• 대한한의학방제학회지
• /
• 제28권1호
• /
• pp.29-39
• /
• 2020

Objectives : Glucoraphanin is one of the well-known natural glucosinolates found in cruciferous plants. In the present study, we investigated the effects and molecular mechanism of glucoraphanin in dexamethasone-induced skeletal muscle atrophy in vitro model. Methods : The cytotoxic effects of glucoraphanin on C2C12 myoblasts or myotubes were evaluated by MTT assay. The glucoraphanin was evaluated effects in dexamethasone-induced skeletal muscle atrophy in C2C12 myotubes using a real-time PCR, western blots analysis, and immunofluorescence staining of myosin heavy chain. Result : Glucoraphanin had no cytotoxicity on both C2C12 myoblasts or myotubes. Dexamethasone markedly induced muscle atrophy by up-regulating muscle-specific ubiquitin E3 ligase markers, atrogin-1 and MuRF1, and down-regulating MyoD, a myogenic regulatory factor whereas co-treatment of glucoraphanin and dexamethasone dose-dependently inhibited it. Furthermore, decreased expressions of p-Akt, p-FOXO1, and p-FOXO3a induced by dexamethasone were reversed by co-treatment with glucoraphanin and dexamethasone. In addition, dexamethasone obviously reduced myotube diameters, while co-treatment of glucoraphanin and dexamethasone increased those to a similar level as control. Conclusions : These results show that glucoraphanin suppresses dexamethasone-induced muscle atrophy in C2C12 myotubes through activation of Akt/FOXO signaling pathway.

• 한국식품영양학회지
• /
• 제37권5호
• /
• pp.236-243
• /
• 2024

The present study was conducted to investigate effects of rabbit meat extract on energy metabolism and muscle differentiation in C2C12 myotubes. Water extract of rabbit meat (10, 50, 100, and 200 ㎍/ml) was used to treat differentiated C2C12 cells. Reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis were used to determine mRNA or protein levels of energy metabolism-related genes. Total adenosine triphosphate (ATP) content was also measured. Treatment with rabbit meat extract significantly increased expression levels of muscle differentiation markers (myogenin and myosin heavy chain) and mitochondrial biogenesis regulators (PGC1α, NRF1, and TFAM) in C2C12 myotubes compared to non-treated control. Additionally, rabbit meat extract activated phosphorylation of AMPK and acetyl-coA carboxylase (ACC). Rabbit meat extract significantly increased ATP contents in myotubes. These results suggest that rabbit meat extract has the potential to improve energy metabolism in skeletal muscles.

• 대한한방내과학회지
• /
• 제41권4호
• /
• pp.599-611
• /
• 2020

Objectives: The aim of this study was to evaluate the effects of Hwanggi-tang on glucose digestion, uptake, and metabolism in murine C2C12 myotubes. Methods: Hwanggi-tang was prepared according to the Dong-ui-bo-gam (≪東醫寶鑑≫) prescription by 70% ethanol extraction. The effect on glucose digestion was examined by determining the inhibitory effect of Hwanggi-tang on α-glucosidase activity. We also compared and verified the gene and protein expression of genes related to glucose uptake in C2C12 myotubes treated with Hwanggi-tang or insulin. Glucose metabolism was assessed by the expression levels of associated enzymes. Results: Hwanggi-tang caused a dose-dependent inhibition of α-glucosidase activity, induced glucose uptake by activation of the PI3K/Akt/mTOR pathway in the insulin signaling pathway, and promoted glucose oxidation and β-oxidation. Conclusions: Hwanggi-tang exerts an anti-diabetic effect on murine myotubes by inhibiting glucose digestion and inducing glucose uptake and consumption.

• 대한본초학회지
• /
• 제37권4호
• /
• pp.31-38
• /
• 2022

Objectives : In this study, we investigated the synergistic protective effects of medicinal herbal mixture (HME) including Mori Ramulus (MR), Acanthopanacis Cortex (AC), Eucommiae Cortex (EC), and Black soybean (BS) in C2C12 cells, mouse myoblasts. Methods : Effects of HME on cell viability of C2C12 myoblasts were monitored by MTT assay. Anti-atrophic activity of HME was determined in myoblasts and myotubes under oxidative stress by H₂O₂. C2C12 myoblasts were differentiated into myotubes in a medium containing 2% horse serum for 6 days. After that, we measured that expression of MyoD and myogenine, the myogenic regulatory factors, to identify the mechanism of inhibiting muscle atophy after HME treatment. In addition, suppression of phosphorylation of Akt, FoxO3a and MARF-1, transcription factors of degradation proteins were analyzed via western blotting. Results : As a result of MTT, HME there was no show cytotoxicity up to a concentration of 1 mg/ml. The cytoprotective effects on oxidative stressed myoblast and myotube was better in HME extract than those of MR, AC, EU, and BS, respectively. HME treatment in Myotube induced by oxidative stress after H₂O₂ treatment increased Myo D, Myogenine activation, and Akt, FoxO3a phosphorylation and decreased expression of MuRF-1. As the results, HME has synergistic effects on protection against proteolysis of C2C12 myotubes through activation of the Akt signaling pathway under oxidative stress. Conclusions : These results suggest that HME may also be useful as a preventing and treating material for skeletal muscle atrophy caused by age-related diseases.

• 생명과학회지
• /
• 제28권4호
• /
• pp.435-443
• /
• 2018

AMPK는 세포 내 에너지 균형을 조절하는 조절자 및 에너지 센서이며, 특히 골격근에서는 muscle-specific ubiquitin ligases의 조절을 통한 근육 단백질 분해를 증가시키는 것으로 알려져 있다. 한편 상엽은 다양한 약리학적 효능을 가지는 전통약재 중 하나이지만 근위축과 관련된 효능에 대해서는 거의 알려져 있지 않다. 본 연구에서는 C2C12 myotubes에서 AMPK 활성제인 AICAR가 유발하는 근위축 및 관련 유전자의 발현과 함께 상엽 에탄올 추출물(ethanol extracts of Mori Folium, EEMF)이 유발하는 근위축 억제 효능에 대해서 조사하였다. 먼저 C2C12 myoblasts에 AICAR를 처리하였을 경우 AMPK 활성화가 유발되었으며, 하위 단계에 있는 FoxO3a의 발현 증가와 함께 muscle-specific ubiquitin ligases인 MAFbx/atrogin-1 및 MuRF1의 발현 증가와 muscle-specific transcription factors인 MyoD 및 myogenin의 발현 감소가 유발되었다. 또한 분화가 유발된 C2C12 myotubes에 세포독성이 없는 조건의 AICAR를 처리하였을 경우 근위축이 유발되었으며, EEMF는 AMPK 불활성화 및 FoxO3a 발현 억제를 유발함으로서 AICAR 처리에 의한 근위축을 억제하는 것으로 나타났다. 본 연구 결과에서 AICAR에 의한 AMPK 활성화가 근위축을 유발한다는 것을 알 수 있었으며, EEMF는 AMPK signaling pathway를 통하여 AICAR에 의한 근위축을 억제한다는 것을 알 수 있었다.

• Journal of Microbiology and Biotechnology
• /
• 제34권3호
• /
• pp.495-505
• /
• 2024

Gromwell (Lithospermum erythrorhizon, LE) can mitigate obesity-induced skeletal muscle atrophy in C2C12 myotubes and high-fat diet (HFD)-induced obese mice. The purpose of this study was to investigate the anti-skeletal muscle atrophy effects of LE and the underlying molecular mechanism. C2C12 myotubes were pretreated with LE or shikonin, and active component of LE, for 24 h and then treated with 500 μM palmitic acid (PA) for an additional 24 h. Additionally, mice were fed a HFD for 8 weeks to induced obesity, and then fed either the same diet or a version containing 0.25% LE for 10 weeks. LE attenuated PA-induced myotubes atrophy in differentiated C2C12 myotubes. The supplementation of LE to obese mice significantly increased skeletal muscle weight, lean body mass, muscle strength, and exercise performance compared with those in the HFD group. LE supplementation not only suppressed obesity-induced skeletal muscle lipid accumulation, but also downregulated TNF-α and atrophic genes. LE increased protein synthesis in the skeletal muscle via the mTOR pathway. We observed LE induced increase of mitochondrial biogenesis and upregulation of oxidative phosphorylation related genes in the skeletal muscles. Furthermore, LE increased the expression of peroxisome proliferator-activated receptor-gamma coactivator-1 alpha and the phosphorylation of adenosine monophosphate-activated protein kinase. Collectively, LE may be useful in ameliorating the detrimental effects of obesity-induced skeletal muscle atrophy through the increase of protein synthesis and mitochondrial biogenesis of skeletal muscle.

• 생명과학회지
• /
• 제26권4호
• /
• pp.387-397
• /
• 2016

본 연구는 17-DMAG이 골격근에서 autophagy에 관여하는 가를 조사하기 위해, C2C12세포와 마우스 골격근에서 17-DMAG (Hsp90 억제제/Hsp72 활성제)을 처치하는 그룹과 autophagy 억제제(Bafilomycin 또는 colchicine)를 처치하는 그룹과 처치하지 않는 그룹을 동시에 두고 autophagy flux를 측정하였다. C2C12 배양세포에서 17-DMAG이 Hsp90 억제/hsp72 활성화시켰으며 Akt-mTOR 신호체계를 유의하게 감소시켰지만(p<0.05) autophagy marker 단백질인 LC3 II와 p62를 증가시키지 않았다. in vivo 모델의 경우 17-DMAG 처치가 배양세포에서 발견된 것처럼 Hsp90억제/hsp72를 활성화시켰고 Akt-mTOR 신호체계를 유의하게 감소시켰다(p<0.05). 반면 LC3 II와 p62 단백질 수준은 autophagy 억제제(colchicine) 처치 수준보다 더 높게 증가되었다. 이는 17-DMAG이 골격근에서 autophagy를 증가시키지만 C2C12 배양세포에서는 autophagy의 활성화가 제한적임을 암시한다. 현재 이러한 in vitro와 in vivo 모델에서의 차이는 불분명하다.

• The Korean Journal of Physiology and Pharmacology
• /
• 제23권6호
• /
• pp.539-547
• /
• 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.

• 한국식품영양학회지
• /
• 제37권5호
• /
• pp.273-281
• /
• 2024

Skeletal muscle is an organ that regulates biological metabolic energy. Its dysfunction causes decline of body functions and disability, thus deteriorating the overall quality of life. Various materials are being developed with an anti-sarcolytic effect. However, anti-sarcolytic effect of Sinomenium acutum rhizomes extract (SAE) remains unclear. Therefore, this study aimed to investigate anti-muscle atrophy effects of SAE and its alkaloids, including sinomenine (SIN), magnoflorine (MF), acutumine (ACU), and N-ferultyramine (NFT) isolated from SAE, on dexamethasone (Dex)-induced myotubules. C2C12 myogenic cells differentiated for 6 days were treated with 1 mM Dex for 24 hours. Induction of muscular atrophy was confirmed by a decrease in myogenin expression. We found that Dex increased expression levels of muscle-specific ubiquitin ligases MuRF1 and MAFbx/atrogin-1. However, mRNA and protein levels of these muscle-specific ubiquitin ligases were significantly reduced by cotreatment with SIN, MF, and NFT in myotubes. Glucose uptake reduced by Dex in myotubules were also restored by SIN, MF, and NFT treatments. These results suggest that SIN, MF, and NFT can reduce muscle wasting and enhance glucose uptake in Dex-treated myotubes, highlighting their potential as therapeutic agents to prevent muscle atrophy.

• 대한본초학회지
• /
• 제34권6호
• /
• pp.79-89
• /
• 2019

Objective : This study investigated the anti-diabetic effects of DM1, a herbal mixture with Atractylodis Rhizoma, Anemarrhenae Rhizoma, and Cinnamomi Cortex in high fat diet (HFD)-induced diabetic mice and the mechanism in C2C12 mouse skeletal muscle cells. Methods : The C57B/6 mice were fed high fat for 12 weeks, and then administrated DM1 extract (500 mg/kg, p.o.) for 4 weeks. The changes of body weight, calorie and water intakes, fasting blood glucose levels and the serum levels of glucose, insulin, triglyceride, HDL-cholesterol, AST and ALT were measured in mice. The histological changes of liver and pancreas tissues were also observed by H&E stain. C2C12 myoblasts were differentiated into myotubes and then treated with DM1 extract (0.5, 1, and 2 mg/㎖) for 24 hr. The expression of myosin heavy chain (MHC), PGC1α, Sirt1 and NRF1, and the AMPK phosphorylation were determined in the myotubes by western blot, respectively. Results : The DM1 extract administration significantly decreased the calorie and water intakes, glucose, triglyceride, AST and ALT levels and increased insulin and HDL-cholesterol in HFD-induced diabetic mice. DM1 extract inhibited lipid accumulation in liver tissue and improved glucose tolerance. In C2C12 myotubes, DM1 treatment increased the expression of MHC, PGC1α, Sirt-1, NRF-1 and the AMPK phosphorylation. Conclusion : In our results indicate that DM1 can improve diabetic symptoms by decreasing the obesity, glucose tolerance and fatty liver in HFD-induced diabetic mice, and responsible mechanism is might be related with energy enhancement.