*2.1. Animal Experiments*

All animal experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health (Bethesda, MD, USA). The Animal Use Committee of Toyohashi SOZO University (A2016003, A2017002) approved the procedures of animal experiments in this study. Male C57BL/6J mice aged 8-week old were used. To investigate the effects of lactate on skeletal muscle hypertrophy or regeneration, mice were randomly divided into control (C) and lactate-administered (L) groups (*n* = 24), or cardio-toxin (CTX)-injected (CX) and lactate-administered after CTX injection (LX) groups (*n* = 28). All mice were housed in a clean room controlled at approximately 23 ◦C with a 12/12 h light-dark cycle. Solid diet and water were provided ad libitum.

Muscle injury-regeneration cycle was induced by injecting 0.1 mL cardiotoxin (CTX, 10 μmol/L in physiological saline, Sigma-Aldrich, St. Louis, MO, USA) of Naja naja atra venom into right tibialis anterior (TA) muscles in the CX and LX groups. After epilation of right hind limb with a commercial hair remover for human, injection of CTX into right TA muscle was performed using a 27-gauge needle. During this procedure, all mice were under anesthesia with intraperitoneal injection of sodium pentobarbital (50 mg/kg) [23].

In the L and LX group, sodium lactate (1 mol/L in distilled water, 8.9 mL/g body weight, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima, Japan) was administered to the mice by using an oral sonde 5 days a week for 2 weeks after CTX injection. The dose (or amount) of lactate was selected considering the previous rat study [18]. The same volume of ultrapure water was administered to the C and CX groups. In a pilot study, the changes of blood lactate concentration, which was collected from the tail vein of mice after oral lactate administration, were evaluated using the Lactate Pro2 blood lactate test meter (ARKRAY, Inc., Kyoto, Japan). Before the administration of lactate, the blood lactate concentration of mice (*n* = 7) was 2.9 ± 0.2 mmol/L (Table 1). Two hours after the lactate administration, blood lactate level significantly increased up to 4.1 ± 0.3 mmol/L. Similar to this observation, the oral administration of lactic acid to rats led to a rise in the blood level [24].


**Table 1.** Blood lactate concentration of mice following orally administered sodium lactate.

Pre: before oral lactate administration (base line); 2 h, 6 h, and 24 h: 2, 6, and 24 h after the lactate administration. Values are means ± SEM. *n* = 7. † and §: *p* < 0.05 vs. Pre and 2 h, respectively.

Mice were sacrificed by cervical dislocation under anesthesia with intraperitoneal injection of sodium pentobarbital (50 mg/kg) 1 and 2 weeks after CTX injection. Immediately after the sacrification, right TA muscle was excised. Dissected TA muscles were rapidly weighed and frozen in isopentane cooled in liquid nitrogen. All samples were then stored at −80 ◦C until analyses.

Serial transverse cryo-sections (8-μm thick) of the samples were cut at −20 ◦C and immediately mounted onto glass slides. Sections were stained to analyze the cross-sectional area (fiber CSA) of muscle fibers by hematoxylin and eosin (H&E), and the profiles of Pax7-positive nuclei by a standard immuno-histochemical technique [23]. Monoclonal anti-Pax7 antibody (Developmental Studies Hybridoma Bank, Iowa, IA, USA) was used for the detection of muscle satellite cells [3]. The sections were fixed in 4% paraformaldehyde, and were then post-fixed in ice-cooled methanol. After blocking by using 1% Roche blocking reagen<sup>t</sup> (Roche Diagnostic, Penzberg, Germany), sections were incubated with the primary antibodies for Pax7 and rabbit polyclonal anti-laminin (Z0097, DakoCytomation, Glostrup, Denmark). Following an incubation period at 4 ◦C, sections were incubated with secondary antibodies for Cy3-conjugated anti-mouse IgG (Jackson Immuno Research, West Grove, PA, USA) and with fluorescein isothiocyanate-conjugated anti-rabbit IgG (Sigma-Aldrich) at room temperature. Nuclear counterstaining was performed in a solution of <sup>4</sup>,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich). The number of Pax7-positive nuclei located within the laminin-positive basal membrane per muscle fiber (approximately 250 fibers) from each muscle was calculated. Using H&E stained sections, mean fiber CSA was measured from approximately 250 fibers of each muscle using the National Institutes of Health Image J 1.38X (NIH, Bethesda, MD, USA) software for Windows.

#### *2.2. Cell Culture Experiments*

Mouse myoblast-derived C2C12 cells (6 × 10<sup>4</sup> cells/well) were cultured on 6-well culture plates, coated with type I collagen (Biocoat, Corning, NY, USA). Cells were maintained in 2 mL of growth medium that consisted of Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, Yokohama, Japan) supplemented with 10% heat-inactivated fetal bovine serum containing high glucose (4.5 g/<sup>L</sup> glucose, 4.0 mM L-glutamine, without sodium pyruvate) for proliferation. During the third day of the proliferation phase (at ~80% confluence), the culture medium was then changed to the same amount of di fferentiation medium, which consisted of DMEM supplemented with 2% heat-inactivated horse serum containing low glucose (1.0 g/<sup>L</sup> glucose, 4.0 mM L-glutamine, and 110 mg/<sup>L</sup> sodium pyruvate) for di fferentiation, as was described previously [19]. Every 2 days, cells were replenished with fresh di fferentiation medium and cultures were maintained for 5 days. All cells were maintained at 37 ◦C, under a humidified atmosphere with 5% CO2 and 95% air.

Sodium lactate was administered into the conditioned medium throughout the di fferentiation phase. The concentration of lactate was set at 20 mM in the conditioned culture medium, which took into account previous studies using skeletal muscle cells [19,25]. Ultrapure water alone was added to the conditioning medium for the control group (*n* = 6 well in each group).

The images of myotubes at the 5th day of the di fferentiation phase were visualized at x40 magnification using a calibrated color imaging camera (DP12, Olympus, Tokyo, Japan) set up to a phase contrast light microscope (CK40, Olympus). In order to measure the myotube diameter, we used a modified method of a previous study [26]. We first randomly selected fields of view from 6 wells of each condition. Using Image J, the diameters of at least 100 myotubes in each well were measured at three randomly selected portions taken along the length of the myotube. Then, the average diameter of a myotube was calculated as the mean of three measurements. The myotubes were also

used for the evaluation of myotube length and the myo-nuclei number as described below. Cells were fixed with 4% paraformaldehyde. After blocking, cells were incubated with the primary antibodies for skeletal myosin (M4276, Sigma-Aldrich). Cells were then incubated with the secondary antibodies for Cy3-conjugated anti-mouse IgG. Then nuclei were counterstained with DAPI. Since a muscle cell containing 3 or more nuclei was considered to be a myotube [27], the myotube length and myonuclei number in di fferentiated myotubes (>2 myonuclei) were measured using Image J.

In addition, the myotubes at the 5th day of di fferentiation were used to analyze muscular protein content as described below. In order to extract protein from C2C12 cells, the cells were lysed in a cell lysis reagen<sup>t</sup> (CelLyticTM-M, Sigma-Aldrich), in accordance with the previously reported method [19]. The cells in each well were rinsed twice in 1 mL of ice-cooled phosphate-bu ffered saline. The cells of each well were then scraped into 0.3 mL of cell lysis reagen<sup>t</sup> on ice. The cell lysate was sonicated and centrifuged at 20,000 *g* at 4 ◦C for 10 min. The supernatant was collected for the analysis of protein content. Protein content in the supernatant was determined using the Bradford technique (protein Assay kit, Bio-Rad, Hercules, CA, USA) and bovine serum albumin (Sigma-Aldrich) as the standard.

## *2.3. Animal Experiments*

All values were expressed as means ± SEM. In animal experiments, the statistically significant level of blood lactate concentration was analyzed using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test. Other significant levels were tested using a two-way (lactate administration and time) ANOVA for multiple comparisons followed by the Tukey-Kramer test. When a significant interaction between main factors was observed, one-way ANOVA followed by the Tukey-Kramer test was performed. Statistically significant levels in cell experiments were evaluated using unpaired Student's *t*-test following F-test. The significance level was accepted at *p* < 0.05.
