• Journal of Ginseng Research
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• v.41 no.4
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• pp.608-614
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• 2017

Background: Ginsenoside Rg1 belongs to protopanaxatriol-type ginsenosides and has diverse pharmacological activities. In this report, we investigated whether Rg1 could upregulate muscular stem cell differentiation and muscle growth. Methods: C2C12 myoblasts, MyoD-transfected 10T1/2 embryonic fibroblasts, and HEK293T cells were treated with Rg1 and differentiated for 2 d, subjected to immunoblotting, immunocytochemistry, or immunoprecipitation. Results: Rg1 activated promyogenic kinases, p38MAPK (mitogen-activated protein kinase) and Akt signaling, that in turn promote the heterodimerization with MyoD and E proteins, resulting in enhancing myogenic differentiation. Through the activation of Akt/mammalian target of rapamycin pathway, Rg1 induced myotube growth and prevented dexamethasone-induced myotube atrophy. Furthermore, Rg1 increased MyoD-dependent myogenic conversion of fibroblast. Conclusion: Rg1 upregulates promyogenic kinases, especially Akt, resulting in improvement of myoblast differentiation and myotube growth.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.6
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• pp.539-547
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• 2019

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced $[Ca2^{+}]_i$ transient and reduced sarcoplasmic reticulum (SR) $Ca^{2+}$ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR $Ca^{2+}-ATPase$ subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises $Ca^{2+}$ signaling by downregulating the expression of DHPR and SERCA proteins.

• Journal of Life Science
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• v.24 no.12
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• pp.1284-1290
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• 2014

Fatty acid transporter protein 1 (FATP1) is highly expressed in skeletal muscle and modulates fatty acid uptake and metabolism. However, the influence of insulin-like growth factor-I (IGF-I), a master regulator of skeletal muscle cells, on FATP1 in skeletal muscle cells has not been demonstrated. To investigate the effect of IGF-I on FATP1 and the expression of the IGFBP5 protein, differentiated C2C12 murine skeletal muscle cells were treated with 20 ng/ml of IGF-I at different time points. The results showed that IGF-I increased FATP1 and IGFBP5 protein expression in a time-dependent manner. To determine whether this induction of FATP1 by the IGF-I treatment was regulated pretranslationally, the mRNA level of FATP1 was measured by real-time quantitative PCR. The IGF-I treatment resulted in very rapid induction of the FATP1 mRNA transcript in C2C12 myotubes. FATP1 mRNA increased 169% and 132% after 24 and 48 h of the IGF-I treatment, respectively, and it returned to control levels after 72 h of the treatment, suggesting that the FATP1 gene is regulated pretranslationally by IGF-I in skeletal muscle cells. This is the first evidence that IGF-I can regulate the expression of FATP1. In conclusion, IGF-I induced rapid transcriptional modification of the FATP1 gene in C2C12 skeletal muscle cells and had modulating effects on fatty acid uptake proteins and oxidative proteins.

• Journal of Life Science
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• v.25 no.10
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• pp.1098-1102
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• 2015

FABPpm (plasma membrane-bound fatty acid binding protein ) is highly expressed in skeletal muscle. The principal role of this protein is modulating fatty acid uptake and metabolism. The influence of insulin-like growth factor-I (IGF-I), which is a major regulator of skeletal muscle cells, on FABPpm in skeletal muscle cells has not been investigated. To determine the effect of IGF-I on the expression of FABPpm, differentiated C2C12 murine skeletal muscle cells were treated with 20 ng/ml of IGF-I for different times. IGF-I increased the expression of FABPpm in a time-dependent manner. The mRNA level of FABPpm was measured by real-time quantitative PCR to determine whether the IGF-1-induced induction of FABPpm was regulated pretranslationally. The IGF-I treatment resulted in very rapid induction of the FABPpm mRNA transcript in the C2C12 myotubes. After 24 and 48 hr of the IGF-I treatment, FABPpm mRNA increased 130 and 179%, respectively. The increase in the protein expression returned to control levels after 72 hr of the IGF-I treatment, suggesting that IGF-1 regulated the FABPpm gene pretranslationally in skeletal muscle cells. This is the first evidence that IGF-I has a modulatory effect on the expression of FABPpm. In conclusion, IGF-I induced rapid transcriptional modification of the FABPpm gene in C2C12 skeletal muscle cells and exerted modulatory effects on FABPpm.

• Journal of Life Science
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• v.28 no.4
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• pp.435-443
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• 2018

AMP-activated protein kinase (AMPK) functions as a metabolic master through regulating and restoring cellular energy balance. In skeletal muscle, AMPK increases myofibril protein degradation through the expression of muscle-specific ubiquitin ligases. Mori Folium, the leaf of Morus alba, is a traditional medicinal herb with various pharmacological functions; however, the effects associated with muscle atrophy have not been fully identified. In this study, we confirmed the effects of AMPK activation by examining the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMPK, on the induction of atrophy and expression of atrophy-related genes in C2C12 myotubes. We also investigated the effects of the ethanol extract of Mori Folium (EEMF) on the recovery of AICAR-induced muscle atrophy in C2C12 myotubes. It was found that exposure to AICAR resulted in the stimulation of Forkhead box O3a (FOXO3a); an up-regulation of muscle-specific ubiquitin ligases such as Muscle Atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), and a down-regulation of muscle-specific transcription factors, such as MyoD and myogenin; with the activation of AMPK. In addition, AICAR without cytotoxicity indicated a decrease in diameter of C2C12 myotubes. However, treatment with EEMF significantly suppressed AICAR-induced muscle atrophy of C2C12 myotubes in a dose-dependent manner as confirmed by a decrease in myotube diameter, which is associated with a reversed stimulation of FOXO3a by the inhibition of AMPK activation. These results indicate that the activation of AMPK by AICAR induces muscle atrophy, and EEMF has preeminent effects on the inhibition of AICAR-induced muscle atrophy through the AMPK signaling pathway.

• BMB Reports
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• v.51 no.7
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• pp.350-355
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• 2018

HnRNPK is a multifunctional protein that participates in chromatin remodeling, transcription, RNA splicing, mRNA stability and translation. Here, we uncovered the function of hnRNPK in regulating the proliferation and differentiation of myoblasts. hnRNPK was mutated in the C2C12 myoblast cell line using the CRISPR/Cas9 system. A decreased proliferation rate was observed in hnRNPK-mutated cells, suggesting an impaired proliferation phenotype. Furthermore, increased G2/M phase, decreased S phase and increased sub-G1 phase cells were detected in the hnRNPK-mutated cell lines. The expression analysis of key cell cycle regulators indicated mRNA of Cyclin A2 was significantly increased in the mutant myoblasts compared to the control cells, while Cyclin B1, Cdc25b and Cdc25c were decreased sharply. In addition to the myoblast proliferation defect, the mutant cells exhibited defect in myotube formation. The myotube formation marker, myosin heavy chain (MHC), was decreased sharply in hnRNPK-mutated cells compared to control myoblasts during differentiation. The deficiency in hnRNPK also resulted in the repression of Myog expression, a key myogenic regulator during differentiation. Together, our data demonstrate that hnRNPK is required for myoblast proliferation and differentiation and may be an essential regulator of myoblast function.

• Journal of Ginseng Research
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• v.42 no.1
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• pp.116-121
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• 2018

Background: Black ginseng (BG) has greatly enhanced pharmacological activities relative to white or red ginseng. However, the effect and molecular mechanism of BG on muscle growth has not yet been examined. In this study, we investigated whether BG could regulate myoblast differentiation and myotube hypertrophy. Methods: BG-treated C2C12 myoblasts were differentiated, followed by immunoblotting for myogenic regulators, immunostaining for a muscle marker, myosin heavy chain or immunoprecipitation analysis for myogenic transcription factors. Results: BG treatment of C2C12 cells resulted in the activation of Akt, thereby enhancing hetero-dimerization of MyoD and E proteins, which in turn promoted muscle-specific gene expression and myoblast differentiation. BG-treated myoblasts formed larger multinucleated myotubes with increased diameter and thickness, accompanied by enhanced Akt/mTOR/p70S6K activation. Furthermore, the BG treatment of human rhabdomyosarcoma cells restored myogenic differentiation. Conclusion: BG enhances myoblast differentiation and myotube hypertrophy by activating Akt/mTOR/p70S6k axis. Thus, our study demonstrates that BG has promising potential to treat or prevent muscle loss related to aging or other pathological conditions, such as diabetes.

• Journal of Korean Medicine for Obesity Research
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• v.14 no.2
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• pp.55-62
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• 2014

Objective: The prevalence of metabolic syndrome and type 2 diabetes is increasing worldwide. Mitochondrial dysfunction is known to be involved in insulin resistance and obesity, researches have been increasing highly. Astragali Radix extract (ARE) or its main components have been shown to perform comparably to insulin by significantly reducing blood glucose levels in animal models however, the influence on mitochondrial dysfunction are not well understood. Methods: ARE (0.2, 0.5 and 1.0 mg/ml) or metformin (2.5 mM) were treated in C2C12 after 6 day-differentiation. The expressions of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and phosphorylation AMPK, peroxisome proliferators-activated receptror ${\gamma}$ coactivator $1{\alpha}$ ($PGC1{\alpha}$), nuclear respiratory factors 1 (NRF1), mitochondrial transcription factor (Tfam) and myosin heavy chain were detected with western blotting or polymerase chain reaction analysis. The morphological changes were also investigated. Results: ARE dose dependently increased phosphorylation of AMPK and respectively activated mRNA expressions of $PGC1{\alpha}$, NRF1 and Tfam which are mitochondrial biogenesis regulators. Furthermore, there were some morphologic differences of differentiated cells between ARE treatment and control. Conclusions: This study suggests that ARE has the potential to increase muscle mitochondrial function by activating AMPK and $PGC1{\alpha}$.

• Journal of Ginseng Research
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• v.46 no.3
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• pp.444-453
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• 2022

Background: Compound K (CK) is among the protopanaxadiol (PPD)-type ginsenoside group, which produces multiple pharmacological effects. Herein, we examined the effects of CK on muscle atrophy under hyperlipidemic conditions along with its pro-myogenic effects. Further, the molecular pathways underlying the effects of CK on skeletal muscle have been justified. Methods: C2C12 myotubes were treated with palmitate and CK. C2C12 myoblasts were differentiated using CK for 4-5 days. For the in vivo experiments, CK was administered to mice fed on a high-fat diet for 8 weeks. The protein expression levels were analyzed using western blotting analysis. Target protein suppression was performed using small interfering (si) RNA transfection. Histological examination was performed using Jenner-Giemsa and H&E staining techniques. Results: CK treatment attenuated ER stress markers, such as eIF2a phosphorylation and CHOP expression and impaired myotube formation in palmitate-treated C2C12 myotubes and skeletal muscle of mice fed on HFD. CK treatment augmented AMPK along with autophagy markers in skeletal muscle cells in vitro and in vivo experiments. AMPK siRNA or 3-MA, an autophagy inhibitor, abrogated the impacts of CK in C2C12 myotubes. CK treatment augmented p38 and Akt phosphorylation, leading to an enhancement of C2C12 myogenesis. However, AMPK siRNA abolished the effects of CK in C2C12 myoblasts. Conclusion: These findings denote that CK prevents lipid-induced skeletal muscle apoptosis via AMPK/autophagy-mediated attenuation of ER stress and induction of myoblast differentiation. Therefore, we may suggest the use of CK as a potential therapeutic approach for treating muscle-wasting conditions associated with obesity.

• Journal of Ginseng Research
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• v.47 no.6
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• pp.726-734
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• 2023

Background: Skeletal muscles play a key role in physical activity and energy metabolism. The loss of skeletal muscle mass can cause problems related to metabolism and physical activity. Studies are being conducted to prevent such diseases by increasing the mass and regeneration capacity of muscles. Ginsenoside Rg5 has been reported to exhibit a broad range of pharmacological activities. However, studies on the effects of Rg5 on muscle differentiation and growth are scarce. Methods: To investigate the effects of Rg5 on myogenesis, C2C12 myoblasts were induced to differentiate with Rg5, followed by immunoblotting, immunostaining, and qRT-PCR for myogenic markers and promyogenic signaling (p38MAPK). Immunoprecipitation confirmed that Rg5 increased the interaction between MyoD and E2A via p38MAPK. To investigate the effects of Rg5 on prevention of muscle mass loss, C2C12 myotubes were treated with dexamethasone to induce muscle atrophy. Immunoblotting, immunostaining, and qRT-PCR were performed for myogenic markers, Akt/mTOR signaling for protein synthesis, and atrophy-related genes (Atrogin-1 and MuRF1). Results: Rg5 promoted C2C12 myoblast differentiation through phosphorylation of p38MAPK and MyoD/E2A heterodimerization. Furthermore, Rg5 stimulated C2C12 myotube hypertrophy via phosphorylation of Akt/mTOR. Phosphorylation of Akt induces FoxO3a phosphorylation, which reduces the expression of Atrogin-1 and MuRF1. Conclusion: This study provides an understanding of how Rg5 promotes myogenesis and hypertrophy and prevents dexamethasone-induced muscle atrophy. The study is the first, to the best of our knowledge, to show that Rg5 promotes muscle regeneration and to suggest that Rg5 can be used for therapeutic intervention of muscle weakness and atrophy, including cancer cachexia.