All chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) onless otherwise stated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from AMRESCO (Solon, OH, USA). Antibodies against myosin heavy chain (MyHC; sc-20641), MyoD (sc-760), myogenin (sc-576), phosphorylated (p-)Akt (Ser473; sc-7985-R), Akt1/2/3 (sc-8312), anti-rabbit IgG-horseradish peroxidase (HRP)-conjugated antibody (sc-2054), and anti-mouse IgG-HRP-conjugated antibody (sc-2031) were obtained from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). Antibodies against eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1; no. 9452), p-4E-BP1 (Thr37/46; no. 2855), mammalian target of rapamycin (mTOR; no. 2983), p-mTOR (Ser2488; no. 5536), and p-p70 S6 kinase (Thr389; no. 9234) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). β-actin antibody was purchased from Sigma-Aldrich, and polyclonal antibody against p70 S6 kinase (bs-6370R) was obtained from Bioss, Inc. (Woburn, MA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from WelGENE, Inc. (Daegu, Korea), and horse serum (HS) was from Invitrogen Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from GE Healthcare Life Sciences (Logan, UT, USA). A creatine kinase (CK) enzymatic assay kit (MaxDiscovery^® Creatine Kinase Enzymatic Assay kit) was purchased from Bioo Scientific Corp. (Austin, TX, USA). Polyvinylidene difluoride (PVDF) membranes were obtained from Merck Millipore (Billerica, MA, USA).

An ethanol (EtOH) extract of loquat leaf (hereafter termed LE) was prepared as previously described in the study by Jung et al (22) with some modifications, and was provided by the Marine Bio-industry Development Center (Busan, Korea). The yield of LE based on the dried weight of the loquat leaf was 4.68%. The final concentration of UA in the LE was 104.14 mg/g. LE was dissolved in EtOH as a 5 mg/ml stock solution. The stock solution was stored at −20°C and diluted with medium to the desired concentration prior to use.

Male Sprague-Dawley rats (aged 5 and 18–19 months) were obtained from Samtako (Osan, Korea). The rats were maintained under controlled environmental conditions (23±1°C, 50±10% relative humidity, 12 h/12 h light/dark cycle) with ad libitum access to water and a basal diet (FORMULA M07; FEEDLAB, Guri, Korea). After an acclimation period (1 week), the rats were randomly divided into 4 study groups (4–6 animals per group) as follows: i) young rats not administered LE (Y-Con group); ii) young rats administered LE (Y-LE group); iii) aged rats not administered LE (O-Con group); and iv) aged rats administered LE (O-LE group).

Animals in the LE-treated groups were administered LE mixed in their food (based on food intake, daily dose of LE=50 mg/kg of body weight). During the experiments, forelimb grip strength was determined using a grip strength meter equipped with a T-shaped pull bar (Columbus Instruments, Columbus, OH, USA). After the scheduled experiment, the rats were sacrificed by decapitation and soleus and gastrocnemius muscles were quickly removed. Muscle weights were measured immediately, and muscles were divided for biological analysis and histological examination. The animal protocol used in the present study was reviewed and approved by the Pusan National University Institutional Animal Care and Use Committee (PNU-IACUC; Approval no. PNU 2013-0461) with respect to ethical issues and scientific care.

Medial portions of soleus muscles were fixed in 10% formalin solution for 24 h, routinely embedded in paraffin blocks, transversely sectioned (3-µm-thick sections) and stained with hematoxylin and eosin (H&E).

To analyze the muscle fiber CSA, images of the soleus muscle were captured using a microscope (Axiovert 100; Zeiss, Göettingen, Germany). The CSA of each muscle fiber in each field was measured using ImageJ software (version 1.49m, National Institutes of Health, Bethesda, MD, USA). The CSA was examined in 3 rats from each group.

The cytosolic fraction of the muscle homogenate was used for the CK activity assay. Briefly, the tissues were homogenized in homogenate buffer containing 50 mM HEPES (pH 7.4), 10 mM KCl, 2 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 20 mM β-glycerophosphate, 20 mM sodium fluoride, 2 mM sodium orthovanadate, 1 mM pepstatin, 2 mM leupeptin and 5 mM aprotinin, and the homogenates obtained were placed on ice for 15 min. Nonidet P-40 (NP-40; 10%, 125 µl/1 ml homogenate) solution was then added, mixed for 15 sec, and the mixture was then centrifuged at 14,000 x g for 2 min. The supernatants obtained were used as cytosolic fractions.

For the CK assay using the C2C12 cells, the cells were collected, washed with Dulbecco's phosphate-buffered saline (DPBS) and then lysed with lysis buffer [40 mM Tris (pH. 8.0), 120 mM NaCl, 0.5% NP-40, 100 µg/ml PMSF and complete protease inhibitor] and stored at −70°C until use. CK activity was determined using 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 lysate or homogenate in a microplate. CK activity was immediately measured 2 times at 5-min time intervals, at 340 nm, using a multi-well reader (GENios; Tecan Austria GmbH, Grödig, Austria). The assay was performed in duplicate. The average 5-min increase in absorbance was multiplied by 2,186 (conversion factor) to obtain the CK activity (IU/l).

C2C12 murine myoblasts were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). For all experiments, the C2C12 cells were cultured from 4 to 9 passages to 70–80% confluence in growth medium (GM) containing DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were maintained in humidified 95% air and 5% CO₂ at 37°C.

For differentiation, the cells were plated at an approximate initial density of 1×10⁵ cells/well in 6-well culture plates and grown in GM. When the cells reached 80–90% confluence, the GM was removed and the cells were washed with DPBS and fed differentiation medium (DM) containing DMEM supplemented with 2% HS to induce differentiation. To examine the effects of LE on myogenic differentiation, LE was added to the DM. The medium was changed every other day until day 6, and the LE was replaced with each medium change.

Cell viability was evaluated by measuring the mitochondrial-dependent conversion of the yellow tetrazolium salt, MTT, to purple formazan crystals by metabolically active cells. Briefly, the C2C12 cells were seeded and induced to differentiate as described above. At the end of the differentiation period, the cells were incubated with 0.5 mg/ml MTT at 37°C for 2 h. Subsequently, the MTT was removed and the formazan crystals were dissolved in dimethyl sulfoxide. The absorbance was measured at 540 nm using a multi-well reader (Thermo Fisher Scientific, Vantaa, Finland).

Following treatment, the cells were harvested and washed with cold DPBS. The cells were lysed in lysis buffer. Following centrifugation, the supernatant was collected and the protein concentration was determined using protein assay reagents (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were boiled for 5 min in 2X Laemmli sample buffer (Bio-Rad). The protein samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 6–15% acrylamide gels and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 buffer (TBST; 20 mM Tris, 100 mM NaCl, pH 7.5 and 0.1% Tween-20) for 1 h, incubated with various primary antibodies at 4°C overnight, washed 3 times with TBST buffer and then incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Antigen-antibody complexes were detected using an enhanced chemiluminescence (ECL) detection system (GE Healthcare Life Sciences, Piscataway, NJ, USA). Densitometric analysis (for optical density) was performed using Fluorchem SP AlphaEase^® FC (version 6.0.0) software (Alpha Innotech, San Leandro, CA, USA), normalized to actin or other control proteins, and expressed as a fold change compared with the untreated controls.

Data are expressed as the means ± SEM, and analyzed using GraphPad Prism software (version 5.02, GraphPad Software, Inc., La Jolla, CA, USA). Treatments were compared by one-way ANOVA followed by Tukey's post hoc test for pairwise comparisons. P-values <0.05 were considered to indicate statistically significant differences.

Changes in body weight were measured every 5 days, and food and water intake were measured every 3 days over the entire experimental period. No adverse effects were observed in behavior, cleanliness and in the appearance of hair and eyes. No significant differences in food and water intake were observed between the beginning and the end of the treatment period for each experimental group (data not shown). As shown in Fig. 1A, the young-aged groups, either the control or the group fed LE, continuously gained body weight during the experimenatl period, whereas the old-aged groups showed insignificant changes in body weight.

We then examined the effects of LE supplementation on age-associated muscle function in rats. To monitor and quantify muscle function, we conducted a forelimb grip strength test using a grip strength meter. Grip strength was measured in each rat once every 5 days. As expected, the old-aged groups showed a significantly lower grip strength than the young-aged groups at the start of the experimental period (day 0, Fig. 1B). Within the age-matched experimental groups, no significant differences in muscle strength were observed on day 0 of the experimental period (Fig. 1B). After 35 days of LE supplementation, the mean grip strength of the old-aged control group (O-Con) decreased by 33% compared with that of the young-aged control group (Y-Con, Fig. 1C). Following LE supplementation, only an 18% decrease in grip strength was observed in the old-aged group supplemented with LE (O-LE group) compared with the Y-Con group. LE supplementation resulted in a significantly increased grip strength in the O-LE group compared with the O-Con group. The grip strength of the rats in the Y-LE group tended to slightly increase, although this change was not significant compared to the rats in the Y-Con group (Fig. 1C). These results suggest that LE supplementation has beneficial effects on muscle strength.

Grip strength decreased in the aged rats in comparison with the young ones, and we then wished to determine whether this decline in grip strength is associated with muscle mass. To determine muscle mass, the weights of the soleus and gastrocnemius muscles were measured immediately after biopsy. Aging resulted in a significant decrease in muscle mass in both the gastrocnemius (Fig. 2A) and soleus muscle (Fig. 2B). Moreover, gastrocnemius muscle mass in the O-LE group tended to increase; however, this trend was not significant compared with the O-Con group (Fig. 2A).

As regards the soleus muscle, the supplementation of LE significantly increased muscle mass in the rats in the O-LE group compared with their age-matched counterparts (Fig. 2B). However, LE intake did not significantly affect the mass of either muscle in the young-aged group (Fig. 2A and B). As the decrease in muscle-specific CK activity may be a major contributor to the loss of muscle function associated with aging (23), we assessed the effects of LE supplementation on CK activity in muscles from young and aged rats. As shown in Fig. 2C, CK activity in the young rats tended to increase slightly, although this change was not statistically significant. By contrast, aging resulted in a significant decrease in muscle CK activity (Fig. 2C). Moreover, LE intake significantly increased CK activity in the O-LE group compared with the O-Con group. Taken together, these findings suggest that LE supplementation improves muscle function in aged rats.

In order to investigate the effects of LE on age-associated muscle weakness, we performed histological analysis on the medial sections of soleus muscles using H&E staining. As shown in Fig. 3A, muscle fibers in the Y-Con group were in intimate contact in muscle bundles. LE supplementation to young rats also produced similar histological results as those observed in the rats in the Y-Con group. In the aged rats, the amount of connective tissue increased compared with that in young rats (Fig. 3A, arrowhead). Thus, connective tissue surrounding the muscle fibers (endomysium) and fiber bundles (perimysium) was observed in the soleus muscle of the O-Con group compared with the young groups; however, these histological changes were attenuated by LE supplementation (Fig. 3A). We also examined the effects of aging and LE intake on muscle CSA. The CSA in the Y-LE group did not differ from that in the Y-Con group (Fig. 3B). Moreover, the CSA in the aged rats (O-Con group) was lower than that in the Y-Con group, although the difference was not statistically significant (Fig. 3B). In the aged rats, LE supplementation increased the CSA slightly, although not significantly (Fig. 3B).

To investigate the effects of LE on muscle regeneration, we examined whether LE affects the myogenic differentiation of C2C12 myoblasts. Since it normally takes up to 6 days for C2C12 myoblasts to fully differentiate into myotubes, we first assessed the effects of LE on C2C12 cell viability by MTT assay under identical conditions of differentiation. As shown in Fig. 4A, LE (0.25–2.5 µg/ml) did not induce the significant cell death of myoblasts.

We then assessed whether LE affects the differentiation of myoblasts into myotubes. For this purpose, we used DM containing 2% HS. The undifferentiated C2C12 cells (Fig. 4B, panel GM) were flat, fusiform or star-shaped. The myotubes began to appear 3–4 days following the induction of differentiation (data not shown). After 6 days of incubation in DM, the C2C12 cells became differentiated (Fig. 4B, panel LE 0). The myotubes exhibited thick and fusiform structures, and they were elongated in 3–4 directions. We found that treatment with LE promoted the differentiation of the C2C12 myoblasts (Fig. 4B). When the LE-treated cells (panels LE 0.1–2.5) were compared with the DM-treated cells (panel LE 0) the myotubes from the LE-treated cells were more stretched and longer in shape with syncytia and nuclei and were more abundant in number than those in the DM-treated cells. Furthermore, thick and Y-shaped (or spindly ring-shaped) myotubes were also occasionally observed in the 2.5 µg/ml LE-treated cells.

To further confirm the effects of LE on myogenic differentiation, we measured the expression of the differentiation marker, MyHC, which is the major structural protein in myotubes (24). As shown in Fig. 5A, the LE-treated C2C12 cells exhibited an increased expression of MyHC in a concentration-dependent manner compared with the untreated control cells.

We then assessed CK activity, which is generally accepted as an indicator of the differentiation state of muscle cells (25). Since CK activity gradually increased until reaching peak levels on day 6 post-differentiation (data not shown), we measured CK activity on day 6 of differentiation. As shown in Fig. 5B, CK activity was induced 2.9-fold by DM alone (0 mg/ml bar) in comparison with GM. In addition, compared to treatment with DM alone (0 mg/ml bar), treatment with LE significantly enhanced CK activity in a concentration-dependent manner (Fig. 5B). Therefore, these results suggest that LE enhances myoblast differentiation into myotubes by increasing CK activity and upregulating MyHC expression.

To elucidate the mechanisms of myogenic differentiation induced by LE, we examined the effects of LE on the levels of myogenic regulatory factors (MRFs). Western blot analysis (Fig. 5C) clearly indicated that treatment with LE increased the expression of MyoD in C2C12 cells in a concentration-dependent manner. In addition, it was evident that LE induced the expression of myogenin (Fig. 5C). Overall, these results suggest that LE promotes myogenic differentiation through the upregulation of MyoD and myogenin.

Phosphatidylinositol 3-kinase (PI3K)/Akt and its downstream mTOR pathway have been shown to play an important role in skeletal myogenesis (26,27). Thus, we examined whether LE can modulate the activation of the PI3K/Akt/mTOR signaling pathway in C2C12 cells. We found that LE induced the phosphorylation of Akt, p70 S6K and 4E-BP1 in a concentration-dependent manner (Fig. 6). The effects of LE on 4E-BP1 activation, however, were clearly evident and occurred in a concentration-dependent manner, whereas those on Akt and p70 S6K were not so prominent. Of note, treatment with LE induced both the phosphorylation and expression of Akt. By contrast, no significant change in the expression of mTOR was observed in the LE-treated C2C12 cells (Fig. 6). Taken together, these results indicate that LE enhances myogenic differentiation through the PI3K/Akt/mTOR signaling pathway; however, the effects of LE on signaling molecules vary.