Mechanical stretch activates glycometabolism-related enzymes via estrogen in C2C12 myoblasts

Moderate exercise improves glycometabolic disorder and type 2 diabetes mellitus in menopausal females. So far, the effect of exercise-induced estrogen on muscular glycometabolism is not well defined. The current study was designed to explore the effect of mechanical stretch-induced estrogen on glycometabolism in mouse C₂C₁₂ myoblasts. The mouse C₂C₁₂ myoblasts in vitro were assigned randomly to the control (C), stretch (S), and stretch plus aromatase inhibitor anastrozole (SA) groups. Cells in the S group were stretched by the Flexcell FX-5000™ system (15% magnitude, 1 Hz frequency, and 6-hr duration) whereas those in the SA group were treated with 400 μg/ml anastrozole before the same stretching. Glucose uptake, estradiol levels, PFK-1 levels, and oxygen consumption rate were determined, and the expression of HK, PI3K, p-AKT, AKT, and GLUT4 proteins were semiquantified with western blot analysis. Compared to the control, the estradiol level, oxygen consumption rate, expression of HK, PI3K, and PFK-1 proteins, the ratio of p-AKT to AKT, and the ratio of GLUT4 in the cell membrane to that in the whole cell were higher in the S group. On the other hand, the estradiol level, glucose uptake, expression of PFK-1 and GLUT4 proteins, oxygen consumption rate, expression of HK protein, and the ratio of p-AKT/AKT were lower in the myoblasts in the SA group than those in the S group. The level of estradiol was positively correlated with glucose uptake (p < .01, r = .818). Therefore, mechanical stretch-induced estrogen increased the expression of glycometabolism-related enzymes and proteins in the mouse C₂C₁₂ myoblasts.     

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.     

Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology

Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the alpha-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease.     

脉冲电磁场对C2C12 成肌细胞增殖影响的实验研究

目的:研究不同强度脉冲电磁场干预对成肌细胞增殖的影响。方法:10Hz脉冲低频电磁场刺激经复苏后培养贴壁良好的C2C12成肌细胞,根据不同磁场强度和作用时间将其分为A、B、C组,无磁场干预的为对照组。采用RT-q PCR检测不同磁场强度下成肌细胞标记基因Myf5、Myo D及Pax7的m RNA的表达。结果:经RT-q PCR检测三种基因的表达情况,Myf5 m RNA在1.5 m T磁场强度下照射第五天表达最高;0.5 m T磁场强度下Myf5 m RNA的表达与对照组相比无统计学意义(P>0.05);1.0 m T磁场强度下Myf5 m RNA表达与对照组比较差异具有统计学意义(P<0.05);1.5 m T磁场强度下Myf5 m RNA表达与对照组比较差异具有统计学意义(P<0.05)。对照组Myo D m RNA的表达要比磁场作用下表达要高。三个磁场强度下Myo D m RNA表达与对照组相比均无统计学意义(P>0.05)。0.5 m T、1.0 m T磁场强度下Pax7 m RNA的表达要比对照组要高,与对照组相比具有统计学意义(P<0.05);1.5 m T磁场强度下Pax7 m RNA表达与对照组相比无统计学意义(P>0.05)。结论 :1.5 m T脉冲电磁场强度下作用5天对体外培养的成肌细胞Myf5 m RNA标记基因增殖促进作用最强。     

Stretch activation of GTP-binding proteins in C2C12 myoblasts

Mechanical stimulation has been proposed as a fundamental determinant of muscle physiology. The mechanotransduction of strain and strain rate in C2C12 myoblasts were investigated utilizing a radiolabeled GTP analogue to detect stretch-induced GTP-binding protein activation. Cyclic uniaxial strains of 10% and 20% at a strain rate of 20% s(-1) rapidly (within 1 min) activated a 25-kDa GTPase (183 +/- 17% and 186 +/- 19%, respectively), while 2% strain failed to elicit a response (109 +/- 11%) relative to controls. One, five, and sixty cycles of 10% strain elicited 187 +/- 20%, 183 +/- 17%, and 276 +/- 38% increases in activation. A single 10% stretch at 20% s(-1), but not 0.3% s(-1), resulted in activation. Insulin activated the same 25-kDa band in a dose-dependent manner. Western blot analysis revealed a panel of GTP-binding proteins in C2C12 myoblasts, and tentatively identified the 25-kDa GTPase as rab5. In separate experiments, a 40-kDa protein tentatively identified as Galpha(i) was activated (240 +/- 16%) by 10% strain at 1 Hz for 15 min. These results demonstrate the rapid activation of GTP-binding proteins by mechanical strain in myoblasts in both a strain magnitude- and strain rate-dependent manner.     

Glucosamine induces rapid desensitization of glucose transport in isolated adipocytes by increasing GlcN-6-P levels

We have examined the hypothesis that glucosamine (GlcN) can rapidly induce insulin resistance through an allosteric mechanism. When insulin-treated adipocytes were exposed to 2mM GlcN, glucose uptake was rapidly reduced by approximately 60% with a T(1/2) of 2 min. We also observed an increase in intracellular GlcN-6-P (at 5 min) from undetectable levels to approximately 260 nmol/g. Continued GlcN treatment resulted in additional accumulation of GlcN-6-P (>1200 nmol/g at 2h), but caused no further decrease in glucose uptake. Although the acute inhibitory action of GlcN could be completely reversed by removing extracellular GlcN, a slow and progressive decrease in insulin-stimulated glucose transport was observed with longer treatment times (T(1/2) of 45 min, 62% loss by 5h). From these data, we conclude that: (1) GlcN elevates intracellular GlcN-6-P levels within minutes, resulting in desensitization of the glucose transport system through allosteric inhibition of hexokinase; (2) prolonged treatment elevates GlcN-6-P to levels that cannot be effectively lowered by cell washing; and (3) residual levels of GlcN-6-P continue to allosterically inhibit glucose uptake, resulting in a slower rate of desensitization that is temporally similar to glucose-induced desensitization, but mechanistically different.     

The microRNA-127-3p directly targeting Vamp2 in C2C12 myoblasts

MicroRNAs (miRNAs) have been reported that can regulate skeletal muscle growth and development. Previously, we demonstrated that miR-127-3p were differently expressed in skeletal muscle and muscle cells. However, the molecular mechanism of miR-127-3p regulation of skeletal myogenesis are not well elucidated. In this study, we transfected miR-127-3p into C2C12 cells, and found miR-127-3p induces myogenesis by targeting Vamp2. Moreover, the regulatory mechanism of Vamp2 in myoblasts proliferation and differentiation was further confirmed. In conclusion, our data providedevidences that miR-127-3p reciprocally regulated myoblasts proliferation and differentiation through directly targeting Vamp2.     

中胚叶叉头-1在骨成形蛋白-2诱导肌胚细胞C2C12转化成骨细胞过程中的作用

为了研究中胚叶叉头-1(MFH-1)基因在骨骼形成和细胞分化中的作用,利用基因重组、杂交瘤技术制作MFH-1单克隆抗体, 利用蛋白质印迹和RNA印迹分析观察了骨成形蛋白-2 (BMP-2)诱导小鼠肌胚细胞C2C12表达MFH-1、产生碱性磷酸酶和骨钙蛋白.小鼠肌胚细胞C2C12低水平地表达内源性MFH-1蛋白以及导入小鼠MFH-1 cDNA的人膀胱癌细胞HTB9也表达小鼠MFH-1蛋白,这种蛋白质定位于细胞核中.用BMP-2处理后, MFH-1蛋白和mRNA在C2C12细胞中的表达显著地增加.用反义MFH-1序列转染小鼠肌胚细胞C2C12可降低内源性MFH-1水平, BMP-2不能诱导导入反义MFH-1序列的肌胚细胞C2C12产生MFH-1蛋白,也不能诱导碱性磷酸酶(ALP)活性和骨钙蛋白量的增加.结果表明, BMP-2诱导的MFH-1蛋白在调节肌胚细胞C2C12向成骨细胞分化方面起关键作用.     

过表达磷脂酶D3影响成肌细胞中Akt磷酸化水平

磷脂酶D(PLD)催化卵磷脂(Phosphatidylc holine,PC)水解产生胆碱(Choline)和磷脂酸(Phosphatidic acid,PA),其代谢产物参与调控细胞内许多生理和生化过程。在过表达磷脂酶D3(PLD3)的成肌细胞(C2C12细胞)中,研究了PLD3对胰岛素刺激后Akt通路激活的影响。研究结果表明,PLD3过表达细胞的Akt磷酸化水平比对照组低,并且不受胰岛素浓度变化的调控。虽然PLD3过表达细胞中Akt磷酸化水平随胰岛素刺激时间的延长而有所增加,但磷酸化总水平比对照组低。磷脂酶D抑制剂丁-1醇能够抑制对照组胰岛素刺激下Akt磷酸化,却不能抑制PLD3过表达细胞的Akt磷酸化,并且PLD3过表达细胞Akt磷酸化水平比对照组高6倍。用磷脂酸(PA)做刺激时,对照组的Akt磷酸化明显增加,而PLD3过表达细胞株的Akt磷酸化没有显著变化;用PA和胰岛素同时刺激时,PLD3过表达株和对照组的Akt磷酸化均比PA单独刺激时降低。这说明PLD3的过表达抑制成肌细胞内胰岛素信号的传导。     

Efficient transfection of mouse-derived C2C12 myoblasts using a matrigel basement membrane matrix

Myogenic cell lines have been used widely in the study of myogenic differentiation, muscle regeneration and homeostasis, but, myoblasts and myotubes are difficult to transfect using conventional techniques. We have used liposome-based transfection method to introduce a green fluorescence protein (GFP)-expressing plasmid into Matrigel basement membrane matrix-coated C2C12 mouse myoblast cells. Myoblasts adhered and proliferated more rapidly on a Matrigel; thus, a dramatic increase in transfection efficiency can be obtained compared to Matrigel-untreated cells. Transfection efficiency was determined by counting fluorescent and total cells from six random fields for each condition. This protocol results in efficient (up to 60–70%) transfection of C2C12 myoblasts, high levels of GFP expression and low rate of cell death (10%). This technique is rapid, reliable, uses a lipid-based transfection reagent, and yields high transfection rates in a previously hard-to-transfect cell type.     

miR-143-3p促进C2C12成肌细胞分化

MicroRNAs (miRNAs) 是一类小非编码RNA,近年研究发现其在骨骼肌发育调控中发挥重要作用.为探明miR-143-3p在C2C12成肌细胞分化中的调控作用,采用 real-time PCR 检测了miR-143-3p在小鼠各组织及C2C12成肌细胞分化过程中的表达;使用miR-143-3p 的模拟物和特异性抑制剂分别处理细胞,采用 real-time PCR 和 Western印迹分别检测成肌因子 MyoG和成肌标志基因 MyHC mRNA和蛋白水平的变化;用免疫荧光染色的方法观察肌管的形成.结果显示,miR-143-3p在小鼠各组织中均有表达,并且随着细胞分化表达量逐渐增加;C2C12成肌细胞过表达 miR-143-3p,与对照组相比,成肌调控因子MyoG和成肌标志基因MyHC 的mRNA和蛋白表达均显著升高,肌管数量明显增多;抑制剂处理结果显示,细胞分化被显著抑制.检测miR-143-3p对MyHC各亚型表达的影响发现,miR-143-3p表达的变化并不直接影响MyHC各亚型的表达.以上结果说明, miR-143-3p在骨骼肌和成肌细胞中均有表达,能够促进C2C12成肌细胞分化,但并不直接调控MyHCs的表达.     

Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration

Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicine     

Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts

Summary Our previous studies have demonstrated that expression of growth-associated genes is regulated by the adhesive state of the cell. To understand the role of cell adhesion in regulating the switch from growth to differentiation, we are studying the differentiation of mouse myoblasts into multinucleated contractile myotubes. In this report, we describe a novel means of culturing C₂C₁₂ myoblasts that permits an analysis of the role of cell adhesion in regulating the sequential induction of muscle-specific genes that control myogenesis. Suspension of an asynchronous, proliferating population of myoblasts in a viscous gel of methylcellulose dissolved in medium containing 20% serum induces growth arrest in G₀ phase of the cell cycle without a concomitant induction of muscle-specific genes. Reattachment to a solid substratum in 20% serum, 0.5nM bFGF, or 10 nM IGF-1 rapidly activates entry of the quiescent cells into G₁ followed by a synchronous progression of the cell population through into S phase. bFGF or IGF-1 added separately facilitate only one passage through the cell cycle, whereas 20% serum or the two growth factors added together support multiple cell divisions. Adhesion of suspended cells in DMEM alone or with 3 nM IGF-1 induces myogenesis as evidenced by the synthesis of myogenin and myosin heavy chain (MHC) proteins followed by fusion into myotubes. bFGF completely inhibits this differentiation process even in the presence of myogenic doses of IGF-1. Addition of 3 nM IGF-1 to quiescent myoblasts maintained in suspension culture in serum-free conditions does not induce myogenin or MHC expression. Thus, adhesion is a requirement for the induction of muscle gene expression in mouse myoblasts. The development of a muscle cell culture environment in which proliferating myoblasts can be growth arrested in G₀ without activating muscle-specific gene expression provides a means of analyzing the synchronous activation of either the myogenic or growth programs and how adhesion affects each process, respectively. Supported by training grant T32-HL07035     

PUGNAc induces protein ubiquitination in C2C12 myotube cells

O‐linked β‐N‐acetylglucosaminylation (O‐GlcNAcylation) regulates many cellular processes including the cell cycle, cell signaling, and protein trafficking. Dysregulation of O‐GlcNAcylation may be involved in the development of insulin resistance and type 2 diabetes. Therefore, it is necessary to identify cellular proteins that are induced by elevated O‐GlcNAcylation. Here, using adenosine 5′‐triphosphate affinity chromatography, we employed a proteomic approach in order to identify differentially expressed proteins in response to treatment with the O‐GlcNAcase inhibitor, O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), in mouse C2C12 myotube cells. Among 205 selected genes, we identified 68 nucleotide‐binding proteins, 14 proteins that have adenosinetriphosphatase activity, and 10 proteins with ligase activity. Upregulation of proteins, including ubiquitin‐activating enzyme E1, proteasome subunit 20S, cullin‐associated NEDD8‐dissociated protein 1, ezrin, and downregulation of the protein nucleoside diphosphate kinase B, were confirmed by western blot analysis. In particular, we found that the protein ubiquitination level in C2C12 cells was increased by PUGNAc treatment. This is the first report of quantitative proteomic profiles of myotube cells after treatment with PUGNAc, and our results demonstrate the potential to enhance understanding of the relationship between insulin resistance, O‐GlcNAc, and PUGNAc in the future. Copyright © 2015 John Wiley & Sons, Ltd.     

Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family

The muscle regulatory factors (MRF) gene family regulate muscle fibre development. Several hormones and drugs also affect muscle development. Glucocorticoids are the only drugs reported to have a beneficial effect on muscle degenerative disorders. We investigated the glucocorticoid-related effects on C2C12 myoblast proliferation rate, morphological differentiation, and subsequent mRNA expression patterns of the MRF genes. C2C12 cells were incubated with the glucocorticoids dexamethasone or alpha-methyl-prednisolone. Both glucocorticoids showed comparable effects. Glucocorticoid treatment of C2C12 cells during the proliferative phase reduced the proliferation rate of the cells dose dependently, especially during the third and fourth day of culture, increased MyoD1, myf-5, and MRF4 mRNA levels, and reduced myogenin mRNA level, compared to untreated control cells. Thus, the mRNA level of proliferation-specific MyoD1 and myf-5 expression does not seem to associate with C2C12 myoblast proliferation rate. Glucocorticoid treatment of C2C12 cells during differentiation reduced the differentiation capacity dose dependently, which is accompanied by a dose dependent reduction of myogenin mRNA level, and increased MyoD1, myf-5, and MRF4 mRNA levels compared to untreated control cells. Therefore, we conclude that glucocorticoid treatment reduces differentiation of C2C12 myoblasts probably through reduction of differentiation-specific myogenin mRNA level, while inducing higher mRNA levels of proliferation-associated MRF genes.