanagawa, Japan) and cared for according to the Guide for the Care
and Use of Laboratory Animals based on the Declaration of Helsinki.
Rats were housed individually in an animal facility under controlled
conditions (12:12-h light-dark cycle). Animals were acclimated for 23
days, which included ad libitum access to food and water and a
purified normal diet (CRF-1; Oriental Yeast, Tokyo, Japan). After 23
days of acclimatization, the animals were randomly assigned to three
groups: sedentary control (Sed, n 10), exercise training (Ex, n
12) and exercise training plus LC compound (LCEx, n 13). During
the 4 wk of the experiment, individually housed rats were given the
purified normal diet (CRF-1) at 13 g/day.
Exercise training and oral administration of LC supplement
protocol. Rats ran on a treadmill (TREDMILL TK-175, Japan) for 30
min at 20 m/min without incline every other day for 4 wk. Exercise
intensity was kept constant during the training period. Then 48 h after
the final bout of exercise training, gastrocnemius (GA) and tibialis
anterior (TA) muscles were quickly removed, weighed, rinsed in
ice-cold saline, and frozen in liquid nitrogen.
In the LCEx group, sodium lactate (Wako, 195-05965; 1,000 mg/kg
body wt) and caffeine (Wako, 031 06792; 36 mg/kg body wt) were
administered to the rats by using an oral sonde every day for 4 wk. In
the LC compound, sodium lactate and caffeine were dissolved in
sterile purified water (sodium lactate: 200 mg/ml; caffeine: 7.2 mg/
ml), and all other excipients were excluded. The same volume of
sterile purified water was administered to the Sed and Ex groups as
that in the vehicle control group.
Blood lactate level. Rats were run on a treadmill for 30 min at 20
m/min without incline. Before treadmill exercise, the rats orally
received a LC supplement (n 4) and water (n 4). After treadmill
exercise, lactate concentration was measured from tail blood using the
Lactate Pro2 blood lactate test meter (ARKRAY; Kyoto, Japan).
Western blotting. C2C12 cells were washed with PBS and directly
dissolved in heated SDS-PAGE sample buffer. Aliquots of the extracts were subjected to SDS-PAGE and transferred to a nitrocellulose
membrane. Tissue samples (30 mg each) were homogenized in 2 ml

Oishi Y et al.

743

of buffer containing 10 mM Tris (pH 7.4), 1% NP-40, 1% sodium

deoxycholate, 0.1% SDS, 150 mM sodium chloride, 5 mM EDTA, 1
mM PMSF, a protease inhibitor mixture (Sigma-Aldrich, St. Louis,
MO), and phosphatase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Samples were incubated on ice for 20 min. Tissues
were then homogenized using a Potter-Elvehjem tissue homogenizer
(AS ONE, Osaka, Japan) on ice with 10 15 gentle strokes on a
motor-driven pestle at 2,500 rpm. The protein extracts (10 g) were
subjected to SDS-PAGE and transferred to a nitrocellulose membrane.
Proteins were probed with antibodies against paired box protein
(Pax7; ab34360; Abcam, Cambridge, United Kingdom), myogenic
differentiation factor (MyoD; sc32758; Santa Cruz Biotechnology,
Santa Cruz, CA), myogenin (sc12732; Santa Cruz Biotechnology),
proliferating cell nuclear antigen (PCNA; P8825; Sigma), cyclin-D1
(SAB4502602; Sigma), myostatin (Mstn; sc34781; Santa Cruz Biotechnology), follistatin (Fst; av42287; Sigma), mTOR (2983; Cell
Signaling, Danvers, MA), phosphorylated-mTOR (5536; Cell Signaling), P70S6K (2708; Cell Signaling), phosphorylated-P70S6K (9205
Cell Signaling), and GAPDH (G9545; Sigma). The immunoblotted
membranes were scanned with ImageQuant LAS-4000 (GE Healthcare Life Science, Fairfield, CT) luminescent image analyzer, and the
optical density of each specific band was analyzed with ImageQuant
TL software (GE Healthcare). Analyzed bands (n 9 and n 8 for
C2C12 and rats, respectively) were normalized to GAPDH.
Immunocytochemistry. For the immunostaining of Ki67, a marker
of proliferating cells, differentiated C2C12 cells cultured on the
coverslips were fixed with PBS containing 3.7% paraformaldehyde
for 15 min at room temperature. Fixed cells were permeabilized in
0.2% Triton X-100/PBS for 10 min and blocked with 1% BSA/PBS.
Cells were then incubated with primary antibodies against Ki67
(Abcam; ab15580), myosin (skeletal, fast; M4276; Sigma), and MyoD
(sc32758; Santa Cruz Biotechnology) overnight, washed with PBS,
and incubated for 1 h with secondary antibody conjugated with Alexa
488 or 633 (Molecular Probes, Grand Island, NY). Furthermore,
Hoechst 33342 (Molecular Probes) was used for nucleic acid staining.
After they were washed with PBS, cells on the coverslips were
mounted and observed under a fluorescence microscope (Biorevo,
BZ-9000; Keyence, Osaka, Japan). For each condition, 18 pictures of
cells (n 6, three pictures for each coverslip) were taken with a 20
objective, and the total number of nuclei (10,000 nuclei in total
under each condition) was counted using a BZ-II image analysis
application (Keyence). The number of Ki67-positive nuclei was measured, and the ratio of Ki67-positive cells to the total number of nuclei
(except for the nuclei located within MHC-positive myotubes) was
calculated. In addition, the differentiation index (%), defined as the
percentage of MHC-positive nuclei divided by total nuclei (42), was
also calculated.
DNA content and myofibrillar protein concentration measurement.
Frozen muscles were powdered, and genomic DNA was extracted
using a DNA extraction kit (NucleoSpin Tissue; MACHEREYNAGEL, Dren, Germany) by following the manufacturers instructions. A quantitative determination of DNA concentration was performed using NanoDrop 2000 spectrophotometers (Thermo Fisher
Scientific, Waltham, MA).
Procedures to determine the myofibrillar protein concentration
were carried out as described by Tsutaki et al. (47).
Statistical analysis. Data were tested for normality (chi-squared
test) and equality of variance (Fisher-Snedecor F-test). When both
conditions were met, an unpaired t-test or one-way analysis of
variance was performed, as appropriate. When the normality or
equality of variance conditions was not met, the variables were
analyzed using the Welch t-test, Mann-Whitney U-test, or KruskalWallis test, as appropriate. The Bonferroni/Dunn or Steel-Dwass post
hoc test was used as needed, and the significance level was set at P
0.05. All results are presented as means SE.

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Lactate-Caffeine Supplement Increases Muscle Mass

RESULTS

Oishi Y et al.

LC

Con

Protein content
(Arbitrary Units)

60kDa

*
1

0
Con

LC

MyoD
50kDa

LC

Con

2 40kDa
Protein content
(Arbitrary Units)

Effect of lactate, caffeine, or LC on satellite cell activity in

C2C12 muscle cells. Initially, we examined whether lactate,
caffeine, or LC treatment in C2C12 cells would affect satellite
cell activity. Because activated satellite cells show increased
expression of MyoD [one of the markers of satellite cell
proliferation (25)] and myogenin [a marker of skeletal muscle
differentiation (11)], we assessed the expression of these proteins. As shown in Fig. 1C, lactate significantly increased
myogenin protein levels relative to control. On the other hand,
LC significantly increased Pax7 and MyoD protein levels
relative to control, in addition to myogenin (Fig. 1, AC);
however, caffeine did not increase Pax7, MyoD, or myogenin
expression (data not shown). Furthermore, LC significantly
increased MyoD protein levels relative to lactate (Fig. 1B).
Effect of lactate, caffeine, or LC on the cell cycle in C2C12
muscle cells. We also examined whether lactate, caffeine, or
LC treatment would affect the cell cycle in C2C12 cells. No
treatment affected PCNA, a useful indicator that satellite cells
have entered into the cell cycle, or cyclin-D1, an important cell
cycle regulator (22, 27) (data not shown). However, the LC
treatment resulted in a significant increase in Ki67, a marker of
cellular proliferation, relative to control (Fig. 2B). In addition,
the LC treatment resulted in a significant increase in the
differentiation index (Fig. 2C).
Effect of lactate, caffeine, or LC on muscle anabolic signals
in C2C12 muscle cells. We next assessed the protein expression of Fst, an essential protein for muscle fiber formation and
growth (38), and Mstn, a transforming growth factor- superfamily member and a negative regulator of skeletal muscle
growth (37). Lactate significantly increased Fst expression and
decreased Mstn expression relative to control (Fig. 3, A and B),
whereas caffeine did not alter Fst or Mstn expression. LC also
significantly increased Fst expression but did not alter the
expression of Mstn (Fig. 3, A and B).
We also examined whether lactate, caffeine, or LC treatment
in C2C12 cells could enhance anabolic markers such as
mTOR, Akt, and P70S6K phosphorylation. Compared with the
control, caffeine significantly increased the phosphorylation of
mTOR and Akt by 1.5-fold and 1.7-fold, respectively (P
0.01). On the other hand, lactate significantly increased phosphorylation of P70S6K, a critical regulator of exercise-induced
muscle protein synthesis and training-induced hypertrophy
(Fig. 4B). LC significantly increased both mTOR and P70S6K
phosphorylation (Fig. 4, A and B) but did not change Akt
phosphorylation (data not shown).
Effects of exercise training and the LC compound on muscle
weight, DNA content, and myofibrillar protein concentration in
rat skeletal muscle. During the 4 wk of the experiment, no rats
showed any abnormalities such as diarrhea or a decrease in
body weight, and there was no significant difference in food
consumption between the groups (13 gday1individual1). In
the basal condition (before treadmill running), blood lactate
levels were 2.38 0.18 mM. Immediately after treadmill
exercise, blood lactate concentration in the Ex group was
1.93 0.16 mM, while in the LCEx group was 7.85 2.63
mM. The blood lactate level of the LCEx group was significantly higher than in the Ex group. No significant differences
in body weight were found between any groups after 4 wk of
the experiment. The Ex group increased muscle mass normal-

Pax7

**
1

0
Con

LC

myogenin
40kDa

2
Protein content
(Arbitrary Units)

744

LC

Con

30kDa

**

**

LC

0
Con

Fig. 1. Effect of lactate or lactate-caffeine on satellite cell activity in C2C12

muscle cells. Protein expression of paired box protein (Pax7; A), myogenic
differentiation factor (MyoD; B), and myogenin (C) is shown with representative immunoblots. C2C12 cells were incubated with media containing 10
mM sodium lactate 5 mM caffeine (LC) or 10 mM sodium lactate (L) for
6 h. L increased myogenin expression compared with control (Con). LC
significantly increased Pax7, MyoD, and myogenin expression compared with
Con, and increased MyoD expression compared with L. *P 0.05, **P
0.01 vs. Con.

ized to total body weight in GA and TA relative to the Sed

group. However, the LCEx group increased muscle mass normalized to body weight in GA and TA relative to both the Sed
and Ex groups (Table 1).
The myofibrillar protein concentration was not changed by
exercise training (Ex) or by exercise with the LC compound
(LCEx; data not shown). The LCEx group experienced a significant increase in the total DNA content of TA muscle compared with the Sed group (Table 2).

J Appl Physiol doi:10.1152/japplphysiol.00054.2014 www.jappl.org

Lactate-Caffeine Supplement Increases Muscle Mass

745

Oishi Y et al.

number of activated satellite cells in vivo. There was a significant increase in myogenin expression in GA and TA in the
LCEx group relative to the Sed and Ex groups (Fig. 5).
Effect of exercise training and the LC compound on Fst and
Mstn expression in rat skeletal muscle. Next, we assessed the
protein expression of Fst and Mstn in GA and TA. There was
a significant increase in Fst expression in the LCEx group
compared with GA in both the Sed and Ex groups (Fig. 6A).
There was significantly increased Fst expression in TA in the
LCEx group relative to the Sed group (Fig. 6B). Mstn expression in TA was decreased in the LCEx group relative to the Sed
group (Fig. 7B), but this was not seen in GA (Fig. 7A).

**

The main findings of this study were that LC treatment

increased satellite cell activity and anabolic signals in C2C12
cells and that low-intensity exercise with a mixed lactate and
caffeine compound increased muscle mass, satellite cell activity, and anabolic signals in vivo.
In our in vitro study, we found that lactate and LC directly
affect muscle cells to increase satellite cell activity as well as
mTOR and P70S6K phosphorylation (Fig. 3). We had previously suggested that lactate elicits a large number of responses
that coordinate metabolism in skeletal muscle cells as a functional adaptation to exercise (24). These responses include

Fst

A
40kDa

Fig. 2. Effects of lactate or LC on the cell cycle in C2C12 muscle cells. A:

differentiated C2C12 cells were immunostained for Ki67 (green), myosin
heavy chain (MHC; red), and Hoechst 33342 (blue). Bar, 100 m. B: the
number of Ki67-positive nuclei was measured and the ratio of Ki67-positive
nuclei to total number of nuclei, except for the nuclei located within MHCpositive myotubes, was calculated. C: differentiation index (%), defined as the
percentage of MHC-positive nuclei divided by total nuclei, was calculated.
*P 0.05, **P 0.01 vs. Con.

Protein content
(Arbitrary Units)

Differentiation Index (%)

Ratio of Ki67 positive

nuclei to total nuclei not
located within myotubes

DISCUSSION

30kDa

**
**

0
Con

Protein content
(Arbitrary Units)

LC

Mstn

Effects of exercise training and the LC compound on satellite cell activity in rat skeletal muscle. The expression of Pax7
in GA muscle was not changed by exercise training (Ex) or
exercise with the LC compound (LCEx; data not shown). In TA
muscle, the LCEx group tended to experience an increase in
Pax7 (P 0.07), but there was no alteration in the expression
of Pax7 in the Ex group (data not shown). The expression of
MyoD in GA and TA muscles was not changed by exercise
training (Ex) or exercise with administered LC compound
(LCEx; data not shown). Although the expression of MyoD is
one of the markers used to identify the activated and proliferating satellite cells in adult skeletal muscle (25), not only
satellite cells but also all myonuclei and undefined myogenic
cells express MyoD (27), which could mask the increased

LC

Con

50kDa

LC

Con

0
Con

LC

Fig. 3. Effect of lactate or LC on follistatin (Fst) and myostatin (Mstn)

expression in C2C12 muscle cells. Protein expression of Fst (A) and Mstn (B)
is shown with representative immunoblots. C2C12 cells were incubated with
media containing LC and L for 6 h. L significantly increased Fst expression
and decreased Mstn expression relative to Con. LC significantly increased Fst
expression relative to Con. *P 0.05, **P 0.01 vs. Con.

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746

Lactate-Caffeine Supplement Increases Muscle Mass

LC

220kDa

DNA content, g/muscle

220kDa

LC

p-P70S6K / t-P70S6K
LC

Con

2
Protein content
(Arbitrary Units)

60kDa
60kDa

**

**

LC

0
Con

Fig. 4. Effect of lactate or LC on anabolic signals in C2C12 muscle cells.

Protein expression of p-mTOR (A) and p-P70S6K (B) is shown with representative immunoblots. C2C12 cells were incubated with LC and L for 6 h. L
significantly increased p-P70S6K compared with Con. LC significantly increased p-mTOR and p-P70S6K expression relative to Con. *P 0.05, **P
0.01 vs. Con.

upregulation of the components of calcium signaling pathways.

Although, in our previous study, the lactate transcriptome in L6
skeletal muscle cells did not include upregulation of anabolic
signals or satellite cell activity (24), our current study demonstrated that lactate and LC could increase satellite cell activity
and anabolic signals in C2C12 cells at the protein level.
Although we did not clarify the molecular mechanisms underlying the direct effects of lactate and LC on muscle cells in the
present study, calcium signals are candidate mediators of
increases in satellite cell activity and anabolic signals. Lactate
may activate the calcium/calmodulin-activated serine-threonine phosphatases calcineurin and myogenin (24). Calcineurin
dephosphorylates nuclear factor of activated T cell (NFAT).
Calcineurin/NFAT is implicated in the regulation of satellite
cell proliferation (25). Moreover, calcineurin/NFAT increases
Table 1. Effects of exercise training and lactate-caffeine
supplement on body weight and muscle weight
Body weight, g
GA, mg/100 g body wt
TA, mg/100 g body wt

LCEx

163.0 17.0

200.7 13.1

218.1 10.6*

Sed

Ex

LCEx

244 3.65
556 4.82
168 1.28

237 2.14
584 5.57**
175 2.14*

234 2.64
607 5.28**,##
183 1.42**,##

Values are means SE. GA, gastrocnemius; TA, tibialis anterior; Sed,
sedentary control; Ex, exercise training; LCEx, exercise training lactatecaffeine (LC) compound, *P 0.05, **P 0.01 vs. Sed, ##P 0.01 vs. Ex.

myogenin

GA
40kDa

2
Protein content
(Arbitrary Units)

Ex

myogenic factor 5 expression, which is in the muscle regulatory factor family (20). Caffeine is an inhibitor of phosphodiesterase, increasing intracellular cAMP and actually activating
the protein kinase cascade driven by cAMP (including Akt) in
this study (3). The results of our cell culture study and previous
studies allowed us to postulate that lactate and caffeine may
complement one another to promote muscle anabolism and
could effectively increase muscle mass by increasing satellite
cell activity and anabolic signals. Therefore, we assessed the
effect of low-intensity exercise training and the mixed lactate
and caffeine compound on satellite cell activity and anabolic
signaling in vivo.
As expected, we found that the Ex group increased muscle
mass normalized to total body weight of GA and TA, whereas
the LCEx group significantly increased muscle mass normalized to body weight in GA and TA relative to both the Sed and
Ex groups (Table 1). Indeed, we did not find increased satellite
cell activity in the Ex group, whereas the LCEx group signifi-

0
Con

Sed

Values are means SE. *P 0.05 vs. Sed.

Sed

Ex

30kDa

LCEx

*#

Sed

40kDa

Ex

LCEx

myogenin

TA

Protein content
(Arbitrary Units)

Protein content
(Arbitrary Units)

Oishi Y et al.

Table 2. Effects of exercise training and the lactate-caffeine

supplement on DNA content in TA muscle

p-mTOR / t-mTOR
Con

Sed

Ex

30kDa

LCEx

** #

2
1
0
Sed

Ex

LCEx

Fig. 5. Effects of exercise training and the LC compound on myogenin protein

expression. The expression of myogenin in gastrocnemius (GA; A) and tibialis
anterior (TA; B) muscle is shown with representative immunoblots. Exercise
training LC compound (LCEx) significantly increased myogenin relative to
sedentary control (Sed) and exercise training (Ex) in GA and TA. *P 0.05,
**P 0.01 vs. Sed. #P 0.05 vs. Ex.

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Lactate-Caffeine Supplement Increases Muscle Mass

Fst
40kDa

Protein content
(Arbitrary Units)

Ex

LCEx

30kDa

*#

Sed

40kDa

Protein content
(Arbitrary Units)

LCEx

Fst

TA
2

Ex

Sed

Sed

Ex

30kDa

LCEx

**

Sed

Ex

747

Oishi Y et al.

creased Mstn; however, Mstn levels returned to normal levels

24 h after the exercise bout had ceased. Because we dissected
muscle 48 h after the final bout of exercise training, it is
conceivable that Mstn expression had normalized.
Additionally, there was a significant increase in the total
DNA content of TA muscle in the LCEx group compared with
the Sed group (Table 2). In line with this observation, we found
that LC treatment resulted in a significant increase in Ki67, a
marker of cellular proliferation, relative to control in C2C12
cells. Although we found unchanged PCNA and cyclin-D1 in
the Western blot analysis, it is conceivable that, slightly increased, these cell cycle markers could be masked by the basal
expression in whole muscle homogenates. In addition, we
found that LC treatment resulted in a significant increase in the
differentiation index relative to control in C2C12 cells. This
dual effect of LC treatment on both the proliferation and
differentiation of skeletal muscle cells may be similar to the
effect of IGFs (9, 18, 42). Also, in a human study, McKay et
al. (36) demonstrated that acute damaging muscle-lengthening
contractions upregulated both MyoD and myogenin gene expressions at 4 h postexercise, suggesting a physiological relevance of the dual effect (i.e., proliferation and differentiation)
in myogenesis. The present study suggests that administration
of the LC compound could effectively increase muscle mass
concomitant with elevated myonuclei, even with endurance
exercise training, by means of activated satellite cells and
anabolic signals in skeletal muscle.

LCEx

Fig. 6. Effects of exercise training and the LC compound on Fst protein

expression. The expression of Fst in GA (A) and TA (B) muscles is shown with
representative immunoblots. LCEx significantly increased Fst in GA relative to
both the Sed and Ex groups. LCEx significantly increased Fst relative to Sed in
TA. *P 0.05, **P 0.01 vs. Sed. #P 0.05 vs. Ex.

cantly increased myogenin expression. Some previous studies

have reported that endurance training increased satellite cell
activity. Enns and Tiidus (19) showed that running downhill at
a 13.5 grade and speed of 17 m/min for a total of 90 min
increased satellite cell activity in rats. However, Enns and
Tiidus (19) used downhill running that induced muscle damage, which typically augments satellite cell activity. Charifi et
al. (5) also reported that endurance training increased the
number of satellite cells in elderly men. However, this endur O2 peak, which is high
ance training intensity was 8595% V
enough to cause muscle damage. From these previous studies,
satellite cell activity may be increased by high-intensity training-induced muscle damage.
Likewise, there was no increase in Fst expression in the Ex
group relative to the Sed group. On the other hand, we found
that myogenin and Fst protein expression were significantly
increased in the LCEx group relative to both the Sed and Ex
groups, representing an anabolic response to exercise training.
From these results, the LC compound may increase satellite
cell activity and anabolic signals more effectively than lowintensity exercise alone. In accordance with the Fst response,
Mstn is decreased by low- or moderate-intensity exercise in
humans (26). In the present study, Mstn was decreased in only
TA in the LCEx group, whereas there was an increase in Fst in
both GA and TA. Matsakas et al. (34) and Louis et al. (32)
reported that endurance training and resistance training de-

Mstn

GA
50kDa

Sed

Ex

LCEx

2
Protein content
(Arbitrary Units)

GA

Ex
Ex

Sed
Sed

LCEx
LCEx

Mstn

TA
50kDa

Sed

Ex

LCEx

2
Protein content
(Arbitrary Units)

Sed
Sed

Ex

LCEx
LCEx

Fig. 7. Effects of exercise training and the LC compound on Mstn protein

expression. The expression of Mstn in GA (A) and TA (B) muscles is shown
with representative immunoblots. LCEx significantly decreased Mstn protein
expression in TA relative to Sed. *P 0.05 vs. Sed.

J Appl Physiol doi:10.1152/japplphysiol.00054.2014 www.jappl.org

748

Lactate-Caffeine Supplement Increases Muscle Mass

The mechanism underlying increased muscle mass by the

LC compound might be a direct effect of lactate and caffeine
on the skeletal muscle, as we have seen in vitro. In our animal
study, exercise intensity was 20 m/min for 30 min; although
the LCEx group increased satellite cell activity, this increase is
not high enough to produce muscle damage in rats. Previous
studies have shown that the average lactate concentration after
treadmill running exercise for 25 min at a speed of 20 m/min
is 3.8 mM (8, 14). In our study, the lactate concentration of
the Ex group was 1.93 0.16 mM, while that of the LCex
group was 7.85 2.63 mM immediately after the treadmill
running. Lake et al. (31) showed that blood caffeine concentration after 400 mg caffeine supplementation was 0.61 g/ml
in humans. In an animal study, administration of 30 mg
caffeine to rats resulted in increased blood caffeine concentrations up to 9.9 g/ml (40). In this study, the LC compound
contained 9 mg caffeine; therefore, the blood caffeine level is
estimated to increase to 50 M. Although we did not measure caffeine levels after LC compound administration, we
propose that the LC compound directly increased satellite cell
activity and anabolic signals. Therefore, endogenously produced lactate and muscle contraction-induced calcium signals,
in response to resistance exercise and/or high-intensity exercise
(15, 44), may partially explain why anabolic signaling is
enhanced and muscle hypertrophy follows these types of exercise training.
A limitation of the present study is that we did not analyze
calcium signals in either the in vivo or in vitro experiments.
Therefore, we do not know whether caffeine could have
biologically or physiologically affected intracellular calcium
levels in the present study. Furthermore, the downstream molecular actions of lactate in the control of skeletal muscle
growth are not known. In addition to the direct effect of lactate
on the muscle growth, a secondary effect of lactate acting
through growth hormone (GH) should be considered. McArdle
disease patients are unable to produce lactate in response to
exercise and cannot produce exercise-induced GH. Therefore,
lactate might play some role in the exercise-induced GH
response (21). GH plays a pivotal role in satellite cell proliferation and differentiation (23). Moreover, GH-stimulated
IGF-I increases satellite cell activity and muscle mass (2).
Although we cannot eliminate a secondary effect of GH in
response to augmented lactate in vivo, our cell study suggests
a direct effect of lactate on skeletal muscle growth. Although
we did not have control groups that received lactate, caffeine,
or both without exercise, supplements such as these (at least,
the LC compound) would be expected to have similar effects in
sedentary rats. In addition, the inclusion of the positive control
for the supplement, such as one of the anabolic steroids, should
have bolstered the significance of the study. Furthermore,
measurements of protein synthesis and breakdown should also
have been needed to determine whether the LC compound is
physiologically relevant. Another limitation of the present
study is that we did not measure the cross-sectional area via
histochemistry; although we found that the muscle mass normalized to body weight was increased, we could not determine
whether the increased muscle mass was due to muscle hypertrophy or hyperplasia. In addition, because the rats were not
perfused with saline before dissecting out the muscles, we
could not exclude a small contamination of connective tissue
and other cells (e.g., endothelial cells, resident mast cells,

Oishi Y et al.

lymphocytes, etc.) in the whole muscle homogenates for the

Western blot analyses and the measurement of DNA content.
Furthermore, we used adult rats in the present study, and
sarcopenia is associated with aging. Thus, it would be more
relevant to examine the hypertrophic effects of low-intensity
exercise with LC supplementation in older rats. Future studies
will be needed to address the mechanism by which lactate and
calcium exert control over skeletal muscle growth.
In conclusion, we found for the first time that treatment with
LC compound increases satellite cell activity and anabolic
signals in C2C12 cells. Furthermore, low-intensity exercise
with LC compound increases muscle mass, satellite cell activity, and anabolic signals. These results suggest that administration of LC compound could effectively increase muscle
mass concomitant with elevated myonuclei, even with lowintensity exercise, by means of activated satellite cells and
anabolic signals in skeletal muscle. Therefore, the study of LC
compound should provide insight into the development of
strategies against muscle wasting and loss of function associated with a wide range of neuromuscular diseases such as
sarcopenia.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Yusuke Ono for valuable comments on
the manuscript and Masahiro Okumura for technical support in conducting the
experiments.
GRANTS
This work was supported in part by a Grant-in-Aid for Young Scientists (A,
B; 26702029 and 22700652, respectively, to T.H.) from the Japan Society for
Promotion of Science.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: Y.O., H. Tsukamoto, K. Hirotsu, and T.H. performed
experiments; Y.O., H. Tsukamoto, and K. Hirotsu analyzed data; Y.O. prepared figures; Y.O. drafted manuscript; Y.O., H. Tsukamoto, T.Y., K. Hirotsu,
M.S., K.U., H. Tomi, K. Higashida, N.I., and T.H. approved final version of
manuscript; T.Y., K. Hirotsu, N.I., and T.H. interpreted results of experiments;
K.U., K. Higashida, N.I., and T.H. edited and revised manuscript; T.H.
conception and design of research.
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