• Biomedical Science Letters
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• v.12 no.3
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• pp.201-208
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• 2006

PI3-kinase activity through p85, the regulatory subunit of class IA PI3-kinase, is indispensable for the growth, differentiation, and survival of skeletal muscle cells, but little is known about the function of other regulatory subunits such as p55 and p50. We examined the subcellular localization and the expression of the regulatory subunits of class IA PI3-kinase in L6 myoblasts. Both p55 and p50 as well as p85 were expressed in L6 myoblasts. Whereas p85 was localized at both cytosolic and nuclear tractions, p55 and p50 were localized at only the nuclear traction. During the differentiation of L6 myoblasts, the protein concentrations of both p55 and p50 were decreased but that of p85 was not significantly changed. Menadione-induced oxidative stress induced the translocation of p85 from cytosol to nucleus and the increase of p55 expression. These results suggest that the regulatory subunits of class IA PI3-kinase play an important role in L6 myoblasts.

• Journal of Life Science
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• v.30 no.9
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• pp.772-782
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• 2020

Skeletal muscle differentiation or myogenesis is important to maintain muscle mass and metabolic homeostasis. Muscle-specific microRNAs (miRNAs) are known to play a critical role in skeletal myogenic differentiation. In this study, we examined the expression profiling of miRNAs during myogenic differentiation in rat L6 myoblasts using rat miRNA microarrays. We identified the upregulated expression of miR-128 as well as several well-known myogenic miRNAs, including miR-1, miR-133b, and miR-206. We additionally confirmed the increased expression of miR-128 observed on microarray through quantitative real-time PCR (qRT-PCR), which showed similarly upregulated expression of both primary miR-128 and mature miR-128, consistent with the microarray findings. Furthermore, transfection of miR-128 into rat L6 myoblasts induced gene expression of myogenic markers such as muscle creatine kinase (MCK), myogenin, and myosin heavy chain (MHC). Protein expression of MHC was increased as well. Inhibition of miR-128 by inhibitory peptide nucleic acids (PNAs) blocked the expression of those myogenic markers. In addition, the transfection of miR-128 into rat L6 myoblasts enhanced the phosphorylation of Erk and Akt proteins stimulated by insulin, while simultaneously reversing the inhibited phosphorylation of Erk and Akt due to insulin resistance. These findings suggest that miR-128 may play important roles in myogenic differentiation and insulin signaling.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.6
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• pp.697-703
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• 2018

Myoblast fusion depends on mitochondrial integrity and intracellular $Ca^{2+}$ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with $[Ca^{2+}]_i$ regulation in normal and mitochondrial DNA-depleted(${\rho}0$) L6 myoblasts. The ${\rho}0$ myoblasts showed impaired myotube formation. The inwardly rectifying $K^+$ current ($I_{Kir}$) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated $Ca^{2+}$ channel and $Ca^{2+}$-activated $K^+$ channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the $I_{Kir}$. The ${\rho}0$ myoblasts showed depolarized resting membrane potential and higher basal $[Ca^{2+}]_i$. Our results demonstrated the specific downregulation of $I_{Kir}$ by dysfunctional mitochondria. The resultant depolarization and altered $Ca^{2+}$ signaling might be associated with impaired myoblast fusion in ${\rho}0$ myoblasts.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.11
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• pp.1408-1413
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• 2017

The aim of this study was to investigate the antioxidant activities and protective effects of hot water extract from Curcuma longa L. (CLW) on oxidative stress-induced C2C12 myoblasts. Antioxidant activities of CLW were evaluated based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities. Protective effects of CLW on oxidative stress-induced C2C12 myoblasts were determined based on cytotoxicity, $H_2O_2$ protective activity, and intracellular reactive oxygen species (ROS) level. DPPH and ABTS radical scavenging activities represented by $SC_{50}$ were $188.5{\pm}3.0{\mu}g/mL$ and $92.0{\pm}0.9{\mu}g/mL$, respectively. Using C2C12 myoblasts, CLW treatment increased cell viability against oxidative stress-induced cell death. Further, CLW treatment reduced the intracellular ROS level in cells treated with $H_2O_2$. These results suggest that CLW might have the capability to protect oxidative stress-induced C2C12 myoblasts.

• BMB Reports
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• v.40 no.6
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• pp.921-927
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• 2007

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.6
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• pp.491-496
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• 2009

Skeletal muscle atrophy is a common phenomenon during the prolonged muscle disuse caused by cast immobilization, extended aging states, bed rest, space flight, or other factors. However, the cellular mechanisms of the atrophic process are poorly understood. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK) in the expression of muscle-specific RING finger 1 (MuRF1) during atrophy of the rat gastrocnemius muscle. Histological analysis revealed that cast immobilization induced the atrophy of the gastrocnemius muscle, with diminution of muscle weight and cross-sectional area after 14 days. Cast immobilization significantly elevated the expression of MuRF1 and the phosphorylation of p38 MAPK. The starvation of L6 rat skeletal myoblasts under serum-free conditions induced the phosphorylation of p38 MAPK and the characteristics typical of cast-immobilized gastrocnemius muscle. The expression of MuRF1 was also elevated in serum-starved L6 myoblasts, but was significantly attenuated by SB203580, an inhibitor of p38 MAPK. Changes in the sizes of L6 myoblasts in response to starvation were also reversed by their transfection with MuRF1 small interfering RNA or treatment with SB203580. From these results, we suggest that the expression of MuRF1 in cast-immobilized atrophy is regulated by p38 MAPK in rat gastrocnemius muscles.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.6
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• pp.375-379
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• 2003

$Ca^{2+}$ influx appears to be important for triggering myoblast fusion. It remains, however, unclear how $Ca^{2+}$ influx rises prior to myoblast fusion. Recently, several studies suggested that NMDA receptors may be involved in $Ca^{2+}$ mobilization of muscle, and that $Ca^{2+}$ influx is mediated by NMDA receptors in C2C12 myoblasts. Here, we report that other types of ionotropic glutamate receptors, non-NMDA receptors (AMPA and KA receptors), are also involved in $Ca^{2+}$ influx in myoblasts. To explore which subtypes of non-NMDA receptors are expressed in C2C12 myogenic cells, RT-PCR was performed, and the results revealed that KA receptor subunits were expressed in both myoblasts and myotubes. However, AMPA receptor was not detected in myoblasts but expressed in myotubes. Using a $Ca^{2+}$ imaging system, $Ca^{2+}$ influx mediated by these receptors was directly measured in a single myoblast cell. Intracellular $Ca^{2+}$ level was increased by KA, but not by AMPA. These results were consistent with RT-PCR data. In addition, KA-induced intracellular $Ca^{2+}$ increase was completely suppressed by treatment of nifedifine, a L-type $Ca^{2+}$ channel blocker. Furthermore, KA stimulated myoblast fusion in a dose-dependent manner. CNQX inhibited not only KA-induced myoblast fusion but also spontaneous myoblast fusion. Therefore, these results suggest that KA receptors are involved in intracellular $Ca^{2+}$ increase in myoblasts and then may play an important role in myoblast fusion.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.10
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• pp.1508-1513
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• 2006

Myostatin (MSTN) and insulin-like growth factors (IGFs) are a known inhibitor and stimulators of proliferation and differentiation of muscle cells, respectively. The present study was performed to investigate the relationship of MSTN-induced growth inhibition to expression of the IGF system components and myogenin, a muscle cell-specific transcription factor, in rat L6 myoblasts. The L6 cells transfected with a green fluorescent protein-MSTN plasmid expression construct had a 47% less cell number than mock-transfected cells after 3-d serum-free culture, accompanied by delayed differentiation which was suggested by inhibited aggregation of cells. Moreover, cells transfected with the expression construct had decreased expression of IGF-II and myogenin genes, but not IGF-I or its receptor genes, as examined by reverse transcription-polymerase chain reaction. The reduced mitosis of the L6 cells transfected with the MSTN-expression construct increased following an addition of either IGF-I or IGF-II to the culture medium, but not to the level of mock-transfected cells. By contrast, myogenin gene expression in these cells increased after the addition of either IGF to the level of mock-transfected cells. Collectively, these results suggest that the inhibitory effect of MSTN on L6 cell proliferation and differentiation is likely to be partly mediated by serially suppressed expression of IGF-II and myogenin genes, not IGF-I gene.

• Journal of Life Science
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• v.23 no.9
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• pp.1163-1169
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• 2013

Insulin induces glucose transporter 4 (GLUT4) translocation to the muscle cell surface. As fagopyritol has insulin-like effects, the effects of fagopyritol on GLUT4 translocation and filamentous (F) actin remodeling in L6-GLUT4myc skeletal muscle cells were investigated. Fagopyritol significantly increased plasma membrane GLUT4 levels compared with the basal control in L6-GLUT4myc myoblast cells. Phosphatidylinositol (PI) 3-kinase inhibitor (LY294002) treatment prevented GLUT4 translocation to the plasma membrane in the myoblasts. Fagopyritol treatment apparently stimulates F-actin remodeling in myoblasts. In addition, fagopyritol treatment induced GLUT4 translocation and F-actin remodeling in myotubes. Taken together, these results suggest that fagopyritol promotes GLUT4 translocation and F-actin remodeling by activating the PI 3-kinase-dependent signaling pathway.

• Molecules and Cells
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• v.21 no.2
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• pp.294-301
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• 2006

We have previously reported that phosphorylation of eukaryotic elongation factor 2 (eEF2) is related to the differentiation of chick embryonic muscle cells in culture. In the present study, we found that eEF2 phosphorylation declined shortly after induction of differentiation of L6 myoblasts, when the cells prepare for terminal differentiation by withdrawing from the cell cycle. This decrease in phosphorylation was prevented by inhibitors of phosphoinositide 3-kinase (PI3-kinase) that strongly inhibit myoblast differentiation. We hypothesized that PI3-kinase plays an important role in myoblast differentiation by regulating eEF2 phosphorylation in the early stages of differentiation. To test this hypothesis, myoblasts were synchronized at in $G_2/M$ and cultured in fresh differentiation medium (DM) or growth medium (GM). In DM the released cells accumulated in $G_0$/$G_1$ while in GM they progressed to S phase. In addition, cyclin D1 was more rapidly degraded in DM than in GM, and eEF2 phosphorylation decreased more. Inhibitors of PI3-kinase increased eEF2 phosphorylation, but PI3-kinase became more activated when eEF2 phosphorylation declined. These results suggest that the regulation of L6 myoblast differentiation by PI3-kinase is related to eEF2 phosphorylation.