• 약학회지
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• 제40권6호
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• pp.734-738
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• 1996

The development of the alpha2 isoform of (Na,K)ATPase which is high affinity ouabain receptors was studied in the differentiating nonfusing muscle cell line BC3H-1. T he differentiation process of BC3H-1 cell line was confirmed by 2-dexy-D-[$^3$H] glucose uptake experiment and the quantity of the expression of ${\alpha}_2$ isoform was measured using a whole cell [$^3$H] ouabain-binding assay. Undifferentiated growing BC3H-1 cells, myoblasts, exhibited low levels of insulin-stimulated glucose uptake and [$^3$H] ouabain-binding sites. In contrast, differentiated BC3H-1 cells, myocytes, had a 5.6-fold increase in insulin-stimulated glucose uptake and 5-fold increase in [$^3$H] ouabain-binding sites. Scatchard analysis showed that myocytes developed more [$^3$H] ouabain-binding sites than myoblasts vath a dissociation constant (kd) of 6${\times}10^{-8}$M and capacity of 6.l${\times}10^{-5}$ sites/cell. Therefore. it seems that myoblasts express low levels of ${\alpha}_2$ subunit and probably the majority of ${\alpha}_1$ subunit, whereas myocytes express high levels of ${\alpha}_2$ isoform. The results indicate that the expression of ${\alpha}_2$ isoform is developmentally regulated during differentiation and that BC3H-1 culture system provides an excellent model for the study of differentiation and mechanism of (Na,K)ATPase action in muscle which requires electrical excitability.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.157-158
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• 2003

A newly synthesized thiazolodinedione derivative, CKD-501, was confirmed to have antihyperglycemic effect in in vivo study. The present study was undertaken to investigate the effect of CKD-501 on glucose transport and its stimulating mechanism in L6-myotubes. L6-myoblasts were cultured and differentiated to myotubes by reducing serum concentration in media from 10％ to 2％. (omitted)

• Animal cells and systems
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• 제11권1호
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• pp.17-22
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• 2007

The effects of antimetabolite 6-AN (6-amino-nicotinamide) on viability and morphology of L6 myoblast cells have been investigated. 6-AN ($100{\mu}M$) induced a time-dependent decrease in cell viability with respect to the untreated control cells. Following 6-AN administration the viability rate started to decline sharply, reaching about 23% of the untreated control cells at 48 h. Inverted phase-contrast microscopy revealed that 6-AN caused characteristic morphological changes such as irregularly elongated and stellate shape of cells, round-shaped nucleus, cytoplasmic vacuolization, irregular cell arrangements and formation of large spaces among cell clusters. The concentrations of ATP and $NAD^{+}$ in the 6-AN treated cells were significantly lower (p < 0.01) than those of the untreated control cells. In contrast, the concentration of AMP was significantly increased by the 6-AN treatment. Activities of catalase, superoxide dismutase and glutathione peroxidase in 6-AN treated cells were significantly higher (p < 0.01) than those of the untreated control cells. The activities of glyceraldehyde-3-phosphate dehydrogenase in 6-AN treated cells were significantly lower (p < 0.01) than those of the untreated control cells. The results suggest that 6-AN caused marked reduction of cell viability and alterations of some important metabolites and enzymes.

• 대한물리의학회지
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• 제14권2호
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• pp.63-70
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• 2019

PURPOSE: While electrical stimulation (ES) is known to be a safe and flexible tool in rehabilitation therapy, it has had limited adoption in muscle regeneration. This study was performed to investigate whether ES can induce myogenic differentiation and to clarify the mechanism underlying the effects of ES on myogenic differentiation. METHODS: This study used rat L6 cell lines as myoblasts for myogenic differentiation. Electric stimulation was applied to the cells using a C-Pace EP culture pacer (IonOptix, Westwood, Ma, USA). The gene expressions of myogenic markers were examined using qPCR and immunochemistry. RESULTS: Our study showed that ES increased the thickness and length of myotubes during myogenic differentiation. It was found that ES increased the expression of myogenic markers, such as MyoD and Myogenin, and also activated the fusion of the myoblast cells. In addition, ES suppressed the expression of small GTPases, which can explain why ES promotes myogenic differentiation. CONCLUSION: We found that ES induced myogenic differentiation by suppressing small GTPases, inhibiting cell division. We suggest that ES-based therapies can contribute to the development of safe and efficient muscle regeneration.

• 대한본초학회지
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• 제29권1호
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• pp.45-52
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• 2014

Objectives : The aim of this study was to investigate the effects water extract of fermented new korean medicinal mixture, combinations of Mori Folium, Adenophorae Radix, Phllostachyos Folium and Citri Pericarpium (F-MAPC), on adipocyte differentiation, adipogenesis and glucose uptake using undiffernentiated 3T3-L1 adipocytes and L6 myoblasts. Methods : Each herb and those mixture were respectively fermented and then extracted with water. We carried on MTT assay for check-up on cell toxicity, Oil Red O staining for determination of cell differentiation and intracelluar adipogenesis. Western blot analysis for measurement of pAMPK and pACC, $C/EBP{\alpha}$, $PPAR{\gamma}$ and Glut-4 protein expressions were performed. Results : F-MAPC showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 preadipocytes without affecting cell toxicity as assessed by measuring fat accumulation, and this effect was 2 fold higher in 0.2 mg/ml F-MAPC than that of the same dose of each fermented herbal extract alone. In addition, these effects were associated with modulation of adipogenic transcription factors, such as $C/EBP{\alpha}$, $PPAR{\gamma}$, as well as stimulated phosphorylations of AMPK and ACC. Translocation of Glut-4 was significantly increased by 10.2% in L6 cells treated with 0.2 mg/ml F-MAPC compared with that of control. Conclusions : These results demonstrate that F-MAPC may be an ideal candidate for therapy of obesity and diabetes by disturbing the differentiation into adipocytes, as well as the inducement of intramuscular glucose uptake from blood.

• Animal cells and systems
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• 제1권1호
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• pp.63-69
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• 1997

We have previously reported that NF-kB is involved in the regulation of nitric oxide synthase gene expression during differentiation of chick embryonic myoblasts. However, how NF-kB is timely activated during myogenesis remains elusive. One of the most prominent events in myogenesis is myoblast membrane fusion, which is accompanied with massive cytoskeletal reorganization. Here we show that the activity of NF-kB markedly increases in L6 rat myogenic cells that have just initiated morphological changes by treating nocodazole, a microtubule-disrupting agent. Furthermore, the induction of NF-kB activation was closely correlated with the myoblast fusion. In addition, a variety of agents that disrupt microtubules stimulated the myoblast fusion as well as the induction of NF-kB activation. In contrast, taxol, a microtubule-stabilizing agent, suppressed the induction of NF-kB activation and inhibited spontaneous differentiation of L6 cells as well. In addition, we found that the NF-KB in the cells consists of p50/p65 heterodimers. These results support the idea that reorganization of microtubule at early stages of differentiation plays a role as a signal for NF-KB activation during myogenesis.

• The Korean Journal of Physiology and Pharmacology
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• 제15권6호
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• pp.363-369
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• 2011

The present study elucidated the effect of the selective inducible nitric oxide synthase (iNOS) inhibitor $N^6$-(1-iminoethyl)-L-lysine (L-NIL) on monosodium urate (MSU) crystal-induced inflammation and edema in mice feet. L-NIL (5 or 10 mg/kg/day) was administered intraperitoneally 4 h before injection of MSU (4 mg) into the soles of mice hindlimb feet. Twenty-four hours after MSU injection, foot thickness was increased by 160% and L-NIL pretreatment reduced food pad swelling in a dose dependent manner. Pretreatment of 10 mg/kg/day L-NIL significantly suppressed the foot pad swelling by MSU. Plasma level of nitric oxide (NO) metabolites and gene expression and protein level of iNOS in feet were increased by MSU, which was suppressed by L-NIL pretreatment. Similar pattern of change was observed in nitrotyrosine level. MSU increased the gene expression of tumor necrosis factor (TNF)-${\alpha}$ and interleukin (IL)-$1{\beta}$ and L-NIL pretreatment suppressed MSU-induced cytokines expression. The mRNA levels of superoxide dismutase and glutathione peroxidase1 were increased by MSU and L-NIL pretreatment normalized the gene expression. Phosphorylation of extracellular signal-regulated kinase 1/2 and p38 was increased by MSU, which was suppressed by L-NIL pretreatment. The mRNA levels of iNOS, TNF-${\alpha}$, and IL-$1{\beta}$ were increased by MSU in human dermal fibroblasts, C2C12 myoblasts, and human fetal osteoblasts in vitro, which was attenuated by L-NIL in a dose dependent manner. This study shows that L-NIL inhibits MSU-induced inflammation and edema in mice feet suggesting that iNOS might be involved in MSU-induced inflammation.

• 한방비만학회지
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• 제15권2호
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• pp.93-103
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• 2015

Objectives: It was investigated the cytoprotective efficacies of isorhamnetin, a flavonoid originally derived from Hippophae rhamnoides L., against oxidative stress-induced apoptosis in C2C12 myoblasts. Methods: The effects of isorhamnetin on cell growth, apoptosis and reactive oxygen species (ROS) generation were evaluated by trypan blue dye exclusion assay, 4',6-diamidino-2-phenylindole staining and flow cytometry. The levels of apoptosis-regulatory and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway-related proteins, and caspase activities (caspase-3 and -9) were determined by Western blot analysis and colorimetric assay, respectively. Results: Our results revealed that treatment with isorhamnetin prior to hydrogen peroxide ($H_2O_2$) exposure significantly increased the C2C12 cell viability and, indicating that the exposure of C2C12 cells to isorhamnetin conferred a protective effect against oxidative stress. Isorhamnetin also effectively attenuated $H_2O_2$-induced apoptosis and ROS generation, which was associated with the restoration of the upregulation of Bax and downregulation of Bcl-2 induced by $H_2O_2$. In addition, $H_2O_2$ enhanced the activation of caspase-9 and -3, and degradation of poly (ADP-ribose)-polymerase, a typical substrate protein of activated caspase-3; however, these events were almost totally reversed by pretreatment with isorhamnetin. Moreover, isorhamnetin increased the levels of heme oxygenase-1, a potent antioxidant enzyme, associated with the induction of Nrf2. Conclusions: Our data indicated that isorhamnetin may potentially serve as an agent for the treatment and prevention of muscle disorders caused by oxidative stress.

• The Korean Journal of Physiology and Pharmacology
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• 제23권6호
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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 Animal Science and Technology
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• 제45권6호
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• pp.891-900
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• 2003

c-fos 유전자는 전사조절인자로서 주로 c-jun family와 결합하여 heterodimers를 형성하며 AP-1 조절 부위를 가지는 유전자들의 전사를 조절하는 것으로 알려져 있다. 이 유전자의 발현은 myoblasts를 비롯한 여러 세포의 분화와 성장을 조절하며 최근 돼지에서 육질에 영향을 미치는 근섬유와 관련된다는 보고가 있다. 본 연구는 소에서 육질과 c-fos 유전자와의 관계를 알아보기 위한 기초자료로서 총 1,443 bp의 mRNA 염기서열을 최초로 소에서 밝혔으며 여러 조직과 기관에서의 발현양상도 살펴보았다. 한우의 c-fos 유전자의 염기서열을 사람, 돼지 및 쥐와 비교하여 본 결과 각각 89.8%, 93.5%와 87.0%의 높은 상동성을 보였다. 이 유전자의 발현은 근육중 갈비에서 가장 많은 발현량을 보였고, 조직에서는 비장에서 가장 많은 발현량을 보이는 것을 알 수 있었다. 이 연구에서 밝혀진 c-fos 유전자는 SNP의 추가분석에 의해 한우에서 육질의 향상과 관련이 있는 후보유전자로 쓰일 수 있을 것으로 사료된다.