• The Korea Journal of Herbology
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• v.34 no.6
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• pp.99-107
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• 2019

Objective : The present study was conducted to investigate the effects of Chaenomelis Fructus (CF) on the regulation of biogenesis in C2C12 mouse skeletal muscle cells. Methods : C2C12 myoblasts were differentiated into myotubes in 2% horse serum-containing medium for 5 days, and then treated with CF extract at different concentrations for 48 hr. The expression of muscle differentiation markers, myogenin and myosin heavy chain (MHC) and mitochondrial biogenesis-regulating factors, peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC1α), sirtuin1 (Sirt1), nuclear respiratory factor1 (NRF1) and transcription factor A, mitochondrial (TFAM), and the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were determined in C2C12 myotubes by reverse transcriptase (RT)-polymerase chain reaction (RT-PCR) and western blot, respectively. The cellular glucose levels and total ATP contents were measured by cellular glucose uptake and ATP assays, respectively. Results : Treatment with CF extract (0.01, 0.02, and 0.05 mg/㎖) significantly increased the expression of MHC protein in C2C12 myotubes compared with non-treated cells. CF extract significantly increased the expression of PGC1α and TFAM in the myotubes. Also, CF extract significantly increased glucose uptake levels and ATP contents in the myotubes. Conclusion : CF extract can stimulate C2C12 myoblasts differentiation into myotubes and increase energy production through upregulation of the expression of mitochondrial biogenetic factors in C2C12 mouse skeletal muscle cell. This suggests that CF can help to improve skeletal muscle function with stimulation of the energy metabolism.

• Journal of Life Science
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• v.27 no.12
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• pp.1479-1485
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• 2017

Muscle atrophy due to aging, starvation, and various chronic diseases leads to a decrease in muscle fiber area and density due to reduced muscle protein synthesis and increased protein breakdown. This study investigated the effect of dexamethasone and hydrogen peroxide on the induction of muscle atrophy and expression of atrophy-related genes in differentiated C2C12 myotubes. C2C12 myoblasts were differentiated into myotubes in differentiation medium. During myoblast differentiation, muscle-specific transcription factors, such as myogenin, and MyoD expression increased. Differentiated C2C12 myotubes exposed to noncytotoxic levels of dexamethasone and hydrogen peroxide showed a decrease in myotube diameter, which was associated with up-regulation of muscle-specific ubiquitin ligases, such as muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), and down-regulation of myogenin and MyoD. These results demonstrated that dexamethasone and hydrogen peroxide induced atrophy through regulation of muscle-specific ubiquitin ligases and muscle-specific transcription factors in C2C12 myotubes. In this study, we confirmed the process of differentiation of C2C12 myoblasts into myotubes in in vitro experiments in the presence of atrophy. This muscle atrophy model of C2C12 cells induced by dexamethasone or hydrogen peroxide seems suited to studies of the mechanism of muscle atrophy suppression and to exploit the experiment for excavating new muscle atrophy.

• Journal of Korean Medicine for Obesity Research
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• v.23 no.2
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• pp.60-68
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• 2023

Objectives: This study was conducted to compare the effects of Hominis placenta (Jahage, J) and wild ginseng (SanSam, S) pharmacopuncture drugs on muscle differentiation and energy metabolism regulation in C2C12 myotubes. Methods: The C2C12 myoblasts were differentiated into myotubes for 5 days by replacing in medium containing 2% horse serum and then treated with J and S pharmacopuncture extract at different concentrations for 24 hr. The expression of myosin heavy chain and energy metabolism-regulating factors, myosin heavy chain (MHC), nuclear respiratory factor-1 (NRF-1), and proliferator-activated receptor γ coactivator-1 alpha (PGC-1α) were determined in C2C12 myotubes by western blot. Additionally, the phosphorylation of AMPK and the expression of mitochondrial biogenesis, including sirtuin 1 (SIRT1) were determined in the myotubes. Results: As a result, treatment with J and S pharmacopuncture extract at 0.1 and 1 mg/mL increased the MHC expression in C2C12 myotubes compared with non-treated cells, but only S pharmacopuncture was shown a significant and distinct increase in the expression. Expression of TFAM and NRF-1 was also shown significant increases in S and J pharmacopuncture in C2C12 myotubes compared to non-treated cells. The phosphorylation of AMPK and the expression of PGC-1α and SIRT1 showed increased expression in S and J pharmacopuncture compared to non-treated cells. The effect of low-dose of J pharmacopuncture on the phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and PGC-1α expression was greater than that of S pharmacopuncture. Conclusions: In conclusion, both J and S pharmacopuncture promote muscle differentiation in C2C12 myoblasts into myotubes and energy metabolism through the AMPK/SIRT1 signaling pathway. This indicates that the pharmacopuncture with tonic herbal medicines can help to improve skeletal muscle function.

• Journal of Korean Medicine for Obesity Research
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• v.15 no.1
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• pp.38-44
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• 2015

Objective: Skeletal muscle is a crucial tissue from the perspectives of mitochondrial dysfunction and insulin resistance, it is formed by myogenesis which is dynamic multistep process to be myotubes. The authors could found that root of Atractylodes macrocephala Koidzumi (Atractylodis Rhizoma Alba, ARA) enhanced glucose and lipid metabolism in C2C12 myotubes via mitochondrial regulation. However its action in myogenesis process is not known. The aim of this work was the study of ARA on proliferation, differentiation and hypertrophy in C2C12 cells. Methods: To study proliferation phase, cells were incubated in growth medium with or without ARA (0.2 or 1.0 mg/ml) for 24 hours. To examine differentiation, at 70% confluence, cells were transferred in differentiation medium both with/without ARA (0.2 or 1.0 mg/ml) for 96 hours. And after 72 hours of differentiation, cells were treated with or without ARA (0.2 or 1.0 mg/ml) for 24 hours, the genesis of hypertrophy in myotubes were analyzed. Results: In proliferation phase, ARA could make difference in morphologic examination. In differentiation phase, it also made morphologic difference furthermore ARA (1.0 mg/ml) increased mRNA expressions of Myogenic regulatory factors and muscle-specific proteins synthesis. In late differentiation, ARA induced hypertrophic morphological changes in neo-formed myotubes. Conclusions: ARA might control cell cycle promoting myogenesis and hypertrophy in C2C12 cells.

• Journal of Korean Medicine for Obesity Research
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• v.15 no.2
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• pp.131-136
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• 2015

Objectives: The prevalence of obesity and metabolic syndrome is increasing worldwide. Regulation of cellular energy metabolis has the potential to be manipulated therapeutically to serve as a target for obesity and insulin resistance. Skeletal muscle is regarded as a target for regulation of energy metabolism and insulin resistance. In this study, the authors investigated the regulatory effect of (Cinnamomum cassia Blume, CCB), Aconitum carmichaeli Debx (ACD), and Benth (Pueraria lobata Benth, PLB) on energy and glucose metabolism in C2C12 myotubes. Methods: The water extracts of CCB, ACD, and PLB (0.5 mg/ml) were treated in differentiated C2C12 myotubes. The expressions of adenosine monophosphate-activated protein kinase (AMPK) and phosphorylation AMPK were detected with western blotting. Glucose metabolism was investigated with glucose uptake assay and glucose consumption assay, total adenosine triphosphate (ATP) content was also analyzed. Results: CCB, ACD, and PLB activated the phophorylation of AMPK, they also increased the glucose metabolism and total ATP contents in C2C12 myotubes. Conclusions: This study suggests that CCB, ACD, and PLB have the potential to increase energy and glucose metabolism in skeletal muscle.

• Journal of Korean Medicine for Obesity Research
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• v.22 no.2
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• pp.93-101
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• 2022

Objectives: This study aimed to observe the anti-diabetic effect and underlying mechanisms of Galgunhwanggumhwangryun-tang (GHH; Gegen-Qinlian-decoction) in the C2C12 myotubes. Methods: GHH (1.0 mg/ml) or metformin (0.75 mM) or insulin (100 nM) were treated in C2C12 myotubes after 4 days differentiation. The glucose uptake was assessed by 2-[N-(7-160 nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose uptake by C2C12 cells. The expression of adenosine monophosphate-activated protein kinase (AMPK) and phosphorylation AMPK (pAMPK) were measured by western blot. We also evaluated gene expression of glucose transporter type 4 (Slc2a4, formerly known as GLUT4), glucokinase (Gk), carnitine palmitoyltransferase IA (Cpt1a), nuclear respiratory factors 1 (Nrf1), mitochondrial transcription factor A (Tfam), and peroxisome proliferator-activated receptor γ coactivator 1α (Ppargc1a) by quantitative real-time polymerase chain reaction. Results: GHH promoted glucose uptake in C2C12 myotubes. The expression of AMPK protein, which plays an essential role in glucose metabolism, was increased by treatment with GHH. GHH treatment tended to increase gene expression of Slc2a4, Gk, and Nrf1 but was not statistically significant. However, GHH significantly improved Tfam and Ppargc1a gene expression in C2C12 myotubes. Conclusions: In summary, GHH treatment promoted glucose uptake in C2C12 myotubes. We suggest that these effects are associated with increased gene expression involved in mitochondrial biosynthesis and oxidative phosphorylation, such as Tfam and Ppargc1a, and increased expression of AMPK protein.

• Molecules and Cells
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• v.44 no.9
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• pp.637-646
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• 2021

Free fatty acids are converted to acyl-CoA by long-chain acyl-CoA synthetases (ACSLs) before entering into metabolic pathways for lipid biosynthesis or degradation. ACSL family members have highly conserved amino acid sequences except for their N-terminal regions. Several reports have shown that ACSL1, among the ACSLs, is located in mitochondria and mainly leads fatty acids to the β-oxidation pathway in various cell types. In this study, we investigated how ACSL1 was localized in mitochondria and whether ACSL1 overexpression affected fatty acid oxidation (FAO) rates in C2C12 myotubes. We generated an ACSL1 mutant in which the N-terminal 100 amino acids were deleted and compared its localization and function with those of the ACSL1 wild type. We found that ACSL1 adjoined the outer membrane of mitochondria through interaction of its N-terminal region with carnitine palmitoyltransferase-1b (CPT1b) in C2C12 myotubes. In addition, overexpressed ACSL1, but not the ACSL1 mutant, increased FAO, and ameliorated palmitate-induced insulin resistance in C2C12 myotubes. These results suggested that targeting of ACSL1 to mitochondria is essential in increasing FAO in myotubes, which can reduce insulin resistance in obesity and related metabolic disorders.

• 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.

• Food Science of Animal Resources
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• v.44 no.4
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• pp.951-965
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• 2024

Lactiplantibacillus plantarum is a valuable potential probiotic species with various proven health-beneficial effects. L. plantarum LM1001 strain was selected among ten strains of L. plantarum based on proteolytic activity on whey proteins. L. plantarum LM1001 produced higher concentrations of total free amino acids and branched-chain amino acids (Ile, Leu, and Val) than other L. plantarum strains. Treatment of C2C12 myotubes with whey protein culture supernatant (1%, 2% and 3%, v/v) using L. plantarum LM1001 significantly increased the expression of myogenic regulatory factors, such as Myf-5, MyoD, and myogenin, reflecting the promotion of myotubes formation (p<0.05). L. plantarum LM1001 displayed β-galactosidase activity but did not produce harmful β-glucuronidase. Thus, the intake of whey protein together with L. plantarum LM1001 has the potential to aid protein digestion and utilization.

• Korean Journal of Agricultural Science
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• v.44 no.4
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• pp.586-594
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• 2017

Mitochondrial activity affects skeletal muscle energy metabolism and phenotype. To address whether mitochondrial activity can modulate muscle phenotype in vitro, protein expression of myosin heavy chain (MyHC) in C2C12 muscle cell lines was investigated after treated with antimycin A, an inhibitor of oxidative phosphorylation in mitochondria. Fully differentiated C2C12 myotubes were administrated with different concentration of antimycin A including 0, 100, 200, 500, 700, and 1000 ng/mL. After 72 h treatment, myosin heavy chain isoform expression and related enzyme activity (lactate dehydrogenase; LDH and creatine kinase) were analyzed. Administration of antimycin A changed expression of MyHC in C2C12 myotubes showing a shift from slow to fast twitching muscle type. Protein expression of MyHC type 2b (fast twitching muscle type) was decreased (P < 0.05) by antimycin A treatment (500, 700, and 1000 ng/mL) when compared with control group. Administration of antimycin A (1000 ng/mL), however, decreased (P < 0.05) MyHC type I (slow twitching muscle type). Interestingly, LDH activity was increased (P < 0.05) by antimycin A treatment. Results from our current study proposed a possibility that skeletal muscle phenotype, including MyHC and LDH activity, can be shifted from slow to fast twitching type by inhibiting the mitochondrial activity in C2C12 myotubes.