Skeletal Muscle Cell Line C2C12 Research Articles

L-VALINE TREATMENT IMPROVES CELLULAR MITOCHONDRIAL FUNCTION

Abstract Branched-chain amino acids (BCAAs) are pivotal for the health of the human body. Leucine, isoleucine, and valine, which make up the BCAAs, are α-amino acids that promote skeletal muscle growth and development. Valine is one of the essential BCAAs required for the synthesis of muscle proteins and the support growth of skeletal muscles. Valine also plays a favorable role in enhancing insulin sensitivity, preserving intestinal health, and optimizing lipid metabolism. Considering the relevance of valine in promoting muscle growth and metabolism, we utilized C2C12 skeletal muscle cell line to investigate the role of valine in regulating mitochondrial functions. C2C12 cells grown on physiological normal glucose media (100mg/dL) were used for the experiments. Cells were treated with a 1.0 mM concentration of valine for 24 h. The effect of valine treatment on gene expression of important mitochondrial-related genes such as PGC-1α, and PGC-1β and mitofusin (MFN1, MFN), and mitochondrial fission 1 (Fis 1) was determined. Further, Oroboros oxygraph-O2K high-resolution respirometer was used to analyze the mitochondrial respiration in the intact C2C12 cells after valine treatment. The results showed increased gene expression of PGC-1α, PGC-1β, and mitochondrial fission and fusin genes after treatment with valine. The basal respiration, leak respiration, as well as maximal capacity of mitochondrial ETC (Electron Transport Chain), were also significantly improved after valine treatment. The findings from this study enhance our understanding of valine as an important nutrient to improve mitochondrial function to drive cellular function and biological processes.

Cysteine-cysteine Chemokine Receptor Type 5 Plays a Critical Role in Exercise Performance by Regulating Mitochondrial Content in Skeletal Muscle.

Cysteine-cysteine chemokine receptor type 5 (CCR5) is thought to play an important role in the trafficking of lymphoid cells but has recently also been associated with AMPK signaling pathways that are implicated in energy metabolism in skeletal muscle. We hypothesized that genetic deletions of CCR5 would alter mitochondria content and exercise performance in mice. CCR5-/- and wild-type mice with the same genetic background were subjected to endurance exercise and grip strength tests. The soleus muscle was stained with immunofluorescence for myosin heavy chain 7 (MYH7) and succinate dehydrogenase (SDH) analysis as well as the expression of genes associated with muscle atrophy and mitochondrial oxidative phosphorylation were measured using qPCR. Although there were no differences in the weight of the soleus muscle between the CCR5-/- group and the wild-type mice, the CCR5-/- mice showed the following muscular dysfunctions: (i) decreased MYH7 percentage and cross-section area, (ii) higher myostatin and atrogin-1 mRNA levels, (iii) dropped expression of mitochondrial DNA-encoded electron respiratory chain genes (cytochrome B, cytochrome c oxidase subunit III, and ATP synthase subunit 6) as well as mitochondrial generation genes (PPARγandPGC-1α), and (iv) lower SDH activity and exercise performance when compared with wild-type mice. In addition, genes associated with mitochondrial biogenesis (PGC-1α, PPARγ, and MFN2) and mitochondrial complex (ND4 and Cytb) were upregulated when the skeletal muscle cell line C2C12 was exposed to cysteine-cysteine chemokine ligand 4 (a ligand of CCR5) in vitro. These findings suggested that attenuation of endurance exercise performance is related to the loss of mitochondrial content and lower SDH activity of soleus muscle in CCR5 knockout mice. The present study provides evidence indicating that the chemokine receptor CCR5 might modulate the skeletal muscle metabolic energy system during exercise.

EFFECT OF L-VALINE TREATMENT ON SIRTUIN (SIRT1 AND SIRT2) ISOFORMS

Abstract L-valine is one of the essential branched-chain amino acids (BCAAs) required for synthesis of proteins in human body. It promotes muscle growth and tissue repair and is important for immune function. Recent data indicate that BCAAs can activate sirtuins expression and elevate mitochondrial biogenesis and fatty acid oxidation in both adipocytes and myotubes thereby increasing life span. Sirtuins are a conserved family of proteins, play a critical role in maintaining metabolic health by deacetylating many target proteins in numerous tissues, and regulate mitochondrial function and the aging process. Due to multiple effect of sirtuins on aging, we sought to determine whether the addition of valine might enhance sirtuin gene expression. We utilized the C2C12 skeletal muscle cell line grown on physiological normal glucose (100mg/dL) media. The cells were treated with two different concentrations of valine (0.5 and 1.0mM) for different time intervals (18 and 24). Gene expression of sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) isoforms were determined by RT-PCR. The results showed increased expression of the sirtuin gene isoforms after treatment with valine. Relative expression varies with in different isoforms of SIRT1 (v1 and v2) and SIRT2 (v1, v2 and v3). Among all, SIRT1 v1 and SIRT2 v1 showed maximum expression as compared to the other isoforms used in the study. Our study showed that adequate supplementation of L-valine enhanced sirtuin gene expression, which may promote healthy muscles and healthy aging.

Atrophic effects by leukemia inhibitory factor (LIF) in 85As2 gastric cancer cell-inoculated cachexia model: <i>in vitro</i> analysis with C2C12 skeletal muscle cell line

Atrophic effects by leukemia inhibitory factor (LIF) in 85As2 gastric cancer cell-inoculated cachexia model: <i>in vitro</i> analysis with C2C12 skeletal muscle cell line

Spexin Promotes the Proliferation and Differentiation of C2C12 Cells In Vitro-The Effect of Exercise on SPX and SPX Receptor Expression in Skeletal Muscle In Vivo.

SPX (spexin) and its receptors GalR2 and GalR3 (galanin receptor subtype 2 and galanin receptor subtype 3) play an important role in the regulation of lipid and carbohydrate metabolism in human and animal fat tissue. However, little is still known about the role of this peptide in the metabolism of muscle. The aim of this study was to determine the impact of SPX on the metabolism, proliferation and differentiation of the skeletal muscle cell line C2C12. Moreover, we determined the effect of exercise on the SPX transduction pathway in mice skeletal muscle. We found that increased SPX, acting via GalR2 and GalR3 receptors, and ERK1/2 phosphorylation stimulated the proliferation of C2C12 cells (p < 0.01). We also noted that SPX stimulated the differentiation of C2C12 by increasing mRNA and protein levels of differentiation markers Myh, myogenin and MyoD (p < 0.01). SPX consequently promoted myoblast fusion into the myotubule (p < 0.01). Moreover, we found that, in the first stage (after 2 days) of myocyte differentiation, GalR2 and GalR3 were involved, whereas in the last stage (day six), the effect of SPX was mediated by the GalR3 isoform. We also noted that exercise stimulated SPX and GalR2 expression in mice skeletal muscle as well as an increase in SPX concentration in blood serum. These new insights may contribute to a better understanding of the role of SPX in the metabolism of skeletal muscle.

Antimicrobial potential and in vitro cytotoxicity study of polyvinyl pyrollidone-stabilised silver nanoparticles synthesised from Lysinibacillus boronitolerans.

The main emphasis herein is on the eco‐friendly synthesis and assessment of the antimicrobial potential of silver nanoparticles (AgNPs) and a cytotoxicity study. Silver nanoparticles were synthesised by an extracellular method using bacterial supernatant. Biosynthesised silver nanoparticles were characterised by UV‐vis spectroscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy, dynamic light scattering, and zeta potential analysis. The synthesised silver nanoparticles exhibited a characteristic peak at 420 nm. TEM analysis depicted the spherical shape and approximately 20 nm size of nanoparticles. Silver nanoparticles carry a charge of −33.75 mV, which confirms their stability. Biogenic polyvinyl pyrrolidone‐coated AgNPs exhibited significant antimicrobial effects against all opportunistic pathogens (Gram‐positive and Gram‐negative bacteria, and fungi). Silver nanoparticles equally affect the growth of both Gram‐positive and Gram‐negative bacteria, with a maximum inhibition zone observed at 22 mm and a minimum at 13 mm against Pseudomonas aeruginosa and Fusarium graminearum, respectively. The minimum inhibitory concentration (MIC) of AgNPs against P. aeruginosa and Staphylococcus aureus was recorded at between 15 and 20 μg/ml. Synthesised nanoparticles exhibited a significant synergistic effect in combination with conventional antibiotics. Cytotoxicity estimates using C2C12 skeletal muscle cell line via 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) test and lactate dehydrogenase assay were directly related to the concentration of AgNPs and length of exposure. On the basis of the MTT test, the IC50 of AgNPs for the C2C12 cell line was approximately 5.45 μg/ml concentration after 4 h exposure.

Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Chronic Kidney Disease

Alterations in mineral metabolism involve changes in serum levels of phosphate as well as its key regulators, fibroblast growth factor 23 (FGF23), vitamin D, parathyroid hormone and klotho. Chronic Kidney Disease (CKD) is a public health epidemic that affects an estimated 37 million Americans, and is associated with elevated serum levels of phosphate (hyperphosphatemia) and FGF23 as well as various pathologies, such vascular calcification, cardiac hypertrophy, and skeletal muscle atrophy. We previously found that elevated FGF23 directly targets cardiac myocytes and contributes to cardiac hypertrophy. Furthermore, elevated phosphate has direct effects on smooth muscle cells and induces vascular calcification. In order to determine whether FGF23 and/or phosphate might also contribute to CKD-associated skeletal muscle atrophy, we tested if skeletal muscle cells can respond to elevations of FGF23 or phosphate. Since the skeletal muscle phenotype in CKD is not well characterized, we analyzed skeletal muscle on a functional, histological, and molecular level in two established mouse models of CKD. We studied the C2C12 skeletal muscle cell line during the differentiation process from myoblasts to myotubes as well as fully differentiated myotubes. Myoblast differentiation was induced with horse serum; cells were co-treated with 25 ng/ml FGF23 or 5 mM phosphate for 5 days, followed by the expression analysis of differentiation markers by qPCR. In a separate study, differentiated C2C12 myotubes were treated with FGF23 or phosphate for 24 hours, followed by the qPCR analysis of atrophy genes (atrogenes), including MT1, Murf1, and Fbox32. Furthermore, we studied mice with global deletion of collagen 4a3 (Col4a3-/-) and wildtype littermates at 10 weeks of age, as well as C57Bl/6 mice receiving an adenine-rich (0.2%) diet or regular chow for 12 weeks. In all mice we studied grip strength, hindlimb circumference by MRI, cross-sectional area of individual muscle fibers that were immuno-labeled with anti-laminin by fluorescence microscopy, and expression levels of atrogenes by qPCR. Phosphate but not FGF23 affected the differentiation process of C2C12 myoblasts and differentiated C2C12 myotubes. Phosphate treatments significantly reduced expression levels of MyoD and MyoG and increased expression of MT1. In both mouse models, grip strength and areas of hindlimb and myotubes were significantly reduced, and expression levels of MT1 and Murf1 were significantly elevated when compared to their controls. FGF23 does not target skeletal muscle cells suggesting that it cannot directly contribute to skeletal muscle atrophy. Elevated phosphate impairs myoblast differentiation and induces atrophy in vitro. We have characterized two CKD mouse models with associated skeletal muscle atrophy, where we will now determine the contribution of hyperphosphatemia to the injury process. Pharmacological approaches targeting phosphate uptake or excretion, or phosphate's direct actions on tissues might alleviate various CKD-associated pathologies.

Structure based analysis of KATP channel with a DEND syndrome mutation in murine skeletal muscle

Developmental delay, epilepsy, and neonatal diabetes (DEND) syndrome, the most severe end of neonatal diabetes mellitus, is caused by mutation in the ATP-sensitive potassium (KATP) channel. In addition to diabetes, DEND patients present muscle weakness as one of the symptoms, and although the muscle weakness is considered to originate in the brain, the pathological effects of mutated KATP channels in skeletal muscle remain elusive. Here, we describe the local effects of the KATP channel on muscle by expressing the mutation present in the KATP channels of the DEND syndrome in the murine skeletal muscle cell line C2C12 in combination with computer simulation. The present study revealed that the DEND mutation can lead to a hyperpolarized state of the muscle cell membrane, and molecular dynamics simulations based on a recently reported high-resolution structure provide an explanation as to why the mutation reduces ATP sensitivity and reveal the changes in the local interactions between ATP molecules and the channel.

Comparative multi-omics analyses reveal differential expression of key genes relevant for parasitism between non-encapsulated and encapsulated Trichinella

Genome assemblies provide a powerful basis of comparative multi-omics analyses that offer insight into parasite pathogenicity, host-parasite interactions, and invasion biology. As a unique intracellular nematode, Trichinella consists of two clades, encapsulated and non-encapsulated. Genomic correlation of the distinct differences between the two clades is still unclear. Here, we report an annotated draft reference genome of non-encapsulated Trichinella, T. pseudospiralis, and perform comparative multi-omics analyses with encapsulated T. spiralis. Genome and methylome analyses indicate that, during Trichinella evolution, the two clades of Trichinella exhibit differential expansion and methylation of parasitism-related multi-copy gene families, especially for the DNase II members of the phospholipase D superfamily and Glutathione S-transferases. Further, methylome and transcriptome analyses revealed divergent key excretory/secretory (E/S) genes between the two clades. Among these key E/S genes, TP12446 is significantly more expressed across three life stages in T. pseudospiralis. Overexpression of TP12446 in the mouse C2C12 skeletal muscle cell line could induce inhibition of myotube formation and differentiation, further indicating its key role in parasitism of T. pseudospiralis. This multi-omics study provides a foundation for further elucidation of the mechanism of nurse cell formation and immunoevasion, as well as the identification of pharmacological and diagnostic targets of trichinellosis.

Vitamin D/Vitamin D Receptor Signaling Attenuates Skeletal Muscle Atrophy by Suppressing Renin-Angiotensin System.

The nutritional level of vitamin D may affect musculoskeletal health. We have reported that vitamin D is a pivotal protector against tissue injuries by suppressing local renin-angiotensin system (RAS). This study aimed to explore the role of the vitamin D receptor (VDR) in the protection against muscle atrophy and the underlying mechanism. A cross-sectional study on participants (n= 1034) in Shanghai (China) was performed to analyze the association between vitamin D level and the risk of low muscle strength as well as to detect the circulating level of angiotensin II (Ang II). In animal studies, dexamethasone (Dex) was applied to induce muscle atrophy in wild-type (WT) and VDR-null mice, and the mice with the induction of muscle atrophy were treated with calcitriol for 10 days. The skeletal muscle cell line C2C12 and the muscle satellite cells were applied in in vitro studies. The increased risk of low muscle strength was correlated to a lower level of vitamin D (adjusted odds ratio [OR] 0.58) accompanied by an elevation in serum Ang II level. Ang II impaired the myogenic differentiation of C2C12 myoblasts as illustrated by the decrease in the area of myotubes and the downregulation of myogenic factors (myosin heavy chain [MHC] and myogenic differentiation factor D [MyoD]). The phenotype of muscle atrophy induced by Dex and Ang II was aggravated by VDR ablation in mice and in muscle satellite cells, respectively, and mediated by RAS and its downstream phosphatidylinositol 3-kinase/protein kinase B/forkhead box O1 (PI3K/Akt/FOXO1) signaling. Calcitriol treatment exhibited beneficial effects on muscle function as demonstrated by the increased weight-loaded swimming time, grip strength, and fiber area, and improved fiber type composition via regulating ubiquitin ligases and their substrates MHC and MyoD through suppressing renin/Ang II axis. Taken together, VDR protects against skeletal muscle atrophy by suppressing RAS. Vitamin D could be a potential agent for the prevention and treatment of skeletal muscle atrophy. © 2021 American Society for Bone and Mineral Research (ASBMR).

Goshajinkigan, a Traditional Japanese Medicine, Suppresses Voltage-Gated Sodium Channel Nav1.4 Currents in C2C12 Cells.

Goshajinkigan (GJG) is a traditional Japanese Kampo medicine used clinically to treat muscle pain in Japan. However, its underlying mechanism remains unclear. Since voltage-gated sodium channel (Nav) 1.4 is involved in skeletal muscle contraction, we investigated the possibility that GJG may affect Nav1.4 currents. By using an electrophysiological technique on skeletal muscle cell line C2C12, we found that GJG suppresses Nav1.4 currents in C2C12 cells. It is suggested that GJG may improve skeletal muscle stiffness or cramps by inhibiting abnormal Nav1.4 excitation. GJG may act as a Nav1.4 blocker and may be useful to treat muscle stiffness and clamps as well as easing the pain.

17β-estradiol upregulates oxytocin and the oxytocin receptor in C2C12 myotubes.

BackgroundThe endocrinology of skeletal muscle is highly complex and many issues about hormone action in skeletal muscle are still unresolved. Aim of the work is to improve our knowledge on the relationship between skeletal muscle and 17β-estradiol.MethodsThe skeletal muscle cell line C2C12 was treated with 17β-estradiol, the oxytocin peptide and a combination of the two hormones. The mRNA levels of myogenic regulatory factors, myosin heavy chain, oxytocin, oxytocin receptor and adipogenic factors were analysed in C2C12 myotubes.ResultsIt was demonstrated that C2C12 myoblasts and myotubes express oxytocin and its receptor, in particular the receptor levels physiologically increase in differentiated myotubes. Myotubes treated with 17β-estradiol overexpressed oxytocin and oxytocin receptor genes by approximately 3- and 29-fold, respectively. A decrease in the expression of fatty acid binding protein 4 (0.62-fold), a fat metabolism-associated gene, was observed in oxytocin-treated myotubes. On the contrary, fatty acid binding protein 4 was upregulated (2.66-fold) after the administration of the combination of 17β-estradiol and oxytocin. 17β-estradiol regulates oxytocin and its receptor in skeletal muscle cells and they act in a synergic way on fatty acid metabolism.DiscussionOxytocin and its receptor are physiologically regulated along differentiation. 17β-estradiol regulates oxytocin and its receptor in skeletal muscle cells. 17β-estradiol and oxytocin act in a synergic way on fatty acid metabolism. A better understanding of the regulation of skeletal muscle homeostasis by estrogens and oxytocin peptide could contribute to increase our knowledge of muscle and its metabolism.

MicroRNA-29a induces insulin resistance by targeting PPARδ in skeletal muscle cells.

Intrauterine growth retardation (IUGR) induces metabolic syndrome, which is often characterized by insulin resistance (IR), in adults. Previous research has shown that microRNAs (miRNAs or miRs) play a role in the target genes involved in this process, but the mechanisms remain unclear. In the present study, we examined miRNA profiles using samples of skeletal muscles from both IUGR and control rat offspring whose mothers were fed either a protein-restricted diet or a diet which involved normal amounts of protein during pregnancy, respectively. miR-29a was found to be upregulated in the skeletal muscles of IUGR offspring. The luciferase reporter assay confirmed the direct interaction between miR-29a and peroxisome proliferator-activated receptor δ (PPARδ). Overexpression of miR-29a in the skeletal muscle cell line C2C12 suppressed the expression of its target gene PPARδ, which, in turn, influenced the expression of its coactivator, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Thus, PPARδ/PGC-1α-dependent signals together reduced insulin-dependent glucose uptake and adenosine triphosphate (ATP) production. Overexpression of miR-29a also caused a decrease in levels of glucose transporter 4 (GLUT4), the most important glucose transporter in skeletal muscle, which partially induced a decrease insulin-dependent glucose uptake. These findings provide evidence for a novel micro-RNA-mediated mechanism of PPARδ regulation, and we also noted the IR-promoting actions of miR-29a in skeletal muscles of IUGR.

Amygdalin isolated from Semen Persicae (Tao Ren) extracts induces the expression of follistatin in HepG2 and C2C12 cell lines.

BackgroundThe Chinese medicine formulation ISF-1 (also known as Bu-Yang-Huan-Wu-Tang) for post-stroke rehabilitation could increase the expression of growth-regulating protein follistatin by approximately 4-fold. This study aims to identify the active compounds of ISF-1 for the induction of follistatin expression.MethodsActive compounds in ISF-1 responsible for induction of follistatin were identified by a bioactivity-guided fractionation procedure involving liquid-liquid extraction, HPLC separation and RT-PCR detection. The aqueous extracts of seven ISF-1 ingredients including Semen Persicae (Tao Ren) and the S. Persicae-derived fractions were assayed for the induction of follistatin mRNA expression in human hepatocarcinoma HepG2 cells by RT-PCR. The concentrations of isolated compounds were proportionally normalized to the reported IC50 concentration (5.8 mg/mL) of the formulation ISF-1 in HepG2. The active fractions were characterized by reverse-phase HPLC on a C18 column and identified by mass spectrometry.ResultsThree ingredients of ISF-1, namely S. Persicae (Tao Ren), Pheretima (Di Long), and Flos Carthami (Hong Hua), induced the expression of follistatin mRNA. Among these, the ingredient S. Persicae were the most active, and amygdalin from S. Persicae extract was identified as a novel follistatin inducer. Amygdalin stimulated the growth of skeletal muscle cell line C2C12 cells in a concentration-dependent manner.ConclusionsAmygdalin isolated from S. Persicae extract in ISF-1 through a bioactivity-guided fractionation procedure induced the expression of follistatin in HepG2 and C2C12 cell lines.

Alignment of Skeletal Muscle Cells Cultured in Collagen Gel by Mechanical and Electrical Stimulation

For in vitro tissue engineering of skeletal muscle, alignment and fusion of the cultured skeletal muscle cells are required. Although the successful alignment of skeletal muscle cells cultured in collagen gel has been reported using a mechanical force, other means of aligning cultured skeletal muscle cells have not been described. However, skeletal muscle cells cultured in a two-dimensional dish have been reported to align in a uniform direction when electrically stimulated. The purpose of this study is to determine if skeletal muscle cells cultured three-dimensionally in collagen gels can be aligned by an electrical load. By adding direct current to cells of the C2C12 skeletal muscle cell line cultured in collagen gel, it was possible to align C2C12 cells in a similar direction. However, the ratio of alignment was better when mechanical force was used as the means of alignment. Thus for tissue engineering of skeletal muscle cells, electrical stimulation may be useful as a supplementary method.

The peculiar apoptotic behavior of skeletal muscle cells.

Apoptosis plays an active role in maintaining skeletal muscle homeostasis. Its deregulation is involved in several skeletal muscle disorders such as dystrophies, myopathies, disuse and sarcopenia. The aim of this work was to study in vitro the apoptotic behavior induced by etoposide, staurosporine and hydrogen peroxide in the C2C12 skeletal muscle cell line, comparing myoblast vs myotube sensitivity, investigated by means of morphological and cytofluorimetric analyses. Myotubes appeared more resistant than myoblasts to apoptotic induction. In myoblasts treated with etoposide, nuclei with chromatin condensation were observed, in the presence of a diffuse DNA fragmentation, as shown by confocal microscopy. The latter also appeared in myotubes, where apoptotic and normal nuclei coexisted inside the same syncytium. After staurosporine treatment, myobalsts evidenced late apoptotic features and a high number of TUNEL-positive nuclei. Secondary necrosis appeared in myotubes, where myonuclei with cleaved DNA again coexisted with normal myonuclei. After H₂O₂ exposure, myotubes, differently from myoblasts, showed a poor sensitivity to cell death. Intriguingly, autophagic granules appeared abundantly in myotubes after each treatment. In myotubes, mitochondria were better preserved than in myoblasts since those which were damaged were probably degraded through autophagic processes. These findings demonstrate a scarce sensitivity of myotubes to apoptotic stimuli due to acquisition of an apoptosis-resistant phenotype during differentiation. The presence of nuclear-dependent "territorial" death domains in the syncytium could explain a slower death of myotubes compared to mononucleated cells. In addition, autophagy could preserve and protect muscle cell integrity against chemical stimuli, making C2C12 cells, in particular myotubes, more resistant to apoptosis induction.

Non-classical localization of androgen receptor in the C2C12 skeletal muscle cell line

The classical model of testosterone action has been traditionally described as being mediated by the androgen receptor (AR) localized exclusively in the nucleus. However, there is increasing functional evidence for extranuclear localization of AR. We present biochemical and immunological data supporting mitochondrial and microsomal localization of AR in the C2C12 skeletal muscle cell line. As a first approach AR was detected by immunoblotting, using specific antibodies after subcellular fractionation, not only in nucleus and cytosol, but also in mitochondria and microsomes. We then established [3H] testosterone binding characteristics in total homogenates and subcellular fractions. Specific and saturable [3H] testosterone binding sites were detected in mitochondria and microsomes. Immunolocalization of the non-classical AR was also confirmed using confocal microscopy. Sucrose gradient fractionation demonstrated the presence of the AR in lipid rafts and caveolae. Besides, the AR interacts physically with Caveolin-1, association that is lost after testosterone treatment. Accordingly, Western blot analysis revealed a decrease of AR expression in the microsomal fraction after testosterone treatment, suggesting translocation of the membrane AR to another subcellular compartment. The non-classical distribution of native pools of AR in skeletal muscle cells suggests an alternative mode of AR localization/function.

T.P.41 The role of the transcriptional factor Pax3 on myogenesis and the effect on the expression of myogenic regulatory factors

<h2>Abstract</h2> Pax3 is a member of paired box (Pax) family and considered to play an important role in the initial stage of skeletal muscle development. However, its precise function of myogenesis <i>in vitro</i> and <i>in vivo</i> remains unknown. For example, how Pax3 affect the expression of myogenic regulatory factors (MRFs) has not yet been clarified. We have investigated the effect of TSA, one of most potent HDACIs, on myogenesis using the C2C12 skeletal muscle cell line. We found that the change of the expression of Pax3 was not directly correlated with other MRFs (Hagiwara et al., BBRC (2011)). To investigate the role of the transcriptional factor Pax3 on myogenesis and its effect on the expression of MRFs, we performed Pax3 cDNA transfection to the C2C12 cell line by using Effectene© Transfection Reagent (QIAGEN). We analyzed the effect of Pax3 transfection by immunostaining, Western blot, and microarray analyses. In Pax3 transfected C2C12 cells, the formation of myotube is decreased compared to non-transfected cells. At 48h after Pax3 transfection, the expression MyoD was reduced by Western blot analysis. By microarray analysis, the expression MyoD was reduced and the expression of the cell cycling regulators were changed most significantly. These results suggest that Pax3 inhibit muscle differentiation by suppressing the expression of MyoD and enhancing the expression of the cell cycling regulators. Pax3 may have a role to preserve muscle cell precursors undifferentiated.

Myricetin attenuates hyperinsulinemia-induced insulin resistance in skeletal muscle cells

Previous studies have shown that hyperinsulinemia is not only a marker of insulin resistance, but also the causative factor of peripheral tissue insulin resistance. It also has been suggested that prolonged high-dose insulin treatment can mimic the effects of hyperinsulinemia and exacerbate insulin resistance in skeletal muscle cells. However, how to prevent or reverse insulin resistance induced by hyperinsulinemia remains largely unclear. In the past few decades, the use of myricetin as an anti-diabetic agent has gained much attention, but little information is available regarding the effects of myricetin on glucose uptake and hyperinsulinemia-induced insulin resistance in skeletal muscle cells. The present study focuses on the effect of myricetin on insulin signaling in skeletal muscle cell line C2C12 myotubes. Initially, the effect of myricetin under normal condition was determined. We found that myricetin’s enhancement in glucose uptake coincided with both protein kinase B (Akt) and AMP-activated protein kinase (AMPK) activities. After that, the role of hyperinsulinemia was investigated. It was showed that prolonged high-dose insulin treatment inhibited both Akt and AMPK activities. As the results, the low-dose insulin stimulation of glucose uptake was inhibited by hyperinsulinemia. However, the treatment of myricetin improved low-dose insulin-stimulated glucose uptake in the hyperinsulinemic state, and this effect essentially depended on the AMPK signal pathway. Together, our data suggest a putative link between hyperinsulinemia and insulin resistance in C2C12 myotubes, and the myricetin treatment stimulates glucose uptake and attenuates insulin resistance.

1α,25(OH)2D3‐dependent modulation of Akt in proliferating and differentiating C2C12 skeletal muscle cells

We previously reported that 1α,25-dihydroxy-vitamin D(3) [1α,25(OH)(2)D(3)] induces non-transcriptional rapid responses through activation of Src and MAPKs in the skeletal muscle cell line C2C12. In the present study we investigated the modulation of Akt by the secosteroid hormone in C2C12 cells at proliferative stage (myoblasts) and at early differentiation stage. In proliferating cells, 1α,25(OH)(2)D(3) activates Akt by phosphorylation in Ser473 in a time-dependent manner (5-60 min). When these cells were pretreated with methyl-beta-cyclodextrin to disrupt caveolae microdomains, hormone-induced activation of Akt was suppressed. Similar results were obtained by siRNA silencing of caveolin-1 expression, further indicating that hormone effects on cell membrane caveolae are required for downstream signaling. PI3K and p38 MAPK, but not ERK1/2, participate in 1α,25(OH)(2)D(3) activation of Akt in myoblasts. The involvement of p38 MAPK in Akt phosphorylation by the hormone probably occurs through MAPK-activated protein kinase 2 (MK2), which is activated by the steroid. In addition, the participation of Src in Akt phosphorylation by 1α,25(OH)(2)D(3) was demonstrated using the inhibitor PP2 and antisense oligodeoxynucleotides that suppress Src expression. We also observed that PI3K participates in hormone-induced proliferation. During the early phase of C2C12 cell differentiation 1α,25(OH)(2)D(3) also increases Akt phosphorylation and activates Src. Of relevance, Src and PI3K are involved in Akt activation and in MHC and myogenin increased expression by 1α,25(OH)(2)D(3). Altogether, these data suggest that 1α,25(OH)(2)D(3) upregulates Akt through Src, PI(3)K, and p38 MAPK to stimulate myogenesis in C2C12 cells.