Ursolic acid, L-leucine and monoclonal antibody against β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ursolic acid was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C prior to the experiments and dilutions were made in culture medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) was obtained from Amresco (Solon, OH, USA). Antibodies against myosin heavy chain (MHC; sc-20641), MyoD (sc-760), myogenin (sc-576), phospho-Akt (Ser 473; sc-7985-R), and Akt1/2/3 (sc-8312) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against 4E-BP1 (cat. no. 9452), phospho-4E-BP1 (cat. no. 2855) and phospho-p70S6K (cat. no. 9234) were purchased from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibody against p70S6K (bs-6370R) was obtained from Bioss (Woburn, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Welgene Inc. (Daegu, Korea) and horse serum (HS) was from Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS) and penicillin-streptomycin were purchased from HyClone (Logan, UT, USA). The creatine kinase enzymatic assay kit (MaxDiscovery™ Creatine Kinase Enzymatic Assay kit) was purchased from Bioo Scientific Corp. (Austin, TX, USA).

The murine myoblast cell line, C2C1,2 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37°C in humidified 95% air and 5% CO₂ in growth medium (GM), containing DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at up to 70–80% confluence. During proliferation, the cells were seeded at 2×10⁵ cells/well in 6-well culture plates and grown in GM. When the cells reached approximately 90–100% confluence, the GM was removed and replaced with differentiation medium (DM), containing DMEM supplemented with 2% horse serum (HS) and ursolic acid, leucine or a combination of ursolic acid and leucine. The medium was changed every 2 days until day 6. The effects of ursolic acid, leucine or their combination on cell viability were determined by MTT assay, which is based on the conversion of MTT to MTT-formazan by mitochondrial enzymes.

The cells were washed with phosphate-buffered saline (PBS) and then lysed with lysis buffer [40 mM Tris (pH 8.0), 120 mM NaCl, 0.5% NP-40, 2 μg/ml aprotinin, 2 μg/ml leupeptine and 100 μg/ml phenymethyl-sulfonyl fluoride (PMSF)], and complete protease inhibitor and stored at −70°C until use. CK activity was determined by a CK enzymatic assay kit (Bioo Scientific Corp.), according to the manufacturer’s instructions. Briefly, 250 μl of CK reagent were added to 5 μl of cell lysate in a microplate. Subsequently, CK activity was immediately measured 2 times with a 5-min time interval at 340 nm. The assay was performed in duplicate. The average 5 min absorbance increase was multiplied by 2,186 (conversion factor) to obtain the CK activity (IU/l).

Following treatment, the cells were harvested and washed with cold PBS and then lysed in lysis buffer. Following centrifugation, the supernatant was collected and the protein concentration was determined using protein assay reagents (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were denatured by boiling at 100°C for 5 min in 2X Laemmli sample buffer (Bio-Rad Laboratories). The protein samples were separated on 6–15% SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 buffer (TBS-T; 20 mM Tris, 100 mM NaCl, pH 7.5 and 0.1% Tween-20) for 1 h at room temperature. The membranes were then incubated with various primary antibodies at 4°C overnight. After being thoroughly washed with TBS-T buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Antigen-antibody complexes were detected by the Enhanced chemiluminescence (ECL) detection system (GE Healthcare, Piscataway, NJ, USA).

For immunofluorescence analysis to evaluate myogenic differentiation, the cells were cultured on gelatin-coated coverslips. For this, the coverslips were soaked in 1 M HCl for 24 h, rinsed 3 times with distilled water and then rinsed 3 times with 95% ethanol. The coverslips were submerged in gelatin solution (0.1 mg/ml) for 5 min, air-dried and placed into 6-well culture plates. Finally, the culture plates with coverslips were sterilized under a UV lamp for at least 2 h. The cells (2×10⁵/well) were seeded and cultured overnight. The cells were then rinsed with PBS, fixed with 4% formalin and 0.3% glutaraldehyde for 20 min, washed with PBS, incubated in PBS supplemented with 0.2 % Triton X-100 and 2% normal goat serum for 1 h at 4°C. The cultures were then incubated overnight with primary antibody against MHC (1:200), followed by rhodamine-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The samples were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) to prevent fading. The samples were examined under a confocal laser scanning microscope (Zeiss, Göettingen, Germany).

The images were randomly selected from at least 3 regions from each well. The fusion index was calculated based on the scoring of random regions with stained nuclei and myotubes.

Total RNA was prepared using TRIzol reagent (Invitrogen). cDNA for the PCR template was synthesized using TOPscript™ RT DryMIX(dT18) (Enzynomics, Daejeon, Korea). For PCR, specific primers were used for the analysis of the expression for the following molecules: MyoD forward, 5′-AGTGAATGAGGCCTTCGAGA-3′ and reverse, 5′-CTGGGTTCCCTGTTCTGTGT-3′ (514 bp); myogenin forward, 5′-ACCAGGAGCCCCACTTCTAT-3′ and reverse, 5′-ACGATGGACGTAAGGGAGTG-3′ (583 bp); GAPDH forward, 5′-ACTCCACTCACGGCAAATTC-3′ and reverse, 5′-CCTTCCACAATGCCAAAGTT-3′ (370 bp). Subsequently, the PCR reactions were performed in a Mastercycler gradient unit (Eppendorf, Hamburg, Germany) using PCR PreMix (Bioneer Corp., Daejeon, Korea). Each PCR cycle was 94°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min. The numbers of cycles of PCR were 26 for MyoD, 27 for myogenin and 25 for GAPDH. The amplified PCR products (7 μl) were electrophoresed on 1.2% agarose gels and visualized by ethidium bromide staining.

The results were expressed as the means ± SD of 3 separate experiments and analyzed using the Student’s t-test and ANOVA. Values of P<0.05 or P<0.01 were considered to indicate statistically significantly differences.

To determine the effects of ursolic acid and leucine on the viability of C2C12 cells, an MTT assay was performed. The C2C12 cells were cultured in DM and treated with ursolic acid (0–10 μM) or leucine (0–500 μM) for 6 days. Cell viability was not affected by treatment with 0–2.5 μM ursolic acid and 0–100 μM leucine. However, treatment with >2.5 μM of ursolic acid and >100 μM of leucine exerted cytotoxic effects (Fig. 1B and C). To investigate whether the combination of ursolic acid and leucine would promote the differentiation of C2C12 myoblasts, the cells were treated with various concentrations of ursolic acid and leucine (at concentrations that had not affected cell viability: ursolic acid, 0.5 and 2.5 μM; leucine, 10 and 50 μM). To confirm the effects of these combinations on cell viability, an MTT assay was conducted again. Treatment with various combinations of ursolic acid and leucine (Fig. 1D), similar to the treatments with either agent alone, had no effect on viability of the C2C12 cells.

Our first hypothesis in the present study was that the combination of ursolic acid and leucine would promote greater myogenic cell differentiation than either ursolic acid or leucine alone. As shown in Fig. 2, both ursolic acid and leucine induced morphological changes in the C2C12 cells. When the ursolic acid- or leucine-treated groups were compared with the untreated groups (GM and DM), the cell shapes differed greatly (Fig. 2 A–F). After 3–4 days of differentiation, the cells seemed to lose their typical triangular morphology and the cell shape gradually changed into a new elongated shape. Furthermore, the differentiation-promoting effects of the different combinations of the agents were even more pronounced. The myotubes that formed in the cells treated with combinations of ursolic acid and leucine were more stretched and longer in shape than those in the cells treated with each agent alone (Fig. 2G–J). In particular, in the cells that were treated with the combination of 0.5 μM ursolic acid and 10 μM leucine, the myotubes showed a spindly ring pattern, indicating muscle hypertrophy and maturation (Fig. 2G).

CK activity was used as an indicator of differentiation. Compared to the GM control, CK activity levels in the cells that were treated with ursolic acid or/and leucine for 6 days were increased (Fig. 3). However, the CK activity level in the cells treated with either agent alone (ursolic acid or leucine) was lower than that in the cells treated with the various combinations of ursolic acid and leucine. The level of CK activity in the cells treated with the combination of 0.5 μM ursolic acid and 10 μM leucine was significantly increased by 50% compared to the DM control during differentiation, and this combination led to the most significant increase in CK activity. The second most significant increase in the level of CK activity was observed in the cells treated with the combination of 2.5 μM ursolic acid and 10 μM leucine.

MHC is considered as a marker of the differentiated state, particularly in the late differentiation of myoblasts. First, western blot analysis was performed to detect the changes in the protein expression levels of MHC over time. The protein levels of MHC gradually increased over 6 days, with the highest levels being observed on day 6 (Fig. 4A). Subsequently, in order to confirm whether the combination of ursolic acid and leucine promotes the differentiation of C2C12 cells as opposed to treatment with each agent alone, the protein level of MHC was evaluated. As expected, treatment with the combination of 0.5 μM ursolic acid and 10 μM leucine induced a significant increase in the expression level of MHC (Fig. 4B). Compared to the DM control, MHC expression increased by almost 2-fold. In addition, compared to the cells treated with the agents alone (0.5 μM ursolic acid or 10 μM leucine), the protein expression of MHC increased by approximately 3- and 2-fold in the cells treated with the combination of 0.5 μM ursolic acid and 10 μM leucine, respectively (Fig. 4C).

To analyze the changes occurring in the nuclear arrangement of myotubes during the late phase of differentiation, immunofluorescence staining using antibody against MHC was performed. As shown in Fig. 5A, after 6 days of differentiation, most of the untreated cells (controls) that expressed MHC were mononuclear; however, in the presence of 0.5 μM ursolic acid or 10 μM leucine, myotubes containing 2 and more nuclei were observed. The number of myotubes containing 4 or more nuclei was highest in the cells treated with the combination of 0.5 μM ursolic acid and 10 μM leucine. In the cells treated with the combination treatment, the myotubes were characterized by a particular arrangement of the nuclei, forming a ring, a morphological marker of muscle maturation. With the combination treatment, the highest density of MHC-expressing cells and a greater number of elongated cells were observed, some of which fused into myotubes (Fig. 5A). The fusion index was also found to be significantly higher (>2-fold) in the cells treated with the combination of 0.5 μM ursolic acid and 10 μM leucine, compared to the untreated cells (Fig. 5B).

In our experiments, MHC was used as a marker of late differentiation. In order to determine the effects of ursolic acid and leucine on early differentiation, MyoD and myogenin were used as biomarkers. First, quantitative analysis of the results from western blot analysis further revealed time-dependent (on day 1, 2, 4 and 6) changes in the expression of the early differentiation markers, MyoD and myogenin. As shown in Fig. 6A and B, MyoD was predominantly expressed on day 1 (Fig. 6B), while myogenin was predominantly expressed on day 2 (Fig. 6C). Compared with the untreated controls, the protein expression of MyoD in the cells treated with the combination of 0.5 μM ursolic acid and 10 μM leucine increased by >2.5-fold (Fig. 6D and E). The epxression of myogenin was also increased, although the increase was not as notable as that of MyoD (Fig. 6D and F). In addition, the mRNA levels of the muscle regulatory factors, MyoD and myogenin, were elevated in response to the combination treatment (Fig. 6G). A sharp increase in the mRNA level of MyoD was observed in the cells treated with the combination of ursolic acid and leucine (Fig. 6H). The mRNA levels of myogenin were also increased, although the increase was not as notable as that of MyoD (Fig. 6I).

Protein synthesis occurs during embryonic development, cell growth, cell differentiation and aging. In previous studies, signaling pathways, such as the IGF-1, PI3K/Akt, and mTOR signaling cascades, have been shown to play a major role in protein synthesis by targeting several components of the translation machinery (27,28). To confirm the effects of the combination of ursolic acid and leucine on the activation of the mTOR signaling pathway, western blot analysis of S6K1 and 4E-BP1, which are two key downstream targets of the mTOR signaling pathway (29), was conducted. In addition, the phosphorylation of 4E-BP1 and S6K1 are crucial activation steps necessary for the downstream event (30). Treatment with the combination of ursolic acid and leucine increased the phosphorylation of mTOR and that of the downstream targets, 4E-BP1 and S6K1, as shown in Fig. 7.