Vigeo Promotes Myotube Differentiation and Protects Dexamethasone-Induced Skeletal Muscle Atrophy via Regulating the Protein Degradation, AKT/mTOR, and AMPK/Sirt-1/PGC1α Signaling Pathway In Vitro and In Vivo

Vigeo was purchased from Panax Bio Co., Ltd. (Nonsan, Republic of Korea). Vigeo was prepared using the traditional Korean fermentation method described in detail in our previous study [12]. Briefly, dried ESM (135 g), AJN (78 g), and AJK (78 g) were extracted in hot water for 3 h in a Kyungseo extractor (COSMOS-660; Kyungseo E&P Co., Ltd., Inchoeon, Republic of Korea). Korean traditional fermentation starter, nuruk (1 kg), was mixed with fresh yeast (4 g) and distilled water (1.5 L); the mixture was fermented for 96 h. Then, popped rice (3 kg) and a combined extract (hot water extract) were mixed and fermented at 26 °C for 15 days. The nuruk fermented extract mixture (Vigeo) was freeze-dried and then dissolved in distilled water (DW) according to the experimental concentration. All experiments were performed with the same batch of Vigeo, and a patent for the composition of matter and manufacture method has been registered with the Korean Intellectual Property Office (Patent No.: 10-1512230-0000).

All animal procedures conformed to Korea’s Animal Welfare Legislation with efforts made to minimize the number of animals and their discomfort. All of the animal experiments were conducted under the Approval of Institutional Animal Committee of Wonkwang University (approval no. WKU23-78); 8-week-old male ICR mice (weighing 33–36 g, n = 20) were purchased from Samtako (Daejeon, Republic of Korea) and housed in a temperature (22–24 °C) and humidity (55–60%) controlled facility on a 12 h light/dark cycle. All mice were provided ad libitum access to water and a standard irradiated chow diet (SAM #31; Samtako Bio-Korea Inc., Osan, Korea). The experimental mice were randomly assigned to one of the following four groups using a computerized random number generator: control (n = 5), Dex (25 mg/kg)-induced (n = 5), Dex + Vigeo Low (200 mg/kg, n = 5), and Dex + Vigeo High (500 mg/kg, n = 5). The staff who assign and manage groups of mice are different from the researchers who evaluate their effectiveness. Mice and tissue samples were labeled to ensure that researchers evaluating treatment effectiveness and analyzing results were not aware of differences among groups. Muscle atrophy was induced in mice by intraperitoneal injection of dexamethasone daily for 8 d [13]. Vigeo and DW were orally administered daily for 8 days. At the end of the experiment, the mice were euthanized via cervical dislocation; gastrocnemius (GA), soleus (SOL), tibialis anterior (TA), and extensor digitorum longus (EDL) muscle were collected for subsequent experiments. All animal studies did not specifically control for potential confounders. Mice were closely monitored throughout the experiment to assess their health status. During the experiment, no experimental units or data points were excluded for any of the animals because there were no significant changes in body weight, responses, or behaviors following drug administration.

The C2C12 myoblasts, a mouse myoblast cell line, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in growth medium (GM), Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Grand Island, NY,USA) with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S) antibiotic. C2C12 cells (5 × 10³ cells/well) were plated in 48-well plates and were grown to 80–90% confluence for 2–3 days. To induce differentiation of C2C12 cells from myoblasts to myotubes, the growth medium was replaced from GM to differential medium (DM) containing 2% horse serum and further cultured for 5–6 days. The medium was replaced every 3 days. After differentiation, myotubes were further cultured with Dex (100 μM) for 48 h at DM 5 days.

Cell viability was measured using a Cell Proliferation Kit II to confirm the potential cytotoxic effects of Vigeo. C2C12 cells (1 × 10³ cells/well) were seeded in 96-well plates with various concentrations of Vigeo and incubated for 5 days. Further, 50 μL of XTT reagent was added to each well, followed by another incubation for 40 min. Cell cytotoxicity was determined by measuring the absorbance at 540 nm in four replicates using a multi-well plate reader (Epoch; BioTek Instruments, Winooski, VT, USA).

Total RNA from C2C12 myotubes or gastrocnemius muscle was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. cDNA prepared using the Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific) was used as a template for qRT-PCR. qRT-PCR was performed to investigate the expression of the following interested genes: musGAPDH for 5′-TCA AGA AGG TGG TGA AGC AG-3′, musGAPDH rev 5′-AGT GGG AGT TGC TGT TGA AGT-3′, musMyoD for 5′-CGC TCC AAC TCT GAT-3′, musMyoD rev 5′-TAG TAG GCG GTG TCG TAG CC-3′, musMyogenin for 5′-CTA CAG GCC TTG CTC AGC T-3′, musMyogenin rev 5′-AGA TTG TGG GCG TCT GTA GG-3′, musMyHC1 for 5′-GCC CAG TGG AGG ACA AAA TA-3′, musMyHC1 rev 5′-TCT ACG TGC TCC TCA GCA T-3′, musAtrogin-1 for5′- CTG GAT TGG AAG AAG ATG TA-3′, musAtrogin-1 rev 5′-CTT GAG GGG AAA GTG AGA CG-3′ musMuRF1 for 5′-CCT ACT TGC TCC TTG TGC-3′, musMuRF1 rev 5′-TCC TGC TCC TGC GTG AT-3′. All data were normalized with GAPDH. The amplification parameters consisted of 40 cycles of an initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. The 2^−ΔΔct method was used for quantification [14].

C2C12 cells or gastrocnemius muscle tissues were subjected to cold lysis buffer and whole protein lysates were extracted. About 20–30 μg of protein in each well was run by sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 6–15% followed by transfer to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 60 min at 37 °C with skim milk of 5% in TBST buffer and the membranes were incubated overnight with the following primary antibodies: anti-MyHC1, anti-MyoD, anti-myogenin, anti-atrogin-1, anti-MuRF-1, anti- silent information regulator 1 (Sirt-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti- peroxisome proliferator-activated receptor-γ co-activator-1α (PGC1α; Proteintech, Rosemont, IL, USA); anti-β-actin (Sigma-Aldrich, St. Rouis, MO, USA); anti-GAPDH, anti-p-ACC, anti-ACC, anti-p-AMP-activated protein kinase (AMPK), anti-AMPK, anti-p-AKT, anti-AKT, anti-p-mTOR, anti-mTOR, anti-p-70-kDa ribosomal S6 protein kinase (p70S6K1), anti-p70S6K1, anti-p-eIF4E-binding protein (4EBP1), and anti-4EBP1 (Cell Signaling Technology, Beverly, MA, USA). Secondary antibodies of anti-rabbit and anti-mouse (Enzo Life Sciences, New York, NY, USA) were incubated for 1 h. Specific protein signals were detected using WestGlow^TM FEMTO ECL Chemiluminescent substrate (Biomax, Guri-si, Gyeonggi-do, Republic of Korea).

C2C12 cells of 5 × 10³ on 48well were grown in DM for 5 d in the presence or absence of Vigeo and exposed to Dex (100 μM) for 48 h to induce atrophy. The cells were fixed with 3.7% formaldehyde in PBS for 15 min and permeabilized with Tween 20 for 10 min. Myoblasts and myotubes were identified by MyHC1 immunofluorescence staining (Santa Cruz Biotechnology). Nuclei were visualized by blue DAPI staining (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were stained with Alexa Fluor^™ 488 goat anti-Mouse IgG (H + L) (Invitrogen Life Technologies, Carlsbad, CA, USA) in the dark at room temperature.

The muscle samples were scanned using a mCT scanner (NFR-Polaris-G90; NanoFocusRay, Jeoju, Republic of Korea). Images were acquired at 50 kVp, 55 μA, 6 μm, and 133 mSec, and 720 views were present. The final reconstructed images were converted into a digital imaging and communication-in-medicine (DICOM) file. The mCT data files were quantified using the Xelis 3D-Image calculator software 2.0 (Infinitt, Seoul, Republic of Korea).

Gastrocnemius muscle tissues were isolated and fixed with 10% neutral buffered formalin for 1 day. Paraffin sections 5 μm-thick were sliced and stained with hematoxylin and eosin (H&E). After H&E staining, representative images were taken on a Nikon Ts2 microscope (Nikon; Shinagawaku, Tokyo, Japan) for use in the cross-sectional area (CSA) analysis. The CSAs of individual myofibers were measured using NIS Elements version 5.3 software (Nikon), which can quantify the area within myofibers. After all CSA measurements were collected, CSA distribution graphs were generated by binning all data to produce a parametric distribution in control groups and then comparing experimental distributions using the same binning.

The sample size estimation was performed a priori using the G power program (https://clinicalc.com/stats/samplesize.aspx↗, accessed on 12 September 2023). All experiments were examined at least three times, and the data were expressed as the mean ± Standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test and Student’s t-test. Differences were considered statistically significant at p < 0.05. Residual diagnostics were conducted to check the normality and linearity assumptions.

According to several in vivo studies, dexamethasone, a synthetic glucocorticoid (GC), can induce muscle atrophy which reduces muscle mass, strength, and function [10,11]. Muscle disease progresses owing to the inhibition of protein synthesis and stimulation of protein degradation in skeletal muscle cells [5]. To determine whether Vigeo affects muscle atrophy in vivo, we used a Dex-induced muscle atrophy model with concomitant administration of Vigeo or DW in mice. The body weight of the Dex-treated mice was significantly lower than that of the control mice, suggesting that atrophy was normally induced by Dex treatment. The addition of Vigeo after Dex treatment tended to increase the body weight compared to that in the Dex-only group, suggesting the possibility that Dex-induced muscle atrophy could be improved by Vigeo treatment (Figure 1A). Additionally, to investigate the protective effects of Vigeo against Dex-induced muscle atrophy, we confirmed the properties of the gastrocnemius (GA), soleus (SOL), tibialis anterior (TA), and extensor digitorum longus (EDL) muscles in Dex-induced atrophy model mice using mCT imaging analysis. According to the mCT analysis method for leg muscle, the total muscle volume parameter and mCT images obtained using mCT analysis showed a prominent decrease in the Dex-injected mice compared to the control group (19.6% decrease; Figure 1D); however, it significantly increased in both the Vigeo low (250 mg/kg) and high (500 mg/kg) groups (26% and 20% increase for low and high doses, respectively; Figure 1C,D) against the Dex-injected group. Similarly, the weight of the GA muscle per body weight in the Dex-treated group was lower than that in the control group. Conversely, the GA muscle weights per body weight of the Dex + low Vigeo-supplemented group and the Dex + high Vigeo-supplemented group significantly recovered the muscle weight of the Dex-treated group. Weight gain in the SOL, TA, and EDL muscles per body weight also showed a similar weight change pattern in the Dex + Vigeo low and high dose treatments although not significantly different (Figure 1B).

To confirm the protective effect of Vigeo against Dex-induced muscle atrophy, we performed a histological analysis of GA muscles. Dex treatment suppresses protein synthesis and promotes proteolysis [15,16]. Muscle atrophy induced by Dex results in a decrease in muscle fibers and their area. As shown in Figure 2A, muscle fibers and the CSA of muscle fibers in the Dex-induced group decreased compared to those in the control group, and increased in the Dex + Vigeo groups (200 and 500 mg/kg) (Figure 2A,B). Specifically, the distribution of muscle fiber diameter showed that the number of small muscle fiber areas (CSA range: 200–1400 μm²) increased in the Dex group compared to that in the control group. In contrast, the Dex group showed a significantly decreased distribution tendency in the large muscle fiber area (CSA range: 1400–1800 μm²) compared to the control group. However, Vigeo produced a similar muscle distribution pattern to the control group, with an improved number of large muscle fiber regions (Figure 2C).

Dex treatment leads to muscle atrophy, which is achieved through the process of a protein degradation system [15]. To understand how Vigeo affects the molecular signaling mechanisms underlying Dex-induced muscle atrophy, we altered the RNA and protein expression levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1, which are markers of muscle proteolysis. We measured mRNA and protein levels in the GA muscles. Dex markedly increased the mRNA expression of proteolytic markers, including atrogin-1 and MuRF-1, compared to the control group. These increases showed that mRNA expression was effectively reversed by low and high doses of Vigeo (Figure 3A). Protein expression also showed an increase in the expression of Dex-induced proteolysis marker proteins; however, the expression was diminished in the Dex + Vigeo low and Dex + Vigeo high groups (Figure 3B). The pattern of change in protein expression is presented in the band quantification graph (Figure 3C). These findings suggest that Vigeo can improve muscle mass by regulating the degradation pathways during muscle atrophy.

Based on the effects of Vigeo on Dex-induced muscle atrophy in in vivo models, as shown in Figure 1, Figure 2 and Figure 3, we validated how Vigeo fundamentally affects myoblast differentiation in the C2C12 culture system in vitro. Before myoblast differentiation, a toxicity test was conducted to assess the effects of Vigeo on myoblast growth. C2C12 cells were cultured in growth medium containing various concentrations of Vigeo (0, 10, 25, and 50 μg/mL) for 5 days. No significant differences in cell proliferation were observed after Vigeo treatment during the growing process (Figure 4A). To further investigate the influence of Vigeo on myoblast differentiation, C2C12 cells were cultured in DM supplemented with various concentrations of Vigeo (0, 10, 25, or 50 μg/mL) for 5 days. To evaluate differences in myotube shape, length, and width, we performed immunofluorescence staining and subsequent measurements. Vigeo increased the number of fused giant myotubes (three or more nuclei) in a dose-dependent manner (Figure 4B,C). Furthermore, Vigeo increased myotube diameter in terms of length and width in a dose-dependent manner compared to the control group (Figure 4B,C). We investigated the effect of Vigeo on the expression of marker genes that regulate myotube formation at each stage of myoblast differentiation. C2C12 cells were cultured in GM for 3 d, followed by each 2, 4, and 6-d incubation in DM in the presence of 50 μg/mL concentrations of Vigeo. Myoblasts exhibited a marked increase in mRNA and protein expression levels of MyHC1, MyoD, and myogenin during myogenic differentiation. The mRNA and protein expression of myogenic marker genes, including MyHC1, myogenin, and MyoD, significantly increased compared to those in the control group from DM4 to DM6 day with the addition of Vigeo (Figure 4D,E). These results indicated that treatment with Vigeo improved the expression of markers and promoted an increase in the size and number of myotubes.

Muscle atrophy is induced by an imbalance in protein lysis and degradation and is characterized by an increased level of protein degradation. Muscle breakdown mainly involves protein degradation via the ubiquitin-proteasome pathway in muscle cells [4,5]. Atrogin-1 and MuRF-1 are muscle-specific ubiquitin ligases and muscle atrophy markers [16]. Further, we determined whether the increase in the expression of these differentiation markers was due to the effect of Vigeo on protein degradation markers. The degradation markers atrogin-1 and MuRF-1, which increased during myoblast differentiation, were significantly decreased in mRNA and protein expression upon the addition of Vigeo (Figure 5A,B). Collectively, these results indicated that Vigeo promoted myoblast differentiation by inhibiting the expression of muscle-specific ubiquitin ligase markers.

Based on the in vivo experimental data showing that Vigeo has a positive effect on Dex-induced muscle atrophy, we examined whether the same tendency was observed in C2C12 cells in vitro. C2C12 cells were treated and cultured with Vigeo (0, 10, 25, and 50 μg/mL) at several concentrations for 5 d in DM. No significant differences were observed in cell proliferation in the presence of Vigeo compared to the vehicle in XTT cell proliferation analysis (Figure 4A). Further, we examined the effects of Vigeo on Dex-induced myotube atrophy in C2C12 cells. C2C12 cells were differentiated from myoblasts into myotubes in 2% HS DM media incubation for 5 d and followed further incubation with Dex treatment of 100 μM for 48 h. After treatment with Dex, we observed and analyzed the fusion index of differentiated myotubes under the conditions indicated in Figure 6A. The elongated myotubes were represented by MyHC green-immunofluorescence-stained cells and blue-stained DAPI in the elongated myotubes showed as fused myotubes; only myotubes formed by the fusion of three or more nuclei were counted. Dex treatment resulted in severely atrophied myotube formation, which was distinct from the thick, well-formed myotubes observed in control cells. Contrary to the Dex-only treated group, the cells treated with Dex + Vigeo (50 μg/kg) showed improved myotube differentiation, resulting in the recovery of the width and length of the myotubes (Figure 6A,B). To compare the protective effects of Vigeo against Dex-induced muscle cell atrophy, we verified the expression of genes related to myoblast differentiation, mitochondrial function, protein synthesis, energy metabolism, and muscle cell degradation in C2C12 myotubes. Dex-treated cells showed remarkably reduced myoblast differentiation, which was confirmed to result in decreased expression of myoblast differentiation-related genes, such as MyHC1, MyoD, and myogenin. However, the expression of muscle cell differentiation factors decreased after Dex treatment, and the expression of differentiation markers recovered in response to Vigeo treatment (Figure 6C). In skeletal muscle cells, mitochondrial biogenesis is one of the important parts of maintaining healthy muscle cells, and among them, the PGC1α is a key regulator of mitochondrial biogenesis and function [17] and is commonly activated by AMPK and Sirt-1 [18,19]. As muscle cell differentiation progressed, the expression of genes involved in mitochondrial biogenesis increased, and the expression of AMPK, Sirt-1, and PGC1α decreased following Dex treatment. The inhibition of myoblast differentiation was expected to be due to a Dex-induced decrease in mitochondrial biogenesis and this decrease was reversed through increased expression of AMPK, Sirt-1, and PGC1, α, which was strongly restored by Vigeo treatment (Figure 6D–E). Skeletal muscle mass and differentiation are regulated by protein synthesis via the mTOR pathway [20]. To evaluate the effects of Vigeo on muscle protein synthesis in response to Dex stress, changes related to the Akt/mTOR pathway were measured in C2C12 cells. Dex treatment inhibited the phosphorylation of Akt and the mammalian target of rapamycin (mTOR) compared with the control. However, the Dex + Vigeo group showed a significant increase in Akt and mTOR phosphorylation (Figure 6F). To further determine the effect of Vigeo on muscle anabolic signaling downstream of Akt/mTOR signaling [21], the phosphorylation of 4EBP1 [22] and p70S6K [23] was detected in C2C12 myotubes. The Dex-treated group also showed that the phosphorylation of p70S6K and 4EBP1 was significantly decreased in C2C12 myotubes. However, the phosphorylation levels of p70S6K and 4EBP1 were slightly increased in the Dex + Vigeo group compared to those in the Dex group (Figure 6F). Finally, concurrent restraint of atrogin-1 and MuRF-1 resulted in myotube hypertrophy, which mainly reflects the upregulation of protein synthesis and prevents Dex-induced myotube atrophy [24]. We cultured myotubes to determine whether Dex-induced muscle loss was affected by the myoblast lytic mechanism induced by the addition of Vigeo. In C2C12 cells, both mRNA and protein expression of the muscle proteolysis markers atrogin-1 and MuRF-1 were significantly increased compared to those in the control due to additional Dex treatment. However, the expression of these two muscle-specific degradation factors was significantly decreased by Vigeo treatment, indicating a tendency to compensate for muscle loss (Figure 6G,H). Together, these results demonstrate that Vigeo prevents muscle atrophy and promotes myoblast differentiation by suppressing Dex-induced proteolysis and regulating mitochondrial biogenesis and energy metabolism of muscle cells in C2C12 myotubes.