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 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 exercise for 60 min at 70% V̇o 2 peak could increase Akt, 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 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.

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 Kruskal-Wallis 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.

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.

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 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 μg) were subjected to SDS-PAGE and transferred to a nitrocellulose membrane.

In the LC Ex 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.

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 (LC Ex , n = 13). During the 4 wk of the experiment, individually housed rats were given the purified normal diet (CRF-1) at 13 g/day.

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.

Next, we assessed the protein expression of Fst and Mstn in GA and TA. There was a significant increase in Fst expression in the LC Ex group compared with GA in both the Sed and Ex groups ( Fig. 6 A ). There was significantly increased Fst expression in TA in the LC Ex group relative to the Sed group ( Fig. 6 B ). Mstn expression in TA was decreased in the LC Ex group relative to the Sed group ( Fig. 7 B ), but this was not seen in GA ( Fig. 7 A ).

The expression of Pax7 in GA muscle was not changed by exercise training (Ex) or exercise with the LC compound (LC Ex ; data not shown). In TA muscle, the LC Ex 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 (LC Ex ; 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 number of activated satellite cells in vivo. There was a significant increase in myogenin expression in GA and TA in the LC Ex group relative to the Sed and Ex groups ( Fig. 5 ).

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 g·day −1 ·individual −1 ). 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 LC Ex group was 7.85 ± 2.63 mM. The blood lactate level of the LC Ex 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 normalized to total body weight in GA and TA relative to the Sed group. However, the LC Ex group increased muscle mass normalized to body weight in GA and TA relative to both the Sed and Ex groups ( Table 1 ).

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. 4 B ). LC significantly increased both mTOR and P70S6K phosphorylation ( Fig. 4 , A and B ) but did not change Akt phosphorylation (data not shown).

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 μm. 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 MHC-positive 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.

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. 2 B ). In addition, the LC treatment resulted in a significant increase in the differentiation index ( Fig. 2 C ).

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. 1 C , 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 , A–C ); however, caffeine did not increase Pax7, MyoD, or myogenin expression (data not shown). Furthermore, LC significantly increased MyoD protein levels relative to lactate ( Fig. 1 B ).

DISCUSSION

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 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 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 LC Ex 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 LC Ex group significantly 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 endurance training intensity was 85–95% V̇o 2 peak, which is high 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 LC Ex 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 low-intensity 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 LC Ex 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 decreased 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 LC Ex 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.

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 LC Ex 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 LC ex 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 μg/ml in humans. In an animal study, administration of ∼30 mg caffeine to rats resulted in increased blood caffeine concentrations up to 9.9 μg/ml (40). In this study, the LC compound contained 9 mg caffeine; therefore, the blood caffeine level is estimated to increase to ∼50 μM. 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, 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 low-intensity 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.