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Sample records for c2c12 myotubes studied

  1. Cytoprotective Effect of Hispidin against Palmitate-Induced Lipotoxicity in C2C12 Myotubes

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    Jun Myoung Park

    2015-03-01

    Full Text Available It is well known that Phellinus linteus, which produces hispidin and its derivatives, possesses antioxidant activities. In this study, we investigated whether hispidin has protective effects on palmitate-induced oxidative stress in C2C12 skeletal muscle cells. Our results showed that palmitate treatment in C2C12 myotubes increased ROS generation and cell death as compared with the control. However, pretreatment of hispidin for 8 h improved the survival of C2C12 myotubes against palmitate-induced oxidative stress via inhibition of intracellular ROS production. Hispidin also inhibited palmitate-induced apoptotic nuclear condensation in C2C12 myotubes. In addition, we found that hispidin can suppress cleavage of caspase-3, expression of Bax, and NF-κB translocation. Therefore, these results suggest that hispidin is capable of protecting C2C12 myotubes against palmitate-induced oxidative stress.

  2. β‐Taxilin participates in differentiation of C2C12 myoblasts into myotubes

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    Sakane, Hiroshi; Makiyama, Tomohiko; Nogami, Satoru; Horii, Yukimi [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan); Akasaki, Kenji [Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Hiroshima 729-0292 (Japan); Shirataki, Hiromichi, E-mail: hiro-sh@dokkyomed.ac.jp [Department of Molecular and Cell Biology, Graduate school of Medicine, Dokkyo Medical University, 880 Kitakobayashi, Mibu-town, Tochigi 321-0293 (Japan)

    2016-07-15

    Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that β-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of β-taxilin in myogenesis. In C2C12 cells, knockdown of β-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that β-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of β-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that β-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes. - Highlights: • β‐Taxilin is progressively expressed during differentiation of C2C12 cell. • Knockdown of β-taxilin impaired C2C12 myotube formation. • β‐Taxilin interacted with dysbindin. • Knockdown of dysbindin promoted C2C12 myotube formation. • The function of β-taxilin in C2C12 myotube formation depends on dysbindin.

  3. Oxidative stress-induced metabolic changes in mouse C2C12 myotubes studied with high-resolution 13C, 1H, and 31P NMR spectroscopy

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    Straadt, Ida K; Young, Jette F; Petersen, Bent O

    2010-01-01

    In this study, stress in relation to slaughter was investigated in a model system by the use of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy for elucidating changes in the metabolites in C2C12 myotubes exposed to H(2)O(2)-induced stress. Oxidative stress resulted in lower...... levels of several metabolites, mainly amino acids; however, higher levels of alanine were apparent in the (13)C spectra after incubation with [(13)C(1)]glucose. In the (13)C spectra [(13)C(3)]lactate tended to increase after exposure to increasing concentrations of H(2)O(2); conversely, a tendency...

  4. Apoptosis in differentiating C2C12 muscle cells selectively targets Bcl-2-deficient myotubes.

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    Schöneich, Christian; Dremina, Elena; Galeva, Nadezhda; Sharov, Victor

    2014-01-01

    Muscle cell apoptosis accompanies normal muscle development and regeneration, as well as degenerative diseases and aging. C2C12 murine myoblast cells represent a common model to study muscle differentiation. Though it was already shown that myogenic differentiation of C2C12 cells is accompanied by enhanced apoptosis in a fraction of cells, either the cell population sensitive to apoptosis or regulatory mechanisms for the apoptotic response are unclear so far. In the current study we characterize apoptotic phenotypes of different types of C2C12 cells at all stages of differentiation, and report here that myotubes of differentiated C2C12 cells with low levels of anti-apoptotic Bcl-2 expression are particularly vulnerable to apoptosis even though they are displaying low levels of pro-apoptotic proteins Bax, Bak and Bad. In contrast, reserve cells exhibit higher levels of Bcl-2 and high resistance to apoptosis. The transfection of proliferating myoblasts with Bcl-2 prior to differentiation did not protect against spontaneous apoptosis accompanying differentiation of C2C12 cells but led to Bcl-2 overexpression in myotubes and to significant protection from apoptotic cell loss caused by exposure to hydrogen peroxide. Overall, our data advocate for a Bcl-2-dependent mechanism of apoptosis in differentiated muscle cells. However, downstream processes for spontaneous and hydrogen peroxide induced apoptosis are not completely similar. Apoptosis in differentiating myoblasts and myotubes is regulated not through interaction of Bcl-2 with pro-apoptotic Bcl-2 family proteins such as Bax, Bak, and Bad.

  5. Ethanol decreases agrin-induced acetylcholine receptor clustering in C2C12 myotube culture.

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    Owen, David B; Chamberlain, Kevin T; Shishido, Sonia; Grow, Wade A

    2010-03-01

    We investigated the effect of ethanol on skeletal muscle development using C2C12 cell culture. The ethanol concentrations of 10mM, 25mM, and 100mM, were tested because plasma samples of alcohol-dependent individuals fall within this range. We assessed two specific events in skeletal muscle development, the fusion of myoblasts to form myotubes and the acetylcholine receptor (AChR) clustering associated with neuromuscular synapse formation. We report that ethanol does not effect myotube formation or the viability of myoblasts or myotubes in C2C12 cell culture. However, ethanol does effect AChR clustering on C2C12 myotubes. As motor neurons approach skeletal muscle during development, agrin is released by motor neurons and induces AChR clustering on muscle fibers. In our experiments, agrin was applied to cell cultures during the period when myoblasts fuse to form myotubes. In cell cultures exposed to ethanol during myotube formation, agrin-induced AChR clustering was decreased compared to untreated cultures. In cell cultures exposed to ethanol during myoblast proliferation, with ethanol removed during myotube formation, agrin-induced AChR clustering was unaffected. We conclude that exposure to a physiologically relevant concentration of ethanol during the specific period of myotube formation decreases agrin-induced AChR clustering. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  6. Downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes.

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    Huang, Shujuan; Huang, Suling; Wang, Xi; Zhang, Qingli; Liu, Jia; Leng, Ying

    2017-01-01

    Dysregulation of lipid metabolism in skeletal muscle is involved in the development of insulin resistance. Mutations in lipin-1, a key lipid metabolism regulator leads to significant systemic insulin resistance in fld mice. However, the function of lipin-1 on lipid metabolism and insulin sensitivity in skeletal muscle is still unclear. Herein we demonstrated that downregulation of lipin-1 in C2C12 myotubes by siRNA transfection suppressed insulin action, characterized by reduced insulin stimulated Akt phosphorylation and glucose uptake. Correspondingly, decreased lipin-1 expression was observed in palmitate-induced insulin resistance in C2C12 myotubes, suggested that lipin-1 might play a role in the etiology of insulin resistance in skeletal muscle. The insulin resistance induced by lipin-1 downregulation was related to the disturbance of lipid homeostasis. Lipin-1 silencing reduced intracellular DAG and TAG levels, but elevated ceramide accumulation in C2C12 myotubes. Moreover, the impaired insulin stimulated Akt phosphorylation and glucose uptake caused by lipin-1 silencing could be blocked by the pretreatment with SPT inhibitor myriocin, ceramide synthase inhibitor FB1, or PP2A inhibitor okadaic acid, suggested that the increased ceramide accumulation might be responsible for the development of insulin resistance induced by lipin-1 silencing in C2C12 myotubes. Meanwhile, decreased lipin-1 expression also impaired mitochondrial function in C2C12 myotubes. Therefore, our study suggests that lipin-1 plays an important role in lipid metabolism and downregulation of lipin-1 induces insulin resistance by increasing intracellular ceramide accumulation in C2C12 myotubes. These results offer a molecular insight into the role of lipin-1 in the development of insulin resistance in skeletal muscle.

  7. Regulation of nonmuscle myosin II during 3-methylcholanthrene induced dedifferentiation of C2C12 myotubes

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    Dey, Sumit K.; Saha, Shekhar; Das, Provas; Das, Mahua R.; Jana, Siddhartha S., E-mail: bcssj@iacs.res.in

    2014-08-01

    3-Methylcholanthrene (3MC) induces tumor formation at the site of injection in the hind leg of mice within 110 days. Recent reports reveal that the transformation of normal muscle cells to atypical cells is one of the causes for tumor formation, however the molecular mechanism behind this process is not well understood. Here, we show in an in vitro study that 3MC induces fragmentation of multinucleate myotubes into viable mononucleates. These mononucleates form colonies when they are seeded into soft agar, indicative of cellular transformation. Immunoblot analysis reveals that phosphorylation of myosin regulatory light chain (RLC{sub 20}) is 5.6±0.5 fold reduced in 3MC treated myotubes in comparison to vehicle treated myotubes during the fragmentation of myotubes. In contrast, levels of myogenic factors such as MyoD, Myogenin and cell cycle regulators such as Cyclin D, Cyclin E1 remain unchanged as assessed by real-time PCR array and reverse transcriptase PCR analysis, respectively. Interestingly, addition of the myosin light chain kinase inhibitor, ML-7, enhances the fragmentation, whereas phosphatase inhibitor perturbs the 3MC induced fragmentation of myotubes. These results suggest that decrease in RLC{sub 20} phosphorylation may be associated with the fragmentation step of dedifferentiation. - Highlights: • 3-Methylcholanthrene induces fragmentation of C2C12-myotubes. • Dedifferentiation can be divided into two steps – fragmentation and proliferation. • Fragmentation is associated with rearrangement of nonmuscle myosin II. • Genes associated with differentiation and proliferation are not altered during fragmentation. • Phosphorylation of myosin regulatory light chain is reduced during fragmentation.

  8. Sarcolipin expression is repressed by endoplasmic reticulum stress in C2C12 myotubes.

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    Takahashi, Nobuhiko; Kimura, Atsushi P; Naito, Sumiyoshi; Yoshida, Mika; Kumano, Osamu; Suzuki, Takeshi; Itaya, Satoshi; Moriya, Mitsuru; Tsuji, Masahiro; Ieko, Masahiro

    2017-11-01

    Sarcolipin is a transmembrane protein expressed in the sarco/endoplasmic reticulum of skeletal and atrial muscles in large animals. Sarcolipin plays crucial roles in heat production through modifying the function of sarco/endoplasmic reticulum Ca2+ ATPase, thereby being involved in thermogenesis and systemic metabolism. In skeletal muscle, endoplasmic reticulum (ER) stress has been implicated in several conditions, such as insulin resistance, muscle diseases, and hypo/hyper-contraction. Here, we investigated the effect of ER stress on sarcolipin expression in skeletal muscle cells, C2C12 myotubes. First, gene expression of sarcolipin was confirmed in the cells during myogenesis. Then, ER stress was induced in C2C12 myotubes by treatment with tunicamycin or thapsigargin. Sarcolipin messenger RNA (mRNA) and protein expression were significantly reduced by ER stress induction. The reduction was independent of inositol-requiring element 1 (IRE1), which is activated by ER stress and has potent endonuclease activity, when evaluated by treatment with an IRE1 inhibitor, 4μ8C. On the other hand, sarcolipin mRNA stability was reduced under the ER stress when evaluated by treatment with actinomycin D. In conclusion, these results show that ER stress represses sarcolipin expression due to changes in mRNA stability in C2C12 myotubes.

  9. Cytoprotective Role of Nrf2 in Electrical Pulse Stimulated C2C12 Myotube.

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    Masaki Horie

    Full Text Available Regular physical exercise is central to a healthy lifestyle. However, exercise-related muscle contraction can induce reactive oxygen species and reactive nitrogen species (ROS/RNS production in skeletal muscle. The nuclear factor-E2-related factor-2 (Nrf2 transcription factor is a cellular sensor for oxidative stress. Regulation of nuclear Nrf2 signaling regulates antioxidant responses and protects organ structure and function. However, the role of Nrf2 in exercise- or contraction-induced ROS/RNS production in skeletal muscle is not clear. In this study, using differentiated C2C12 cells and electrical pulse stimulation (EPS of muscle contraction, we explored whether Nrf2 plays a role in the skeletal muscle response to muscle contraction-induced ROS/RNS. We found that EPS (40 V, 1 Hz, 2 ms stimulated ROS/RNS accumulation and Nrf2 activation. We also showed that expression of NQO1, HO-1 and GCLM increased after EPS-induced muscle contraction and was remarkably suppressed in cells with Nrf2 knockdown. We also found that the antioxidant N-acetylcysteine (NAC significantly attenuated Nrf2 activation after EPS, whereas the nitric oxide synthetase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME did not. Furthermore, Nrf2 knockdown after EPS markedly decreased ROS/RNS redox potential and cell viability and increased expression of the apoptosis marker Annexin V in C2C12 myotubes. These results indicate that Nrf2 activation and expression of Nrf2 regulated-genes protected muscle against the increased ROS caused by EPS-induced muscle contraction. Thus, our findings suggest that Nrf2 may be a key factor for preservation of muscle function during muscle contraction.

  10. C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene

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    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Sun, Wenxing [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); College of Public Health, Nantong University, Nantong 226019 (China); Gao, Ying [College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095 (China); Zhang, Lifan [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Chen, Jie, E-mail: jiechen@njau.edu.cn [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China)

    2016-03-25

    Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes. - Highlights: • C2C12 myotubes inhibited proliferation and differentiation of 3T3-L1 preadipocytes. • C2C12 myotubes arrested cell cycle of 3T3-L1 preadipocytes. • C2C12 myotubes induced apoptosis of 3T3-L1 preadipocytes. • C2C12 inhibit 3T3-L1 cells by reducing the expression of glucocorticoid receptor gene.

  11. Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes

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    Hughes, M. A.; Downs, R. M.; Webb, G. W.; Crocker, C. L.; Kinsey, S.T.; Baumgarner, Bradley L.

    2017-01-01

    Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1–10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our ...

  12. Metabolic profiling of heat or anoxic stress in mouse C2C12 myotubes using multinuclear magnetic resonance spectroscopy

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    Straadt, Ida K; Young, Jette F; Petersen, Bent O

    2010-01-01

    In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of (13)C, (1)H, and (31)P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [(13)C]-glucose. Both the (13)C and the (1)H NMR...... spectra showed reduced levels of the amino acids alanine, glutamate, and aspartate after heat or anoxic stress. The decreases were smallest at 42 degrees C, larger at 45 degrees C, and most pronounced after anoxic conditions. In addition, in both the (1)H and the (31)P NMR spectra, decreases in the high...

  13. Permethrin alters adipogenesis in 3T3-L1 adipocytes and causes insulin resistance in C2C12 myotubes.

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    Kim, Jonggun; Park, Yooheon; Yoon, Kyong Sup; Clark, J Marshall; Park, Yeonhwa

    2014-09-01

    Pyrethroids are a class of insecticides structurally derived from the naturally occurring insecticides called pyrethrins. Along with emerging evidence that exposure to insecticides is linked to altered weight gain and glucose homeostasis, exposure to pyrethroids has been linked to altered blood glucose levels in humans. Thus, the purpose of this study was to determine the role of permethrin on lipid and glucose metabolisms. Permethrin was treated to 3T3-L1 adipocytes and C2C12 myoblasts to determine its role in lipid and glucose metabolisms, respectively. Permethrin treatment resulted in increased expression of key markers of adipogenesis and lipogenesis in adipocytes. Permethrin significantly reduced insulin-stimulated glucose uptake in myotubes. This is the first report on the role of permethrin in altered lipid metabolism in adipocytes and impaired glucose homeostasis in myotubes. These results may help elucidate fundamental underlying mechanisms between insecticide exposure, particularly permethrin, and potential risk of developing obesity and its comorbidities. © 2014 Wiley Periodicals, Inc.

  14. DHA Inhibits Protein Degradation More Efficiently than EPA by Regulating the PPARγ/NFκB Pathway in C2C12 Myotubes

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    Yue Wang

    2013-01-01

    Full Text Available This study was conducted to evaluate the mechanism by which n-3 PUFA regulated the protein degradation in C2C12 myotubes. Compared with the BSA control, EPA at concentrations from 400 to 600 µM decreased total protein degradation (P0.05. Interestingly, EPA and DHA both still decreased the total protein degradation, although PPARγ knockdown attenuated the suppressive effects of EPA and DHA on the total protein degradation (P<0.01. These results revealed that DHA inhibits protein degradation more efficiently than EPA by regulating the PPARγ/NF-κB pathway in C2C12 myotubes.

  15. Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes.

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    Hughes, M A; Downs, R M; Webb, G W; Crocker, C L; Kinsey, S T; Baumgarner, Bradley L

    2017-04-01

    Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1-10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our analysis revealed that caffeine promoted a 40% increase in autolysosome formation and a 25% increase in autophagic flux. In contrast, caffeine treatment did not significantly increase the expression of the skeletal muscle specific ubiquitin ligases MAFbx and MuRF1 or 20S proteasome activity. Caffeine treatment significantly reduced mTORC1 signaling, total protein synthesis and myotube diameter in a CaMKKβ/AMPK-dependent manner. Further, caffeine promoted a CaMKII-dependent increase in myostatin mRNA expression that did not significantly contribute to the caffeine-dependent reduction in protein synthesis. Our results indicate that short-term caffeine exposure significantly reduced skeletal myotube diameter by increasing autophagic flux and promoting a CaMKKβ/AMPK-dependent reduction in protein synthesis.

  16. Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes.

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    Shaw, Andy; Jeromson, Stewart; Watterson, Kenneth R; Pediani, John D; Gallagher, Iain J; Whalley, Tim; Dreczkowski, Gillian; Brooks, Naomi; Galloway, Stuart D; Hamilton, D Lee

    2017-06-01

    Mutations in the gene that encodes the principal l-carnitine transporter, OCTN2, can lead to a reduced intracellular l-carnitine pool and the disease Primary Carnitine Deficiency. l-Carnitine supplementation is used therapeutically to increase intracellular l-carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake, we hypothesized that AMPK-activating compounds and insulin would increase l-carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level, and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase l-carnitine uptake at 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), berberine (100 μM), DNP (500 μM)], or directly activate AMPK [AICAR (250 μM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 μM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 μM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM caffeine, suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits l-carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role. Copyright © 2017 the American Physiological Society.

  17. Melatonin protects against uric acid-induced mitochondrial dysfunction, oxidative stress, and triglyceride accumulation in C2C12myotubes.

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    Maarman, Gerald J; Andrew, Brittany M; Blackhurst, Dee M; Ojuka, Edward O

    2017-04-01

    Excess uric acid has been shown to induce oxidative stress, triglyceride accumulation, and mitochondrial dysfunction in the liver and is an independent predictor of type-2 diabetes. Skeletal muscle plays a dominant role in type 2 diabetes and presents a large surface area to plasma uric acid. However, the effects of uric acid on skeletal muscle are underinvestigated. Our aim was therefore to characterize the effects of excessive uric acid on oxidative stress, triglyceride content, and mitochondrial function in skeletal muscle C 2 C 12 myotubes and assess how these are modulated by the antioxidant molecule melatonin. Differentiated C 2 C 12 myotubes were exposed to 750 µM uric acid or uric acid + 10 nM melatonin for 72 h. Compared with control, uric acid increased triglyceride content by ~237%, oxidative stress by 32%, and antioxidant capacity by 135%. Uric acid also reduced endogenous ROUTINE respiration, complex II-linked oxidative phosphorylation, and electron transfer system capacities. Melatonin counteracted the effects of uric acid without further altering antioxidant capacity. Our data demonstrate that excess uric acid has adverse effects on skeletal muscle similar to those previously reported in hepatocytes and suggest that melatonin at a low physiological concentration of 10 nM may be a possible therapy against some adverse effects of excess uric acid. NEW & NOTEWORTHY Few studies have investigated the effects of uric acid on skeletal muscle. This study shows that hyperuricemia induces mitochondrial dysfunction and triglyceride accumulation in skeletal muscle. The findings may explain why hyperuricemia is an independent predictor of diabetes. Copyright © 2017 the American Physiological Society.

  18. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

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    Kamolrat, Torkamol [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom); Gray, Stuart R., E-mail: s.r.gray@abdn.ac.uk [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom)

    2013-03-22

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using {sup 3}H-labelled phenylalanine. Protein breakdown was measured using {sup 3}H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion.

  19. Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

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    Lomax Michael A

    2007-03-01

    Full Text Available Abstract Background The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis Results Incubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA, relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p Conclusion In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

  20. Infectious prions accumulate to high levels in non proliferative C2C12 myotubes.

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    Allen Herbst

    Full Text Available Prion diseases are driven by the strain-specific, template-dependent transconformation of the normal cellular prion protein (PrP(C into a disease specific isoform PrP(Sc. Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared to animals. Using non-replicating cells allows the accumulation of higher levels of PrP(Sc and, thus, greater amounts of infectivity. Here, we infect non-proliferating muscle fiber myotube cultures prepared from differentiated myoblasts. We demonstrate that prion-infected myotubes generate substantial amounts of PrP(Sc and that the level of infectivity produced in these post-mitotic cells, 10(5.5 L.D.50/mg of total protein, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. Mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.

  1. Cyclic stretch facilitates myogenesis in C2C12 myoblasts and rescues thiazolidinedione-inhibited myotube formation

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    Ya-Ju eChang

    2016-03-01

    Full Text Available Thiazolidinedione (TZD, a specific peroxisome proliferator-activated receptor r (PPARr agonist, was developed to control blood glucose in diabetes patients. However, several side effects were reported that increased the risk of heart failure. We used C2C12 myoblasts to investigate the role of PPARs and their transcriptional activity during myotube formation. The role of mechanical stretch during myogenesis was also explored by applying cyclic stretch to the differentiating C2C12 myoblasts with 10% strain deformation at 1 Hz. The myogenesis medium (MM, composed of Dulbecco’s modified Eagle’s medium with 2% horse serum, facilitated myotube formation with increased myosin heavy chain and a-smooth muscle actin (a-SMA protein expression. The PPAR protein and PPAR response element (PPRE promoter activity decreased during MM induction. Cyclic stretch further facilitated the myogenesis in MM with increased a-SMA and decreased PPARr protein expression and inhibited PPRE promoter activity. Adding a PPARr agonist (TZD to the MM stopped the myogenesis and restored the PPRE promoter activity, whereas a PPARr antagonist (GW9662 significantly increased the myotube number and length. During the myogenesis induction, application of cyclic stretch rescued the inhibitory effects of TZD. These results provide novel perspectives for mechanical stretch to interplay and rescue the dysfunction of myogenesis with the involvement of PPARr and its target drugs.

  2. Promotion of Glucose Uptake in C2C12 Myotubes by Cereal Flavone Tricin and Its Underlying Molecular Mechanism.

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    Kim, Sohyun; Go, Gwang-Woong; Imm, Jee-Young

    2017-05-17

    The effect of tricin, a methylated flavone widely distributed in cereals, on glucose uptake and the underlying molecular mechanism was investigated using C2C12 myotubes. Tricin significantly increased glucose uptake in C2C12 myotubes, regardless of the absence (1.4-fold at 20 μM) or presence (1.6-fold at 20 μM) of insulin. The GLUT4 expression on the plasma membrane was increased 1.6-fold after tricin treatment (20 μM) in the absence of insulin. Tricin treatment significantly activated the insulin-dependent cell signaling pathway, including the activation of insulin receptor substrate-1 (IRS1), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and AKT substrate of 160 kDa (AS160). The oral administration of tricin (64 and 160 mg kg -1 of body weight day -1 ) also significantly lowered blood glucose levels in glucose-loaded C57BL/6 mice (p < 0.05). These results suggest that tricin has great potential to be used as a functional agent for glycemic control.

  3. Dehydroepiandrosterone activates AMP kinase and regulates GLUT4 and PGC-1α expression in C2C12 myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Yokokawa, Takumi [Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto (Japan); Sato, Koji [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Iwanaka, Nobumasa [The Graduate School of Science and Engineering, Ritsumeikan University, Shiga (Japan); Honda, Hiroki [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Higashida, Kazuhiko [Faculty of Sport Science, Waseda University, Saitama (Japan); Iemitsu, Motoyuki [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan); Hayashi, Tatsuya [Laboratory of Sports and Exercise Medicine, Graduate School of Human and Environmental Studies, Kyoto University, Kyoto (Japan); Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Graduate School of Sport & Health Science, Ritsumeikan University, Shiga (Japan)

    2015-07-17

    Exercise and caloric restriction (CR) have been reported to have anti-ageing, anti-obesity, and health-promoting effects. Both interventions increase the level of dehydroepiandrosterone (DHEA) in muscle and blood, suggesting that DHEA might partially mediate these effects. In addition, it is thought that either 5′-adenosine monophosphate-activated protein kinase (AMPK) or peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mediates the beneficial effects of exercise and CR. However, the effects of DHEA on AMPK activity and PGC-1α expression remain unclear. Therefore, we explored whether DHEA in myotubes acts as an activator of AMPK and increases PGC-1α. DHEA exposure increased glucose uptake but not the phosphorylation levels of Akt and PKCζ/λ in C2C12 myotubes. In contrast, the phosphorylation levels of AMPK were elevated by DHEA exposure. Finally, we found that DHEA induced the expression of the genes PGC-1α and GLUT4. Our current results might reveal a previously unrecognized physiological role of DHEA; the activation of AMPK and the induction of PGC-1α by DHEA might mediate its anti-obesity and health-promoting effects in living organisms. - Highlights: • We assessed whether dehydroepiandrosterone (DHEA) activates AMPK and PGC-1α. • DHEA exposure increased glucose uptake in C2C12 myotubes. • The phosphorylation levels of AMPK were elevated by DHEA exposure. • DHEA induced the expression of the genes PGC-1α and GLUT4. • AMPK might mediate the anti-obesity and health-promoting effects of DHEA.

  4. New gene targets of PGC-1α and ERRα co-regulation in C2C12 myotubes.

    Science.gov (United States)

    Nsiah-Sefaa, Abena; Brown, Erin L; Russell, Aaron P; Foletta, Victoria C

    2014-12-01

    As a transcriptional coactivator, PGC-1α contributes to the regulation of a broad range of metabolic processes in skeletal muscle health and disease; however, there is limited information about the genes it transcriptionally regulates. To identify new potential gene targets of PGC-1α regulation, mouse C2C12 myotubes were screened by microarray analysis following PGC-1α overexpression. Genes with an mRNA expression of 2.5-fold or more (P genes were singled out if they had no previous connection to PGC-1α regulation or characterization in skeletal muscle, or were unannotated with no known function. Following confirmation of their regulation by PGC-1α using qPCR analysis, eight genes were focused on for further investigation (Akr1b10, Rmnd1, 1110008P14Rik, 1700021F05Rik, Mtfp1, Mrm1, Oxnad1 and Cluh). Bioinformatics indicated a number of the genes were linked to a range of metabolic-related functions including fatty acid oxidation, oxido-reductase activity, and mitochondrial remodeling and transport. Treating C2C12 myotubes for 6 h with AICAR, a known activator of AMP kinase and inducer of Pgc-1α gene expression, increased the mRNA levels of both Pgc-1α (P genes to contain either a consensus or near consensus response elements for the estrogen-related receptor α (ERRα), a key transcription factor-binding partner of PGC-1α in skeletal muscle. Furthermore, knockdown of endogenous ERRα levels partially or completely blocked the induction of gene expression of all genes by PGC-1α, while each gene was significantly upregulated in the presence of a constitutively active form of ERRα (P regulation of these genes by PGC-1α and its signaling pathway in skeletal muscle.

  5. Metabolic and morphological alterations induced by proteolysis-inducing factor from Walker tumour-bearing rats in C2C12 myotubes

    Directory of Open Access Journals (Sweden)

    Tisdale Michael J

    2008-01-01

    Full Text Available Abstract Background Patients with advanced cancer suffer from cachexia, which is characterised by a marked weight loss, and is invariably associated with the presence of tumoral and humoral factors which are mainly responsible for the depletion of fat stores and muscular tissue. Methods In this work, we used cytotoxicity and enzymatic assays and morphological analysis to examine the effects of a proteolysis-inducing factor (PIF-like molecule purified from ascitic fluid of Walker tumour-bearing rats (WF, which has been suggested to be responsible for muscle atrophy, on cultured C2C12 muscle cells. Results WF decreased the viability of C2C12 myotubes, especially at concentrations of 20–25 μg.mL-1. There was an increase in the content of the pro-oxidant malondialdehyde, and a decrease in antioxidant enzyme activity. Myotubes protein synthesis decreased and protein degradation increased together with an enhanced in the chymotrypsin-like enzyme activity, a measure of functional proteasome activity, after treatment with WF. Morphological alterations such as cell retraction and the presence of numerous cells in suspension were observed, particularly at high WF concentrations. Conclusion These results indicate that WF has similar effects to those of proteolysis-inducing factor, but is less potent than the latter. Further studies are required to determine the precise role of WF in this experimental model.

  6. C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.

    Science.gov (United States)

    Cooper, S T; Maxwell, A L; Kizana, E; Ghoddusi, M; Hardeman, E C; Alexander, I E; Allen, D G; North, K N

    2004-07-01

    We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development. Copyright 2004 Wiley-Liss, Inc.

  7. Rapamycin has a biphasic effect on insulin sensitivity in C2C12 myotubes due to sequential disruption of mTORC1 and mTORC2

    Directory of Open Access Journals (Sweden)

    Lan eYe

    2012-09-01

    Full Text Available Rapamycin, an inhibitor of mTOR complex 1 (mTORC1, improves insulin sensitivity in acute studies in vitro and in vivo by disrupting a negative feedback loop mediated by S6 kinase. We find that rapamycin has a clear biphasic effect on insulin sensitivity in C2C12 myotubes, with enhanced responsiveness during the first hour that declines to almost complete insulin resistance by 24-48 hours. We and others have recently observed that chronic rapamycin treatment induces insulin resistance in rodents, at least in part due to disruption of mTORC2, an mTOR-containing complex that is not acutely sensitive to the drug. Chronic rapamycin treatment may also impair insulin action via the inhibition of mTORC1-dependent mitochondrial biogenesis and activity, which could result in a buildup of lipid intermediates that are known to trigger insulin resistance. We confirmed that rapamycin inhibits expression of PGC-1α, a key mitochondrial transcription factor, and acutely reduces respiration rate in myotubes. However, rapamycin did not stimulate phosphorylation of PKCθ, a central mediator of lipid-induced insulin resistance. Instead, we found dramatic disruption of mTORC2, which coincided with the onset of insulin resistance. Selective inhibition of mTORC1 or mTORC2 by shRNA-mediated knockdown of specific components (Raptor and Rictor, respectively confirmed that mitochondrial effects of rapamycin are mTORC1-dependent, whereas insulin resistance was recapitulated only by knockdown of mTORC2. Thus, mTORC2 disruption, rather than inhibition of mitochondria, causes insulin resistance in rapamycin-treated myotubes, and this system may serve as a useful model to understand the effects of rapamycin on mTOR signaling in vivo.

  8. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Young Woo [Cell Therapy and Tissue Engineering Center, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin [Biomedical Research Institute, Lifeliver Co., Ltd., Suwon (Korea, Republic of); Park, Won Jin [Dr. Park' s Aesthetic Clinic, Seoul (Korea, Republic of); Kong, Jee Hyun; Shim, Kwang Yong [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Lee, Jong In, E-mail: oncochem@yonsei.ac.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of); Kim, Hyun Soo, E-mail: khsmd@unitel.co.kr [Department of Hematology-Oncology, Wonju College of Medicine, Yonsei Univ., Wonju (Korea, Republic of)

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  9. In vitro assessment of the combined effect of eicosapentaenoic acid, green tea extract and curcumin C3 on protein loss in C2C12 myotubes.

    Science.gov (United States)

    Mirza, Kamran A; Luo, Menghua; Pereira, Suzette; Voss, Anne; Das, Tapas; Tisdale, Michael J

    2016-09-01

    EPA has been clinically shown to reduce muscle wasting during cancer cachexia. This study investigates whether curcumin or green tea extract (GTE) enhances the ability of low doses of eicosapentaenoic acid (EPA) to reduce loss of muscle protein in an in vitro model. A low dose of EPA with minimal anti-cachectic activity was chosen to evaluate any potential synergistic effect with curcumin or GTE. Depression of protein synthesis and increase in degradation was determined in C2C12 myotubes in response to tumour necrosis factor-α (TNF-α) and proteolysis-inducing factor (PIF). EPA (50 μM) or curcumin (10 μg ml(-1)) alone had little effect on protein degradation caused by PIF but the combination produced complete inhibition, as did the combination with GTE (10 μg ml(-1)). In response to TNF-α (25 ng ml(-1))-induced protein degradation, EPA had a small, but not significant effect on protein degradation; however, when curcumin and GTE were combined with EPA, the effect was enhanced. EPA completely attenuated the depression of protein synthesis caused by TNF-α, but not that caused by PIF. The combination of EPA with curcumin produced a significant increase in protein synthesis to both agents. GTE alone or in combination with EPA had no effect on the depression of protein synthesis by TNF-α, but did significantly increase protein synthesis in PIF-treated cells. Both TNF-α and PIF significantly reduced myotube diameter from 17 to 13 μm for TNF-α (23.5%) and 15 μm (11.8%) for PIF However the triple combination of EPA, curcumin and GTE returned diameters to values not significantly different from the control. These results suggest that either curcumin or GTE or the combination could enhance the anti-catabolic effect of EPA on lean body mass.

  10. CHARACTERIZATION OF P2-PURINOCEPTOR MEDIATED CYCLIC-AMP FORMATION IN MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; DUIN, M; DENHERTOG, A; NELEMANS, A

    1 The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2Cl2 myotubes. 2 ATP (100 muM) and ATPgammaS (100 muM) caused a sustained increase in cyclic AMP content of the cells,

  11. APPL1 promotes glucose uptake in response to mechanical stretch via the PKCζ-non-muscle myosin IIa pathway in C2C12 myotubes.

    Science.gov (United States)

    Saito, Tsugumichi; Okada, Shuichi; Shimoda, Yoko; Tagaya, Yuko; Osaki, Aya; Yamada, Eijiro; Shibusawa, Ryo; Nakajima, Yasuyo; Ozawa, Atsushi; Satoh, Tetsurou; Mori, Masatomo; Yamada, Masanobu

    2016-11-01

    Expression of adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1 (APPL1) promoted glucose transporter 4 (GLUT4) translocation and glucose uptake in adipose and muscle tissues in response to stimulation with insulin, adiponectin, or exercise. In response to mechanical stretch, knockdown of APPL1 in C2C12 myotubes suppressed glucose uptake. APPL1-induced increased glucose uptake was mediated by protein kinase C (PKC) ζ but not AKT, AMPK, or calmodulin-dependent protein kinase. In myotubes overexpressing APPL1, PKCζ was phosphorylated and translocated to the plasma membrane (PM) in response to mechanical stretch. Phosphorylated PKCζ co-immunoprecipitated with protein phosphatase 2A (PP2A) under basal conditions, but dissociated upon myotube stretching. Moreover, stretch-induced phosphorylated PKCζ co-immunoprecipitated with non-muscle myosin IIa. Blebbistatin, an inhibitor of myosin II ATPase activity, suppressed APPL1-mediated stretch-induced glucose uptake and PKCζ translocation. Taken together these data demonstrate that in response to mechanical stretch, APPL1 enhances glucose uptake by modulating the activation and localization of PKCζ, as well as its functional interaction with both PP2A and myosin IIa. These findings support a new function for non-muscle myosin IIa in differentiated myotubes. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Expressional studies of the aldehyde oxidase (AOX1) gene during myogenic differentiation in C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamli, Majid Rasool; Kim, Jihoe; Pokharel, Smritee; Jan, Arif Tasleem [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Lee, Eun Ju [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Bovine Genome Resources Bank, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Choi, Inho, E-mail: inhochoi@ynu.ac.kr [School of Biotechnology, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Bovine Genome Resources Bank, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of)

    2014-08-08

    Highlights: • AOX1 contributes to the formation of myotube. • Silencing of AOX1 reduces myotube formation. • AOX1 regulates MyoG gene expression. • AOX1 contributes to myogenesis via H{sub 2}O{sub 2}. - Abstract: Aldehyde oxidases (AOXs), which catalyze the hydroxylation of heterocycles and oxidation of a wide variety of aldehydic compounds, have been present throughout evolution from bacteria to humans. While humans have only a single functional aldehyde oxidase (AOX1) gene, rodents are endowed with four AOXs; AOX1 and three aldehyde oxidase homologs (AOH1, AOH2 and AOH3). In continuation of our previous study conducted to identify genes differentially expressed during myogenesis using a microarray approach, we investigated AOX1 with respect to its role in myogenesis to conceptualize how it is regulated in C2C12 cells. The results obtained were validated by silencing of the AOX1 gene. Analysis of their fusion index revealed that formation of myotubes showed a marked reduction of up to 40% in AOX1{sub kd} cells. Expression of myogenin (MYOG), one of the marker genes used to study myogenesis, was also found to be reduced in AOX1{sub kd} cells. AOX1 is an enzyme of pharmacological and toxicological importance that metabolizes numerous xenobiotics to their respective carboxylic acids. Hydrogen peroxide (H{sub 2}O{sub 2}) produced as a by-product in this reaction is considered to be involved as a part of the signaling mechanism during differentiation. An observed reduction in the level of H{sub 2}O{sub 2} among AOX1{sub kd} cells confirmed production of H{sub 2}O{sub 2} in the reaction catalyzed by AOX1. Taken together, these findings suggest that AOX1 acts as a contributor to the process of myogenesis by influencing the level of H{sub 2}O{sub 2}.

  13. Pyropia yezoensis peptide PYP1‑5 protects against dexamethasone‑induced muscle atrophy through the downregulation of atrogin1/MAFbx and MuRF1 in mouse C2C12 myotubes.

    Science.gov (United States)

    Lee, Min-Kyeong; Kim, Young-Min; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2017-06-01

    Skeletal muscle atrophy refers to the decline in muscle mass and strength that occurs under various conditions, including aging, starvation, cancer and other cachectic diseases. Muscle atrophy caused by aging, known as sarcopenia, primarily occurs after 50 years of age. Muscle atrophy‑related genes, including atrogin1/muscle atrophy F‑box (MAFbx) and muscle RING finger 1 (MuRF1), are expressed early in the muscle atrophy process, and their expression precedes the loss of muscle mass. The present study investigated the potential anti‑atrophic effects of the Pyropia yezoensis peptide PYP1‑5. The MTS assay did not detect cytotoxic effects of PYP1‑5 on C2C12 mouse myoblast cells. Subsequently, the anti‑atrophic effects of PYP1‑5 on skeletal muscle cells was examined by treating C2C12 myotubes with 100 µM dexamethasone (DEX) and/or 500 ng/ml PYP1‑5 for 24 h. Compared with the control, myotube diameter was reduced in DEX‑treated cells, whereas PYP1‑5 treatment protected against DEX‑induced muscle atrophy. MAFbx and MuRF1 protein and mRNA expression levels were detected by western blot analysis and reverse transcription‑quantitative polymerase chain reaction, respectively. The results demonstrated that PYP1‑5 significantly reduced the expression of atrogin1/MAFbx and MuRF1. Therefore, data from the present study suggest that PYP1‑5 inhibits the expression of atrogin1/MAFbx and MuRF1 in C2C12 cells, and these characteristics may be of value in the development of anti‑atrophy functional foods.

  14. Pyropia yezoensis peptide PYP1-5 protects against dexamethasone-induced muscle atrophy through the downregulation of atrogin1/MAFbx and MuRF1 in mouse C2C12 myotubes

    Science.gov (United States)

    Lee, Min-Kyeong; Kim, Young-Min; Kim, In-Hye; Choi, Youn-Hee; Nam, Taek-Jeong

    2017-01-01

    Skeletal muscle atrophy refers to the decline in muscle mass and strength that occurs under various conditions, including aging, starvation, cancer and other cachectic diseases. Muscle atrophy caused by aging, known as sarcopenia, primarily occurs after 50 years of age. Muscle atrophy-related genes, including atrogin1/muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), are expressed early in the muscle atrophy process, and their expression precedes the loss of muscle mass. The present study investigated the potential anti-atrophic effects of the Pyropia yezoensis peptide PYP1-5. The MTS assay did not detect cytotoxic effects of PYP1-5 on C2C12 mouse myoblast cells. Subsequently, the anti-atrophic effects of PYP1-5 on skeletal muscle cells was examined by treating C2C12 myotubes with 100 µM dexamethasone (DEX) and/or 500 ng/ml PYP1-5 for 24 h. Compared with the control, myotube diameter was reduced in DEX-treated cells, whereas PYP1-5 treatment protected against DEX-induced muscle atrophy. MAFbx and MuRF1 protein and mRNA expression levels were detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. The results demonstrated that PYP1-5 significantly reduced the expression of atrogin1/MAFbx and MuRF1. Therefore, data from the present study suggest that PYP1-5 inhibits the expression of atrogin1/MAFbx and MuRF1 in C2C12 cells, and these characteristics may be of value in the development of anti-atrophy functional foods. PMID:28393223

  15. Permethrin decreased insulin-stimulated AKT phosphorylation dependent on extracellular signal-regulated kinase-1 (ERK), but not AMP-activated protein kinase α (AMPKα), in C2C12 myotubes.

    Science.gov (United States)

    Sun, Quancai; Peng, Ye; Qi, Weipeng; Kim, Yoo; Clark, John M; Kim, Daeyoung; Park, Yeonhwa

    2017-11-01

    Previously 10 μM permethrin (38.7% cis and 59.4% trans isomers), a pyrethroid insecticide widely used in agriculture and household products for pest control, was reported to reduce insulin-stimulated glucose uptake and phosphorylation of protein kinase B (p-AKT) in C2C12 mouse myotubes. The underlying mechanisms on how permethrin decreases insulin-stimulated AKT phosphorylation, however, are unknown. Thus, the goal of this study was to determine the possible mechanism(s) through which permethrin reduced insulin-stimulated AKT phosphorylation in C2C12 myotubes. Permethrin treatment, at 10 μM, decreased insulin-stimulated membrane glucose transporter type 4 (GLUT4) and AKT phosphorylation, and increased insulin receptor substrate 1 (IRS1) Ser307 phosphorylation in the presence of insulin. The inactivation of AKT by permethrin was independent of AMPKα. ERK inactivation by U0126, however, restored insulin-stimulated AKT phosphorylation, which was decreased by permethrin treatment. These results suggest that permethrin decreased insulin-stimulated AKT phosphorylation via ERK activation, but not by AMPKα inactivation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Inulin Increases Glucose Transport in C2C12 Myotubes and HepG2 Cells via Activation of AMP-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Pathways

    Science.gov (United States)

    Yun, Hee; Lee, Jong Hwa; Park, Chang Eun; Kim, Min-Jung; Min, Byung-Il; Bae, Hyunsu; Choe, Wonchae; Kang, Insug; Kim, Sung-Soo

    2009-01-01

    Abstract Inulin, a naturally occurring, functional food ingredient found in various edible plants, has been reported to exert potential health benefits, including decreased risk of colonic diseases, non–insulin-dependent diabetes, obesity, osteoporosis, and cancer. However, the mechanism of the antidiabetic activity of inulin has not yet been elucidated. In this study, we showed that inulin increased the uptake of glucose in C2C12 myotubes, which was associated with both AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways, but both of these pathways appeared to transmit their signals in an independent manner. Moreover, we found that inulin was able to increase the uptake of glucose in C2C12 myotubes in which insulin resistance was induced by exposing cells to high glucose concentrations. The identical effects of inulin were also observed in HepG2 hepatoma cells. Collectively, we report the antidiabetic activity of inulin and further demonstrate for the first time that such activity is associated with AMPK and PI3-K activation. PMID:19857065

  17. Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes.

    Science.gov (United States)

    Hurst, S E; Minkin, S C; Biggerstaff, J; Dhar, M S

    2012-01-01

    Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and p44/42. Additionally, glucose transporter-1 (GLUT1) was significantly upregulated; no changes in glucose transporter-4 (GLUT4) expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

  18. Transient Silencing of a Type IV P-Type ATPase, Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes

    Directory of Open Access Journals (Sweden)

    S. E. Hurst

    2012-01-01

    Full Text Available Atp10c is a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C, Atp10c expression was altered in vitro in C2C12 skeletal muscle myotubes by transient transfection with an Atp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs, p38, and p44/42. Additionally, glucose transporter-1 (GLUT1 was significantly upregulated; no changes in glucose transporter-4 (GLUT4 expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate that Atp10c regulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

  19. Eburicoic Acid, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects in Palmitate-Treated C2C12 Myotubes and in High-Fat Diet-Fed Mice

    Directory of Open Access Journals (Sweden)

    Cheng-Hsiu Lin

    2017-11-01

    Full Text Available This study was designed to investigate the antidiabetic and antihyperlipidemic effects and mechanisms of eburicoic acid (TRR; one component of Antrodia camphorata in vitro and in an animal model for 14 weeks. Expression levels of membrane glucose transporter type 4 (GLUT4; phospho-5′-adenosine monophosphate-activated protein kinase (AMPK/total AMPK; and phospho-Akt/total-Akt in insulin-resistant C2C12 myotube cells were significantly decreased by palmitate; and such decrease was prevented and restored by TRR at different concentrations. A group of control (CON was on low-fat diet over a period of 14 weeks. Diabetic mice; after high-fat-diet (HFD induction for 10 weeks; were randomly divided into six groups and were given once a day oral gavage doses of either TRR (at three dosage levels; fenofibrate (Feno (at 0.25 g/kg body weight; metformin (Metf (at 0.3 g/kg body weight; or vehicle (distilled water (HF group over a period of 4 weeks and still on HFD. Levels of glucose; triglyceride; free fatty acid (FFA; insulin; and leptin in blood were increased in 14-week HFD-fed mice as compared to the CON group; and the increases were prevented by TRR, Feno, or Metf as compared to the HF group. Moreover, HFD-induction displayed a decrease in circulating adiponectin levels, and the decrease was prevented by TRR, Feno, or Metf treatment. The overall effect of TRR is to decrease glucose and triglyceride levels and improved peripheral insulin sensitivity. Eburicoic acid, Feno, and Metf displayed both enhanced expression levels of phospho-AMPK and membrane expression levels of GLUT4 in the skeletal muscle of HFD-fed mice to facilitate glucose uptake with consequent enhanced hepatic expression levels of phospho-AMPK in the liver and phosphorylation of the transcription factor forkhead box protein O1 (FOXO1 but decreased messenger RNA (mRNA of phosphenolpyruvate carboxykinase (PEPCK to inhibit hepatic glucose production; resulting in lowered blood glucose

  20. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kodaka, Manami [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Yang, Zeyu [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Ultrasound, Shengjing Hospital of China Medical University, Shenyang (China); Nakagawa, Kentaro; Maruyama, Junichi [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Xu, Xiaoyin [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Sarkar, Aradhan; Ichimura, Ayana [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Nasu, Yusuke [Department of Breast Surgery, The Second Affiliated Hospital of Wenzhou Medical University, Wenzhou (China); Ozawa, Takeaki [Department of Chemistry, School of Science, The University of Tokyo, Tokyo (Japan); Iwasa, Hiroaki [Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo (Japan); Ishigami-Yuasa, Mari [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Shigeru [Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); Kagechika, Hiroyuki [Chemical Biology Screening Center, Tokyo Medical and Dental University, Tokyo (Japan); Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo (Japan); and others

    2015-08-15

    The development of the efficient screening system of detecting compounds that promote myogenesis and prevent muscle atrophy is important. Mouse C2C12 cells are widely used to evaluate myogenesis but the procedures of the assay are not simple and the quantification is not easy. We established C2C12 cells expressing the N-terminal green fluorescence protein (GFP) and the C-terminal GFP (GFP1–10 and GFP11 cells). GFP1–10 and GFP11 cells do not exhibit GFP signals until they are fused. The signal intensity correlates with the expression of myogenic markers and myofusion. Myogenesis-promoting reagents, such as insulin-like growth factor-1 (IGF1) and β-guanidinopropionic acid (GPA), enhance the signals, whereas the poly-caspase inhibitor, z-VAD-FMK, suppresses it. GFP signals are observed when myotubes formed by GFP1–10 cells are fused with single nuclear GFP11 cells, and enhanced by IGF1, GPA, and IBS008738, a recently-reported myogenesis-promoting reagent. Fusion between myotubes formed by GFP1–10 and GFP11 cells is associated with the appearance of GFP signals. IGF1 and GPA augment these signals, whereas NSC23766, Rac inhibitor, decreases them. The conditioned medium of cancer cells suppresses GFP signals during myogenesis and reduces the width of GFP-positive myotubes after differentiation. Thus the novel split GFP-based assay will provide the useful method for the study of myogenesis, myofusion, and atrophy. - Highlights: • C2C12 cells expressing split GFP proteins show GFP signals when mix-cultured. • The GFP signals correlate with myogenesis and myofusion. • The GFP signals attenuate under the condition that muscle atrophy is induced.

  1. MicroRNA, miR-374b, directly targets Myf6 and negatively regulates C2C12 myoblasts differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Zhiyuan; Sun, Xiaorui; Xu, Dequan; Xiong, Yuanzhu; Zuo, Bo, E-mail: zuobo@mail.hzau.edu.cn

    2015-11-27

    Myogenesis is a complex process including myoblast proliferation, differentiation and myotube formation and is controlled by myogenic regulatory factors (MRFs), MyoD, MyoG, Myf5 and Myf6 (also known as MRF4). MicroRNA is a kind of ∼22 nt-long non-coding small RNAs, and act as key transcriptional or post-transcriptional regulators of gene expression. Identification of miRNAs involved in the regulation of muscle genes could improve our understanding of myogenesis process. In this study, we investigated the regulation of Myf6 gene by miRNAs. We showed that miR-374b specifically bound to the 3'untranslated region (UTR) of Myf6 and down-regulated the expression of Myf6 gene at both mRNA and protein level. Furthermore, miR-374b is ubiquitously expressed in the tissues of adult C57BL6 mouse, and the mRNA abundance increases first and then decreases during C2C12 myoblasts differentiation. Over-expression of miR-374b impaired C2C12 cell differentiation, while inhibiting miR-374b expression by 2′-O-methyl antisense oligonucleotides promoted C2C12 cell differentiation. Taken together, our findings identified miR-374b directly targets Myf6 and negatively regulates myogenesis. - Highlights: • MiR-374b directly targets 3′UTR of Myf6. • MiR-374b negatively regulates Myf6 in C2C12 cells. • MiR-374b abundance significiently changes during C2C12 cells differentiation. • MiR-374b negatively regulates C2C12 cells differentiation.

  2. Glycogenome expression dynamics during mouse C2C12 myoblast differentiation suggests a sequential reorganization of membrane glycoconjugates

    Directory of Open Access Journals (Sweden)

    Dupuy Fabrice

    2009-10-01

    Full Text Available Abstract Background Several global transcriptomic and proteomic approaches have been applied in order to obtain new molecular insights on skeletal myogenesis, but none has generated any specific data on glycogenome expression, and thus on the role of glycan structures in this process, despite the involvement of glycoconjugates in various biological events including differentiation and development. In the present study, a quantitative real-time RT-PCR technology was used to profile the dynamic expression of 375 glycogenes during the differentiation of C2C12 myoblasts into myotubes. Results Of the 276 genes expressed, 95 exhibited altered mRNA expression when C2C12 cells differentiated and 37 displayed more than 4-fold up- or down-regulations. Principal Component Analysis and Hierarchical Component Analysis of the expression dynamics identified three groups of coordinately and sequentially regulated genes. The first group included 12 down-regulated genes, the second group four genes with an expression peak at 24 h of differentiation, and the last 21 up-regulated genes. These genes mainly encode cell adhesion molecules and key enzymes involved in the biosynthesis of glycosaminoglycans and glycolipids (neolactoseries, lactoseries and ganglioseries, providing a clearer indication of how the plasma membrane and extracellular matrix may be modified prior to cell fusion. In particular, an increase in the quantity of ganglioside GM3 at the cell surface of myoblasts is suggestive of its potential role during the initial steps of myogenic differentiation. Conclusion For the first time, these results provide a broad description of the expression dynamics of glycogenes during C2C12 differentiation. Among the 37 highly deregulated glycogenes, 29 had never been associated with myogenesis. Their biological functions suggest new roles for glycans in skeletal myogenesis.

  3. MiRNA-199a-3p Regulates C2C12 Myoblast Differentiation through IGF-1/AKT/mTOR Signal Pathway

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    Long Jia

    2013-12-01

    Full Text Available MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3' untranslated regions (3' UTRs of messenger RNAs (mRNAs. Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.

  4. 4,4'-Dichlorodiphenyltrichloroethane (DDT) and 4,4'-dichlorodiphenyldichloroethylene (DDE) inhibit myogenesis in C2C12 myoblasts.

    Science.gov (United States)

    Kim, Jonggun; Park, Min Young; Kim, Yoo; Yoon, Kyong Sup; Clark, John Marshall; Park, Yeonhwa; Whang, Kwang-Youn

    2017-12-01

    Most countries have banned the use of 4,4'-dichlorodiphenyltrichloroethane (DDT). However, owing to its extremely high lipophilic characteristics, DDT and its metabolite 4,4'-dichlorodiphenyldichloroethylene (DDE) are ubiquitous in the environment and in many types of food. The positive correlation between exposure to insecticides, including DDT and DDE, and weight gain, resulting in impaired energy metabolism in offspring following perinatal DDT and DDE exposure, was previously reported. Therefore the influence of DDT and DDE on myogenesis using C2C12 myoblasts was investigated in this study. DDT and DDE decreased myotube formation dose- and time-dependently. Among myogenic regulatory factors, DDT and DDE mainly decreased MyoD1 and Myf5 expression. DDT and DDE treatment also altered Myostatin expression, phosphorylation of protein kinase B, p70 ribosomal protein S6 kinase, forkhead box O protein 3 and mammalian target of rapamycin, resulting in attenuation of myotube formation. These results may have significant implications for understanding the effects of developmental exposure of DDT and DDE on myogenesis and development of obesity and type 2 diabetes later in life. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/β-Catenin Signaling

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    Qiangling Zhang

    2015-08-01

    Full Text Available Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

  6. Three-dimensional co-culture of C2C12/PC12 cells improves skeletal muscle tissue formation and function.

    Science.gov (United States)

    Ostrovidov, Serge; Ahadian, Samad; Ramon-Azcon, Javier; Hosseini, Vahid; Fujie, Toshinori; Parthiban, S Prakash; Shiku, Hitoshi; Matsue, Tomokazu; Kaji, Hirokazu; Ramalingam, Murugan; Bae, Hojae; Khademhosseini, Ali

    2017-02-01

    Engineered muscle tissues demonstrate properties far from native muscle tissue. Therefore, fabrication of muscle tissues with enhanced functionalities is required to enable their use in various applications. To improve the formation of mature muscle tissues with higher functionalities, we co-cultured C2C12 myoblasts and PC12 neural cells. While alignment of the myoblasts was obtained by culturing the cells in micropatterned methacrylated gelatin (GelMA) hydrogels, we studied the effects of the neural cells (PC12) on the formation and maturation of muscle tissues. Myoblasts cultured in the presence of neural cells showed improved differentiation, with enhanced myotube formation. Myotube alignment, length and coverage area were increased. In addition, the mRNA expression of muscle differentiation markers (Myf-5, myogenin, Mefc2, MLP), muscle maturation markers (MHC-IId/x, MHC-IIa, MHC-IIb, MHC-pn, α-actinin, sarcomeric actinin) and the neuromuscular markers (AChE, AChR-ε) were also upregulated. All these observations were amplified after further muscle tissue maturation under electrical stimulation. Our data suggest a synergistic effect on the C2C12 differentiation induced by PC12 cells, which could be useful for creating improved muscle tissue. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  7. Folic acid is necessary for proliferation and differentiation of C2C12 myoblasts.

    Science.gov (United States)

    Hwang, Seong Y; Kang, Yong J; Sung, Bokyung; Jang, Jung Y; Hwang, Na L; Oh, Hye J; Ahn, Yu R; Kim, Hong J; Shin, Jin H; Yoo, Mi-Ae; Kim, Cheol M; Chung, Hae Y; Kim, Nam D

    2017-05-04

    Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one-carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle-specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and Western blot analyses revealed that DNA damage was more increased in folic acid-deficient medium-treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid-deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells. © 2017 Wiley Periodicals, Inc.

  8. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  9. Developmental Changes is Expression of Beta-Adrenergic Receptors in Cultures of C2C12 Skeletal Muscle Cells

    Science.gov (United States)

    Young, Ronald B.; Bridge, K. Y.; Vaughn, J. R.

    2000-01-01

    beta-Adrenergic receptor (bAR) agonists have been reported to modulate growth in several mammalian and avian species, and bAR agonists presumably exert their physiological action on skeletal muscle cells through this receptor. Because of the importance of bAR regulation on muscle protein metabolism in muscle cells, the objectives of this study were to determine the developmental expression pattern of the bAR population in C2C12 skeletal muscle cells, and to analyze changes in both the quantity and isoform expression of the major muscle protein, myosin. The number of bAR in mononucleated C2C12 cells was approximately 8,000 bAR per cell, which is comparable with the population reported in several other nonmuscle cell types. However, the bar population increased after myoblast fusion to greater than 50,000 bAR per muscle cell equivalent. The reasons for this apparent over-expression of bAR in C2C12 cells is not known. The quantity of myosin also increased after C2C12 myoblast fusion, but the quantity of myosin was less than that reported in primary muscle cell cultures. Finally, at least five different isoforms of myosin heavy chain could be resolved in C2C12 cells, and three of these exhibited either increased or decreased developmental regulation relative to the others. Thus, C2C12 myoblasts undergo developmental regulation of bAR population and myosin heavy chain isoform expression.

  10. N-methyl-D-aspartate (NMDA) impairs myogenesis in C2C12 cells.

    Science.gov (United States)

    Auh, Q-SChick; Park, Kyung-Ran; Lee, Myeong-Ok; Hwang, Mi-Jin; Kang, Soo-Kyung; Hong, Jung-Pyo; Yun, Hyung-Mun; Kim, Eun-Cheol

    2017-09-01

    N-methyl-d-aspartate (NMDA) is expressed in sensory neurons and plays important roles in peripheral pain mechanisms. The aim of this study was to examine the effects and molecular mechanisms of NMDA on C2C12 myoblast proliferation and differentiation. Cytotoxicity and differentiation were examined by the MTT assay, reverse transcription-polymerase chain reaction, and immunofluorescence. NMDA had no cytotoxicity (10-500 μM) and inhibited myoblastic differentiation of C2C12 cells, as assessed by F-actin immunofluorescence and levels of mRNAs encoding myogenic markers such as myogenin and myosin heavy-chain 2. It inhibited phosphorylation of mammalian target of rapamycin (mTOR) by inactivating mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38. It induced reactive oxygen species production. Furthermore, NMDA-suppressed expression of F-actin was reversed by adding the antioxidant N-acetylcysteine. Collectively, these results indicate that NMDA impairs myogenesis or myogenic differentiation in C2C12 cells through the mTOR/MAPK signaling pathways and may lead to skeletal muscle degeneration. Muscle Nerve 56: 510-518, 2017. © 2016 Wiley Periodicals, Inc.

  11. Expression of Thyroid Stimulating Hormone Receptor mRNA in Mouse C2C12 Skeletal Muscle Cells

    Directory of Open Access Journals (Sweden)

    Jung Hun Ohn

    2013-06-01

    Full Text Available BackgroundWe analyzed whether thyroid stimulating hormone receptor (TSH-R is expressed in a skeletal muscle cell line and if TSH has influence on the differentiation of muscle cells or on the determination of muscle fiber types.MethodsTSH-R gene expression was detected with nested real-time polymerase chain reaction (RT-PCR in C2C12, a mouse skeletal muscle cell line. The effect of TSH on myotube differentiation was assessed by microscopic examination of myotube formation and through the measurement of expression of muscle differentiation markers, i.e., myogenin and myoD, and muscle type-specific genes, i.e., MyHC1, MyHC2a, and MyHC2b, with quantitative RT-PCR before and after incubation of C2C12 myotube with TSH.ResultsTSH-R was expressed in the mouse skeletal muscle cell line. However, treatment with TSH had little effect on the differentiation of muscle cells, although the expression of the muscle differention marker myogenin was significantly increased after TSH treatment. Treatment of TSH did not affect the expression of muscle type-specific genes.ConclusionTSH-R is expressed in a mouse skeletal muscle cell line, but the role of TSH receptor signaling in skeletal muscle needs further investigation.

  12. Internalization and fate of silica nanoparticles in C2C12 skeletal muscle cells: evidence of a beneficial effect on myoblast fusion.

    Science.gov (United States)

    Poussard, Sylvie; Decossas, Marion; Le Bihan, Olivier; Mornet, Stéphane; Naudin, Grégoire; Lambert, Olivier

    2015-01-01

    The use of silica nanoparticles for their cellular uptake capability opens up new fields in biomedical research. Among the toxicological effects associated with their internalization, silica nanoparticles induce apoptosis that has been recently reported as a biochemical cue required for muscle regeneration. To assess whether silica nanoparticles could affect muscle regeneration, we used the C2C12 muscle cell line to study the uptake of fluorescently labeled NPs and their cellular trafficking over a long period. Using inhibitors of endocytosis, we determined that the NP uptake was an energy-dependent process mainly involving macropinocytosis and clathrin-mediated pathway. NPs were eventually clustered in lysosomal structures. Myoblasts containing NPs were capable of differentiation into myotubes, and after 7 days, electron microscopy revealed that the NPs remained primarily within lysosomes. The presence of NPs stimulated the formation of myotubes in a dose-dependent manner. NP internalization induced an increase of apoptotic myoblasts required for myoblast fusion. At noncytotoxic doses, the NP uptake by skeletal muscle cells did not prevent their differentiation into myotubes but, instead, enhanced the cell fusion.

  13. Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication

    Directory of Open Access Journals (Sweden)

    Caterina Morabito

    2017-01-01

    Full Text Available Extremely low-frequency electromagnetic fields (ELF-EMFs can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction.

  14. Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication.

    Science.gov (United States)

    Morabito, Caterina; Steimberg, Nathalie; Rovetta, Francesca; Boniotti, Jennifer; Guarnieri, Simone; Mazzoleni, Giovanna; Mariggiò, Maria A

    2017-01-01

    Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction.

  15. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation and cell differentiation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann

    have shown that m-calpain is necessary for myoblast fusion leading to the formation of muscle fibers and that inhibition of this enzyme restricts myotube formation. Whether there is a link between stretchor load induced signaling and m-calpain expression and activation is not known. Using a magnetic...... documented and has been shown to affect transcription of specific gene sequences, protein synthesis, the immune system and increase in Ca2+ influx. The past 10 years has seen a dramatic increase in the understanding of how proteolytic enzymes such as calpains can affect the growth of muscle. In vivo studies...... bead stimulation assay and a C2C12 mouse myoblast cell population, we have found that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, an enzyme found to be required for muscle cell fusion. After a short period of stimulation, m-calpain relocates into focal...

  16. The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome

    Science.gov (United States)

    Gundry, Rebekah L.; Raginski, Kimberly; Tarasova, Yelena; Tchernyshyov, Irina; Bausch-Fluck, Damaris; Elliott, Steven T.; Boheler, Kenneth R.; Van Eyk, Jennifer E.; Wollscheid, Bernd

    2009-01-01

    Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes. PMID:19656770

  17. Uniaxial cyclic strain of human adipose–derived mesenchymal stem cells and C2C12 myoblasts in coculture

    Directory of Open Access Journals (Sweden)

    James M Dugan

    2014-03-01

    Full Text Available Tissue engineering skeletal muscle in vitro is of great importance for the production of tissue-like constructs for treating tissue loss due to traumatic injury or surgery. However, it is essential to find new sources of cells for muscle engineering as efficient in vitro expansion and culture of primary myoblasts are problematic. Mesenchymal stem cells may be a promising source of myogenic progenitor cells and may be harvested in large numbers from adipose tissue. As skeletal muscle is a mechanically dynamic tissue, we have investigated the effect of cyclic mechanical strain on the myogenic differentiation of a coculture system of murine C2C12 myoblasts and human adipose–derived mesenchymal stem cells. Fusion of mesenchymal stem cells with nascent myotubes and expression of human sarcomeric proteins was observed, indicating the potential for myogenic differentiation of human mesenchymal stem cells. Cyclic mechanical strain did not affect the fusion of mesenchymal stem cells, but maturation of myotubes was perturbed.

  18. Enigma homolog 1 promotes myogenic gene expression and differentiation of C2C12 cells.

    Science.gov (United States)

    Ito, Jumpei; Takita, Masatoshi; Takimoto, Koichi; Maturana, Andrés D

    2013-06-07

    The Enigma homolog (ENH) gene generates several splicing variants. The initially identified ENH1 possesses one PDZ and three LIM domains, whereas ENH2~4 lack the latter domains. The splicing switch from ENH1 to LIM-less ENHs occurs during development/maturation of skeletal and heart muscles. We examined for the roles of ENH splicing variants in muscle differentiation using C2C12 cells. Cells stably expressing ENH1 exhibited significantly higher MyoD and myogenin mRNA levels before differentiation and after 5 days in low serum-differentiating medium than mock-transfected cells. ENH1-stable transformants also retained the ability to exhibit elongated morphology with well-extended actin fibers following differentiation. In contrast, cells stably expressing ENH3 or ENH4 did not show myotube-like morphology or reorganization of actin fibers following culture in the differentiating medium. Transient overexpression of ENH1 using adenovirus supported the increased expression of muscle marker mRNAs and the formation of well-organized stress fibers, whereas ENH4 overexpression prevented these morphological changes. Furthermore, specific suppression of ENH1 expression by RNAi caused a significant reduction in MyoD mRNA level and blocked the morphological changes. These results suggest that ENH1 with multiple protein-protein interaction modules is essential for differentiation of striated muscles, whereas ectopic expression of LIM-less ENH disrupts normal muscle differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Biocompatible 3D printed polymers via fused deposition modelling direct C2C12 cellular phenotype in vitro.

    Science.gov (United States)

    Rimington, Rowan P; Capel, Andrew J; Christie, Steven D R; Lewis, Mark P

    2017-08-22

    The capability to 3D print bespoke biologically receptive parts within short time periods has driven the growing prevalence of additive manufacture (AM) technology within biological settings, however limited research concerning cellular interaction with 3D printed polymers has been undertaken. In this work, we used skeletal muscle C2C12 cell line in order to ascertain critical evidence of cellular behaviour in response to multiple bio-receptive candidate polymers; polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), polyethylene terephthalate (PET) and polycarbonate (PC) 3D printed via fused deposition modelling (FDM). The extrusion based nature of FDM elicited polymer specific topographies, within which C2C12 cells exhibited reduced metabolic activity when compared to optimised surfaces of tissue culture plastic, however assay viability readings remained high across polymers outlining viable phenotypes. C2C12 cells exhibited consistently high levels of morphological alignment across polymers, however differential myotube widths and levels of transcriptional myogenin expression appeared to demonstrate response specific thresholds at which varying polymer selection potentiates cellular differentiation, elicits pre-mature early myotube formation and directs subsequent morphological phenotype. Here we observed biocompatible AM polymers manufactured via FDM, which also appear to hold the potential to simultaneously manipulate the desired biological phenotype and enhance the biomimicry of skeletal muscle cells in vitro via AM polymer choice and careful selection of machine processing parameters. When considered in combination with the associated design freedom of AM, this may provide the opportunity to not only enhance the efficiency of creating biomimetic models, but also to precisely control the biological output within such scaffolds.

  20. Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Gia-Ming [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Present address: The University of Chicago, Section of Hematology/Oncology, 900 E. 57th Street, Room 7134, Chicago, IL 60637 (United States); Bain, Lisa J., E-mail: lbain@clemson.edu [Environmental Toxicology Graduate Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States)

    2012-05-01

    Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20 nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20 nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (∼ 2-fold) to the myogenin promoter at the transcription start site (− 40 to + 42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (− 40 to + 42). -- Highlights: ► Igf-1 expression is decreased in C2C12 cells after 20 nM arsenite exposure. ► Arsenic exposure alters histone remodeling on the myogenin promoter. ► Glp expression, a H3–K9 methyltransferase, was increased in arsenic-exposed cells. ► Ezh2

  1. Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways.

    Science.gov (United States)

    Pijet, Maja; Pijet, Barbara; Litwiniuk, Anna; Pajak, Beata; Gajkowska, Barbara; Orzechowski, Arkadiusz

    2013-02-01

    Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T(202/)Y(204)P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y(705)P-STAT3) and MEK (T(202/)Y(204)P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S(473)) and GSK-3β (S(9)) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S(9)P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y(216)P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Fibroblast Growth Factor 21 Promotes C2C12 Cells Myogenic Differentiation by Enhancing Cell Cycle Exit

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    Xinyi Liu

    2017-01-01

    Full Text Available Fibroblast growth factor 21 (FGF21, a secretion protein, functions as a pivotal regulator of energy metabolism and is being considered as a therapeutic candidate in metabolic syndromes. However, the roles of FGF21 in myogenic differentiation and cell cycle remain obscure. In this study, we investigated the function of FGF21 in myogenesis and cell cycle exit using C2C12 cell line. Our data showed that the expression of myogenic genes as well as cell cycle exit genes was increased after FGF21 overexpression, and FGF21 overexpression induces cell cycle arrest. Moreover, cell cycle genes were decreased in FGF21 overexpression cells while they were increased in FGF21 knockdown cells. Further, FGF21/P53/p21/Cyclin-CDK has been suggested as the key pathway for cell cycle exit mediated by FGF21 in C2C12 cells. Also, we deduce that FGF21 promotes the initiation of myogenic differentiation mainly through enhancing cell cycle exit of C2C12 cells. Taken together, our results demonstrated that FGF21 promotes cell cycle exit and enhances myogenic differentiation of C2C12 cells. This study provided new evidence that FGF21 promotes myogenic differentiation, which could be useful for better understanding the roles of FGF21 in myogenesis.

  3. Overexpression of Striated Muscle Activator of Rho Signaling (STARS) Increases C2C12 Skeletal Muscle Cell Differentiation.

    Science.gov (United States)

    Wallace, Marita A; Della Gatta, Paul A; Ahmad Mir, Bilal; Kowalski, Greg M; Kloehn, Joachim; McConville, Malcom J; Russell, Aaron P; Lamon, Séverine

    2016-01-01

    Skeletal muscle growth and regeneration depend on the activation of satellite cells, which leads to myocyte proliferation, differentiation and fusion with existing muscle fibers. Skeletal muscle cell proliferation and differentiation are tightly coordinated by a continuum of molecular signaling pathways. The striated muscle activator of Rho signaling (STARS) is an actin binding protein that regulates the transcription of genes involved in muscle cell growth, structure and function via the stimulation of actin polymerization and activation of serum-response factor (SRF) signaling. STARS mediates cell proliferation in smooth and cardiac muscle models; however, whether STARS overexpression enhances cell proliferation and differentiation has not been investigated in skeletal muscle cells. We demonstrate for the first time that STARS overexpression enhances differentiation but not proliferation in C2C12 mouse skeletal muscle cells. Increased differentiation was associated with an increase in the gene levels of the myogenic differentiation markers Ckm, Ckmt2 and Myh4, the differentiation factor Igf2 and the myogenic regulatory factors (MRFs) Myf5 and Myf6. Exposing C2C12 cells to CCG-1423, a pharmacological inhibitor of SRF preventing the nuclear translocation of its co-factor MRTF-A, had no effect on myotube differentiation rate, suggesting that STARS regulates differentiation via a MRTF-A independent mechanism. These findings position STARS as an important regulator of skeletal muscle growth and regeneration.

  4. Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12

    Directory of Open Access Journals (Sweden)

    Christo J. Botha

    2017-01-01

    Full Text Available Geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern Africa. The toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. Because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. The objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (C2C12 using methyl-thiazol-tetrazolium (MTT and lactate dehydrogenase (LDH assays, annexin V and propidium iodide (PI flow cytometry and transmission electron microscopy (TEM. Mouse myoblasts were exposed to 2.0 mM, 2.5 mM and 5.0 mM geigerin for 24, 48 and 72 h. A concentration-dependent cytotoxic response was observed. Apoptosis was detected by means of annexin V flow cytometry during the first 24 h and apoptotic bodies were also visible on TEM. According to the LDH and PI flow cytometry results, myoblast cell membranes were not injured. We concluded that the murine myoblast cell line (C2C12 is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts.

  5. DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts.

    Science.gov (United States)

    Kanomata, Kazuhiro; Kokabu, Shoichiro; Nojima, Junya; Fukuda, Toru; Katagiri, Takenobu

    2009-06-01

    Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.

  6. ZnO nanoparticles augment ALT, AST, ALP and LDH expressions in C2C12 cells

    OpenAIRE

    Pandurangan, Muthuraman; Kim, Doo Hwan

    2015-01-01

    The present study aimed to investigate the effect of ZnO nanoparticles on alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme expressions in C2C12 cells. ZnO nanoparticles are widely used in the several cosmetic lotions and other biomedical products. Several studies report on ZnO nanoparticle mediated cytotoxicity. However, there are no reports on the effect of ZnO nanoparticles on ALT, AST, ALP and LDH enzyme expressions...

  7. Response of C2C12 Myoblasts to Hypoxia: The Relative Roles of Glucose and Oxygen in Adaptive Cellular Metabolism

    Directory of Open Access Journals (Sweden)

    Wei Li

    2013-01-01

    Full Text Available Background. Oxygen and glucose are two important nutrients for mammalian cell function. In this study, the effect of glucose and oxygen concentrations on C2C12 cellular metabolism was characterized with an emphasis on detecting whether cells show oxygen conformance (OC in response to hypoxia. Methods. After C2C12 cells being cultured in the levels of glucose at 0.6 mM (LG, 5.6 mM (MG, or 23.3 mM(HG under normoxic or hypoxic (1% oxygen condition, cellular oxygen consumption, glucose consumption, lactate production, and metabolic status were determined. Short-term oxygen consumption was measured with a novel oxygen biosensor technique. Longer-term measurements were performed with standard glucose, lactate, and cell metabolism assays. Results. It was found that oxygen depletion in normoxia is dependent on the glucose concentration in the medium. Cellular glucose uptake and lactate production increased significantly in hypoxia than those in normoxia. In hypoxia the cellular response to the level of glucose was different to that in normoxia. The metabolic activities decreased while glucose concentration increased in normoxia, while in hypoxia, metabolic activity was reduced in LG and MG, but unchanged in HG condition. The OC phenomenon was not observed in the present study. Conclusions. Our findings suggested that a combination of low oxygen and low glucose damages the viability of C2C12 cells more seriously than low oxygen alone. In addition, when there is sufficient glucose, C2C12 cells will respond to hypoxia by upregulating anaerobic respiration, as shown by lactate production.

  8. Activated Integrin-Linked Kinase Negatively Regulates Muscle Cell Enhancement Factor 2C in C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Zhenguo Dong

    2015-01-01

    Full Text Available Our previous study reported that muscle cell enhancement factor 2C (MEF2C was fully activated after inhibition of the phosphorylation activity of integrin-linked kinase (ILK in the skeletal muscle cells of goats. It enhanced the binding of promoter or enhancer of transcription factor related to proliferation of muscle cells and then regulated the expression of these genes. In the present investigation, we explored whether ILK activation depended on PI3K to regulate the phosphorylation and transcriptional activity of MEF2C during C2C12 cell proliferation. We inhibited PI3K activity in C2C12 with LY294002 and then found that ILK phosphorylation levels and MEF2C phosphorylation were decreased and that MCK mRNA expression was suppressed significantly. After inhibiting ILK phosphorylation activity with Cpd22 and ILK-shRNA, we found MEF2C phosphorylation activity and MCK mRNA expression were increased extremely significantly. In the presence of Cpd22, PI3K activity inhibition increased MEF2C phosphorylation and MCK mRNA expression indistinctively. We conclude that ILK negatively and independently of PI3K regulated MEF2C phosphorylation activity and MCK mRNA expression in C2C12 cells. The results provide new ideas for the study of classical signaling pathway of PI3K-ILK-related proteins and transcription factors.

  9. Graphene oxide increases the viability of C2C12 myoblasts microencapsulated in alginate.

    Science.gov (United States)

    Ciriza, J; Saenz del Burgo, L; Virumbrales-Muñoz, M; Ochoa, I; Fernandez, L J; Orive, G; Hernandez, R M; Pedraz, J L

    2015-09-30

    Cell microencapsulation represents a great promise for long-term drug delivery, but still several challenges need to be overcome before its translation into the clinic, such as the long term cell survival inside the capsules. On this regard, graphene oxide has shown to promote proliferation of different cell types either in two or three dimensions. Therefore, we planned to combine graphene oxide with the cell microencapsulation technology. We first studied the effect of this material on the stability of the capsules and next we analyzed the biocompatibility of this chemical compound with erythropoietin secreting C2C12 myoblasts within the microcapsule matrix. We produced 160 μm-diameter alginate microcapsules with increasing concentrations of graphene oxide and did not find modifications on the physicochemical parameters of traditional alginate microcapsules. Moreover, we observed that the viability of encapsulated cells within alginate microcapsules containing specific graphene oxide concentrations was enhanced. These results provide a relevant step for the future clinical application of graphene oxide on cell microencapsulation. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. File list: InP.Myo.10.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Myo.10.AllAg.C2C12 mm9 Input control Muscle C2C12 SRX1482291,SRX262224,SRX26222...695944 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Myo.10.AllAg.C2C12.bed ...

  11. File list: NoD.Myo.50.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Myo.50.AllAg.C2C12 mm9 No description Muscle C2C12 SRX683676,DRX000201,SRX68367...020490,DRX020492 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Myo.50.AllAg.C2C12.bed ...

  12. File list: His.Myo.20.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Myo.20.AllAg.C2C12 mm9 Histone Muscle C2C12 SRX1482269,SRX1482270,SRX1482268,SR...04,SRX143610,SRX115555,SRX103216 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Myo.20.AllAg.C2C12.bed ...

  13. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

    Directory of Open Access Journals (Sweden)

    Yan Shen

    Full Text Available We previously demonstrated that cinnamon extract (CE ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4 translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK.

  14. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

    Science.gov (United States)

    Shen, Yan; Honma, Natsumi; Kobayashi, Katsuya; Jia, Liu Nan; Hosono, Takashi; Shindo, Kazutoshi; Ariga, Toyohiko; Seki, Taiichiro

    2014-01-01

    We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK.

  15. L-carnitine protects C2C12 cells against mitochondrial superoxide overproduction and cell death

    Science.gov (United States)

    Le Borgne, Françoise; Ravaut, Gaétan; Bernard, Arnaud; Demarquoy, Jean

    2017-01-01

    AIM To identify and characterize the protective effect that L-carnitine exerted against an oxidative stress in C2C12 cells. METHODS Myoblastic C2C12 cells were treated with menadione, a vitamin K analog that engenders oxidative stress, and the protective effect of L-carnitine (a nutrient involved in fatty acid metabolism and the control of the oxidative process), was assessed by monitoring various parameters related to the oxidative stress, autophagy and cell death. RESULTS Associated with its physiological function, a muscle cell metabolism is highly dependent on oxygen and may produce reactive oxygen species (ROS), especially under pathological conditions. High levels of ROS are known to induce injuries in cell structure as they interact at many levels in cell function. In C2C12 cells, a treatment with menadione induced a loss of transmembrane mitochondrial potential, an increase in mitochondrial production of ROS; it also induces autophagy and was able to provoke cell death. Pre-treatment of the cells with L-carnitine reduced ROS production, diminished autophagy and protected C2C12 cells against menadione-induced deleterious effects. CONCLUSION In conclusion, L-carnitine limits the oxidative stress in these cells and prevents cell death. PMID:28289521

  16. Nanoparticle-mediated intracellular lipid accumulation during C2C12 cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp [Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Haniu, Hisao, E-mail: hhaniu@shinshu-u.ac.jp [Institute of Carbon Science and Technology, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2011-03-25

    Research highlights: {yields} HTT2800 has a significant effect on intracellular lipid accumulation. {yields} HTT2800 reduced muscle-specific genes and led to the emergence of adipocyte-related genes. {yields} HT2800 converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells. -- Abstract: In this report, we sought to elucidate whether multiwall carbon nanotubes are involved in the modulation of the proliferation and differentiation of the skeletal muscle cell line C2C12. Skeletal muscle is a major mass peripheral tissue that accounts for 40% of total body weight and 50% of energy consumption. We focused on the differentiation pathway of myoblasts after exposure to a vapor-grown carbon fiber, HTT2800, which is one of the most highly purified carbon nanotubes. This treatment leads in parallel to the expression of a typical adipose differentiation program. We found that HTT2800 stimulated intracellular lipid accumulation in C2C12 cells. We have also shown by quantified PCR analysis that the expression of adipose-related genes was markedly upregulated during HTT2800 exposure. Taken together, these results suggest that HTT2800 specifically converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells.

  17. Insulin sensitizing effects of oligomannuronate-chromium (III complexes in C2C12 skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Cui Hao

    Full Text Available BACKGROUND: It was known that the insulin resistance in skeletal muscle is a major pathogenic factor in diabetes mellitus. Therefore prevention of metabolic disorder caused by insulin resistance and improvement of insulin sensitivity are very important for the therapy of type 2 diabetes. In the present study, we investigated the ability of marine oligosaccharides oligomannuronate and its chromium (III complexes from brown alga to enhance insulin sensitivity in C2C12 skeletal muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that oligomannuronate, especially its chromium (III complexes, enhanced insulin-stimulated glucose uptake and increased the mRNA expression of glucose transporter 4 (GLUT4 and insulin receptor (IR after their internalization into C2C12 skeletal muscle cells. Additionally, oligosaccharides treatment also significantly enhanced the phosphorylation of proteins involved in both AMP activated protein kinase (AMPK/acetyl-CoA carboxylase (ACC and phosphoinositide 3-kinase (PI3K/protein kinase B (Akt signaling pathways in C2C12 cells, indicating that the oligosaccharides activated both the insulin signal pathway and AMPK pathways as their mode of action. Moreover, oligosaccharides distributed to the mitochondria after internalization into C2C12 cells and increased the expression of transcriptional regulator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, carnitine palmitoyl transferase-1 (CPT-1, and phosphorylated acetyl-CoA carboxylase (p-ACC, which suggested that the actions of these oligosaccharides might be associated with mitochondria through increasing energy expenditure. All of these effects of marine oligosaccharides were comparable to that of the established anti-diabetic drug, metformin. In addition, the treatment with oligosaccharides showed less toxicity than that of metformin. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that oligomannuonate and its chromium (III complexes improved

  18. The mouse C2C12 myoblast cell surface N-linked glycoproteome: identification, glycosite occupancy, and membrane orientation.

    Science.gov (United States)

    Gundry, Rebekah L; Raginski, Kimberly; Tarasova, Yelena; Tchernyshyov, Irina; Bausch-Fluck, Damaris; Elliott, Steven T; Boheler, Kenneth R; Van Eyk, Jennifer E; Wollscheid, Bernd

    2009-11-01

    Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and beta-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.

  19. Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/β-catenin pathway.

    Science.gov (United States)

    Huang, Jian; Romero-Suarez, Sandra; Lara, Nuria; Mo, Chenglin; Kaja, Simon; Brotto, Leticia; Dallas, Sarah L; Johnson, Mark L; Jähn, Katharina; Bonewald, Lynda F; Brotto, Marco

    2017-10-01

    We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1. We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca2+ signaling and the Wnt

  20. Methylcobalamin promotes proliferation and migration and inhibits apoptosis of C2C12 cells via the Erk1/2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Okamoto, Michio [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Tanaka, Hiroyuki, E-mail: tanahiro-osk@umin.ac.jp [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Kiyoshi [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kuroda, Yusuke [Department of Orthopaedic Surgery, Kansai Rosai Hospital, 3-1-69 Inabaso, Amagasaki, Hyogo 660-8511 (Japan); Nishimoto, Shunsuke; Murase, Tsuyoshi; Yoshikawa, Hideki [Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2014-01-17

    Highlights: •Methylcobalamin activated the Erk1/2 signaling pathway in C2C12 cells. •Methylcobalamin promoted the proliferation and migration in C2C12 cells. •C2C12 cell apoptosis during differentiation was inhibited by methylcobalamin. -- Abstract: Methylcobalamin (MeCbl) is a vitamin B12 analog that has some positive effects on peripheral nervous disorders. Although some previous studies revealed the effects of MeCbl on neurons, its effect on the muscle, which is the final target of motoneuron axons, remains to be elucidated. This study aimed to determine the effect of MeCbl on the muscle. We found that MeCbl promoted the proliferation and migration of C2C12 myoblasts in vitro and that these effects are mediated by the Erk1/2 signaling pathway without affecting the activity of the Akt signaling pathway. We also demonstrated that MeCbl inhibits C2C12 cell apoptosis during differentiation. Our results suggest that MeCbl has beneficial effects on the muscle in vitro. MeCbl administration may provide a novel therapeutic approach for muscle injury or degenerating muscle after denervation.

  1. Graphene oxide-stimulated myogenic differentiation of C2C12 cells on PLGA/RGD peptide nanofiber matrices

    Science.gov (United States)

    Shin, Y. C.; Lee, J. H.; Kim, M. J.; Hong, S. W.; Oh, J.-W.; Kim, C.-S.; Kim, B.; Hyun, J. K.; Kim, Y.-J.; Han, D.-W.

    2015-07-01

    During the last decade, much attention has been paid to graphene-based nanomaterials because they are considered as potential candidates for biomedical applications such as scaffolds for tissue engineering and substrates for the differentiation of stem cells. Until now, electrospun matrices composed of various biodegradable copolymers have been extensively developed for tissue engineering and regeneration; however, their use in combination with graphene oxide (GO) is novel and challenging. In this study, nanofiber matrices composed of poly(lactic-co-glycolic acid, PLGA) and M13 phage with RGD peptide displayed on its surface (RGD peptide-M13 phage) were prepared as extracellular matrix (ECM)-mimicking substrates. RGD peptide is a tripeptide (Arg-Gly-Asp) found on ECM proteins that promotes various cellular behaviors. The physicochemical properties of PLGA and RGD peptide-M13 phage (PLGA/RGD peptide) nanofiber matrices were characterized by atomic force microscopy, Fourier-transform infrared spectroscopy and thermogravimetric analysis. In addition, the growth of C2C12 mouse myoblasts on the PLGA/RGD peptide matrices was examined by measuring the metabolic activity. Moreover, the differentiation of C2C12 mouse myoblasts on the matrices when treated with GO was evaluated. The cellular behaviors, including growth and differentiation of C2C12 mouse myoblasts, were substantially enhanced on the PLGA/RGD peptide nanofiber matrices when treated with GO. Overall, these findings suggest that the PLGA/RGD peptide nanofiber matrices can be used in combination with GO as a novel strategy for skeletal tissue regeneration.

  2. File list: Pol.Myo.50.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Myo.50.AllAg.C2C12 mm9 RNA polymerase Muscle C2C12 SRX142527,SRX101682,SRX10168...3,SRX142518,SRX062103,SRX062102,SRX1176761,SRX1176745,SRX1176759,SRX1176747 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Myo.50.AllAg.C2C12.bed ...

  3. File list: ALL.Myo.20.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.20.AllAg.C2C12 mm9 All antigens Muscle C2C12 SRX1482269,SRX262223,SRX262224...,SRX683673,DRX000355,SRX103216,SRX373248,SRX039345,SRX695944,DRX000353 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Myo.20.AllAg.C2C12.bed ...

  4. File list: ALL.Myo.50.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Myo.50.AllAg.C2C12 mm9 All antigens Muscle C2C12 SRX1482269,SRX262223,SRX262224...,SRX115557,SRX115559,SRX062124,DRX020490,DRX020492,SRX022849,SRX022851 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Myo.50.AllAg.C2C12.bed ...

  5. File list: Pol.Myo.05.AllAg.C2C12 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Myo.05.AllAg.C2C12 mm9 RNA polymerase Muscle C2C12 SRX142527,SRX142518,SRX10168...3,SRX101682,SRX062102,SRX062103,SRX1176745,SRX1176761,SRX1176747,SRX1176759 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Myo.05.AllAg.C2C12.bed ...

  6. Nerve growth factor stimulation of ERK1/2 phosphorylation requires both p75NTR and α9β1 integrin and confers myoprotection towards ischemia in C2C12 skeletal muscle cell model.

    Science.gov (United States)

    Ettinger, Keren; Lecht, Shimon; Arien-Zakay, Hadar; Cohen, Gadi; Aga-Mizrachi, Shlomit; Yanay, Nurit; Saragovi, H Uri; Nedev, Hinyu; Marcinkiewicz, Cezary; Nevo, Yoram; Lazarovici, Philip

    2012-12-01

    The functions of nerve growth factor (NGF) in skeletal muscles physiology and pathology are not clear and call for an updated investigation. To achieve this goal we sought to investigate NGF-induced ERK1/2 phosphorylation and its role in the C2C12 skeletal muscle myoblasts and myotubes. RT-PCR and western blotting experiments demonstrated expression of p75(NTR), α9β1 integrin, and its regulator ADAM12, but not trkA in the cells, as also found in gastrocnemius and quadriceps mice muscles. Both proNGF and βNGF induced ERK1/2 phosphorylation, a process blocked by (a) the specific MEK inhibitor, PD98059; (b) VLO5, a MLD-disintegrin with relative selectivity towards α9β1 integrin; and (c) p75(NTR) antagonists Thx-B and LM-24, but not the inactive control molecule backbone Thx. Upon treatment for 4 days with either anti-NGF antibody or VLO5 or Thx-B, the proliferation of myoblasts was decreased by 60-70%, 85-90% and 60-80% respectively, indicative of trophic effect of NGF which was autocrinically released by the cells. Exposure of myotubes to ischemic insult in the presence of βNGF, added either 1h before oxygen-glucose-deprivation or concomitant with reoxygenation insults, resulted with about 20% and 33% myoprotection, an effect antagonized by VLO5 and Thx-B, further supporting the trophic role of NGF in C2C12 cells. Cumulatively, the present findings propose that proNGF and βNGF-induced ERK1/2 phosphorylation in C2C12 cells by functional cooperation between p75(NTR) and α9β1 integrin, which are involved in myoprotective effects of autocrine released NGF. Furthermore, the present study establishes an important trophic role of α9β1 in NGF-induced signaling in skeletal muscle model, resembling the role of trkA in neurons. Future molecular characterization of the interactions between NGF receptors in the skeletal muscle will contribute to the understanding of NGF mechanism of action and may provide novel therapeutic targets. Copyright © 2012 Elsevier Inc. All

  7. Static magnetic fields inhibit proliferation and disperse subcellular localization of gamma complex protein3 in cultured C2C12 myoblast cells.

    Science.gov (United States)

    Kim, SeungChan; Im, Wooseok

    2010-05-01

    Magnetic fields may delay the rate of cell cycle progression, and there are reports that magnetic fields induce neurite outgrowth in cultured neuronal cells. To demonstrate whether magnetic field also effects on myoblast cells in cell growth, C2C12 cell lines were cultured and 2000G static magnetic field was applied. After 48 h of incubation, both the WST-1 assay (0.01 magnetic fields inhibit the proliferation of cultured C2C12 cells. Immunocytochemistry for alpha and tubulin gamma complex protein (TUBA and GCP3) was made and applying a static magnetic field-dispersed tubulin GCP3 formation, a intracellular apparatus for tubulin structuring in cell division. This protein expression was not altered by western blot. This study indicates that applying a static magnetic field alters the subcellular localizing of GCP3, and may delay the cell growth in cultured C2C12 myoblast cells.

  8. Effect of alkyl glycerophosphate on the activation of peroxisome proliferator-activated receptor gamma and glucose uptake in C2C12 cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp [Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Haniu, Hisao [Department of Orthopedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Matsuda, Yoshikazu [Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Ina-machi, Saitama 362-0806 (Japan)

    2013-04-12

    Highlights: •Alkyl-LPA specifically interacts with PPARγ. •Alkyl-LPA treatments induces lipid accumulation in C2C12 cells. •Alkyl-LPA enhanced glucose uptake in C2C12 cells. •Alkyl-LPA-treated C2C12 cells express increased amounts of GLUT4 mRNA. •Alkyl-LPA is a novel therapeutic agent that can be used for the treatment of obesity and diabetes. -- Abstract: Studies on the effects of lipids on skeletal muscle cells rarely examine the effects of lysophospholipids. Through our recent studies, we identified select forms of phospholipids, such as alkyl-LPA, as ligands for the intracellular receptor peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ is a nuclear hormone receptor implicated in many human diseases, including diabetes and obesity. We previously showed that alkyl-LPA is a specific agonist of PPARγ. However, the mechanism by which the alkyl-LPA–PPARγ axis affects skeletal muscle cells is poorly defined. Our objective in the present study was to determine whether alkyl-LPA and PPARγ activation promotes glucose uptake in skeletal muscle cells. Our findings indicate that PPARγ1 mRNA is more abundant than PPARγ2 mRNA in C2C12 cells. We showed that alkyl-LPA (3 μM) significantly activated PPARγ and increased intracellular glucose levels in skeletal muscle cells. We also showed that incubation of C2C12 cells with alkyl-LPA led to lipid accumulation in the cells. These findings suggest that alkyl-LPA activates PPARγ and stimulates glucose uptake in the absence of insulin in C2C12 cells. This may contribute to the plasma glucose-lowering effect in the treatment of insulin resistance.

  9. Bromopropane compounds inhibit osteogenesis by ERK-dependent Runx2 inhibition in C2C12 cells.

    Science.gov (United States)

    Jeong, Hyung Min; Choi, You Hee; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2014-02-01

    Bromopropane (BP) is a halogenated alkan compound used in various industries as chemical intermediates, extraction solvents, and degreasing compounds. Halogenated alkan compounds can damage the nervous system, immune system, and hematopoietic and reproductive functions in animals and humans. However, the effect of BPs on bone formation has not yet been examined. This study examined the effects of BPs on osteoblast differentiation and analyzed the mechanisms involved in C2C12, mesenchymal stem cells. BPs dose dependently reduced the alkaline phosphatase activity, expression levels and promoter activity of bone marker genes. Additionally, 1,2-dibromopropane (1,2-DBP) significantly reduced the levels and transcriptional activity of Runx2 and Osterix, major bone transcription factors, in BMP2 induced C2C12 cells. Furthermore, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) were significantly inhibited by 1,2-DBP. These results demonstrate that BPs inhibit osteoblast differentiation by suppressing Runx2 and Osterix through the ERK/JNK pathway.

  10. IGF-1 attenuates hypoxia-induced atrophy but inhibits myoglobin expression in C2C12 skeletal muscle myotubes

    NARCIS (Netherlands)

    Peters, Eva L.; van der Linde, Sandra M.; Vogel, Ilse S.P.; Haroon, Mohammad; Offringa, Carla; de Wit, Gerard M.J.; Koolwijk, Pieter; van der Laarse, Willem J.; Jaspers, Richard T.

    2017-01-01

    Chronic hypoxia is associated with muscle wasting and decreased oxidative capacity. By contrast, training under hypoxia may enhance hypertrophy and increase oxidative capacity as well as oxygen transport to the mitochondria, by increasing myoglobin (Mb) expression. The latter may be a feasible

  11. Catalytic activity of nuclear PLC-beta(1) is required for its signalling function during C2C12 differentiation.

    Science.gov (United States)

    Ramazzotti, Giulia; Faenza, Irene; Gaboardi, Gian Carlo; Piazzi, Manuela; Bavelloni, Alberto; Fiume, Roberta; Manzoli, Lucia; Martelli, Alberto M; Cocco, Lucio

    2008-11-01

    Here we report that PLC-beta(1) catalytic activity plays a role in the increase of cyclin D3 levels and induces the differentiation of C2C12 skeletal muscle cells. PLC-beta(1) mutational analysis revealed the importance of His(331) and His(378) for the catalysis. The expression of PLC-beta(1) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that PLC-beta(1) activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-beta(1) is a crucial regulator of the mouse cyclin D3 gene. We show that after insulin treatment cyclin D3 mRNA levels are lower in cells overexpressing the PLC-beta(1) catalytically inactive form in comparison to wild type cells. We describe a novel signalling pathway elicited by PLC-beta(1) that modulates AP-1 activity. Gel mobility shift assay and supershift performed with specific antibodies indicate that the c-jun binding site is located in a cyclin D3 promoter region specifically regulated by PLC-beta(1) and that c-Jun binding activity is significantly increased by insulin and PLC-beta(1) overexpression. Mutation of AP-1 site decreased the basal cyclin D3 promoter activity and eliminated its induction by insulin and PLC-beta(1). These results hint at the fact that PLC-beta(1) catalytic activity signals a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation.

  12. Characterization and Functional Analysis of Extracellular Vesicles and Muscle-Abundant miRNAs (miR-1, miR-133a, and miR-206 in C2C12 Myocytes and mdx Mice.

    Directory of Open Access Journals (Sweden)

    Yasunari Matsuzaka

    Full Text Available Duchenne muscular dystrophy (DMD is a progressive neuromuscular disorder. Here, we show that the CD63 antigen, which is located on the surface of extracellular vesicles (EVs, is associated with increased levels of muscle-abundant miRNAs, namely myomiRs miR-1, miR-133a, and miR-206, in the sera of DMD patients and mdx mice. Furthermore, the release of EVs from the murine myoblast C2C12 cell line was found to be modulated by intracellular ceramide levels in a Ca2+-dependent manner. Next, to investigate the effects of EVs on cell survival, C2C12 myoblasts and myotubes were cultured with EVs from the sera of mdx mice or C2C12 cells overexpressing myomiRs in presence of cellular stresses. Both the exposure of C2C12 myoblasts and myotubes to EVs from the serum of mdx mice, and the overexpression of miR-133a in C2C12 cells in presence of cellular stress resulted in a significant decrease in cell death. Finally, to assess whether miRNAs regulate skeletal muscle regeneration in vivo, we intraperitoneally injected GW4869 (an inhibitor of exosome secretion into mdx mice for 5 and 10 days. Levels of miRNAs and creatine kinase in the serum of GW4869-treated mdx mice were significantly downregulated compared with those of controls. The tibialis anterior muscles of the GW4869-treated mdx mice showed a robust decrease in Evans blue dye uptake. Collectively, these results indicate that EVs and myomiRs might protect the skeletal muscle of mdx mice from degeneration.

  13. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Dunham, Ian [EMBL-European Bioinformatics Institute (EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Murai, Kasumi [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Jones, Philip H., E-mail: phj20@cam.ac.uk [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom)

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  14. Potentiated Osteoinductivity via Cotransfection with BMP-2 and VEGF Genes in Microencapsulated C2C12 Cells

    Directory of Open Access Journals (Sweden)

    Yang Shen

    2015-01-01

    Full Text Available Microcapsules with entrapped cells hold great promise for repairing bone defects. Unfortunately, the osteoinductivity of microcapsules has been restricted by many factors, among which the deficiency of functional proteins is a significant priority. We potentiated the osteoinductivity of microencapsulated cells via cotransfection with BMP-2 and VEGF genes. Various tissue-derived mesenchymal stem cells and cell lines were compared for BMP-2 and VEGF cotransfection. Ethidium bromide (EB/Calcein AM staining revealed that all of the cell categories could survive for 4 weeks after microencapsulation. An ELISA assay indicated that all microencapsulated BMP-2 or VEGF transfected cells could secrete gene products constitutively for 1 month. Particularly, the recombinant microencapsulated C2C12 cells released the most desirable level of BMP-2 and VEGF. Further experiments demonstrated that microencapsulated BMP-2 and VEGF cotransfected C2C12 cells generated both BMP-2 and VEGF for 4 weeks. Additionally, the cotransfection of BMP-2 and VEGF in microencapsulated C2C12 cells showed a stronger osteogenic induction against BMSCs than individual BMP-2-transfected microencapsulated C2C12 cells. These results demonstrated that the cotransfection of BMP-2 and VEGF into microencapsulated C2C12 cells is of potent utility for the potentiation of bone regeneration, which would provide a promising clinical strategy for cellular therapy in bone defects.

  15. Retinoid acid-induced microRNA-27b-3p impairs C2C12 myoblast proliferation and differentiation by suppressing α-dystrobrevin

    Energy Technology Data Exchange (ETDEWEB)

    Li, Nan; Tang, Yi; Liu, Bo; Cong, Wei; Liu, Chao, E-mail: liuchao_19760711@yahoo.com; Xiao, Jing, E-mail: xiaoj@dmu.edu.cn

    2017-01-15

    We previously reported that excess retinoic acid (RA) resulted in hypoplastic and derangement of myofilaments in embryonic tongue by inhibiting myogenic proliferation and differentiation through CamKIID pathway. Our further studies revealed that the expression of a series of miRNAs was altered by RA administration in embryonic tongue as well as in C2C12 cells. Thus, if excess RA impairs myogenic proliferation and differentiation through miRNAs is taken into account. In present study, miR-27b-3p was found up-regulated in RA-treated C2C12 cells as in embryonic tongue, and predicted to target the 3′UTR of α-dystrobrevin (DTNA). Luciferase reporter assays confirmed the direct interaction between miR-27b-3p and the 3′UTR of DTNA. MiR-27b-3p mimics recapitulated the RA repression on DTNA expression, C2C12 proliferation and differentiation, while the miR-27b-3p inhibitor circumvented these defects resulting from excess RA. As expected, the effects of siDTNA on C2C12 were coincided with those by RA treatment or miR-27b-3p mimics. Therefore, these findings indicated that excess RA inhibited the myoblast proliferation and differentiation by up-regulating miR-27b-3p to target DTNA, which implied a new mechanism in myogenic hypoplasia. - Highlights: • A mechanism that RA results in tongue deformity by disrupting the myogenesis. • A non-muscle specific miR mediating the RA suppression on tongue myogenesis. • A target gene of non-muscle specific miR involved in RA induced tongue deformity.

  16. Stimulating effect of graphene oxide on myogenesis of C2C12 myoblasts on RGD peptide-decorated PLGA nanofiber matrices.

    Science.gov (United States)

    Shin, Yong Cheol; Lee, Jong Ho; Kim, Min Jeong; Hong, Suck Won; Kim, Bongju; Hyun, Jung Keun; Choi, Yu Suk; Park, Jong-Chul; Han, Dong-Wook

    2015-01-01

    In the field of biomedical engineering, many studies have focused on the possible applications of graphene and related nanomaterials due to their potential for use as scaffolds, coating materials and delivery carriers. On the other hand, electrospun nanofiber matrices composed of diverse biocompatible polymers have attracted tremendous attention for tissue engineering and regenerative medicine. However, their combination is intriguing and still challenging. In the present study, we fabricated nanofiber matrices composed of M13 bacteriophage with RGD peptide displayed on its surface (RGD-M13 phage) and poly(lactic-co-glycolic acid, PLGA) and characterized their physicochemical properties. In addition, the effect of graphene oxide (GO) on the cellular behaviors of C2C12 myoblasts, which were cultured on PLGA decorated with RGD-M13 phage (RGD/PLGA) nanofiber matrices, was investigated. Our results revealed that the RGD/PLGA nanofiber matrices have suitable physicochemical properties as a tissue engineering scaffold and the growth of C2C12 myoblasts were significantly enhanced on the matrices. Moreover, the myogenic differentiation of C2C12 myoblasts was substantially stimulated when they were cultured on the RGD/PLGA matrices in the presence of GO. In conclusion, these findings propose that the combination of RGD/PLGA nanofiber matrices and GO can be used as a promising strategy for skeletal tissue engineering and regeneration.

  17. Mitochondrial hyperfusion during oxidative stress is coupled to a dysregulation in calcium handling within a C2C12 cell model.

    Directory of Open Access Journals (Sweden)

    Calum J Redpath

    Full Text Available Atrial Fibrillation is the most common sustained cardiac arrhythmia worldwide harming millions of people every year. Atrial Fibrillation (AF abruptly induces rapid conduction between atrial myocytes which is associated with oxidative stress and abnormal calcium handling. Unfortunately this new equilibrium promotes perpetuation of the arrhythmia. Recently, in addition to being the major source of oxidative stress within cells, mitochondria have been observed to fuse, forming mitochondrial networks and attach to intracellular calcium stores in response to cellular stress. We sought to identify a potential role for rapid stimulation, oxidative stress and mitochondrial hyperfusion in acute changes to myocyte calcium handling. In addition we hoped to link altered calcium handling to increased sarcoplasmic reticulum (SR-mitochondrial contacts, the so-called mitochondrial associated membrane (MAM. We selected the C2C12 murine myotube model as it has previously been successfully used to investigate mitochondrial dynamics and has a myofibrillar system similar to atrial myocytes. We observed that rapid stimulation of C2C12 cells resulted in mitochondrial hyperfusion and increased mitochondrial colocalisation with calcium stores. Inhibition of mitochondrial fission by transfection of mutant DRP1K38E resulted in similar effects on mitochondrial fusion, SR colocalisation and altered calcium handling. Interestingly the effects of 'forced fusion' were reversed by co-incubation with the reducing agent N-Acetyl cysteine (NAC. Subsequently we demonstrated that oxidative stress resulted in similar reversible increases in mitochondrial fusion, SR-colocalisation and altered calcium handling. Finally, we believe we have identified that myocyte calcium handling is reliant on baseline levels of reactive oxygen species as co-incubation with NAC both reversed and retarded myocyte response to caffeine induced calcium release and re-uptake. Based on these results we

  18. Fatty acids increase glucose uptake and metabolism in C2C12 myoblasts stably transfected with human lipoprotein lipase.

    Science.gov (United States)

    Capell, Warren H; Schlaepfer, Isabel R; Wolfe, Pamela; Watson, Peter A; Bessesen, Daniel H; Pagliassotti, Michael J; Eckel, Robert H

    2010-10-01

    Cellular effects of FFA might differ from those of lipoprotein triglyceride (TG)-derived fatty acids (TGFA). The aim of the current study was to examine the relationship between lipoprotein lipase (LPL) expression, TGFA, or FFA availability and glucose metabolism in the absence of insulin in C2C12 myoblasts. Control myoblasts or myoblasts stably transfected with human lipoprotein lipase (C2/LPL; 15-fold greater LPL activity) were incubated for 12 h in fetal bovine serum-free medium in the absence or presence of Intralipid-20. Intracellular retention of labeled medium glucose was assessed in a subset of experiments. In the presence of Intralipid, medium glucose disappearance was increased in C2/LPL cells but not in control cells. In both cell types, glucose label retention in cellular TG was increased in the presence of Intralipid; incubation with albumin-bound oleate produced similar results. In the presence of Intralipid, the LPL hydrolytic inhibitor tetrahydrolipstatin blocked excess glucose retention in cellular TG but did not significantly decrease glucose disappearance in C2/LPL cells. Changes in glucose transport or hexokinase II did not explain the altered glucose disappearance in C2/LPL cells. Our results suggest that LPL overexpression in these cells leads to chronic metabolic adaptations that alter glucose uptake and retention.

  19. Hybrid Alginate-Protein-Coated Graphene Oxide Microcapsules Enhance the Functionality of Erythropoietin Secreting C2C12 Myoblasts.

    Science.gov (United States)

    Saenz Del Burgo, Laura; Ciriza, Jesús; Acarregui, Argia; Gurruchaga, Haritz; Blanco, Francisco Javier; Orive, Gorka; Hernández, Rosa María; Pedraz, Jose Luis

    2017-03-06

    The beneficial effect of combining alginate hydrogel with graphene oxide (GO) on microencapsulated C2C12-myoblast viability has recently been described. However, the commercially available GO lacks homogeneity in size, this parameter being of high relevance for the cell fate in two-dimensional studies. In three-dimensional applications the capacity of this material for binding different kinds of proteins can result in the reduction of de novo released protein that can effectively reach the vicinity of the microcapsules. Undoubtedly, this could be an important hurdle in its clinical use when combined with alginate-PLL microcapsules. Here, we demonstrate that the homogenization of GO nanoparticles is not a mandatory preparation step in order to get the best of this material upon cell microencapsulation. In fact, when the superficial area of these particles is increased, higher amounts of the therapeutic protein erythropoietin (EPO) are adsorbed on their surface. On the other hand, we have been able to improve even more the favorable effects of this graphene derivative on microencapsulated cell viability by forming a protein biocorona. These proteins block the potential binding sites of EPO and, therefore, enhance the amount of therapeutic drug that is released. Finally, we prove that these hybrid alginate-protein-coated GO-microcapsules are functional in vivo.

  20. Evaluation of an in vitro muscle contraction model in mouse primary cultured myotubes.

    Science.gov (United States)

    Manabe, Yasuko; Ogino, Shinya; Ito, Miyuki; Furuichi, Yasuro; Takagi, Mayumi; Yamada, Mio; Goto-Inoue, Naoko; Ono, Yusuke; Fujii, Nobuharu L

    2016-03-15

    To construct an in vitro contraction model with the primary cultured myotubes, we isolated satellite cells from the mouse extensor digitorum longus. Differentiated myotubes possessed a greater number of sarcomere assemblies and higher expression levels of myosin heavy chain, cytochrome c oxidase IV, and myoglobin than in C2C12 myotubes. In agreement with these results regarding the sarcomere assemblies and protein expressions, the primary myotubes showed higher contractile activity stimulated by the electric pulses than that in the C2C12 myotubes. These data suggest that mouse primary myotubes will be a valuable research tool as an in vitro muscle contraction model. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12

    Science.gov (United States)

    2014-01-01

    Background Excessive saturated fatty acids have been considered to be one of major contributing factors for the dysfunction of skeletal muscle cells as well as pancreatic beta cells, leading to the pathogenesis of type 2 diabetes. Results PA induced cell death in a dose dependent manner up to 1.5 mM, but AA protected substantially lipotoxicity caused by PA at even low concentration of 62 μM, at which monounsaturated fatty acids including palmitoleic acid (POA) and oleic acid (OA) did not protect as much as AA did. Induction of cell death by PA was resulted from mitochondrial membrane potential loss, and AA effectively blocked the progression of apoptosis. Furthermore, AA rescued significantly PA-impaired glucose uptake and -signal transduction of Akt in response to insulin. Based on the observations that polyunsaturated AA generated competently cellular droplets at low concentration within the cytosol of myotubes compared with other monounsaturated fatty acids, and AA-driven lipid droplets were also enhanced in the presence of PA, we hypothesized that incorporation of harmful PA into inert triglyceride (TG) may be responsible for the protective effects of AA against PA-induced lipotoxicity. To address this assumption, C2C12 myotubes were incubated with fluorescent probed-PA analogue 4, 4-difluoro-5, 7-dimethyl-4-boro-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16) in the presence of AA and their subsequent lipid profiles were analyzed. The analyses of lipids on thin layer chromatograpy (TLC) showed that fluorescent PA analogue was rapidly channeled into AA-driven TG droplets. Conclusion Taken together, it is proposed that AA diverts PA into inert TG, therefore reducing the availability of harmful PA into intracellular target molecules. PMID:24521082

  2. Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12.

    Science.gov (United States)

    Cheon, Hyae Gyeong; Cho, Young Sik

    2014-02-12

    Excessive saturated fatty acids have been considered to be one of major contributing factors for the dysfunction of skeletal muscle cells as well as pancreatic beta cells, leading to the pathogenesis of type 2 diabetes. PA induced cell death in a dose dependent manner up to 1.5 mM, but AA protected substantially lipotoxicity caused by PA at even low concentration of 62 μM, at which monounsaturated fatty acids including palmitoleic acid (POA) and oleic acid (OA) did not protect as much as AA did. Induction of cell death by PA was resulted from mitochondrial membrane potential loss, and AA effectively blocked the progression of apoptosis. Furthermore, AA rescued significantly PA-impaired glucose uptake and -signal transduction of Akt in response to insulin.Based on the observations that polyunsaturated AA generated competently cellular droplets at low concentration within the cytosol of myotubes compared with other monounsaturated fatty acids, and AA-driven lipid droplets were also enhanced in the presence of PA, we hypothesized that incorporation of harmful PA into inert triglyceride (TG) may be responsible for the protective effects of AA against PA-induced lipotoxicity. To address this assumption, C2C12 myotubes were incubated with fluorescent probed-PA analogue 4, 4-difluoro-5, 7-dimethyl-4-boro-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16) in the presence of AA and their subsequent lipid profiles were analyzed. The analyses of lipids on thin layer chromatography (TLC) showed that fluorescent PA analogue was rapidly channeled into AA-driven TG droplets. Taken together, it is proposed that AA diverts PA into inert TG, therefore reducing the availability of harmful PA into intracellular target molecules.

  3. Specific knockdown of delta-sarcoglycan gene in C2C12 in vitro causes post-translational loss of other sarcoglycans without mechanical stress.

    Science.gov (United States)

    Honda, Michiyo; Hosoda, Mari; Kanzawa, Nobuyuki; Tsuchiya, Takahide; Toyo-oka, Teruhiko

    2009-03-01

    The precise role of delta-sarcoglycan (SG) that is constitutively expressed in skeletal muscle cells and may serve for maintaining the sarcolemmal integrity has not been identified. The delta-SG protein is at first among SG complex. To specifically identify the role in C(2)C(12) cells during the myogenesis, we screened several RNA interference (RNAi) candidates at first, and knocked down both levels of the mRNA and protein, employing adenovirus-mediated RNAi. We found no morphological alteration at both myoblast and myotube stages by suppression of delta-SG. The specific knockdown of delta-SG accompanied a concomitant decrease of alpha-, beta-, and gamma-SGs preserving normal levels of each transcript. As for the localization, alpha-, beta-, and gamma-SGs were weakly stained on the cell membrane in delta-SG knockdown cells, whereas each SG in control cell was localized both on the cell membrane and myoplasm abundantly. This enhanced post-translational loss would represent similitude of the progression of cardiomuscular diseases in vitro. Different from cardiac muscle cells, skeletal muscle cell culture without muscle contraction may imply that mechanical stress per se is not primarily involved in the progression of limb-girdle muscular dystrophy. Furthermore, we have observed translocation of calpain-2 to cell membrane in delta-SG knockdown cells, suggesting that Ca(2+)-sensitive proteases, calpains closely take part in post-translational proteolysis.

  4. Boehmeria nivea Stimulates Glucose Uptake by Activating Peroxisome Proliferator-Activated Receptor Gamma in C2C12 Cells and Improves Glucose Intolerance in Mice Fed a High-Fat Diet

    Directory of Open Access Journals (Sweden)

    Sung Hee Kim

    2013-01-01

    Full Text Available We examined the antidiabetic property of Boehmeria nivea (L. Gaud. Ethanolic extract of Boehmeria nivea (L. Gaud. (EBN increased the uptake of 2-[N-(nitrobenz-2-oxa-1,3-diazol-4-ylamino]-2-deoxy-d-glucose in C2C12 myotubes. To examine the mechanisms underlying EBN-mediated increase in glucose uptake, we examined the transcriptional activity and expression of peroxisome proliferator-activated receptor gamma (PPAR-γ, a pivotal target for glucose metabolism in C2C12 myotubes. We found that the EBN increased both the transcriptional activity and mRNA expression levels of PPAR-γ. In addition, we measured phosphorylation and expression levels of other targets of glucose metabolism, such as AMP-activated protein kinase (AMPK and protein kinase B (Akt/PKB. We found that EBN did not alter the phosphorylation or expression levels of these proteins in a time- or dose-dependent manner, which suggested that EBN stimulates glucose uptake through a PPAR-γ-dependent mechanism. Further, we investigated the antidiabetic property of EBN using mice fed a high-fat diet (HFD. Administration of 0.5% EBN reduced the HFD-induced increase in body weight, total cholesterol level, and fatty liver and improved the impaired fasting glucose level, blood insulin content, and glucose intolerance. These results suggest that EBN had an antidiabetic effect in cell culture and animal systems and may be useful for preventing diabetes.

  5. Ethanol extract of Prunus mume fruit attenuates hydrogen peroxide-induced oxidative stress and apoptosis involving Nrf2/HO-1 activation in C2C12 myoblasts

    Directory of Open Access Journals (Sweden)

    Ji Sook Kang

    Full Text Available ABSTRACT The fruit of the Prunus mume (Siebold Siebold & Zucc., Rosaceae (Korean name: Maesil has long been used as a health food or valuable medicinal material in traditional herb medicine in Southeast Asian countries. In this study, we determined the potential therapeutic efficacy of the ethanol extract of P. mume fruits (EEPM against H2O2-induced oxidative stress and apoptosis in the murine skeletal muscle myoblast cell line C2C12, and sought to understand the associated molecular mechanisms. The results indicated that exposure of C2C12 cells to H2O2 caused a reduction in cell viability by increasing the generation of intracellular reactive oxygen species and by disrupting mitochondrial membrane permeability, leading to DNA damage and apoptosis. However, pretreatment of the cells with EEPM before H2O2 exposure effectively attenuated these changes, suggesting that EEPM prevented H2O2-induced mitochondria-dependent apoptosis. Furthermore, the increased ex-pression and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2 and up-regulation of heme oxygenase-1 (HO-1, a phase II antioxidant enzyme, were detected in EEPM-treated C2C12 cells. We also found that zinc protoporphyrin IX, an HO-1 inhibitor, attenuated the protective effects of EEPM against H2O2-induced reactive oxygen species accumulation and cytotoxicity. Therefore, these results indicate that the activation of the Nrf2/HO-1 pathway might be involved in the protection of EEPM against H2O2-induced cellular oxidative damage. In conclusion, these results show that EEPM contributes to the prevention of oxidative damage and could be used as a nutritional agent for oxidative stress-related diseases.

  6. A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts

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    Friedmann Theodore

    2009-08-01

    Full Text Available Abstract Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1, relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IκB kinase b (IκBKb and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.

  7. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2008-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. During embryonic development, myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Sti...

  8. Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom

    National Research Council Canada - National Science Library

    Silva, Luciana Miato Gonçalves; Silva, Camila Aparecida Alves da; Silva, Aline da; Vieira, Rodolfo Paula; Mesquita-Ferrari, Raquel Agnelli; Cogo, José Carlos; Zamuner, Stella Regina

    2016-01-01

    .... Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown...

  9. Propolis Ethanol Extract Stimulates Cytokine and Chemokine Production through NF-κB Activation in C2C12 Myoblasts

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    Kohei Washio

    2015-01-01

    Full Text Available Myoblast activation is a triggering event for muscle remodeling. We assessed the stimulatory effects of propolis, a beehive product, on myoblasts. After an 8 h treatment with 100 μg/mL of Brazilian propolis ethanol extract, expression of various chemokines, including CCL-2 and CCL-5, and cytokines, such as IL-6, increased. This propolis-induced cytokine production appears to depend on NF-κB activation, because the IKK inhibitor BMS-345541 repressed mRNA levels of CCL-2 by ~66%, CCL-5 by ~81%, and IL-6 by ~69% after propolis treatment. Supernatant from propolis-conditioned C2C12 cells upregulated RAW264 macrophage migration. The supernatant also stimulated RAW264 cells to produce angiogenic factors, including VEGF-A and MMP-12. Brazilian green propolis therefore causes myoblasts to secrete cytokines and chemokines, which might contribute to tissue remodeling of skeletal muscle.

  10. Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux.

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    Hyunju Kim

    Full Text Available Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2 result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux.

  11. Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux.

    Science.gov (United States)

    Kim, Hyunju; Lee, Kang Il; Jang, Minsu; Namkoong, Sim; Park, Rackhyun; Ju, Hyunwoo; Choi, Inho; Oh, Won Keun; Park, Junsoo

    2016-01-01

    Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagic adapter, accumulated in the autophagosomes. Reactive oxygen species such as hydrogen peroxide (H2O2) result in muscle cell death by inducing excessive autophagic flux. Treatment with conessine inhibited H2O2-induced autophagic flux in C2C12 myoblast cells and also interfered with cell death. Our results indicate that conessine has the potential effect to inhibit muscle cell death by interfering with autophagic flux.

  12. Docosahexaenoyl ethanolamide improves glucose uptake and alters endocannabinoid system gene expression in proliferating and differentiating C2C12 myoblasts

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    Jeffrey eKim

    2014-03-01

    Full Text Available Skeletal muscle is a major storage site for glycogen and a focus for understanding insulin resistance and type-2-diabetes. New evidence indicates that overactivation of the peripheral endocannabinoid system (ECS in skeletal muscle diminishes insulin sensitivity. Specific n-6 and n-3 polyunsaturated fatty acids (PUFA are precursors for the biosynthesis of ligands that bind to and activate the cannabinoid receptors. The function of the ECS and action of PUFA in skeletal muscle glucose uptake was investigated in proliferating and differentiated C2C12 myoblasts treated with either 25µM of arachidonate (AA or docosahexaenoate (DHA, 25µM of EC [anandamide (AEA, 2-arachidonoylglycerol (2-AG, docosahexaenoylethanolamide (DHEA], 1µM of CB1 antagonist NESS0327, and CB2 antagonist AM630. Compared to the BSA vehicle control cell cultures in both proliferating and differentiated myoblasts those treated with DHEA, the EC derived from the n-3 PUFA DHA, had higher 24 h glucose uptake, while AEA and 2-AG, the EC derived from the n-6 PUFA AA, had lower basal glucose uptake. Adenylyl cyclase mRNA was higher in myoblasts treated with DHA in both proliferating and differentiated states while those treated with AEA or 2-AG were lower compared to the control cell cultures. Western blot and qPCR analysis showed higher expression of the cannabinoid receptors in differentiated myoblasts treated with DHA while the opposite was observed with AA. These findings indicate a compensatory effect of DHA and DHEA compared to AA-derived ligands on the ECS and associated ECS gene expression and higher glucose uptake in myoblasts.Key Words: endocannabinoid system •C2C12 myoblasts cannabinoid receptors glucose uptake gene expression DHEA • polyunsaturated fatty acids

  13. Unveiling novel genes upregulated by both rhBMP2 and rhBMP7 during early osteoblastic transdifferentiation of C2C12 cells

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    Sogayar Mari C

    2011-09-01

    Full Text Available Abstract Findings We set out to analyse the gene expression profile of pre-osteoblastic C2C12 cells during osteodifferentiation induced by both rhBMP2 and rhBMP7 using DNA microarrays. Induced and repressed genes were intercepted, resulting in 1,318 induced genes and 704 repressed genes by both rhBMP2 and rhBMP7. We selected and validated, by RT-qPCR, 24 genes which were upregulated by rhBMP2 and rhBMP7; of these, 13 are related to transcription (Runx2, Dlx1, Dlx2, Dlx5, Id1, Id2, Id3, Fkhr1, Osx, Hoxc8, Glis1, Glis3 and Cfdp1, four are associated with cell signalling pathways (Lrp6, Dvl1, Ecsit and PKCδ and seven are associated with the extracellular matrix (Ltbp2, Grn, Postn, Plod1, BMP1, Htra1 and IGFBP-rP10. The novel identified genes include: Hoxc8, Glis1, Glis3, Ecsit, PKCδ, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 and IGFBP-rP10. Background BMPs (bone morphogenetic proteins are members of the TGFβ (transforming growth factor-β super-family of proteins, which regulate growth and differentiation of different cell types in various tissues, and play a critical role in the differentiation of mesenchymal cells into osteoblasts. In particular, rhBMP2 and rhBMP7 promote osteoinduction in vitro and in vivo, and both proteins are therapeutically applied in orthopaedics and dentistry. Conclusion Using DNA microarrays and RT-qPCR, we identified both previously known and novel genes which are upregulated by rhBMP2 and rhBMP7 during the onset of osteoblastic transdifferentiation of pre-myoblastic C2C12 cells. Subsequent studies of these genes in C2C12 and mesenchymal or pre-osteoblastic cells should reveal more details about their role during this type of cellular differentiation induced by BMP2 or BMP7. These studies are relevant to better understanding the molecular mechanisms underlying osteoblastic differentiation and bone repair.

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

    Science.gov (United States)

    Yang, Chuanbin; Li, Xuechen; Rong, Jianhui

    2014-01-01

    The 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. Active 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. Three 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. Amygdalin 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.

  15. α-Actinin involvement in Z-disk assembly during skeletal muscle C2C12 cells in vitro differentiation.

    Science.gov (United States)

    Salucci, S; Baldassarri, V; Falcieri, E; Burattini, S

    2015-01-01

    α-Actinin is involved in the assembly and maintenance of muscle fibers. α-Actinin is required to cross-link actin filaments and to connect the actin cytoskeleton to the cell membrane and it is necessary for the attachment of actin filaments to Z-disks in skeletal muscle fibers and to dense bodies in smooth muscle ones. In addition to its mechanical role, sarcomeric α-actinin interacts with proteins involved in a variety of signaling and metabolic pathways. The aim of this work is to monitor Z-disk formation, in order to clear up the role of sarcomeric α-actinin in undifferentiated stage, after 4 days of differentiation (intermediate differentiation stage) and after 7 days of differentiation (fully differentiated stage). For this purpose, C2C12 murine skeletal muscle cells, grown in vitro, were analyzed at three time points of differentiation. Confocal laser scanner microscopy and transmission electron microscopy have been utilized for α-actinin immunolocalization. Both techniques reveal that in undifferentiated cells labeling appears uniformly distributed in the cytoplasm with punctate α-actinin Z-bodies. Moreover, we found that when differentiation is induced, α-actinin links at first membrane-associated proteins, then it aligns longitudinally across the cytoplasm and finally binds actin, giving rise to Z-disks. These findings evidence α-actinin involvement in sarcomeric development, suggesting for this protein an important role in stabilizing the muscle contractile apparatus. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Fusion and differentiation of murine C2C12 skeletal muscle cells that express Trichinella spiralis p43 protein.

    Science.gov (United States)

    Jasmer, Douglas P; Kwak, Dongmi

    2006-02-01

    The ability of a 43 kDa stichocyte protein from Trichinella spiralis (Tsp43) to interfere with mammalian skeletal muscle gene expression was investigated. A MYC-tagged Tsp43 construct was expressed as a recombinant protein in C2C12 myoblasts. Transfection with low amounts of expression plasmid was required for successful expression of the protein. This construct had apparent toxic effects on transfected myoblasts and ectopic green fluorescent protein expression was suppressed in myoblasts co-transfected with the Tsp43 construct. These effects may result from similarities of Tsp43 to DNase II. Use of the general DNase inhibitor aurintricarboxylic acid (ATA) enhanced expression of MYC-Tsp43 in transfected muscle cells. Myoblasts transfected with Tsp43 did not fuse well when cultured under differentiation conditions without ATA. In contrast, transfected myoblasts transiently cultured with ATA underwent fusion and differentiation. Under short-term differentiation conditions without ATA, unfused myoblasts nevertheless expressed both MYC-Tsp43 and myosin heavy chain. Collectively, the results support that Tsp43 has a role in the T. spiralis life cycle that is distinct from repressing muscle gene expression during the muscle phase of infection. While the function of Tsp43 as a DNase is under debate, the effects of ATA on transfected muscle cells were consistent with this possibility.

  17. Screening with a novel cell-based assay for TAZ activators identifies a compound that enhances myogenesis in C2C12 cells and facilitates muscle repair in a muscle injury model.

    Science.gov (United States)

    Yang, Zeyu; Nakagawa, Kentaro; Sarkar, Aradhan; Maruyama, Junichi; Iwasa, Hiroaki; Bao, Yijun; Ishigami-Yuasa, Mari; Ito, Shigeru; Kagechika, Hiroyuki; Hata, Shoji; Nishina, Hiroshi; Abe, Shinya; Kitagawa, Masanobu; Hata, Yutaka

    2014-05-01

    The transcriptional coactivator with a PDZ-binding motif (TAZ) cooperates with various transcriptional factors and plays various roles. Immortalized human mammalian epithelial MCF10A cells form spheres when TAZ is overexpressed and activated. We developed a cell-based assay using sphere formation by TAZ-expressing MCF10A cells as a readout to screen 18,458 chemical compounds for TAZ activators. Fifty compounds were obtained, and 47 were confirmed to activate the TAZ-dependent TEAD-responsive reporter activity in HEK293 cells. We used the derived subset of compounds as a TAZ activator candidate minilibrary and searched for compounds that promote myogenesis in mouse C2C12 myoblast cells. In this study, we focused on one compound, IBS008738. IBS008738 stabilizes TAZ, increases the unphosphorylated TAZ level, enhances the association of MyoD with the myogenin promoter, upregulates MyoD-dependent gene transcription, and competes with myostatin in C2C12 cells. TAZ knockdown verifies that the effect of IBS008738 depends on endogenous TAZ in C2C12 cells. IBS008738 facilitates muscle repair in cardiotoxin-induced muscle injury and prevents dexamethasone-induced muscle atrophy. Thus, this cell-based assay is useful to identify TAZ activators with a variety of cellular outputs. Our findings also support the idea that TAZ is a potential therapeutic target for muscle atrophy.

  18. Tiam-1, a GEF for Rac1, plays a critical role in metformin-mediated glucose uptake in C2C12 cells.

    Science.gov (United States)

    You, Ga Young; Lee, Jung Ok; Kim, Ji Hae; Kim, Nami; Lee, Soo Kyung; Moon, Ji Wook; Jie, Sha; Lee, Hye Jeong; Kim, Su Jin; Park, Sun Hwa; Kim, Hyeon Soo

    2013-12-01

    Metformin is known to stimulate glucose uptake, but the mechanism for this action is not fully understood. In this study, AMPK activators (AICAR and metformin) increased the expression of T-lymphoma invasion and metastasis-inducing protein-1 (Tiam-1), a Rac1 specific guanine nucleotide exchange factor (GEF), mRNA and protein in skeletal muscle C2C12 cells. Metformin increases the serine-phosphorylation of Tiam-1 by AMPK and induces interaction between Tiam-1 and 14-3-3. Pharmacologic inhibition of AMPK blocks this interaction, indicating that 14-3-3 may be required for induction of Tiam-1 by AMPK. Metformin also increases the phosphorylation of p21-activated kinase 1 (PAK1), a direct downstream target of Rac1, dependent on AMPK. Tiam-1 is down-regulated at high glucose concentrations in cultured cells and in the db/db mouse model of hyperglycemia. Furthermore, Tiam-1 knock-down blocked metformin-induced increase in glucose uptake. These findings suggest that metformin promotes cellular glucose uptake in part through Tiam-1 induction. © 2013. Published by Elsevier Inc. All rights reserved.

  19. Effect of testosterone on the regulation of p53 and p66Shc during oxidative stress damage in C2C12 cells.

    Science.gov (United States)

    Pronsato, Lucía; Milanesi, Lorena

    2016-02-01

    Accumulating evidence indicates that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H2O2)-induced apoptosis in C2C12 muscle cells, at different levels: morphological, physiological, biochemical and molecular. In the present study we observed that H2O2 induces the mitochondrial permeability transition pore (mPTP) opening and exerts p53 activation in a time-dependent way, with a maximum response after 1-2h of treatment. Testosterone treatment, prior to H2O2, reduces not only p53 phosphorylation but also p66Shc expression, activation and its mitochondrial localization, at the same time that it prevents the mPTP opening. Furthermore, testosterone diminishes JNK and PKCβI phosphorylation induced by H2O2 and probably contributing thus, to reduce the activation of p66Shc. Thus, the mPTP opening, p53, JNK and PKCβI activation, as well as p66Shc mRNA increase, induced by oxidative stress, were reduced by testosterone pretreatment. The data presented in this work show some of the components upstream of the classical apoptotic pathway, that are activated during oxidative stress and that are points where testosterone exerts its protective action against apoptosis, exposing some of the puzzle pieces of the intricate network that aged skeletal muscle apoptosis represents. Copyright © 2015. Published by Elsevier Inc.

  20. Efeitos do ultra-som terapêutico contínuo sobre a proliferação e viabilidade de células musculares C2C12 Effects of continuous therapeutic ultrasound on proliferation and viability of C2C12 muscle cells

    Directory of Open Access Journals (Sweden)

    Paola Pelegrineli Artilheiro

    2010-06-01

    Full Text Available O ultra-som terapêutico (US é um recurso bioestimulante utilizado para propiciar reparo muscular de melhor qualidade e menor duração, mas o potencial terapêutico do US contínuo não está totalmente estabelecido. O objetivo deste trabalho foi avaliar o efeito do US contínuo sobre a proliferação e viabilidade de células musculares precursoras (mioblastos C2C12. Mioblastos C2C12 foram cultivados em meio de cultura contendo 10% de soro fetal bovino e irradiados com US contínuo nas freqüências de 1 e 3 MHz nas intensidades de 0,2 e 0,5 W/cm2, durante 2 e 5 minutos. A viabilidade e proliferação celular foram avaliadas após 24, 48 e 72 h de incubação. Grupos não-irradiados serviram como controle. Foram realizados experimentos independentes em cada condição acima, e os dados obtidos submetidos à análise estatística. Os resultados mostram que não houve diferença estatisticamente significativa na proliferação e viabilidade celular entre os mioblastos tratados com US e as culturas controles após os diferentes períodos de incubação, em todos os parâmetros avaliados. Conclui-se que o US contínuo, nos parâmetros avaliados, não foi capaz de alterar a proliferação e viabilidade dos mioblastos.Therapeutic ultrasound (US is a biophysical stimulation resource widely used in order to promote better, faster muscle repair, but the effectiveness of continuous US in treating injuries is not fully established. The aim of the present in vitro study was to assess the effects of continuous ultrasound on viability and proliferation of skeletal muscle precursor cells (C2C12 myoblasts. C2C12 myoblasts were cultured in a medium containing 10% foetal bovine serum and irradiated with continuous ultrasound at 1 and 3 MHz frequencies, at intensities of 0.2 and 0.5 W/cm² for 2 and 5 minutes. Cell viability and proliferation were assessed after different incubation periods (24, 48 and 72 h. Non-irradiated groups served as control and data were

  1. Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells.

    Science.gov (United States)

    Gallea, S; Lallemand, F; Atfi, A; Rawadi, G; Ramez, V; Spinella-Jaegle, S; Kawai, S; Faucheu, C; Huet, L; Baron, R; Roman-Roman, S

    2001-05-01

    Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.

  2. Differential effect of fructose on fat metabolism and clock gene expression in hepatocytes vs. myotubes.

    Science.gov (United States)

    Chapnik, Nava; Rozenblit-Susan, Sigal; Genzer, Yoni; Froy, Oren

    2016-08-01

    In the liver, fructose bypasses the main rate-limiting step of glycolysis at the level of phosphofructokinase, allowing it to act as an unregulated substrate for de novo lipogenesis. It has been reported that consumption of large amounts of fructose increases de novo lipogenesis in the liver. However, the effect of fructose on ectopic deposition of muscle fat has been under dispute. Our aim was to study the effect of fructose on levels of genes and proteins involved in fatty acid oxidation and synthesis in hepatocytes vs. muscle cells. In addition, as fat accumulation leads to disruption of daily rhythms, we tested the effect of fructose treatment on clock gene expression. AML-12 hepatocytes and C2C12 myotubes were treated with fructose or glucose for 2 consecutive 24-h cycles and harvested every 6h. In contrast to glucose, fructose disrupted clock gene rhythms in hepatocytes, but in myotubes, it led to more robust rhythms. Fructose led to low levels of phosphorylated AMP-activated protein kinase (pAMPK) and high levels of LIPIN1 in hepatocytes compared with glucose. In contrast, fructose led to high pAMPK and low LIPIN1 and microsomal triacylglycerol transfer protein (MTTP) levels in myotubes compared with glucose. Analysis of fat content revealed that fructose led to less fat accumulation in myotubes compared to hepatocytes. In summary, fructose shifts metabolism towards fatty acid synthesis and clock disruption in hepatocytes, but not in myotubes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Extracellular vesicles from a muscle cell line (C2C12 enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34

    Directory of Open Access Journals (Sweden)

    Roger D. Madison

    2014-02-01

    Full Text Available Introduction: There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. Background: When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. Methods: We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? Conclusion: Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

  4. Microbial synthesis of gold nanoparticles using the fungus Penicillium brevicompactum and their cytotoxic effects against mouse mayo blast cancer C 2 C 12 cells.

    Science.gov (United States)

    Mishra, Amrita; Tripathy, Suraj Kumar; Wahab, Rizwan; Jeong, Song-Hoon; Hwang, Inho; Yang, You-Bing; Kim, Young-Soon; Shin, Hyung-Shik; Yun, Soon-Il

    2011-11-01

    Microorganisms, their cell filtrates, and live biomass have been utilized for synthesizing various gold nanoparticles. The shape, size, stability as well as the purity of the bio synthesized nanoparticles become very essential for application purpose. In the present study, gold nanoparticles have been synthesized from the supernatant, live cell filtrate, and biomass of the fungus Penicillium brevicompactum. The fungus has been grown in potato dextrose broth which is also found to synthesize gold nanoparticles. The size of the particles has been investigated by Bio-TEM before purification, following purification and after storing the particles for 3 months under refrigerated condition. Different characterization techniques like X-ray diffraction, Fourier transform infrared spectroscopy, and UV-visible spectroscopy have been used for analysis of the particles. The effect of reaction parameters such as pH and concentration of gold salt have also been monitored to optimize the morphology and dispersity of the synthesized gold nanoparticles. A pH range of 5 to 8 has favored the synthesis process whereas increasing concentration of gold salt (beyond 2 mM) has resulted in the formation of bigger sized and aggregated nanoparticles. Additionally, the cytotoxic nature of prepared nanoparticles has been analyzed using mouse mayo blast cancer C(2)C(12) cells at different time intervals (24, 48, and 72 h) of incubation period. The cells are cultivated in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum with antibiotics (streptopenicillin) at 37°C in a 5% humidified environment of CO(2). The medium has been replenished every other day, and the cells are subcultured after reaching the confluence. The viability of the cells is analyzed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method.

  5. Extracellular vesicles from a muscle cell line (C2C12) enhance cell survival and neurite outgrowth of a motor neuron cell line (NSC-34).

    Science.gov (United States)

    Madison, Roger D; McGee, Christopher; Rawson, Renee; Robinson, Grant A

    2014-01-01

    There is renewed interest in extracellular vesicles over the past decade or 2 after initially being thought of as simple cellular garbage cans to rid cells of unwanted components. Although there has been intense research into the role of extracellular vesicles in the fields of tumour and stem cell biology, the possible role of extracellular vesicles in nerve regeneration is just in its infancy. When a peripheral nerve is damaged, the communication between spinal cord motor neurons and their target muscles is disrupted and the result can be the loss of coordinated muscle movement. Despite state-of-the-art surgical procedures only approximately 10% of adults will recover full function after peripheral nerve repair. To improve upon such results will require a better understanding of the basic mechanisms that influence axon outgrowth and the interplay between the parent motor neuron and the distal end organ of muscle. It has previously been shown that extracellular vesicles are immunologically tolerated, display targeting ligands on their surface, and can be delivered in vivo to selected cell populations. All of these characteristics suggest that extracellular vesicles could play a significant role in nerve regeneration. We have carried out studies using 2 very well characterized cell lines, the C2C12 muscle cell line and the motor neuron cell line NSC-34 to ask the question: Do extracellular vesicles from muscle influence cell survival and/or neurite outgrowth of motor neurons? Our results show striking effects of extracellular vesicles derived from the muscle cell line on the motor neuron cell line in terms of neurite outgrowth and survival.

  6. Effect of Excess Gravitational Force on Cultured Myotubes in Vitro

    Directory of Open Access Journals (Sweden)

    Shigehiro Hashimoto

    2013-06-01

    Full Text Available An effect of an excess gravitational force on cultured myoblasts has been studied in an experimental system with centrifugal force in vitro. Mouse myoblasts (C2C12 were seeded on a culture dish of 35 mm diameter, and cultured in the Dulbecco's Modified Eagle's Medium until the sub-confluent condition. To apply the excess gravitational force on the cultured cells, the dish was set in a conventional centrifugal machine. Constant gravitational force was applied to the cultured cells for three hours. Variations were made on the gravitational force (6 G, 10 G, 100 G, 500 G, and 800 G with control of the rotational speed of the rotator in the centrifugal machine. Morphology of the cells was observed with a phasecontrast microscope for eight days. The experimental results show that the myotube thickens day by day after the exposure to the excess gravitational force field. The results also show that the higher excess gravitational force thickens myotubes. The microscopic study shows that myotubes thicken with fusion each other.

  7. ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin-Hua [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Bauman, William A. [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Cardozo, Christopher, E-mail: chris.cardozo@va.gov [National Center of Excellence for the Medical Consequences of Spinal Cord Injury, James J. Peter VA Medical Center, Bronx, NY 10468 (United States); Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Rehabilitation Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States); Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029 (United States)

    2015-08-14

    Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle. - Highlights: • AnkrD1 is upregulated by TNFα and represses NF-κB-induced transcriptional activity. • AnkrD1 binds to p50 subunit of NF-κB and is recruited to NF-κB bound to chromatin. • AnkrD1 mediates a feed-back inhibitory loop

  8. Mechanical Stimulation of C2C12 Cells Increases m-Calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion

    DEFF Research Database (Denmark)

    Grossi, Alberto; Lawson, Moira Ann; Karlsson, Anders H

    to stretch- or load-induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ infux in myocytes. Evidence of the involvement of Ca2+ dependent activity in myoblast fusion, cell membrane and cytoskeleton component reorganization due......Abstract Mechanical Stimulation of C2C12 Cells Increases m-calpain Expression and Activity, Focal Adhesion Plaque Degradation and Cell Fusion A. Grossi, A. H. Karlsson, M. A. Lawson; Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark...

  9. Mechanical stimuli on C2C12 myoblasts affect myoblast differentiation, focal adhesion kinase phosphorylation and galectin-1 expression

    DEFF Research Database (Denmark)

    Grossi, Alberto Blak; Lametsch, Rene; Karlsson, Anders H

    2011-01-01

    Mechanical forces are crucial in the regulation of cell morphology and function. At the cellular level, these forces influence myoblast differentiation and fusion. In this study we applied mechanical stimuli to embryonic muscle cells using magnetic microbeads, a method shown to apply stress...... by mechanical stimulation including Galectin-1, Annexin III, and RhoGDI. In this study we demonstrate how the combination of this method of mechanical stimuli and proteomic analysis can be a powerful tool to detect proteins that are potentially interacting in biochemical pathways or complex cellular mechanisms...... during the process of myoblast differentiation. We determined an increase in expression and changes in cellular localization of Galectin-1, in mechanically stimulated myoblasts. A potential involvement of Galectin-1 in myoblast differentiation is presented....

  10. Development of gas chromatography-flame ionization detection system with a single column and liquid nitrogen-free for measuring atmospheric C2-C12 hydrocarbons.

    Science.gov (United States)

    Liu, Chengtang; Mu, Yujing; Zhang, Chenglong; Zhang, Zhibo; Zhang, Yuanyuan; Liu, Junfeng; Sheng, Jiujiang; Quan, Jiannong

    2016-01-04

    A liquid nitrogen-free GC-FID system equipped with a single column has been developed for measuring atmospheric C2-C12 hydrocarbons. The system is consisted of a cooling unit, a sampling unit and a separation unit. The cooling unit is used to meet the temperature needs of the sampling unit and the separation unit. The sampling unit includes a dehydration tube and an enrichment tube. No breakthrough of the hydrocarbons was detected when the temperature of the enrichment tube was kept at -90 °C and sampling volume was 400 mL. The separation unit is a small round oven attached on the cooling column. A single capillary column (OV-1, 30 m × 0.32 mm I.D.) was used to separate the hydrocarbons. An optimal program temperature (-60 ∼ 170 °C) of the oven was achieved to efficiently separate C2-C12 hydrocarbons. There were good linear correlations (R(2)=0.993-0.999) between the signals of the hydrocarbons and the enrichment amount of hydrocarbons, and the relative standard deviation (RSD) was less than 5%, and the method detection limits (MDLs) for the hydrocarbons were in the range of 0.02-0.10 ppbv for sampling volume of 400 mL. Field measurements were also conducted and more than 50 hydrocarbons from C2 to C12 were detected in Beijing city. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. α-linolenic acid reduces TNF-induced apoptosis in C2C12 myoblasts by regulating expression of apoptotic proteins

    Directory of Open Access Journals (Sweden)

    Felicia Carotenuto

    2016-11-01

    Full Text Available Impaired regeneration and consequent muscle wasting is a major feature of muscle degenerative diseases. Nutritional interventions as adjuvant strategy for preventing such conditions are recently gaining increasing attention. Ingestion of n3-polyunsaturated fatty acids has been suggested to have a positive impact on muscle diseases. We recently demonstrated that the dietary n3-fatty acid, alpha-linolenic acid (ALA, exerts potent beneficial effects in preserving skeletal muscle regeneration in models of muscle dystrophy. To better elucidate the underlying mechanism we investigate here on the expression level of the anti- and pro-apototic proteins, as well as caspase-3 activity, in C2C12 myoblasts challenged with pathological levels of TNF. The results demonstrated that ALA protective effect on C2C12 myoblasts was associated to an increased Bcl-2/Bax ratio. Indeed, the effect of ALA was directed to rescue Bcl-2 expression and decrease Bax expression both affected in an opposite way by TNF treatment. This effect was associated with a decrease in caspase-3 activity by ALA. TNF is a major pro-inflammatory cytokine that is expressed in damaged skeletal muscle, therefore, counteract inflammatory signals in the muscle microenvironment represents a critical strategy to ameliorate skeletal muscle pathologies

  12. Dopamine-Incorporated Dual Bioactive Electroactive Shape Memory Polyurethane Elastomers with Physiological Shape Recovery Temperature, High Stretchability, and Enhanced C2C12 Myogenic Differentiation.

    Science.gov (United States)

    Zhao, Xin; Dong, Ruonan; Guo, Baolin; Ma, Peter X

    2017-09-06

    Soft tissue engineering needs elastic biomaterials not only mimicking the elasticity of soft tissue but also possessing multiple bioactivity to promote cell adhesion, proliferation, and differentiation, which still remain ongoing challenges. Herein, we synthesized a series of dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers by combining the properties of elastomeric poly(citric acid-co-polycaprolactone) (CA-PCL) polyurethane elastomer, bioactive dopamine (DA), and electroactive aniline hexamer (AH). The chemical structures, electroactivity, conductivity, thermal properties, hydrophilicity and hydration ability, mechanical properties, and degradability of the polyurethane elastomers were systematically characterized. The elastomers showed excellent shape fixity ratio and shape recovery ability under physiological conditions. The elastomers' elongation and stress were tailored by the AH content, whereas the hydrophilicity and hydration ability of the elastomers were adjusted by the content of DA and AH, as well as the doping state of AH. The viability and proliferation results of C2C12 cells seeded on the elastomers showed their excellent cytocompatibility. Additionally, by analyzing the protein and gene level, the promotion effect on myogenic differentiation of C2C12 cells by these elastomers compared to that by control groups (PCL80 000, CA-PCL elastomer, and CA-PCL elastomer with the DA segment) was demonstrated. Furthermore, the results from subcutaneous implantation confirmed the elastomers' mild host response in vivo. These results represent that these dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers are promising candidates for soft tissue regeneration that is sensitive to electrical signals.

  13. Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and β2-adrenoceptor mechanisms

    Science.gov (United States)

    Ngala, R A; O'Dowd, J; Wang, S J; Agarwal, A; Stocker, C; Cawthorne, M A; Arch, J R S

    2008-01-01

    Background and purpose: Picomolar concentrations of the β3-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via β2-adrenoceptors. Effects of BRL37344 and β2-adrenoceptor agonists are compared. Experimental approach: Mouse soleus muscles were incubated with 2-deoxy[1-14C]-glucose, [1-14C]-palmitate or [2-14C]-pyruvate, and BRL37344, β2-adrenoceptor agonists and selective β-adrenoceptor antagonists. Formation of 2-deoxy[1-14C]-glucose-6-phosphate or 14CO2 was measured. 2-Deoxy[1-14C]-glucose uptake and β-adrenoceptor mRNA were measured in C2C12 cells. Key results: 10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33–54%. The effect of BRL37344 was prevented by 1 μM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 μM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 st4mulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only β2-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant. Conclusions and implications: Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via β2-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake. PMID:18552870

  14. Metabolic responses to BRL37344 and clenbuterol in soleus muscle and C2C12 cells via different atypical pharmacologies and beta2-adrenoceptor mechanisms.

    Science.gov (United States)

    Ngala, R A; O'Dowd, J; Wang, S J; Agarwal, A; Stocker, C; Cawthorne, M A; Arch, J R S

    2008-10-01

    Picomolar concentrations of the beta3-adrenoceptor agonist BRL37344 stimulate 2-deoxyglucose uptake in soleus muscle via undefined receptors. Higher concentrations alter uptake, apparently via beta2-adrenoceptors. Effects of BRL37344 and beta2-adrenoceptor agonists are compared. Mouse soleus muscles were incubated with 2-deoxy[1-(14)C]-glucose, [1-(14)C]-palmitate or [2-(14)C]-pyruvate, and BRL37344, beta2-adrenoceptor agonists and selective beta-adrenoceptor antagonists. Formation of 2-deoxy[1-(14)C]-glucose-6-phosphate or (14)CO2 was measured. 2-Deoxy[1-(14)C]-glucose uptake and beta-adrenoceptor mRNA were measured in C2C12 cells. 10 pM BRL37344, 10 pM clenbuterol and 100 pM salbutamol stimulated 2-deoxyglucose uptake in soleus muscle by 33-54%. The effect of BRL37344 was prevented by 1 microM atenolol but not by 300 nM CGP20712A or IC3118551, or 1 microM SR59230A; that of clenbuterol was prevented by ICI118551 but not atenolol. 10 nM BRL37344 stimulated 2-deoxyglucose uptake, whereas 100 nM clenbuterol and salbutamol inhibited uptake. These effects were blocked by ICI118551. Similar results were obtained in C2C12 cells, in which only beta2-adrenoceptor mRNA could be detected by RT-PCR. 10 nM BRL37344 and 10 pM clenbuterol stimulated muscle palmitate oxidation. In the presence of palmitate, BRL37344 no longer stimulated 2-deoxyglucose uptake and the effect of clenbuterol was not significant. Stimulation of glucose uptake by 10 pM BRL37344 and clenbuterol involves different atypical pharmacologies. Nanomolar concentrations of BRL37344 and clenbuterol, probably acting via beta2-adrenoceptors, have opposite effects on glucose uptake. The agonists preferentially stimulate fat rather than carbohydrate oxidation, but stimulation of endogenous fat oxidation cannot explain why 100 nM clenbuterol inhibited 2-deoxyglucose uptake.

  15. Testosterone exerts antiapoptotic effects against H2O2 in C2C12 skeletal muscle cells through the apoptotic intrinsic pathway.

    Science.gov (United States)

    Pronsato, Lucía; Boland, Ricardo; Milanesi, Lorena

    2012-03-01

    Experimental data indicate that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H(2)O(2))-induced apoptosis in C2C12 muscle cells. Here we identified molecular events involved in the antiapoptotic effect of testosterone. At short times of exposure to H(2)O(2) cells exhibit a defense response but at longer treatment times cells undergo apoptosis. Incubation with testosterone prior to H(2)O(2) induces BAD inactivation, inhibition of poly(ADP-ribose) polymerase cleavage, and a decrease in BAX levels, and impedes the loss of mitochondrial membrane potential, suggesting that the hormone participates in the regulation of the apoptotic intrinsic pathway. Simultaneous treatment with testosterone, H(2)O(2), and the androgen receptor (AR) antagonist, flutamide, reduces the effects of the hormone, pointing to a possible participation of the AR in the antiapoptotic effect. The data presented allow us to begin to elucidate the mechanism by which the hormone prevents apoptosis in skeletal muscle.

  16. Mining the Secretome of C2C12 Muscle Cells: Data Dependent Experimental Approach To Analyze Protein Secretion Using Label-Free Quantification and Peptide Based Analysis.

    Science.gov (United States)

    Grube, Leonie; Dellen, Rafael; Kruse, Fabian; Schwender, Holger; Stühler, Kai; Poschmann, Gereon

    2018-01-24

    Secretome analysis faces several challenges including detection of low abundant proteins and the discrimination of bona fide secreted proteins from false-positive identifications stemming from cell leakage or serum. Here, we developed a two-step secretomics approach and applied it to the analysis of secreted proteins of C2C12 skeletal muscle cells since the skeletal muscle has been identified as an important endocrine organ secreting myokines as signaling molecules. First, we compared culture supernatants with corresponding cell lysates by mass spectrometry-based proteomics and label-free quantification. We identified 672 protein groups as candidate secreted proteins due to their higher abundance in the secretome. On the basis of Brefeldin A mediated blocking of classical secretory processes, we estimated a sensitivity of >80% for the detection of classical secreted proteins for our experimental approach. In the second step, the peptide level information was integrated with UniProt based protein information employing the newly developed bioinformatics tool "Lysate and Secretome Peptide Feature Plotter" (LSPFP) to detect proteolytic protein processing events that might occur during secretion. Concerning the proof of concept, we identified truncations of the cytoplasmic part of the protein Plexin-B2. Our workflow provides an efficient combination of experimental workflow and data analysis to identify putative secreted and proteolytic processed proteins.

  17. Malonyl coenzyme A affects insulin-stimulated glucose transport in myotubes.

    Science.gov (United States)

    Patil, P B; Minteer, S D; Mielke, A A; Lewis, L R; Casmaer, C A; Barrientos, E J; Ju, J-S; Smith, J L; Fisher, J S

    2007-02-01

    There seems to be an association between increased concentrations of malonyl coenzyme A (malonyl CoA) in skeletal muscle and diabetes and/or insulin resistance. The purpose of the current study was to test the hypothesis that treatments designed to manipulate malonyl CoA concentrations would affect insulin-stimulated glucose transport in cultured C2C12 myotubes. We assessed glucose transport after polyamine-mediated delivery of malonyl CoA to myotubes, after incubation with dichloroacetate (which reportedly increases malonyl CoA levels), or after exposure of myotubes to 2-bromopalmitate, a carnitine palmitoyl transferase I inhibitor. All three of these treatments prevented stimulation of glucose transport by insulin. We also assayed glucose transport after 30 min of inhibition of acetyl coenzyme A carboxylase (ACC), the enzyme which catalyzes the production of malonyl CoA. Three unrelated ACC inhibitors (diclofop, clethodim, and Pfizer CP-640186) all enhanced insulin-stimulated glucose transport. However, none of the treatments designed to manipulate malonyl CoA concentrations altered markers of proximal insulin signaling through Akt. The findings support the hypothesis that acute changes in malonyl CoA concentrations affect insulin action in muscle cells but suggest that the effects do not involve alterations in proximal insulin signaling.

  18. Pannexin Channels Mediate the Acquisition of Myogenic Commitment in C2C12 Reserve Cells Promoted by P2 Receptor Activation

    Directory of Open Access Journals (Sweden)

    Manuel Antonio Riquelme

    2015-05-01

    Full Text Available The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i. Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs as well as connexin (Cx and/or pannexin (Panx hemichannels and channels (Cx HChs and Panx Chs, respectively, which are known to permeate Ca2+. Reserve cells (RCs are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs, did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs and Panx Chs.

  19. Pannexin channels mediate the acquisition of myogenic commitment in C2C12 reserve cells promoted by P2 receptor activation

    Science.gov (United States)

    Riquelme, Manuel A.; Cea, Luis A.; Vega, José L.; Puebla, Carlos; Vargas, Aníbal A.; Shoji, Kenji F.; Subiabre, Mario; Sáez, Juan C.

    2015-01-01

    The acquisition of myoblast commitment to the myogenic linage requires rises in intracellular free Ca2+ concentration ([Ca2+]i). Putative cell membrane pathways involved in these [Ca2+]i increments are P2 receptors (P2Rs) as well as connexin (Cx) and/or pannexin (Panx) hemichannels and channels (Cx HChs and Panx Chs), respectively, which are known to permeate Ca2+. Reserve cells (RCs) are uncommitted myoblasts obtained from differentiated C2C12 cell cultures, which acquire commitment upon replating. Regarding these cells, we found that extracellular ATP increases the [Ca2+]i via P2Rs. Moreover, ATP increases the plasma membrane permeability to small molecules and a non-selective membrane current, both of which were inhibited by Cx HCh/Panx1Ch blockers. However, RCs exposed to divalent cation-free saline solution, which is known to activate Cx HChs (but not Panx Chs), did not enhance membrane permeability, thus ruling out the possible involvement of Cx HChs. Moreover, ATP-induced membrane permeability was inhibited with blockers of P2Rs that activate Panx Chs. In addition, exogenous ATP induced the expression of myogenic commitment and increased MyoD levels, which was prevented by the inhibition of P2Rs or knockdown of Panx1 Chs. Similarly, increases in MyoD levels induced by ATP released by RCs were inhibited by Panx Ch/Cx HCh blockers. Myogenic commitment acquisition thus requires a feed-forward mechanism mediated by extracellular ATP, P2Rs, and Panx Chs. PMID:26000275

  20. Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

    Science.gov (United States)

    Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

  1. Stimulating effect of graphene oxide on myogenesis of C2C12 myoblasts on RGD peptide-decorated PLGA nanofiber matrices

    National Research Council Canada - National Science Library

    Shin, Yong Cheol; Lee, Jong Ho; Kim, Min Jeong; Hong, Suck Won; Kim, Bongju; Hyun, Jung Keun; Choi, Yu Suk; Park, Jong-Chul; Han, Dong-Wook

    2015-01-01

    .... However, their combination is intriguing and still challenging. In the present study, we fabricated nanofiber matrices composed of M13 bacteriophage with RGD peptide displayed on its surface (RGD-M13 phage) and poly...

  2. JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Yuasa, Katsutoshi; Aoki, Natsumi; Hijikata, Takao, E-mail: hijikata@musashino-u.ac.jp

    2015-08-15

    Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes. AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis. - Highlights: • JAZF1 promotes cell cycle progression and proliferation of myoblasts. • JAZF1 retards myogenic differentiation and hypertrophy of myotubes. • JAZF1 transcriptionally represses Mef2C and Mrf4 expression. • JAZF1 has an impact on the phosphorylation of AMPK.

  3. Dual roles of palladin protein in in vitro myogenesis: inhibition of early induction but promotion of myotube maturation.

    Directory of Open Access Journals (Sweden)

    Ngoc-Uyen-Nhi Nguyen

    Full Text Available Palladin is a microfilament-associated phosphoprotein whose function in skeletal muscle has rarely been studied. Therefore, we investigate whether myogenesis is influenced by the depletion of palladin expression known to interfere with the actin cytoskeleton dynamic required for skeletal muscle differentiation. The inhibition of palladin in C2C12 myoblasts leads to precocious myogenic differentiation with a concomitant reduction in cell apoptosis. This premature myogenesis is caused, in part, by an accelerated induction of p21, myogenin, and myosin heavy chain, suggesting that palladin acts as a negative regulator in early differentiation phases. Paradoxically, palladin-knockdown myoblasts are unable to differentiate terminally, despite their ability to perform some initial steps of differentiation. Cells with attenuated palladin expression form thinner myotubes with fewer myonuclei compared to those of the control. It is noteworthy that a negative regulator of myogenesis, myostatin, is activated in palladin-deficient myotubes, suggesting the palladin-mediated impairment of late-stage myogenesis. Additionally, overexpression of 140-kDa palladin inhibits myoblast differentiation while 200-kDa and 90-kDa palladin-overexpressed cells display an enhanced differentiation rate. Together, our data suggest that palladin might have both positive and negative roles in maintaining the proper skeletal myogenic differentiation in vitro.

  4. Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

    Directory of Open Access Journals (Sweden)

    Nielsen Niels

    2010-02-01

    Full Text Available Abstract Background Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. Methods Differentiated mouse myotube cultures (C2C12 were exposed to 5 mM creatine monohydrate (CMH for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH. Results The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while down-regulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the

  5. Application of direct HPTLC-MALDI for the qualitative and quantitative profiling of neutral and acidic glycosphingolipids: the case of NEU3 overexpressing C2C12 murine myoblasts.

    Science.gov (United States)

    Torretta, Enrica; Vasso, Michele; Fania, Chiara; Capitanio, Daniele; Bergante, Sonia; Piccoli, Marco; Tettamanti, Guido; Anastasia, Luigi; Gelfi, Cecilia

    2014-05-01

    Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The dynamic of lipid oxidation in human myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael

    2009-01-01

    Both endogenous and exogenous lipid levels may be regulators of total lipid oxidation in skeletal muscles. We studied the dynamics of lipid oxidation in human myotubes established from healthy, lean subjects exposed to acutely and chronically increased palmitate concentrations. The intramyocellul...... oxidation in human myotubes. A reduced exogenous lipid oxidation, secondary to increased triacylglycerol levels, may redirect free fatty acids into esterification and oxidation from intracellular stores, thereby protecting myotubes from FFA lipotoxic effects....

  7. Metabolic flexibility is conserved in diabetic myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael

    2007-01-01

    The purpose of this study was to test the hypothesis that metabolic inflexibility is an intrinsic defect. Glucose and lipid oxidation were studied in human myotubes established from healthy lean and obese subjects and patients with type 2 diabetes (T2D). In lean myotubes, glucose oxidation...... inflexibility described in obese and diabetic patients is not an intrinsic defect; rather, it is based on an extramuscular mechanism (i.e., the inability to vary extracellular fatty acid concentrations during insulin stimulation). Thus, skeletal muscles are metabolic-flexible per se....

  8. Insulin inhibits AMPK activity and phosphorylates AMPK Ser⁴⁸⁵/⁴⁹¹ through Akt in hepatocytes, myotubes and incubated rat skeletal muscle.

    Science.gov (United States)

    Valentine, Rudy J; Coughlan, Kimberly A; Ruderman, Neil B; Saha, Asish K

    2014-11-15

    Recent studies have highlighted the importance of an inhibitory phosphorylation site, Ser(485/491), on the α-subunit of AMP-activated protein kinase (AMPK); however, little is known about the regulation of this site in liver and skeletal muscle. We examined whether the inhibitory effects of insulin on AMPK activity may be mediated through the phosphorylation of this inhibitory Ser(485/491) site in hepatocytes, myotubes and incubated skeletal muscle. HepG2 and C2C12 cells were stimulated with or without insulin for 15-min. Similarly, rat extensor digitorum longus (EDL) muscles were treated +/- insulin for 10-min. Insulin significantly increased Ser(485/491) p-AMPK under all conditions, resulting in a subsequent reduction in AMPK activity, ranging from 40% to 70%, despite no change in p-AMPK Thr(172). Akt inhibition both attenuated the increase in Ser(485/491) p-AMPK caused by insulin, and prevented the decrease in AMPK activity. Similarly, the growth factor IGF-1 stimulated Ser(485/491) AMPK phosphorylation, and this too was blunted by inhibition of Akt. Inhibition of the mTOR pathway with rapamycin, however, had no effect on insulin-stimulated Ser(485/491) p-AMPK. These data suggest that insulin and IGF-1 diminish AMPK activity in hepatocytes and muscle, most likely through Akt activation and the inhibitory phosphorylation of Ser(485/491) on its α-subunit. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Static stretch promotes MEF2A nuclear translocation and expression of neonatal myosin heavy chain in C2C12 myocytes in a calcineurin- and p38-dependent manner.

    Science.gov (United States)

    Rauch, Cyril; Loughna, Paul T

    2005-03-01

    Although the effects of mechanical stimuli have been studied extensively in fully differentiated skeletal muscle and have been shown to promote changes in phenotype, including altered myosin heavy chain isoform expression, the effects of a change in mechanical environment have been poorly studied at earlier stages of skeletal muscle differentiation. In particular, the early events elicited by mechanical stimuli upon differentiating myocytes have not been investigated. In the present study, the effect of static stretch on the activation of transcriptional factors MEF2A and NFATc1, which have been shown to be involved in the differentiation and phenotype regulation of skeletal muscle, have been examined. Furthermore, putative second messenger signaling pathways that could be involved in the dephosphorylation and hence activation of these factors were also examined. We have demonstrated that static stretch application produces a robust increase in p38 phosphorylation preceding MEF2A, but not NFATc1, nuclear translocation as well as deactivation of GSK-3beta via its phosphorylation. Using SB-203580 and cyclosporine A drugs to inhibit both p38- or/and calcineurin-dependent signals, respectively, we have shown that MEF2A phosphorylation and subsequent nuclear translocation are regulated by p38 and calcineurin in a biphasic, time-dependent manner. Moreover, we also present evidence for another kinase that is involved in the stretch-related signal triggering MEF2A hyperphosphorylation, impairing its nuclear translocation, and that is related to p38. Finally, we have shown that static stretch application overnight promotes neonatal myosin heavy chain expression, which is inhibited by an inactivation of both p38 and calcineurin.

  10. Methionine Regulates mTORC1 via the T1R1/T1R3-PLC?-Ca2+-ERK1/2 Signal Transduction Process in C2C12 Cells

    OpenAIRE

    Yuanfei Zhou; Jiao Ren; Tongxing Song; Jian Peng; Hongkui Wei

    2016-01-01

    The mammalian target of rapamycin complex 1 (mTORC1) integrates amino acid (AA) availability to support protein synthesis and cell growth. Taste receptor type 1 member (T1R) is a G protein-coupled receptor that functions as a direct sensor of extracellular AA availability to regulate mTORC1 through Ca2+ stimulation and extracellular signal–regulated kinases 1 and 2 (ERK1/2) activation. However, the roles of specific AAs in T1R1/T1R3-regulated mTORC1 are poorly defined. In this study, T1R1 and...

  11. Acute myotube protein synthesis regulation by IL-6-related cytokines.

    Science.gov (United States)

    Gao, Song; Durstine, J Larry; Koh, Ho-Jin; Carver, Wayne E; Frizzell, Norma; Carson, James A

    2017-11-01

    IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis. Copyright © 2017 the

  12. Reduced TCA Flux in Diabetic Myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael

    2012-01-01

    The diabetic phenotype is complex, requiring elucidation of key initiating defects. Diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux but at present it is unclear in which part of the TCA cycle the defect is localised. In order to localise the defect we studied ATP...... production in isolated mitochondria from substrates entering the TCA cycle at various points. ATP production was measured by luminescence with or without concomitant ATP utilisation by hexokinase in mitochondria isolated from myotubes established from eight lean and eight type 2 diabetic subjects. The ATP...... production of investigated substrate combinations was significantly reduced in mitochondria isolated from type 2 diabetic subjects compared to lean. However, when ATP synthesis rates at different substrate combinations were normalized to the corresponding individual pyruvate-malate rate...

  13. Conserved regions of the DMD 3' UTR regulate translation and mRNA abundance in cultured myotubes.

    Science.gov (United States)

    Larsen, C Aaron; Howard, Michael T

    2014-08-01

    Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease, is caused by mutations in the DMD gene, which encodes for the protein dystrophin. Its regulation is of therapeutic interest as even small changes in expression of functional dystrophin can significantly impact the severity of DMD. While tissue-specific distribution and transcriptional regulation of several DMD mRNA isoforms has been well characterized, the post-transcriptional regulation of dystrophin synthesis is not well understood. Here, we utilize qRTPCR and a quantitative dual-luciferase reporter assay to examine the effects of isoform specific DMD 5' UTRs and the highly conserved DMD 3' UTR on mRNA abundance and translational control of gene expression in C2C12 cells. The 5' UTRs were shown to initiate translation with low efficiency in both myoblasts and myotubes. Whereas, two large highly conserved elements in the 3' UTR, which overlap the previously described Lemaire A and D regions, increase mRNA levels and enhance translation upon differentiation of myoblasts into myotubes. The results presented here implicate an important role for DMD UTRs in dystrophin expression and delineate the cis-acting elements required for the myotube-specific regulation of steady-state mRNA levels and translational enhancer activity found in the DMD 3' UTR. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Conserved regions of the DMD 3’ UTR regulate translation and mRNA abundance in cultured myotubes

    Science.gov (United States)

    Larsen, C. Aaron; Howard, Michael T.

    2014-01-01

    Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease, is caused by mutations in the DMD gene, which encodes for the protein dystrophin. Its regulation is of therapeutic interest as even small changes in expression of functional dystrophin can significantly impact the severity of DMD. While tissue-specific distribution and transcriptional regulation of several DMD mRNA isoforms has been well characterized, the post-transcriptional regulation of dystrophin synthesis is not well understood. Here, we utilize qRTPCR and a quantitative dual-luciferase reporter assay to examine the effects of isoform specific DMD 5’ UTRs and the highly conserved DMD 3’ UTR on mRNA abundance and translational control of gene expression in C2C12 cells. The 5’ UTRs were shown to initiate translation with low efficiency in both myoblasts and myotubes. Whereas, two large highly conserved elements in the 3’ UTR, which overlap the previously described Lemaire A and D regions, increase mRNA levels and enhance translation upon differentiation of myoblasts into myotubes. The results presented here implicate an important role for DMD UTRs in dystrophin expression and delineate the cis-acting elements required for the myotube-specific regulation of steady-state mRNA levels and translational enhancer activity found in the DMD 3’ UTR. PMID:24928536

  15. 6-Paradol and 6-Shogaol, the Pungent Compounds of Ginger, Promote Glucose Utilization in Adipocytes and Myotubes, and 6-Paradol Reduces Blood Glucose in High-Fat Diet-Fed Mice

    Directory of Open Access Journals (Sweden)

    Chien-Kei Wei

    2017-01-01

    Full Text Available The anti-diabetic activity of ginger powder (Zingiber officinale has been recently promoted, with the recommendation to be included as one of the dietary supplements for diabetic patients. However, previous studies presented different results, which may be caused by degradation and metabolic changes of ginger components, gingerols, shogaols and paradols. Therefore, we prepared 10 ginger active components, namely 6-, 8-, 10-paradols, 6-, 8-, 10-shogaols, 6-, 8-, 10-gingerols and zingerone, and evaluated their anti-hyperglycemic activity. Among the tested compounds, 6-paradol and 6-shogaol showed potent activity in stimulating glucose utilization by 3T3-L1 adipocytes and C2C12 myotubes. The effects were attributed to the increase in 5′ adenosine monophosphate-activated protein kinase (AMPK phosphorylation in 3T3-L1 adipocytes. 6-Paradol, the major metabolite of 6-shogaol, was utilized in an in vivo assay and significantly reduced blood glucose, cholesterol and body weight in high-fat diet-fed mice.

  16. Merosin and laminin in myogenesis; specific requirement for merosin in myotube stability and survival

    DEFF Research Database (Denmark)

    Vachon, P H; Loechel, F; Xu, H

    1996-01-01

    or expression are the causes of some types of congenital muscular dystrophy. However, the precise nature of the functions of merosin in muscle remain unknown. We have developed an in vitro system that exploits human RD and mouse C2C12 myoblastic cell lines and their clonal variants to study the roles of merosin...... in congenital muscular dystrophy caused by merosin deficiency and provide in vitro models to further investigate the molecular mechanisms of this disease....

  17. Skeletal myotube formation enhanced by electrospun polyurethane carbon nanotube scaffolds

    Science.gov (United States)

    Sirivisoot, Sirinrath; Harrison, Benjamin S

    2011-01-01

    Background This study examined the effects of electrically conductive materials made from electrospun single- or multiwalled carbon nanotubes with polyurethane to promote myoblast differentiation into myotubes in the presence and absence of electrical stimulation. Methods and results After electrical stimulation, the number of multinucleated myotubes on the electrospun polyurethane carbon nanotube scaffolds was significantly larger than that on nonconductive electrospun polyurethane scaffolds (5% and 10% w/v polyurethane). In the absence of electrical stimulation, myoblasts also differentiated on the electrospun polyurethane carbon nanotube scaffolds, as evidenced by expression of Myf-5 and myosin heavy chains. The myotube number and length were significantly greater on the electrospun carbon nanotubes with 10% w/v polyurethane than on those with 5% w/v polyurethane. The results suggest that, in the absence of electrical stimulation, skeletal myotube formation is dependent on the morphology of the electrospun scaffolds, while with electrical stimulation it is dependent on the electrical conductivity of the scaffolds. Conclusion This study indicates that electrospun polyurethane carbon nanotubes can be used to modulate skeletal myotube formation with or without application of electrical stimulation. PMID:22072883

  18. Taurine Rescues Cisplatin-Induced Muscle Atrophy In Vitro: A Morphological Study

    Directory of Open Access Journals (Sweden)

    Alessandra Stacchiotti

    2014-01-01

    Full Text Available Cisplatin (CisPt is a widely used chemotherapeutic drug whose side effects include muscle weakness and cachexia. Here we analysed CisPt-induced atrophy in C2C12 myotubes by a multidisciplinary morphological approach, focusing on the onset and progression of autophagy, a protective cellular process that, when excessively activated, may trigger protein hypercatabolism and atrophy in skeletal muscle. To visualize autophagy we used confocal and transmission electron microscopy at different times of treatment and doses of CisPt. Moreover we evaluated the effects of taurine, a cytoprotective beta-amino acid able to counteract oxidative stress, apoptosis, and endoplasmic reticulum stress in different tissues and organs. Our microscopic results indicate that autophagy occurs very early in 50 μM CisPt challenged myotubes (4 h–8 h before overt atrophy but it persists even at 24 h, when several autophagic vesicles, damaged mitochondria, and sarcoplasmic blebbings engulf the sarcoplasm. Differently, 25 mM taurine pretreatment rescues the majority of myotubes size upon 50 μM CisPt at 24 h. Taurine appears to counteract atrophy by restoring regular microtubular apparatus and mitochondria and reducing the overload and the localization of autophagolysosomes. Such a promising taurine action in preventing atrophy needs further molecular and biochemical studies to best define its impact on muscle homeostasis and the maintenance of an adequate skeletal mass in vivo.

  19. Coculture of rat embryonic proprioceptive sensory neurons and myotubes

    NARCIS (Netherlands)

    Copray, S; Liem, R; MantinghOtter, [No Value; Brouwer, N

    1996-01-01

    With the aim to study the cellular mechanisms underlying the process of muscle spindle (re)generation, dorsal root ganglia (DRG) neurons derived from 16-day rat embryos were cocultured with developing myotubes in a compartmentalized culture device. To accomplish the selective survival and neurite

  20. Expression profiling of insulin action in human myotubes

    DEFF Research Database (Denmark)

    Hansen, Lars; Gaster, Michael; Oakeley, Edward J

    2004-01-01

    Myotube cultures from patients with type 2 diabetes mellitus (T2DM) represent an experimental in vitro model of T2DM that offers a possibility to perform gene expression studies under standardized conditions. During a time-course of insulin stimulation (1 microM) at 5.5 mM glucose for 0 (no insul...

  1. Expressed sequence tags for bovine muscle satellite cells, myotube formed-cells and adipocyte-like cells.

    Directory of Open Access Journals (Sweden)

    Eun Ju Lee

    Full Text Available BACKGROUND: Muscle satellite cells (MSCs represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs and adipocyte-like cells (ALCs, we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs. RESULTS: A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248 highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2 during myogenesis and hemoglobin subunit alpha2 (HBA2 during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay. CONCLUSION: In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to

  2. l-glutamine Improves Skeletal Muscle Cell Differentiation and Prevents Myotube Atrophy After Cytokine (TNF-α) Stress Via Reduced p38 MAPK Signal Transduction.

    Science.gov (United States)

    Girven, Matthew; Dugdale, Hannah F; Owens, Daniel J; Hughes, David C; Stewart, Claire E; Sharples, Adam P

    2016-12-01

    Tumour Necrosis Factor-Alpha (TNF-α) is chronically elevated in conditions where skeletal muscle loss occurs. As l-glutamine can dampen the effects of inflamed environments, we investigated the role of l-glutamine in both differentiating C2C12 myoblasts and existing myotubes in the absence/presence of TNF-α (20 ng · ml(-1) ) ± l-glutamine (20 mM). TNF-α reduced the proportion of cells in G1 phase, as well as biochemical (CK activity) and morphological differentiation (myotube number), with corresponding reductions in transcript expression of: Myogenin, Igf-I, and Igfbp5. Furthermore, when administered to mature myotubes, TNF-α induced myotube loss and atrophy underpinned by reductions in Myogenin, Igf-I, Igfbp2, and glutamine synthetase and parallel increases in Fox03, Cfos, p53, and Bid gene expression. Investigation of signaling activity suggested that Akt and ERK1/2 were unchanged, JNK increased (non-significantly) whereas P38 MAPK substantially and significantly increased in both myoblasts and myotubes in the presence of TNF-α. Importantly, 20 mM l-glutamine reduced p38 MAPK activity in TNF-α conditions back to control levels, with a corresponding rescue of myoblast differentiation and a reversal of atrophy in myotubes. l-glutamine resulted in upregulation of genes associated with growth and survival including; Myogenin, Igf-Ir, Myhc2 & 7, Tnfsfr1b, Adra1d, and restored atrophic gene expression of Fox03 back to baseline in TNF-α conditions. In conclusion, l-glutamine supplementation rescued suppressed muscle cell differentiation and prevented myotube atrophy in an inflamed environment via regulation of p38 MAPK. l-glutamine administration could represent an important therapeutic strategy for reducing muscle loss in catabolic diseases and inflamed ageing. J. Cell. Physiol. 9999: 231: 2720-2732, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. Kinetics of lactate and pyruvate transport in cultured rat myotubes

    DEFF Research Database (Denmark)

    von Grumbckow, Lena; Elsner, Peter; Hellsten, Ylva

    1999-01-01

    Skeletal muscle transport of lactate and pyruvate was studied in primary cultures of rat myotubes, applying the pH-sensitive fluorescent indicator 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The initial rate of decrease in intracellular pH (pHi) upon lactate or pyruvate incubation was used......, respectively. Furthermore, it was observed that the two monocarboxylate transporter isoforms present in mature skeletal muscles, MCT1 and MCT4 (formerly called MCT3 (M.C. Wilson, V.N. Jackson, C. Heddle, N.T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O.F. Hutter, A.P. Halestrap, Lactic acid efflux...... from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes....

  4. Reduced TCA Flux in Diabetic Myotubes: Determined by Single Defects?

    Science.gov (United States)

    Gaster, Michael

    2012-01-01

    The diabetic phenotype is complex, requiring elucidation of key initiating defects. Diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux but at present it is unclear in which part of the TCA cycle the defect is localised. In order to localise the defect we studied ATP production in isolated mitochondria from substrates entering the TCA cycle at various points. ATP production was measured by luminescence with or without concomitant ATP utilisation by hexokinase in mitochondria isolated from myotubes established from eight lean and eight type 2 diabetic subjects. The ATP production of investigated substrate combinations was significantly reduced in mitochondria isolated from type 2 diabetic subjects compared to lean. However, when ATP synthesis rates at different substrate combinations were normalized to the corresponding individual pyruvate-malate rate, there was no significant difference between groups. These results show that the primary reduced TCA cycle flux in diabetic myotubes is not explained by defects in specific part of the TCA cycle but rather results from a general downregulation of the TCA cycle.

  5. Distinct Roles of Different Presynaptic and Postsynaptic NCAM Isoforms in Early Motoneuron-Myotube Interactions Required for Functional Synapse Formation.

    Science.gov (United States)

    Hata, Katsusuke; Maeno-Hikichi, Yuka; Yumoto, Norihiro; Burden, Steven J; Landmesser, Lynn T

    2018-01-10

    The neural cell adhesion molecule (NCAM) is expressed both presynaptically and postsynaptically during neuromuscular junction formation. Genetic deletion in mice of all three isoforms (180, 140, and 120 kDa), or just the 180 isoform, suggested that different isoforms played distinct roles in synaptic maturation. Here we characterized in mice of either sex the earliest adhesive contacts between the growth cones of motoneurons and myotubes and their subsequent maturation into functional synapses in cocultures of motoneurons and myotubes, which expressed their normal complement of NCAM isoforms, or were lacking all isoforms either presynaptically or postsynaptically. Growth cone contact with +/+ mouse myotubes resulted in immediate adhesive contacts and the rapid downregulation of growth cone motility. When contacting NCAM -/- myotubes, growth cones touched and retracted/collapsed multiple times and failed to form stable contacts, even after 10 h. Exogenous expression in myotubes of either the 180 or 140 isoform, but not the 120 kDa isoform, rescued the rapid formation of stable contacts, the accumulation of presynaptic and postsynaptic molecules, and functional transmission. When NCAM was absent only in motoneurons, growth cones did not retract upon myotube contact, but, since their motility was not downregulated, they grew off the ends of the myotubes, failing to form synapses. The agrin receptor Lrp4 was strongly downregulated in NCAM-negative myotubes, and motoneuron growth cones did not make stable contacts with Lrp4-negative myotubes. These studies have identified novel roles for presynaptic and postsynaptic NCAM in mediating early cell-cell interactions required for synapse formation. SIGNIFICANCE STATEMENT Although many molecular signals needed to form the functionally effective neuromuscular synapses required for normal movement have been described, the earliest signals that let motoneuron growth cones make stable adhesive contacts with myotubes and cease

  6. Lipid in Skeletal Muscle Myotubes is associated to the Donors’ Insulin Sensitivity and Physical Activity Phenotypes

    Science.gov (United States)

    Bajpeyi, Sudip; Myrland, Cassandra K.; Covington, Jeffrey D.; Obanda, Diana; Cefalu, William T.; Smith, Steven R.; Rustan, Arild C.; Ravussin, Eric

    2013-01-01

    Objective This study investigated the relationship between in-vitro lipid content in myotubes and in-vivo whole body phenotypes of the donors such as insulin sensitivity, intramyocellular lipids (IMCL), physical activity and oxidative capacity. Design and Methods Six physically active donors were compared to 6 sedentary lean and 6 T2DM. Lipid content was measured in tissues and myotubes by immunohistochemistry. Ceramides, triacylglycerols (TAGs) and diacylglycerols (DAGs) were measured by LC-MS-MS and GC-FID. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (80mU/min/m2), maximal mitochondrial capacity (ATPmax) by 31P-MRS, physical fitness by VO2max and physical activity level (PAL) by accelerometers. Results Myotubes cultured from physically active donors had higher lipid content (0.047±0.003 vs. 0.032±0.001 and 0.033±0.001AU; p<0.001) than myotubes from lean and T2DM donors. Lipid content in myotubes was not associated with IMCL in muscle tissue but importantly, correlated with in-vivo measures of ATPmax (r=0.74; p<0.001), insulin sensitivity (r=0.54; p<0.05), type-I fibers (r=0.50; p<0.05) and PAL (r=0.92; p<0.0001). DAGs and ceramides in myotubes were inversely associated with insulin sensitivity (r=−0.55, r=−0.73; p<0.05) and ATPmax (r=−0.74, r=−0.85; p<0.01). Conclusions These results indicate that cultured human myotubes can be used in mechanistic studies to study the in vitro impact of interventions on phenotypes such as mitochondrial capacity, insulin sensitivity, and physical activity. PMID:23818429

  7. Exercise in vivo marks human myotubes in vitro: Training-induced increase in lipid metabolism.

    Directory of Open Access Journals (Sweden)

    Jenny Lund

    Full Text Available Physical activity has preventive as well as therapeutic benefits for overweight subjects. In this study we aimed to examine effects of in vivo exercise on in vitro metabolic adaptations by studying energy metabolism in cultured myotubes isolated from biopsies taken before and after 12 weeks of extensive endurance and strength training, from healthy sedentary normal weight and overweight men.Healthy sedentary men, aged 40-62 years, with normal weight (body mass index (BMI < 25 kg/m2 or overweight (BMI ≥ 25 kg/m2 were included. Fatty acid and glucose metabolism were studied in myotubes using [14C]oleic acid and [14C]glucose, respectively. Gene and protein expressions, as well as DNA methylation were measured for selected genes.The 12-week training intervention improved endurance, strength and insulin sensitivity in vivo, and reduced the participants' body weight. Biopsy-derived cultured human myotubes after exercise showed increased total cellular oleic acid uptake (30%, oxidation (46% and lipid accumulation (34%, as well as increased fractional glucose oxidation (14% compared to cultures established prior to exercise. Most of these exercise-induced increases were significant in the overweight group, whereas the normal weight group showed no change in oleic acid or glucose metabolism.12 weeks of combined endurance and strength training promoted increased lipid and glucose metabolism in biopsy-derived cultured human myotubes, showing that training in vivo are able to induce changes in human myotubes that are discernible in vitro.

  8. Insulin resistance and the mitochondrial link. Lessons from cultured human myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael

    2007-01-01

    In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects and exposed to mitochondrial inhibitors, either affecting the electron transport chain...... lipid uptake. The metabolic phenotype during respiratory uncoupling resembled the above picture, except for an increase in glucose and palmitate oxidation. Antimycin A and oligomycin treatment induced insulin resistance at the level of glucose and palmitate uptake in all three study groups while......, at the level of glycogen synthesis, insulin resistance was only seen in lean myotubes. Primary insulin resistance in diabetic myotubes was significantly worsened at the level of glucose and lipid uptake. The present study is the first convincing data linking functional mitochondrial impairment per se...

  9. Park7 expression influences myotube size and myosin expression in muscle.

    Directory of Open Access Journals (Sweden)

    Hui Yu

    Full Text Available Callipyge sheep exhibit postnatal muscle hypertrophy due to the up-regulation of DLK1 and/or RTL1. The up-regulation of PARK7 was identified in hypertrophied muscles by microarray analysis and further validated by quantitative PCR. The expression of PARK7 in hypertrophied muscle of callipyge lambs was confirmed to be up-regulated at the protein level. PARK7 was previously identified to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The purpose of this study was to investigate the effects of PARK7 in muscle growth and protein accretion in response to IGF1. Primary myoblasts isolated from Park7 (+/+ and Park7 (-/- mice were used to examine the effect of differential expression of Park7. The Park7 (+/+ myotubes had significantly larger diameters and more total sarcomeric myosin expression than Park7 (-/- myotubes. IGF1 treatment increased the mRNA abundance of Myh4, Myh7 and Myh8 between 20-40% in Park7 (+/+ myotubes relative to Park7 (-/-. The level of AKT phosphorylation was increased in Park7 (+/+ myotubes at all levels of IGF1 supplementation. After removal of IGF1, the Park7 (+/+ myotubes maintained higher AKT phosphorylation through 3 hours. PARK7 positively regulates the PI3K/AKT pathway by inhibition of PTEN phosphatase activity in skeletal muscle. The increased PARK7 expression can increase protein synthesis and result in myotube hypertrophy. These results support the hypothesis that elevated expression of PARK7 in callipyge muscle would increase levels of AKT activity to cause hypertrophy in response to the normal IGF1 signaling in rapidly growing lambs. Increasing expression of PARK7 could be a novel mechanism to increase protein accretion and muscle growth in livestock or help improve muscle mass with disease or aging.

  10. Neonatal Satellite Cells Form Small Myotubes In Vitro.

    Science.gov (United States)

    Carvajal Monroy, P L; Grefte, S; Kuijpers-Jagtman, A M; Von den Hoff, J W; Wagener, F A D T G

    2017-03-01

    Although palatal muscle reconstruction in patients with cleft palate takes place during early childhood, normal speech development is often not achieved. We hypothesized that the intrinsic properties of head satellite cells (SCs) and the young age of these patients contribute to the poor muscle regeneration after surgery. First, we studied the fiber type distribution and the expression of SC markers in ex vivo muscle tissue from head (branchiomeric) and limb (somite-derived) muscles from neonatal (2-wk-old) and young (9-wk-old) rats. Next, we cultured SCs isolated from these muscles for 5, 7, and 9 d, and investigated the in vitro expression of SC markers, as well as changes in proliferation, early differentiation, and fusion index (myotube formation) in these cells. In our ex vivo samples, we found that virtually all myofibers in both the masseter (Mass) and the levator veli palatini (LVP) muscles contained fast myosin heavy chain (MyHC), and a small percentage of digastric (Dig) and extensor digitorum longus myofibers also contained slow MyHC. This was independent of age. More SCs were found in muscles from neonatal rats as compared with young rats [17.6 (3.8%) v. 2.3 (1.6%); P form myotubes less efficiently than those from young muscles. These age-dependent differences in stem cell properties urge careful consideration for future clinical applications in patients with cleft palate.

  11. Testosterone enables growth and hypertrophy in fusion impaired myoblasts that display myotube atrophy: deciphering the role of androgen and IGF-I receptors.

    Science.gov (United States)

    Hughes, David C; Stewart, Claire E; Sculthorpe, Nicholas; Dugdale, Hannah F; Yousefian, Farzad; Lewis, Mark P; Sharples, Adam P

    2016-06-01

    We have previously highlighted the ability of testosterone (T) to improve differentiation and myotube hypertrophy in fusion impaired myoblasts that display reduced myotube hypertrophy via multiple population doublings (PD) versus their parental controls (CON); an observation which is abrogated via PI3K/Akt inhibition (Deane et al. 2013). However, whether the most predominant molecular mechanism responsible for T induced hypertrophy occurs directly via androgen receptor or indirectly via IGF-IR/PI3K/Akt pathway is currently debated. PD and CON C2C12 muscle cells were exposed to low serum conditions in the presence or absence of T (100 nM) ± inhibitors of AR (flutamide/F, 40 μm) and IGF-IR (picropodophyllin/PPP, 150 nM) for 72 h and 7 days (early/late muscle differentiation respectively). T increased AR and Akt abundance, myogenin gene expression, and myotube hypertrophy, but not ERK1/2 activity in both CON and PD cell types. Akt activity was not increased significantly in either cell type with T. Testosterone was also unable to promote early differentiation in the presence of IGF-IR inhibitor (PPP) yet still able to promote appropriate later increases in myotube hypertrophy and AR abundance despite IGF-IR inhibition. The addition of the AR inhibitor powerfully attenuated all T induced increases in differentiation and myotube hypertrophy with corresponding reductions in AR abundance, phosphorylated Akt, ERK1/2 and gene expression of IGF-IR, myoD and myogenin with increases in myostatin mRNA in both cell types. Interestingly, despite basally reduced differentiation and myotube hypertrophy, PD cells showed larger T induced increases in AR abundance vs. CON cells, a response abrogated in the presence of AR but not IGF-IR inhibitors. Furthermore, T induced increases in Akt abundance were sustained despite the presence of IGF-IR inhibition in PD cells only. Importantly, flutamide alone reduced IGF-IR mRNA in both cell types across time points, with an observed

  12. Black ginseng activates Akt signaling, thereby enhancing myoblast differentiation and myotube growth

    Directory of Open Access Journals (Sweden)

    Soo-Yeon Lee

    2018-01-01

    Conclusion: BG enhances myoblast differentiation and myotube hypertrophy by activating Akt/mTOR/p70S6k axis. Thus, our study demonstrates that BG has promising potential to treat or prevent muscle loss related to aging or other pathological conditions, such as diabetes.

  13. Triacylglycerol Accumulation is not primarily affected in Myotubes established from Type 2 Diabetic Subjects

    DEFF Research Database (Denmark)

    Gaster, Michael; Beck-Nielsen, Henning

    2006-01-01

    In the present study, we investigated triacylglycerol (TAG) accumulation, glucose and fatty acid (FA) uptake, and glycogen synthesis (GS) in human myotubes from healthy, lean, and obese subjects with and without type 2 diabetes (T2D), exposed to increasing palmitate (PA) and oleate (OA) concentra...

  14. Metabolic switching of human myotubes is improved by n-3 fatty acids

    NARCIS (Netherlands)

    Hessvik, N.P.; Bakke, S.S.; Fredriksson, K.; Boekschoten, M.V.; Fjorkenstad, A.; Koster, G.; Hesselink, M.K.; Kersten, A.H.; Kase, E.T.; Rustan, A.C.; Thoresen, G.H.

    2010-01-01

    The aim of the present study was to examine whether pretreatment with different fatty acids, as well as the liver X receptor (LXR) agonist T0901317, could modify metabolic switching of human myotubes. The n-3 FA eicosapentaenoic acid (EPA) increased suppressibility, the ability of glucose to

  15. A role for phospholipase D3 in myotube formation.

    Directory of Open Access Journals (Sweden)

    Mary Osisami

    Full Text Available Phospholipase D3 (PLD3 is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.

  16. Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jaemin, E-mail: jmj1103@kirams.re.kr [Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720-1762 (United States); Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Conboy, Irina M., E-mail: iconboy@berkeley.edu [Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720-1762 (United States)

    2011-10-14

    Highlights: {yields} PS broadly and persistently trans-locates to the outer leaflet of plasma membrane during myoblast fusion into myotubes. {yields} Robust myotubes are formed when PS liposomes are added exogenously. {yields} PS increases the width of de novo myotubes and the numbers of myonuclei, but not the myotube length. {yields} Annexin V or PS antibody inhibits myotube formation by masking exposed PS. -- Abstract: Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell-cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.

  17. A composite catalyst and its utilization for aromatization of C2-C12 hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Alario, F.; Benazzi, E.; Deves, J.M.; Joly, J.F.

    1994-09-02

    A composite catalyst is proposed for aromatization reactions of hydrocarbons containing from 2 to 12 carbon atoms per molecule, and more especially from 3 to 10 C atoms per molecule; the catalyst contains: a MFI structure zeolite which skeleton is composed of silicon, aluminium and/or gallium and which crystallites external faces have been modified after synthesis; a matrix; at least one doping element chosen among gallium and zinc when the zeolite does not contain gallium in its crystal skeleton; eventually, at least one doping element chosen among gallium and zinc when the zeolite contains gallium in its skeleton; and eventually, at least one element chosen among alkaline and alkaline-earth metals. 2 tabs.

  18. The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome

    OpenAIRE

    Gundry, Rebekah L.; Raginski, Kimberly; Tarasova, Yelena; Tchernyshyov, Irina; Bausch-Fluck, Damaris; Elliott, Steven T.; Boheler, Kenneth R.; Van Eyk, Jennifer E.; Wollscheid, Bernd

    2009-01-01

    Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing ...

  19. Gold Nanoshell-Mediated Remote Myotube Activation.

    Science.gov (United States)

    Marino, Attilio; Arai, Satoshi; Hou, Yanyan; Degl'Innocenti, Andrea; Cappello, Valentina; Mazzolai, Barbara; Chang, Young-Tae; Mattoli, Virgilio; Suzuki, Madoka; Ciofani, Gianni

    2017-03-28

    Mild heat stimulation of muscle cells within the physiological range represents an intriguing approach for the modulation of their functions. In this work, photothermal conversion was exploited to remotely stimulate striated muscle cells by using gold nanoshells (NSs) in combination with near-infrared (NIR) radiation. Temperature increments of approximately 5 °C were recorded by using an intracellular fluorescent molecular thermometer and were demonstrated to efficiently induce myotube contraction. The mechanism at the base of this phenomenon was thoroughly investigated and was observed to be a Ca2+-independent event directly involving actin-myosin interactions. Finally, chronic remote photothermal stimulations significantly increased the mRNA transcription of genes encoding heat shock proteins and sirtuin 1, a protein which in turn can induce mitochondrial biogenesis. Overall, we provide evidence that remote NIR + NS muscle excitation represents an effective wireless stimulation technique with great potential in the fields of muscle tissue engineering, regenerative medicine, and bionics.

  20. Simvastatin Inhibits Glucose Metabolism and Legumain Activity in Human Myotubes

    Science.gov (United States)

    Smith, Robert; Solberg, Rigmor; Jacobsen, Linn Løkken; Voreland, Anette Larsen; Rustan, Arild Christian; Thoresen, G. Hege; Johansen, Harald Thidemann

    2014-01-01

    Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the Musculus obliquus internus abdominis of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with [14C]glucose or [3H]deoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of legumain. Dysregulation of glucose metabolism and decreased legumain activity by simvastatin points out new knowledge about the effects of statins on skeletal muscle, and may contribute to the understanding of the myotoxicity observed by statins. PMID:24416446

  1. (S)-[6]-Gingerol enhances glucose uptake in L6 myotubes by activation of AMPK in response to [Ca2+]i.

    Science.gov (United States)

    Li, Yiming; Tran, Van H; Koolaji, Nooshin; Duke, Colin; Roufogalis, Basil D

    2013-01-01

    The aim of this study was to investigate the mechanism of (S)-[6]-gingerol in promoting glucose uptake in L6 skeletal muscle cells. The effect of (S)-[6]-gingerol on glucose uptake in L6 myotubes was examined using 2-[1,2-3H]-deoxy-D-glucose. Intracellular Ca2+ concentration was measured using Fluo-4. Phosphorylation of AMPKα was determined by Western blotting analysis. (S)-[6]-Gingerol time-dependently enhanced glucose uptake in L6 myotubes. (S)-[6]-Gingerol elevated intracellular Ca2+ concentration and subsequently induced a dose- and time-dependent enhancement of threonine172 phosphorylated AMPKα in L6 myotubes via modulation by Ca2+/calmodulin-dependent protein kinase kinase. The results indicated that (S)-[6]-gingerol increased glucose uptake in L6 skeletal muscle cells by activating AMPK. (S)-[6]-gingerol, a major component of Zingiber officinale, may have potential for development as an antidiabetic agent.

  2. Negative regulation of glucose metabolism in human myotubes by supraphysiological doses of 17β-estradiol or testosterone.

    Science.gov (United States)

    Garrido, Pablo; Salehzadeh, Firoozeh; Duque-Guimaraes, Daniella E; Al-Khalili, Lubna

    2014-09-01

    Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle. Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined. Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17β-estradiol or testosterone augmented phosphorylation GSK3β(Ser9) and PKCδ(Thr505), two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment. An acute exposure to supraphysiological doses of either 17β-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Exercise in vivo marks human myotubes in vitro: Training-induced increase in lipid metabolism

    Science.gov (United States)

    Lund, Jenny; Mudry, Jonathan M.; Langleite, Torgrim M.; Feng, Yuan Z.; Stensrud, Camilla; Brubak, Mari G.; Drevon, Christian A.; Birkeland, Kåre I.; Kolnes, Kristoffer J.; Johansen, Egil I.; Tangen, Daniel S.; Stadheim, Hans K.; Gulseth, Hanne L.; Krook, Anna; Kase, Eili T.; Jensen, Jørgen; Thoresen, G. Hege

    2017-01-01

    Background and aims Physical activity has preventive as well as therapeutic benefits for overweight subjects. In this study we aimed to examine effects of in vivo exercise on in vitro metabolic adaptations by studying energy metabolism in cultured myotubes isolated from biopsies taken before and after 12 weeks of extensive endurance and strength training, from healthy sedentary normal weight and overweight men. Methods Healthy sedentary men, aged 40–62 years, with normal weight (body mass index (BMI) exercise showed increased total cellular oleic acid uptake (30%), oxidation (46%) and lipid accumulation (34%), as well as increased fractional glucose oxidation (14%) compared to cultures established prior to exercise. Most of these exercise-induced increases were significant in the overweight group, whereas the normal weight group showed no change in oleic acid or glucose metabolism. Conclusions 12 weeks of combined endurance and strength training promoted increased lipid and glucose metabolism in biopsy-derived cultured human myotubes, showing that training in vivo are able to induce changes in human myotubes that are discernible in vitro. PMID:28403174

  4. Dissociation of charge movement from calcium release and calcium current in skeletal myotubes by gabapentin.

    Science.gov (United States)

    Alden, Kris J; García, Jesús

    2002-09-01

    The skeletal muscle L-type calcium channel or dihydropyridine receptor (DHPR) plays an integral role in excitation-contraction (E-C) coupling. Its activation initiates three sequential events: charge movement (Q(r)), calcium release, and calcium current (I(Ca,L)). This relationship suggests that changes in Q(r) might affect release and I(Ca,L). Here we studied the effect of gabapentin (GBP) on the three events generated by DHPRs in skeletal myotubes in culture. GBP specifically binds to the alpha(2)/delta(1) subunit of the brain and skeletal muscle DHPR. Myotubes were stimulated with a protocol that included a depolarizing prepulse to inactivate voltage-dependent proteins other than DHPRs. Gabapentin (50 microM) significantly increased Q(r) while decreasing the rate of rise of calcium transients. Gabapentin also reduced the maximum amplitude of the I(Ca,L) (as we previously reported) without modifying the kinetics of activation. Exposure of GBP-treated myotubes to 10 microM nifedipine prevented the increase of Q(r) promoted by this drug, indicating that the extra charge recorded originated from DHPRs. Our data suggest that GBP dissociates the functions of the DHPR from the initial voltage-sensing step and implicates a role for the alpha(2)/delta(1) subunit in E-C coupling.

  5. File list: Oth.Myo.20.AllAg.Myotube [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  16. Transciptional profiling of myotubes from patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Frederiksen, CM; Højlund, K; Hansen, L

    2008-01-01

    previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes. RESULTS: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either....... It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes. METHODS: Using cDNA microarray technology and global pathway analysis with the Gene Map Annotator...... and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have...

  17. Pharmacologic characterization of the Na+ ionophores in L6 myotubes.

    Science.gov (United States)

    Sastre, A; Podleski, T R

    1976-01-01

    We present a pharmacologic characterization of the Na+ ionophores present in L6 myotubes in vitro. Action potentials are abolished by replacement of the external Na+ by Tris. The amplitude of the action potential is generally resistant to high concentrations of tetrodotoxin (10(-5) M) and saxitoxin (10(-6 M), but the effect of these agents is highly variable. Veratridine (10(-4 M) consistently induces, as a short-term effect, a marked prolongation of the falling phase of the action potential. As a long-term effect, veratridine consistently induces a Na+-dependent reduction in the resting potential of the cell. The effects of veratridine on the action potential are not antagonized by tetrodotoxin or saxitoxin. However, the effects of veratridine on the resting potential are strongly antagonized by tetrodotoxin (10(-5) M) and fully inhibited by saxitoxin (10(-6) M). Significantly, under conditions where saxitoxin has fully inhibited the effects of veratridine on the resting potential, the myotubes are capable of generating overshooting action potentials. In contrast to their sensitivity to veratridine, L6 myotubes are insensitive to 10(-5) M alpha-dihydro-grayanotoxin-II. These results are discussed in the contexts of developmental significance and current views about Na+ ionophores. PMID:1063416

  18. Reduced lipid oxidation in myotubes established from obese and type 2 diabetic subjects

    DEFF Research Database (Denmark)

    Gaster, Michael

    2009-01-01

    To date, it is unknown whether reduced lipid oxidation of skeletal muscle of obese and obese type 2 diabetic (T2D) subjects partly is based on reduced oxidation of endogenous lipids. Palmitate (PA) accumulation, total oxidation and lipolysis were not different between myotubes established from lean......, obese and T2D subjects, chronic exposed for PA. Complete oxidation from endogenous PA was reduced in diabetic and obese compared to lean myotubes while exogenous PA oxidation was reduced in diabetic compared to lean myotubes. The complete/incomplete ratio was significantly reduced in diabetic myotubes...... both for endogenous and exogenous lipids. Thus myotubes established from obese and obese T2D subjects express a reduced complete oxidation of endogenous lipids. Two cardinal principles govern the reduced lipid oxidation in obese and diabetic myotubes; firstly, an impaired coupling between endogenous...

  19. Alternative splicing alterations of Ca2+ handling genes are associated with Ca2+ signal dysregulation in myotonic dystrophy type 1 (DM1) and type 2 (DM2) myotubes.

    Science.gov (United States)

    Santoro, Massimo; Piacentini, Roberto; Masciullo, Marcella; Bianchi, Maria Laura Ester; Modoni, Anna; Podda, Maria Vittoria; Ricci, Enzo; Silvestri, Gabriella; Grassi, Claudio

    2014-06-01

    The pathogenesis of myotonic dystrophy type 1 (DM1) and type 2 (DM2) has been related to the aberrant splicing of several genes, including those encoding for ryanodine receptor 1 (RYR1), sarcoplasmatic/endoplasmatic Ca(2+)-ATPase (SERCA) and α1S subunit of voltage-gated Ca(2+) channels (Cav 1.1). The aim of this study is to determine whether alterations of these genes are associated with changes in the regulation of intracellular Ca(2+) homeostasis and signalling. We analysed the expression of RYR1, SERCA and Cav 1.1 and the intracellular Ca(2+) handling in cultured myotubes isolated from DM1, DM2 and control muscle biopsies by semiquantitative RT-PCR and confocal Ca(2+) imaging respectively. (i) The alternative splicing of RYR1, SERCA and Cav 1.1 was more severely affected in DM1 than in DM2 myotubes; (ii) DM1 myotubes exhibited higher resting intracellular Ca(2+) levels than DM2; (iii) the amplitude of intracellular Ca(2+) transients induced by sustained membrane depolarization was higher in DM1 myotubes than in controls, whereas DM2 showed opposite behaviour; and (iv) in both DM myotubes, Ca(2+) release from sarcoplasmic reticulum through RYR1 was lower than in controls. The aberrant splicing of RYR1, SERCA1 and Cav 1.1 may alter intracellular Ca(2+) signalling in DM1 and DM2 myotubes. The differing dysregulation of intracellular Ca(2+) handling in DM1 and DM2 may explain their distinct sarcolemmal hyperexcitabilities. © 2013 British Neuropathological Society.

  20. In Vitro Palmitate Treatment of Myotubes from Postmenopausal Women Leads to Ceramide Accumulation, Inflammation and Affected Insulin Signaling

    DEFF Research Database (Denmark)

    Abildgaard, Julie; Henstridge, Darren C; Pedersen, Anette Tønnes

    2014-01-01

    treatment led to a 108% (CI 95%: 50%; 267%) increase in intramyocellular ceramide in the myotubes from the postmenopausal women (post-myotubes) compared with a 26% (CI 95%: -57%; 96%) increase in myotubes from the premenopausal women (pre-myotubes), (ppost-myotubes had a 22% (CI 95%: 4......, post-myotubes showed a blunted insulin stimulated phosphorylation of AS160 in response to chronic palmitate treatment compared with pre-myotubes (p = 0.02). The increased intramyocellular ceramide content in the post-myotubes was associated with a significantly higher mRNA expression of Serine...... Palmitoyltransferase1 (SPT1) after one day of palmitate treatment (p = 0.03) in post-myotubes compared with pre-myotubes. Our findings indicate that post-myotubes are more prone to develop lipid accumulation and defective insulin signaling following chronic saturated fatty acid exposure as compared to pre-myotubes....

  1. Clonal derivation of a rat muscle cell strain that forms contraction-competent myotubes.

    Science.gov (United States)

    Merrill, G F

    1989-05-01

    A muscle cell strain capable of forming contracting myotubes was isolated from an established rat embryo cell line. The myogenic cells, termed rat myoblast omega or RMo cells, have a diploid complement of chromosomes (n = 42). In the presence of mitogen-containing growth medium, RMo cells proliferated with a cell generation time of about 12 hours. In mitogen-depleted medium, RMo cells withdrew from the cell cycle and formed myotubes that spontaneously contracted. Differentiated RMo cells produced creatine kinase isozymes in a ratio characteristic of skeletal muscle cells. RMo cells were easy to cultivate. Cells proliferated and differentiated equally well on gelatin-coated or noncoated culture dishes, at clonal or mass culture densities, and in all basal media tested. In most experiments, growth medium consisted of horse serum-containing medium supplemented with either chicken embryo extract or FGF activity; cells proliferated equally well in medium containing unsupplemented calf serum. RMo cells differentiated if growth medium was not replenished regularly. Alternatively, differentiation was induceable by incubation in mitogen-depleted medium consisting of basal medium supplemented either with 10(-6) M insulin, 0.5% serum, or 50% conditioned growth medium. RMo cells were competently transformed with cloned exogenous genes. Because it forms functional myofibrils, the RMo cell line constitutes a useful model system for studying the cell biology and biochemistry of proteins involved in contractile apparatus assembly and muscle disease.

  2. Regulation of GLUT4 activity in myotubes by 3-O-methyl-d-glucose.

    Science.gov (United States)

    Shamni, Ofer; Cohen, Guy; Gruzman, Arie; Zaid, Hilal; Klip, Amira; Cerasi, Erol; Sasson, Shlomo

    2017-10-01

    The rate of glucose influx to skeletal muscles is determined primarily by the number of functional units of glucose transporter-4 (GLUT4) in the myotube plasma membrane. The abundance of GLUT4 in the plasma membrane is tightly regulated by insulin or contractile activity, which employ distinct pathways to translocate GLUT4-rich vesicles from intracellular compartments. Various studies have indicated that GLUT4 intrinsic activity is also regulated by conformational changes and/or interactions with membrane components and intracellular proteins in the vicinity of the plasma membrane. Here we show that the non-metabolizable glucose analog 3-O-methyl-d-glucose (MeGlc) augmented the rate of hexose transport into myotubes by increasing GLUT4 intrinsic activity without altering the content of the transporter in the plasma membrane. This effect was not a consequence of ATP depletion or hyperosmolar stress and did not involve Akt/PKB or AMPK signal transduction pathways. MeGlc reduced the inhibitory potency (increased Ki) of indinavir, a selective inhibitor of GLUT4, in a dose-dependent manner. Kinetic analyses indicate that MeGlc induced changes in GLUT4 or GLUT4 complexes within the plasma membrane, which enhanced the hexose transport activity and reduced the potency of indinavir inhibition. Finally, we present a simple kinetic analysis for screening and discovering low molecular weight compounds that augment GLUT4 activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. DNA replication is intrinsically hindered in terminally differentiated myotubes.

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    Deborah Pajalunga

    2010-07-01

    Full Text Available Terminally differentiated (TD cells permanently exit the mitotic cycle while acquiring specialized characteristics. Although TD cells can be forced to reenter the cell cycle by different means, they cannot be made to stably proliferate, as attempts to induce their replication constantly result in cell death or indefinite growth arrest. There is currently no biological explanation for this failure.Here we show that TD mouse myotubes, reactivated by depletion of the p21 and p27 cell cycle inhibitors, are unable to complete DNA replication and sustain heavy DNA damage, which triggers apoptosis or results in mitotic catastrophe. In striking contrast, quiescent, non-TD fibroblasts and myoblasts, reactivated in the same way, fully replicate their DNA, do not suffer DNA damage, and proliferate even in the absence of growth factors. Similar results are obtained when myotubes and fibroblasts are reactivated by forced expression of E1A or cyclin D1 and cdk4.We conclude that the inability of myotubes to complete DNA replication must be ascribed to peculiar features inherent in their TD state, rather than to the reactivation method. On reviewing the literature concerning reactivation of other TD cell types, we propose that similar mechanisms underlie the general inability of all kinds of TD cells to proliferate in response to otherwise mitogenic stimuli. These results define an unexpected basis for the well known incompetence of mammalian postmitotic cells to proliferate. Furthermore, this trait might contribute to explain the inability of these cells to play a role in tissue repair, unlike their counterparts in extensively regenerating species.

  4. Conessine Interferes with Oxidative Stress-Induced C2C12 Myoblast Cell Death through Inhibition of Autophagic Flux

    OpenAIRE

    Hyunju Kim; Kang Il Lee; Minsu Jang; Sim Namkoong; Rackhyun Park; Hyunwoo Ju; Inho Choi; Won Keun Oh; Junsoo Park

    2016-01-01

    Conessine, a steroidal alkaloid isolated from Holarrhena floribunda, has anti-malarial activity and interacts with the histamine H3 receptor. However, the cellular effects of conessine are poorly understood. Accordingly, we evaluated the involvement of conessine in the regulation of autophagy. We searched natural compounds that modulate autophagy, and conessine was identified as an inhibitor of autophagic flux. Conessine treatment induced the formation of autophagosomes, and p62, an autophagi...

  5. Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary porcine myotubes

    DEFF Research Database (Denmark)

    Che, Brita Ngum; Oksbjerg, Niels; Hellgren, Lars

    2013-01-01

    stimulated glucose uptake nor glycogen synthesis in insulin-resistant myotubes generated by excess glucose exposure. CONCLUSIONS: Primary porcine myotubes were established as a model of skeletal muscles for measuring glucose uptake and glycogen synthesis, and we showed that PA can play a role in stimulating...

  6. A cellular modelsystem of differentiated human myotubes

    DEFF Research Database (Denmark)

    Gaster, M; Kristensen, S R; Beck-Nielsen, H

    2001-01-01

    The aim of this study was to select an effective and stable protocol for the differentiation of human satellite cells (Sc) and to identify the optimal time period for the experimental use of differentiated human Sc-cultures. In order to identify the differentiation conditions which give a good su...

  7. Accelerated myotube formation using bioprinting technology for biosensor applications.

    Science.gov (United States)

    Cui, Xiaofeng; Gao, Guifang; Qiu, Yongjun

    2013-03-01

    Muscle-powered, biological, microelectro-mechanical system is promising for actuator and biosensor applications. Functional conjugation between the cells, tissues, and biomolecules to the microdevice is crucial for this application. Bioprinting as an enabling technology possesses the advantages of high throughput, digital control, and highly accurate delivery of various biological factors to the desired locations for numerous applications such as 3D tissue fabrication. We have now evaluated the feasibility of the precise placement of mouse myoblasts onto micro-sized cantilevers. The evenly aligned printed cells fused with each other and formed mature myotubes after only 4 days. In contrast, it took more than 14 days for randomly deposited cells to do so. The printed myotubes were functional and responded to the electrical stimulation synchronously. Furthermore, the integrated Bio-MEMS device responded to the chemical stimulation spontaneously which demonstrated the potential as a functional biosensor. The contractility of the system was recovered quickly after the removal of the chemical stimulation, which indicated the flexibility of this system and the recycling potential.

  8. Involvement of TRPV2 and SOCE in calcium influx disorder in DMD primary human myotubes with a specific contribution of α1-syntrophin and PLC/PKC in SOCE regulation.

    Science.gov (United States)

    Harisseh, Rania; Chatelier, Aurélien; Magaud, Christophe; Déliot, Nadine; Constantin, Bruno

    2013-05-01

    Calcium homeostasis is critical for several vital functions in excitable and nonexcitable cells and has been shown to be impaired in many pathologies including Duchenne muscular dystrophy (DMD). Various studies using murine models showed the implication of calcium entry in the dystrophic phenotype. However, alteration of store-operated calcium entry (SOCE) and transient receptor potential vanilloid 2 (TRPV2)-dependant cation entry has not been investigated yet in human skeletal muscle cells. We pharmacologically characterized basal and store-operated cation entries in primary cultures of myotubes prepared from muscle of normal and DMD patients and found, for the first time, an increased SOCE in DMD myotubes. Moreover, this increase cannot be explained by an over expression of the well-known SOCE actors: TRPC1/4, Orai1, and stromal interaction molecule 1 (STIM1) mRNA and proteins. Thus we investigated the modes of regulation of this cation entry. We firstly demonstrated the important role of the scaffolding protein α1-syntrophin, which regulates SOCE in primary human myotubes through its PDZ domain. We also studied the implication of phospholipase C (PLC) and protein kinase C (PKC) in SOCE and showed that their inhibition restores normal levels of SOCE in DMD human myotubes. In addition, the involvement of TRPV2 in calcium deregulation in DMD human myotubes was explored. We showed an abnormal elevation of TRPV2-dependant cation entry in dystrophic primary human myotubes compared with normal ones. These findings show that calcium homeostasis mishandling in DMD myotubes depends on SOCE under the influence of Ca(2+)/PLC/PKC pathway and α1-syntrophin regulation as well as on TRPV2-dependant cation influx.

  9. Antidiabetic effect of taurine in cultured rat skeletal l6 myotubes.

    Science.gov (United States)

    Cheong, Sun Hee; Chang, Kyung Ja

    2013-01-01

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, is found in all animal cells at millimolar concentrations and has been reported to show various health promoting activities including antidiabetic properties. The beneficial effects of taurine in diabetes mellitus have been known. However, the exact mechanism of hypoglycemic action of taurine is not properly defined. In this study, we investigated antidiabetic effect of taurine in the cell culture system using rat skeletal muscle cells. In cultured rat skeletal L6 myotubes, we studied the effect of taurine (0-100 μM) on glucose uptake to plasma membrane from the aspects of AMP-activated protein kinase (AMPK) signaling. Taurine stimulated glucose uptake in a dose-dependent manner by activating AMPK signaling. From these results, it may suggest that taurine show antidiabetic effect by stimulating insulin-independent glucose uptake in rat skeletal muscle.

  10. Ectopic expression of Msx2 in mammalian myotubes recapitulates aspects of amphibian muscle dedifferentiation

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    Atilgan Yilmaz

    2015-11-01

    Full Text Available In contrast to urodele amphibians and teleost fish, mammals lack the regenerative responses to replace large body parts. Amphibian and fish regeneration uses dedifferentiation, i.e., reversal of differentiated state, as a means to produce progenitor cells to eventually replace damaged tissues. Therefore, induced activation of dedifferentiation responses in mammalian tissues holds an immense promise for regenerative medicine. Here we demonstrate that ectopic expression of Msx2 in cultured mouse myotubes recapitulates several aspects of amphibian muscle dedifferentiation. We found that MSX2, but not MSX1, leads to cellularization of myotubes and downregulates the expression of myotube markers, such as MHC, MRF4 and myogenin. RNA sequencing of myotubes ectopically expressing Msx2 showed downregulation of over 500 myotube-enriched transcripts and upregulation of over 300 myoblast-enriched transcripts. MSX2 selectively downregulated expression of Ptgs2 and Ptger4, two members of the prostaglandin pathway with important roles in myoblast fusion during muscle differentiation. Ectopic expression of Msx2, as well as Msx1, induced partial cell cycle re-entry of myotubes by upregulating CyclinD1 expression but failed to initiate S-phase. Finally, MSX2-induced dedifferentiation in mouse myotubes could be recapitulated by a pharmacological treatment with trichostatin A (TSA, bone morphogenetic protein 4 (BMP4 and fibroblast growth factor 1 (FGF1. Together, these observations indicate that MSX2 is a major driver of dedifferentiation in mammalian muscle cells.

  11. Oleate Prevents Palmitate-Induced Atrophy via Modulation of Mitochondrial ROS Production in Skeletal Myotubes

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    Hojun Lee

    2017-01-01

    Full Text Available Accumulation of saturated fatty acids contributes to lipotoxicity-related insulin resistance and atrophy in skeletal muscle. Conversely, unsaturated fatty acids like docosahexaenoic acid were proven to preserve muscle mass. However, it is not known if the most common unsaturated oleate will protect skeletal myotubes against palmitate-mediated atrophy, and its specific mechanism remains to be elucidated. Therefore, we investigated the effects of oleate on atrophy-related factors in palmitate-conditioned myotubes. Exposure of myotubes to palmitate, but not to oleate, led to an induction of fragmented nuclei, myotube loss, atrophy, and mitochondrial superoxide in a dose-dependent manner. Treatment of oleate to myotubes attenuated production of palmitate-induced mitochondrial superoxide in a dose-dependent manner. The treatment of oleate or MitoTEMPO to palmitate-conditioned myotubes led to inhibition of palmitate-induced mRNA expression of proinflammatory (TNF-α and IL6, mitochondrial fission (Drp1 and Fis1, and atrophy markers (myostatin and atrogin1. In accordance with the gene expression data, our immunocytochemistry experiment demonstrated that oleate and MitoTEMPO prevented or attenuated palmitate-mediated myotube shrinkage. These results provide a mechanism indicating that oleate prevents palmitate-mediated atrophy via at least partial modulation of mitochondrial superoxide production.

  12. Cultured myotubes from skeletal muscle of adult rats. Characterization and action of Anemonia sulcata toxin II.

    Science.gov (United States)

    Tesseraux, I; Gülden, M; Wassermann, O

    1987-08-01

    Mononucleated myogenic cells (satellite cells) were isolated from skeletal muscle of adult rats and grown in culture. These cells replicated and, beginning with the 6th day in culture, they fused and differentiated into multinucleated myotubes, which accumulated creatine kinase and developed cross striation and spontaneous contractions. The differentiation of the excitable membrane and the action of sea anemone toxin ATX II were investigated with microelectrode techniques. Mature myotubes reached a stable membrane potential of -47.3 mV (+/- 6.5 mV) with the 11th day in culture. Action potentials could be generated in all myotubes. During maturation they became faster (increasing rate of rise) and shorter in duration. In spontaneously contracting myotubes spontaneous action potentials were recorded, which were often associated with subthreshold oscillations of membrane potential. ATX II reduced the membrane potential and prolonged the action potential duration with the lowest effective concentrations being 1 nmol/l and 0.5 nmol/l, respectively. Furthermore, ATX II induced electrical activity in quiescent myotubes. After fusion the development of the membrane electrical properties of satellite cell derived muscle cells followed essentially the same pattern as in primary cultures of embryonic myotubes. Electrophysiologically and with respect to their sensitivity to ATX II the mature myotubes resemble denervated muscle fibres.

  13. Effects of inhibition of serine palmitoyltransferase (SPT and sphingosine kinase 1 (SphK1 on palmitate induced insulin resistance in L6 myotubes.

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    Agnieszka Mikłosz

    Full Text Available BACKGROUND: The objective of this study was to examine the effects of short (2 h and prolonged (18 h inhibition of serine palmitoyltransferase (SPT and sphingosine kinase 1 (SphK1 on palmitate (PA induced insulin resistance in L6 myotubes. METHODS: L6 myotubes were treated simultaneously with either PA and myriocin (SPT inhibitor or PA and Ski II (SphK1inhibitor for different time periods (2 h and 18 h. Insulin stimulated glucose uptake was measured using radioactive isotope. Expression of insulin signaling proteins was determined using Western blot analyses. Intracellular sphingolipids content [sphinganine (SFA, ceramide (CER, sphingosine (SFO, sphingosine-1-phosphate (S1P] were estimated by HPLC. RESULTS: Our results revealed that both short and prolonged time of inhibition of SPT by myriocin was sufficient to prevent ceramide accumulation and simultaneously reverse palmitate induced inhibition of insulin-stimulated glucose transport. In contrast, prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3β/GSK3β ratio in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes. CONCLUSION: Myriocin is more effective in restoration of palmitate induced insulin resistance in L6 myocytes, despite of the time of SPT inhibition, comparing to SKII (a specific SphK1 inhibitor. Observed changes in insulin signaling proteins were related to the content of specific sphingolipids, namely to the reduction of ceramide. Interestingly, inactivation of SphK1 augmented the effect of PA induced insulin resistance in L6 myotubes, which was associated with further inhibition of insulin stimulated PKB and GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.

  14. Physical activity is associated with retained muscle metabolism in human myotubes challenged with palmitate

    DEFF Research Database (Denmark)

    Green, C J; Bunprajun, T; Pedersen, B K

    2013-01-01

      The aim of this study was to investigate whether physical activity is associated with preserved muscle metabolism in human myotubes challenged with saturated fatty acids. Human muscle satellite cells were isolated from sedentary or active individuals and differentiated into myocytes in culture....... Metabolic differences were then investigated in the basal state or after chronic palmitate treatment. At basal, myocytes from sedentary individuals exhibited higher CD36 and HSP70 protein expression as well as elevated phosphorylation of c-Jun NH2-terminal kinase (JNK) and insulin receptor substrate 1 (IRS1......) serine(307) compared to myocytes from active individuals. Despite equal lipid accumulation following palmitate treatment, myocytes from sedentary individuals exhibited delayed acetyl coenzyme A carboxylase phosphorylation compared to the active group. Myocytes from sedentary individuals had significantly...

  15. Cortisol Induces Reactive Oxygen Species Through a Membrane Glucocorticoid Receptor in Rainbow Trout Myotubes.

    Science.gov (United States)

    Espinoza, Marlen B; Aedo, Jorge E; Zuloaga, Rodrigo; Valenzuela, Cristian; Molina, Alfredo; Valdés, Juan A

    2017-04-01

    Cortisol is an essential regulator of neuroendocrine stress responses in teleosts. Cortisol predominantly affects target tissues through the genomic pathway, which involves interacting with cytoplasmic glucocorticoid receptors, and thereby, modulating stress-response gene expressions. Cortisol also produces rapid effects via non-genomic pathways, which do not involve gene transcription. Although cortisol-mediated genomic pathways are well documented in teleosts, non-genomic pathways are not fully understood. Moreover, no studies have focused on the contribution of non-genomic cortisol pathways in compensatory stress responses in fish. In this study, rainbow trout (Oncorhynchus mykiss) skeletal myotubes were stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable agent, resulting in an early induction of reactive oxygen species (ROS). This production was not suppressed by transcription or translation inhibitors, suggesting non-genomic pathway involvement. Moreover, myotube preincubation with RU486 and NAC completely suppressed cortisol- and cortisol-BSA-induced ROS production. Subcellular fractionation analysis revealed the presence of cell membrane glucocorticoid receptors. Finally, cortisol-BSA induced a significant increase in ERK1/2 and CREB phosphorylation, as well as in CREB-dependent transcriptional activation of the pgc1a gene expression. The obtained results strongly suggest that cortisol acts through a non-genomic glucocorticoid receptor-mediated pathway to induce ROS production and contribute to ERK/CREB/PGC1-α signaling pathway activation as stress compensation mechanisms. J. Cell. Biochem. 118: 718-725, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Myotubes from severely obese type 2 diabetic subjects accumulate less lipids and show higher lipolytic rate than myotubes from severely obese non-diabetic subjects.

    Directory of Open Access Journals (Sweden)

    Siril S Bakke

    Full Text Available About 80% of patients with type 2 diabetes are classified as overweight. However, only about 1/3 of severely obese subjects have type 2 diabetes. This indicates that several severely obese individuals may possess certain characteristics that protect them against type 2 diabetes. We therefore hypothesized that this apparent paradox could be related to fundamental differences in skeletal muscle lipid handling. Energy metabolism and metabolic flexibility were examined in human myotubes derived from severely obese subjects without (BMI 44±7 kg/m2 and with type 2 diabetes (BMI 43±6 kg/m2. Lower insulin sensitivity was observed in myotubes from severely obese subjects with type 2 diabetes. Lipolysis rate was higher, and oleic acid accumulation, triacylglycerol content, and fatty acid adaptability were lower in myotubes from severely obese subjects with type 2 diabetes compared to severely obese non-diabetic subjects. There were no differences in lipid distribution and mRNA and protein expression of the lipases HSL and ATGL, the lipase cofactor CGI-58, or the lipid droplet proteins PLIN2 and PLIN3. Glucose and oleic acid oxidation were also similar in cells from the two groups. In conclusion, myotubes established from severely obese donors with established type 2 diabetes had lower ability for lipid accumulation and higher lipolysis rate than myotubes from severely obese donors without diabetes. This indicates that a difference in intramyocellular lipid turnover might be fundamental in evolving type 2 diabetes.

  17. Characterization of human myotubes from type 2 diabetic and non-diabetic subjects using complementary quantitative mass spectrometric methods

    DEFF Research Database (Denmark)

    Thingholm, Tine E; Bak, Steffen; Beck-Nielsen, Henning

    2011-01-01

    2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery...... strategy using iTRAQ and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring (SRM) to identify, quantify and validate changes in protein abundance between human myotubes obtained from non-diabetic lean, non-diabetic obese and type 2 diabetic subjects...

  18. Effect of Solanum surattense on mitochondrial enzymes in diabetic rats and in vitro glucose uptake activity in L6 myotubes

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    Muruhan Sridevi

    2015-01-01

    Full Text Available Background: S. surattense is widely used in Siddha medicine for various ailments. Objective: The aim was to evaluate the impact of alcoholic leaf-extract of S. surattense on mitochondrial enzymes in streptozotocin (STZ induced diabetic rats and to study the in vitro muscle glucose uptake activity on L6 myotubes. Materials and Methods: The male albino Wistar rats were randomly divided into five groups of six animals each. Diabetes was induced by intraperitoneal injection of STZ (40 mg/kg body weight. After being confirmed the diabetic rats were treated with alcoholic leaf-extract of S. surattense (100 mg/kg body weight for 45 days. The biochemical estimations (liver mitochondrial enzymes, antioxidants, thiobarbituric acid reactive substances [TBARS] and histopathological studies were performed. Further, the in vitro muscle glucose uptake activity in L6 myotubes and messenger RNA (mRNA expression of glucose transporter-4 (GLUT-4 was performed. Results: In diabetic rats, the activities of liver mitochondrial enzymes were found to be significantly lowered. The mitochondrial TBARS level increased, whereas the activities/level of enzymatic and non-enzymatic antioxidants decreased in diabetic rats. Administration of S. surattense to diabetic rats significantly reversed the above parameters toward normalcy. Furthermore in diabetic rats, the histopathological studies showed growth of adipose tissue and shrinkage of islets in the pancreas, liver showed fatty change with mild inflammation of portal triad, and kidney showed messangial capillary proliferation of glomeruli and fatty infiltration of tubules. Treatment with S. surattense brought back these changes to near normalcy. The extract was analyzed for in vitro muscle glucose uptake activity in L6 myotubes and mRNA expression of GLUT-4 by semi-quantitative reverse transcriptase-polymerase chain reaction. One nano gram per millilitre of S. surattense leaf-extract gave 115% glucose uptake on L6 myotubes

  19. Dietary Flaxseed Mitigates Impaired Skeletal Muscle Regeneration: in Vivo, in Vitro and in Silico Studies

    Science.gov (United States)

    Carotenuto, Felicia; Costa, Alessandra; Albertini, Maria Cristina; Rocchi, Marco Bruno Luigi; Rudov, Alexander; Coletti, Dario; Minieri, Marilena; Di Nardo, Paolo; Teodori, Laura

    2016-01-01

    Background: Diets enriched with n-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to exert a positive impact on muscle diseases. Flaxseed is one of the richest sources of n-3 PUFA acid α-linolenic acid (ALA). The aim of this study was to assess the effects of flaxseed and ALA in models of skeletal muscle degeneration characterized by high levels of Tumor Necrosis Factor-α (TNF). Methods: The in vivo studies were carried out on dystrophic hamsters affected by muscle damage associated with high TNF plasma levels and fed with a long-term 30% flaxseed-supplemented diet. Differentiating C2C12 myoblasts treated with TNF and challenged with ALA represented the in vitro model. Skeletal muscle morphology was scrutinized by applying the Principal Component Analysis statistical method. Apoptosis, inflammation and myogenesis were analyzed by immunofluorescence. Finally, an in silico analysis was carried out to predict the possible pathways underlying the effects of n-3 PUFAs. Results: The flaxseed-enriched diet protected the dystrophic muscle from apoptosis and preserved muscle myogenesis by increasing the myogenin and alpha myosin heavy chain. Moreover, it restored the normal expression pattern of caveolin-3 thereby allowing protein retention at the sarcolemma. ALA reduced TNF-induced apoptosis in differentiating myoblasts and prevented the TNF-induced inhibition of myogenesis, as demonstrated by the increased expression of myogenin, myosin heavy chain and caveolin-3, while promoting myotube fusion. The in silico investigation revealed that FAK pathways may play a central role in the protective effects of ALA on myogenesis. Conclusions: These findings indicate that flaxseed may exert potent beneficial effects by preserving skeletal muscle regeneration and homeostasis partly through an ALA-mediated action. Thus, dietary flaxseed and ALA may serve as a useful strategy for treating patients with muscle dystrophies. PMID:26941581

  20. Skeletal muscle myotubes in severe obesity exhibit altered ubiquitin-proteasome and autophagic/lysosomal proteolytic flux.

    Science.gov (United States)

    Bollinger, Lance M; Powell, Jonathan J S; Houmard, Joseph A; Witczak, Carol A; Brault, Jeffrey J

    2015-06-01

    Whole-body protein metabolism is dysregulated with obesity. The goal of the study was to determine whether activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity. Primary human skeletal muscle (HSkM) cell cultures were utilized since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean women (BMI ≤ 26.0 kg/m(2) ) and 8 women with severe obesity (BMI ≥ 39.0) were examined basally and when stimulated to atrophy (serum and amino acid starvation). HSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin-proteasome system diverged according to obesity status, with a decrease in lean subjects and an increase in HSkM from subjects with obesity. HSkM in obesity also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from individuals with and without obesity under basal and starved conditions. Our data indicate that muscle cells in lean individuals and in those with severe obesity have innate differences in management of protein degradation, which may explain their metabolic differences. © 2015 The Obesity Society.

  1. Cinnamic acid, from the bark of Cinnamomum cassia, regulates glucose transport via activation of GLUT4 on L6 myotubes in a phosphatidylinositol 3-kinase-independent manner.

    Science.gov (United States)

    Lakshmi, Baddireddi Subhadra; Sujatha, Sundaresan; Anand, Singaravelu; Sangeetha, Kadapakkam Nandhabalan; Narayanan, Rangarajan Badri; Katiyar, Chandrakanth; Kanaujia, Anil; Duggar, Rajeev; Singh, Yogendra; Srinivas, Konasubrahmanya; Bansal, Vinay; Sarin, Simi; Tandon, Ruchi; Sharma, Suchitra; Singh, Suchita

    2009-06-01

    Cinnamomum cassia (Family: Lauraceae) is an Ayurvedic medicinal plant used traditionally for the treatment of a number of diseases, including diabetes. The hypoglycemic effect of this plant has been established in vivo. However, the effects of cinnamic acid, isolated from C. cassia, on the insulin signaling cascade in an in vitro model have not been elucidated. Hence, the aim of the present study was to evaluate the anti-diabetic effect of cinnamic acid on glucose transport by L6 myotubes. The mechanism of action of cinnamic acid was determined using specific targets in the insulin signaling pathway, including protein tyrosine phosphatase (PTP) 1B, phosphatidylinositol 3-kinase (PI3-K) and the glucose transporter GLUT4. After differentiation of myoblast to myotubes, the cells were serum deprived for 5 h and then treated with 1 ng/mL cinnamic acid and 50 μmol/L rosiglitazone for 18 h and 100 nmol/L insulin for 20 min for gene expression studies. Expression of GLUT4 mRNA was increased following treatment of L6 myotubes with 1 ng/mL cinnamic acid. Furthermore, cinnamic acid inhibited PTP1B activity (by 96.5%), but had no significant effect on PI3-K activity. On the basis of the results of the present study, we postulate that cinnamic acid isolated from the hydro-alcoholic extract of Cinnamomum cassia activates glucose transport by a PI3-K-independent pathway. However, the detailed mechanism of action requires further analysis.

  2. The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes

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    Cameron-Smith David

    2011-06-01

    Full Text Available Abstract Background The branched-chain amino acid (BCAA leucine has been identified to be a key regulator of skeletal muscle anabolism. Activation of anabolic signalling occurs via the mammalian target of rapamycin (mTOR through an undefined mechanism. System A and L solute carriers transport essential amino acids across plasma membranes; however it remains unknown whether an exogenous supply of leucine regulates their gene expression. The aim of the present study was to investigate the effects of acute and chronic leucine stimulation of anabolic signalling and specific amino acid transporters, using cultured primary human skeletal muscle cells. Results Human myotubes were treated with leucine, insulin or co-treated with leucine and insulin for 30 min, 3 h or 24 h. Activation of mTOR signalling kinases were examined, together with putative nutrient sensor human vacuolar protein sorting 34 (hVps34 and gene expression of selected amino acid transporters. Phosphorylation of mTOR and p70S6K was transiently increased following leucine exposure, independently to insulin. hVps34 protein expression was also significantly increased. However, genes encoding amino acid transporters were differentially regulated by insulin and not leucine. Conclusions mTOR signalling is transiently activated by leucine within human myotubes independently of insulin stimulation. While this occurred in the absence of changes in gene expression of amino acid transporters, protein expression of hVps34 increased.

  3. Experiment list: SRX022861 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ue Diagnosis=NOS 8318912,26.1,58.4,335 GSM559804: Sin3B MT 5 source_name=C2C12 myotubes || origin=C2C12 cell line || genetic backgrou...nd=C3H || cell type=myotube || chip antibody=Sin3B || chip antibody manufacturer=Sa

  4. Experiment list: SRX022854 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ue Diagnosis=NOS 9430628,50.8,52.4,296 GSM559797: Sin3A MT 2 source_name=C2C12 myotubes || origin=C2C12 cell line || genetic backgrou...nd=C3H || cell type=myotube || chip antibody=Sin3A || chip antibody manufacturer=Sa

  5. Mononuclear cells from dedifferentiation of mouse myotubes display remarkable regenerative capability.

    Science.gov (United States)

    Yang, Zhong; Liu, Qiang; Mannix, Robert J; Xu, Xiaoyin; Li, Hongli; Ma, Zhiyuan; Ingber, Donald E; Allen, Paul D; Wang, Yaming

    2014-09-01

    Certain lower organisms achieve organ regeneration by reverting differentiated cells into tissue-specific progenitors that re-enter embryonic programs. During muscle regeneration in the urodele amphibian, postmitotic multinucleated skeletal myofibers transform into mononucleated proliferating cells upon injury, and a transcription factor-msx1 plays a role in their reprograming. Whether this powerful regeneration strategy can be leveraged in mammals remains unknown, as it has not been demonstrated that the dedifferentiated progenitor cells arising from muscle cells overexpressing Msx1 are lineage-specific and possess the same potent regenerative capability as their amphibian counterparts. Here, we show that ectopic expression of Msx1 reprograms postmitotic, multinucleated, primary mouse myotubes to become proliferating mononuclear cells. These dedifferentiated cells reactivate genes expressed by embryonic muscle progenitor cells and generate only muscle tissue in vivo both in an ectopic location and inside existing muscle. More importantly, distinct from adult muscle satellite cells, these cells appear both to fuse with existing fibers and to regenerate myofibers in a robust and time-dependent manner. Upon transplantation into a degenerating muscle, these dedifferentiated cells generated a large number of myofibers that increased over time and replenished almost half of the cross-sectional area of the muscle in only 12 weeks. Our study demonstrates that mammals can harness a muscle regeneration strategy used by lower organisms when the same molecular pathway is activated. © AlphaMed Press.

  6. Fenugreek increases insulin-stimulated creatine content in L6C11 muscle myotubes.

    Science.gov (United States)

    Tomcik, Kristyen A; Smiles, William J; Camera, Donny M; Hügel, Helmut M; Hawley, John A; Watts, Rani

    2017-04-01

    Creatine uptake by muscle cells is increased in the presence of insulin. Accordingly, compounds with insulin-like actions may also augment creatine uptake. The aim of this study was to investigate whether Trigonella foenum-graecum (fenugreek), an insulin mimetic, increases total intracellular creatine levels in vitro. Total cellular creatine content was measured fluorometrically in L6C11 muscle myotubes treated for 1, 4, and 24 h with 0.5 mM creatine (CR), CR and 20 μg/mL fenugreek seed extract (CR + FEN), CR and 100 nM insulin (CR + INS), and CR + INS + FEN (n = 6 per treatment group). Alterations in the expression of the sodium- and chloride-dependent creatine transporter, SLC6A8, and key signaling proteins in the PI3-K/Akt pathway were determined. Compared to control (CON), CR + INS + FEN increased total creatine content after 4 h (P  CON and CR at 1 h (P  CON, CR, and CR + INS at 4 h (P creatine content via a mechanism which is independent of the activity of SLC6A8, suggesting that an alternative mechanism is responsible for the regulation and facilitation of insulin-mediated creatine uptake in skeletal muscle cells.

  7. Ca2+-Dependent Excitation-Contraction Coupling Triggered by the Heterologous Cardiac/Brain DHPR β2a-Subunit in Skeletal Myotubes

    Science.gov (United States)

    Sheridan, David C.; Carbonneau, Leah; Ahern, Chris A.; Nataraj, Priya; Coronado, Roberto

    2003-01-01

    Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) α1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR β1a-subunit. It is unknown whether EC coupling domains present in the β-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit α1S is paired with the heterologous heart/brain DHPR β2a-subunit. Studies were conducted in primary cultured myotubes from β1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The β2a-mediated Ca2+ current recovered in β1 KO myotubes lacking the endogenous DHPR β1a-subunit verified formation of the α1S/β1a pair. In myotube genotypes which express no or low-density L-type Ca2+ currents, namely β1 KO and RyR1 KO, β2a overexpression recovered a wild-type density of nifedipine-sensitive Ca2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca2+ current recovery, there was a drastic reduction of voltage–dependent, skeletal-type EC coupling and emergence of Ca2+ transients triggered by the Ca2+ current. A comparison of β2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca2+-dependent Ca2+ release triggered by the β2a-subunit. In β1 KO and RyR1 KO myotubes, the Ca2+-dependent EC coupling promoted by β2a overexpression had the following characteristics: 1), L-type Ca2+ currents had a wild-type density; 2), Ca2+ transients activated much slower than controls overexpressing β1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca

  8. ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2010-01-01

    To date, it is unknown whether mitochondrial dysfunction in skeletal muscle from subjects with type 2 diabetes is based on primarily reduced mitochondrial mass and/or a primarily decreased mitochondrial ATP synthesis. Mitochondrial mass were determined in myotubes established from eight lean, eig...

  9. Insulin Resistance Is Not Conserved in Myotubes Established from Women with PCOS

    DEFF Research Database (Denmark)

    Eriksen, Mette; Pørneki, Ann Dorte; Skov, Vibe

    2010-01-01

    Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among premenopausal women, who often develop insulin resistance. We tested the hypothesis that insulin resistance in skeletal muscle of patients with polycystic ovary syndrome (PCOS) is an intrinsic defect, by investigating...... the metabolic characteristics and gene expression of in vitro differentiated myotubes established from well characterized PCOS subjects....

  10. Profiling of human myotubes reveals an intrinsic proteomic signature associated with type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Lubna Al-Khalili

    2014-03-01

    Full Text Available The development of insulin resistance and type 2 diabetes (T2D involves a complex array of metabolic defects in skeletal muscle. An in vitro cell culture system excludes the acute effects of external systemic factors existing in vivo. Thus, we aimed to determine whether intrinsic differences in the protein profile exist in cultured myotubes derived from T2D versus normal glucose tolerant (NGT healthy people. Applying two dimensional difference gel electrophoresis technology (2-D DIGE, the abundance of 47 proteins differed in myotubes derived from T2D patients versus NGT donors. Proteins involved in fatty acid and amino acid metabolism, TCA cycle, mitochondrial function, mRNA processing, DNA repair and cell survival showed higher abundance, while proteins associated with redox signaling (PARK7; Parkinson disease 7, glutathione metabolism (glutathione S-transferase, GST, isoforms T1, P1 and M2, and protein dynamics (heat shock protein, HSP, isoform B1 and 90A showed reduced abundance in myotubes derived from T2D versus NGT donors. Consistent with our proteome analysis results, the level of total glutathione was reduced in myotubes obtained from T2D versus NGT donors. Taken together, our data provide evidence for intrinsic differences in the profile of proteins involved in energy metabolism, cellular oxidative stress, protein dynamics and gene regulation in myotubes derived from T2D patients. These differences thereby suggest a genetic or epigenetic influence on protein content level, which can be further investigated to understand the molecular underpinnings of T2D progression and lead to new therapeutic approaches.

  11. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    Energy Technology Data Exchange (ETDEWEB)

    Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  12. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

    Science.gov (United States)

    Goh, Qingnian; Dearth, Christopher L; Corbett, Jacob T; Pierre, Philippe; Chadee, Deborah N; Pizza, Francis X

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Human myotubes from myoblast cultures undergoing senescence exhibit defects in glucose and lipid metabolism

    DEFF Research Database (Denmark)

    Nehlin, Jan O; Just, Marlene; Rustan, Arild C

    2011-01-01

    Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways...... number and could be explained by reduced incorporation into diacyl- and triacylglycerols. The levels of long-chain acyl-CoA esters decreased with increased passage number. Late-passage, non-proliferating, myoblast cultures showed strong senescence-associated β-galactosidase activity indicating...... that the observed metabolic defects accompany the induction of a senescent state. The main function of SCs is regeneration and skeletal muscle-build up. Thus, the metabolic defects observed during aging of SC-derived myotubes could have a role in sarcopenia, the gradual age-related loss of muscle mass and strength....

  14. Reduced TCA flux in diabetic myotubes: A governing influence on the diabetic phenotype?

    DEFF Research Database (Denmark)

    Gaster, Michael

    2009-01-01

    The diabetic phenotype is complex, requiring elucidation of key initiating defects. It is unknown whether the reduced tricarboxylic acid cycle (TCA) flux in skeletal muscle of obese and obese type 2 diabetic (T2D) subjects is of primary origin. Acetate oxidation (measurement of TCA......-flux) was significantly reduced in primary myotube cultures established from T2D versus lean subjects. Acetate oxidation was acutely stimulated by insulin and respiratory uncoupling. Inhibition of TCA flux in lean myotubes by malonate was followed by a measured decline in; acetate oxidation, complete palmitate oxidation......, lipid uptake, glycogen synthesis, ATP content and increased glucose uptake, while glucose oxidation was unaffected. Acute TCA inhibition did not induce insulin resistance. Thus the reduced TCA cycle flux in T2D skeletal muscle may be of primary origin. The diabetic phenotype of increased basal glucose...

  15. Grooved PLGA films incorporated with RGD/YIGSR peptides for potential application on skeletal muscle tissue engineering.

    Science.gov (United States)

    Wang, Peng-Yuan; Wu, Tsung-Han; Tsai, Wei-Bor; Kuo, Wei-Hsuan; Wang, Meng-Jiy

    2013-10-01

    Alignment of myocytes or myotubes is critical for skeletal muscle tissue engineering. In this study, grooved PLGA films (800nm in width of ridge/groove and 600nm in depth) incorporated with RGD or YIGSR peptides were fabricated to evaluate its efficacy for skeletal muscle tissue engineering. The growth and differentiation of C2C12 myoblasts were enhanced by the presentation of RGD or YIGSR compared with the untreated PLGA control. On the other hand, cell morphology was guided by the grooved structure, i.e. alignment of myoblasts and myotubes with the direction of grooves. This study elucidates the effects of both surface biochemical and topographic cues on the proliferation and differentiation of C2C12 myoblasts on biodegradable polymer films. Combination of surface topography and peptide presentation has a great potential in designing scaffolds for skeletal muscle tissue engineering. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Chicoric acid is an antioxidant molecule that stimulates AMP kinase pathway in L6 myotubes and extends lifespan in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Audrey Schlernitzauer

    Full Text Available Chicoric acid (CA is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans. In L6 myotubes, CA was a potent reactive oxygen species (ROS scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK pathway and displayed various features associated with AMPK activation: CA (a enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx and superoxide dismutase (SOD activities, (b favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5-100 μM. These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.

  17. Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes

    Science.gov (United States)

    Bhattacharya, Dipankar; Mehle, Andrew; Kamp, Timothy J.; Balijepalli, Ravi C.

    2015-01-01

    The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes. PMID:26114725

  18. Divergent regulation of insulin-like growth factor binding protein genes in cultured Atlantic salmon myotubes under different models of catabolism and anabolism.

    Science.gov (United States)

    Garcia de la Serrana, Daniel; Fuentes, Eduardo N; Martin, Samuel A M; Johnston, Ian A; Macqueen, Daniel J

    2017-06-01

    Much attention has been given to insulin-like growth factor (Igf) pathways that regulate the balance of skeletal muscle protein synthesis and breakdown in response to a range of extrinsic and intrinsic signals. However, we have a less complete understanding of how the same signals modulate muscle mass upstream of such signalling, through a family of functionally-diverse Igf-binding proteins (Igfbps) that modify the availability of Igfs to the cell receptor Igf1r. We exposed cultured myotubes from Atlantic salmon (Salmo salar L.) to treatments recapturing three catabolic signals: inflammation (interleukin-1β), stress (dexamethasone) and fasting (amino acid deprivation), plus one anabolic signal: recovery of muscle mass post-fasting (supplementation of fasted myotubes with Igf-I and amino acids). The intended phenotype of treatments was confirmed by significant changes in myotube diameter and immunofluorescent staining of structural proteins. We quantified the mRNA-level regulation of the full expressed Igf and Igfbp gene complement across a post-treatment time course, along with marker genes for muscle structural protein synthesis, as well as muscle breakdown, via the ubiquitin-proteasome and autophagy systems. Our results highlight complex, non-overlapping responses of Igfbp family members to the different treatments, suggesting that the profile of expressed Igfbps is differentially regulated by distinct signals promoting similar muscle remodelling phenotypes. We also demonstrate divergent regulation of salmonid-specific gene duplicates of igfbp5b1 and igfbp5b2 under distinct catabolic and anabolic conditions. Overall, this study increases our understanding of the regulation of Igfbp genes in response to signals that promote remodelling of skeletal muscle. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET of Dihydropyridine Receptor (DHPR β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

    Directory of Open Access Journals (Sweden)

    Dipankar Bhattacharya

    Full Text Available The dihydropyridine receptor (DHPR β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1 complex is still debatable. We used fluorescence resonance energy transfer (FRET to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1 myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

  20. Oleate Prevents Palmitate-Induced Atrophy via Modulation of Mitochondrial ROS Production in Skeletal Myotubes

    OpenAIRE

    Lee, Hojun; Lim, Jae-Young; Choi, Seung-Jun

    2017-01-01

    Accumulation of saturated fatty acids contributes to lipotoxicity-related insulin resistance and atrophy in skeletal muscle. Conversely, unsaturated fatty acids like docosahexaenoic acid were proven to preserve muscle mass. However, it is not known if the most common unsaturated oleate will protect skeletal myotubes against palmitate-mediated atrophy, and its specific mechanism remains to be elucidated. Therefore, we investigated the effects of oleate on atrophy-related factors in palmitate-c...

  1. The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts

    Science.gov (United States)

    The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or...

  2. A composite catalyst containing an halogen, a noble metal and at least an additional metal, and its utilization for aromatization of C2-C12 hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Alario, F.; Benazzi, E.; Deves, J.M.

    1994-09-02

    A composite catalyst is proposed for aromatization reactions of hydrocarbons containing from 2 to 12 carbon atoms per molecule, and more especially from 3 to 10 C atoms per molecule; the catalyst contains: a MFI structure zeolite which skeleton is composed of silicon, aluminium and/or gallium and which crystallites external faces have been modified after synthesis; a matrix; at least a noble metal from the platinum family and at least an additional metal chosen among tin, germanium, indium, copper, iron, molybdenum, gallium, thallium, gold, silver, ruthenium, chromium, tungsten and lead; at least an halogen (chlorine); eventually gallium and/or zinc; and eventually, at least one element chosen among alkaline and alkaline-earth metals. 2 tabs.

  3. Testosterone or 17{beta}-estradiol exposure reveals sex-specific effects on glucose and lipid metabolism in human myotubes.

    Science.gov (United States)

    Salehzadeh, Firoozeh; Rune, Anna; Osler, Megan; Al-Khalili, Lubna

    2011-08-01

    Changes in sex hormone levels with aging or illness may lead to metabolic disorders. Moreover, the ratio changes in men versus women may have distinct pathological responses. Since little is known about sex hormone action on muscle metabolism, we examined the role of testosterone or 17β-estradiol (E(2)) in metabolism and investigated whether either hormone may mediate a sex-specific effect. Myotubes from postmenopausal women and age-matched male donors were treated with 10 nM testosterone or E(2) for 4 days, and assays were performed to measure metabolic readouts, signal transduction, and mRNA expression. Testosterone and E(2) treatment enhanced insulin-stimulated glucose incorporation into glycogen and AKT phosphorylation in myotubes from female donors, highlighting a sex-specific role of sex hormone in glucose metabolism. Testosterone treatment increased palmitate oxidation in myotubes from both female and male donors, while E(2) enhanced palmitate oxidation in myotubes from male donors only. Testosterone-mediated increase in palmitate oxidation was attenuated at the presence of androgen receptor antagonist, which may indicate a role of nuclear steroid receptor in muscle lipid oxidation. Testosterone treatment increased mRNA expression of the insulin receptor substrate 2 in myotubes from male and female donors, whereas it increased mRNA expression of glycogen synthase 1 only in myotubes from male donors. E(2) treatment increased pyruvate dehydrogenase kinase 4 mRNA expression in myotubes from female donors. Thus, our data suggest that testosterone or E(2) modulates muscle glucose and lipid metabolism and may play a role in metabolism in a sex-dependent manner.

  4. Polyurethane acrylates as effective substrates for sustained in vitro culture of human myotubes.

    Science.gov (United States)

    Andriani, Yosephine; Chua, Jason Min-Wen; Chua, Benjamin Yan-Jiang; Phang, In Yee; Shyh-Chang, Ng; Tan, Wui Siew

    2017-07-15

    Muscular disease has debilitating effects with severe damage leading to death. Our knowledge of muscle biology, disease and treatment is largely derived from non-human cell models, even though non-human cells are known to differ from human cells in their biochemical responses. Attempts to develop highly sought after in vitro human cell models have been plagued by early cell delamination and difficulties in achieving human myotube culture in vitro. In this work, we developed polyurethane acrylate (PUA) materials to support long-term in vitro culture of human skeletal muscle tissue. Using a constant base with modulated crosslink density we were able to vary the material modulus while keeping surface chemistry and roughness constant. While previous studies have focused on materials that mimic soft muscle tissue with stiffness ca. 12kPa, we investigated materials with tendon-like surface moduli in the higher 150MPa to 2.4GPa range, which has remained unexplored. We found that PUA of an optimal modulus within this range can support human myoblast proliferation, terminal differentiation and sustenance beyond 35days, without use of any extracellular protein coating. Results show that PUA materials can serve as effective substrates for successful development of human skeletal muscle cell models and are suitable for long-term in vitro studies. We developed polyurethane acrylates (PUA) to modulate the human skeletal muscle cell growth and maturation in vitro by controlling surface chemistry, morphology and tuning material's stiffness. PUA was able to maintain muscle cell viability for over a month without any detectable signs of material degradation. The best performing PUA prevented premature cell detachment from the substrate which often hampered long-term muscle cell studies. It also supported muscle cell maturation up to the late stages of differentiation. The significance of these findings lies in the possibility to advance studies on muscle cell biology, disease and

  5. Methanolic extract of Momordica cymbalaria enhances glucose uptake in L6 myotubes in vitro by up-regulating PPAR-γ and GLUT-4.

    Science.gov (United States)

    Kumar, Puttanarasaiah Mahesh; Venkataranganna, Marikunte V; Manjunath, Kirangadur; Viswanatha, Gollapalle L; Ashok, Godavarthi

    2014-12-01

    The present study was undertaken to evaluate the influence of the methanolic fruit extract of Momordica cymbalaria (MFMC) on PPARγ (Peroxisome Proliferator Activated Receptor gamma) and GLUT-4 (Glucose transporter-4) with respect to glucose transport. Various concentrations of MFMC ranging from 62.5 to 500 μg·mL(-1) were evaluated for glucose uptake activity in vitro using L6 myotubes, rosiglitazone was used as a reference standard. The MFMC showed significant and dose-dependent increase in glucose uptake at the tested concentrations, further, the glucose uptake activity of MFMC (500 μg·mL(-1)) was comparable with rosigilitazone. Furthermore, MFMC has shown up-regulation of GLUT-4 and PPARγ gene expressions in L6 myotubes. In addition, the MFMC when incubated along with cycloheximide (CHX), which is a protein synthesis inhibitor, has shown complete blockade of glucose uptake. This indicates that new protein synthesis is required for increased GLUT-4 translocation. In conclusion, these findings suggest that MFMC is enhancing the glucose uptake significantly and dose dependently through the enhanced expression of PPARγ and GLUT-4 in vitro. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  6. Riluzole increases the rate of glucose transport in L6 myotubes and NSC-34 motor neuron-like cells via AMPK pathway activation.

    Science.gov (United States)

    Daniel, Bareket; Green, Omer; Viskind, Olga; Gruzman, Arie

    2013-09-01

    Riluzole is the only approved ALS drug. Riluzole influences several cellular pathways, but its exact mechanism of action remains unclear. Our goal was to study the drug's influence on the glucose transport rate in two ALS relevant cell types, neurons and myotubes. Stably transfected wild-type or mutant G93A human SOD1 NSC-34 motor neuron-like cells and rat L6 myotubes were exposed to riluzole. The rate of glucose uptake, translocation of glucose transporters to the cell's plasma membrane and the main glucose transport regulatory proteins' phosphorylation levels were measured. We found that riluzole increases the glucose transport rate and up-regulates the translocation of glucose transporters to plasma membrane in both types of cells. Riluzole leads to AMPK phosphorylation and to the phosphorylation of its downstream target, AS-160. In conclusion, increasing the glucose transport rate in ALS affected cells might be one of the mechanisms of riluzole's therapeutic effect. These findings can be used to rationally design and synthesize novel anti-ALS drugs that modulate glucose transport in neurons and skeletal muscles.

  7. NMR-Based Metabonomic Investigation of Heat Stress in Myotubes Reveals a Time-Dependent Change in the Metabolites

    DEFF Research Database (Denmark)

    Straadt, Ida K; Young, Jette F; Bross, Peter

    2010-01-01

    NMR-based metabonomics was applied to elucidate the time-dependent stress responses in mouse myotubes after heat exposure of either 42 or 45 degrees C for 1 h. Principal component analysis (PCA) revealed that the gradual time-dependent changes in metabolites contributing to the clustering......, samples returned toward control samples, however, without reaching the control samples even 10 h after stress. The results also indicate that the myotubes efficiently regulate the pH level by release of lactate to the culture medium at a heat stress level of 42 degrees C, which is a temperature level...

  8. Muscle cell cultures in Menkes' disease: copper accumulation in myotubes

    NARCIS (Netherlands)

    van den Berg, G. J.; Kroon, J. J.; Wijburg, F. A.; Sinjorgo, K. M.; Herzberg, N. H.; Bolhuis, P. A.

    1990-01-01

    We present 64Cu uptake studies in cultured muscle cells from a one-year-old patient with Menkes' disease. The cultured muscle cells from the patient showed a five-fold higher 64Cu uptake than control muscle cells. Copper uptake in muscle cells was of the same magnitude as that found in fibroblasts

  9. Eicosapentaenoic acid improves metabolic switching in human myotubes

    NARCIS (Netherlands)

    Hessvik, Nina P.; Bakke, Siril S.; Fredriksson, Katarina; Boekschoten, Mark; Fjorkenstad, Anne; Koster, Gerbrand; Hesselink, Matthijs K.; Kersten, Sander; Kase, Eili T.; Rustan, Arild C.; Thoresen, Hege

    2010-01-01

    Metabolically healthy skeletal muscle is characterized by the ability to switch easily between glucose and fat oxidation, whereas loss of this ability seems to be related to insulin resistance. The aim of this study was to investigate whether different fatty acids (FAs) and the LXR ligand T0901317

  10. Reduced TCA flux in diabetic myotubes: A governing influence on the diabetic phenotype?

    Science.gov (United States)

    Gaster, Michael

    2009-10-02

    The diabetic phenotype is complex, requiring elucidation of key initiating defects. It is unknown whether the reduced tricarboxylic acid cycle (TCA) flux in skeletal muscle of obese and obese type 2 diabetic (T2D) subjects is of primary origin. Acetate oxidation (measurement of TCA-flux) was significantly reduced in primary myotube cultures established from T2D versus lean subjects. Acetate oxidation was acutely stimulated by insulin and respiratory uncoupling. Inhibition of TCA flux in lean myotubes by malonate was followed by a measured decline in; acetate oxidation, complete palmitate oxidation, lipid uptake, glycogen synthesis, ATP content and increased glucose uptake, while glucose oxidation was unaffected. Acute TCA inhibition did not induce insulin resistance. Thus the reduced TCA cycle flux in T2D skeletal muscle may be of primary origin. The diabetic phenotype of increased basal glucose uptake and glucose oxidation, the reduced complete lipid oxidation and increased respiratory quotient, are likely to be adaptive responses to the reduced TCA cycle flux.

  11. N-acetylcysteine protects against bupivacaine-induced myotoxicity caused by oxidative and sarcoplasmic reticulum stress in human skeletal myotubes.

    Science.gov (United States)

    Galbes, Olivier; Bourret, Annick; Nouette-Gaulain, Karine; Pillard, Fabien; Matecki, Stefan; Py, Guillaume; Mercier, Jacques; Capdevila, Xavier; Philips, Alexandre

    2010-09-01

    Local anesthetics offer the benefits of extended analgesia with greater patient satisfaction and faster rehabilitation compared with intravenous morphine. These benefits, however, can be offset by adverse iatrogenic muscle pain. Here, the authors investigate the mechanisms of local anesthetic-induced myotoxicity and assess the protective effect of N-acetylcysteine. The authors used primary cell cultures of human skeletal muscle myoblasts to study local anesthetic adverse effects. Production of reactive oxygen species was investigated in human skeletal myotubes by fluorescence microscopy. Expression of sarcoplasmic/endoplasmic reticulum stress markers and induction of apoptosis were followed by immunofluorescence and Western blot analysis. Finally, the effect of N-acetylcysteine on bupivacaine-induced myotoxicity was investigated in vitro. Bupivacaine sequentially induced reactive oxygen species production, oxidative stress, sarcoplasmic/endoplasmic reticulum stress, and activation of caspases 9 and 7 in human differentiated myoblasts. These iatrogenic effects were prevented by N-acetylcysteine. The authors demonstrated a protective effect of N-acetylcysteine against bupivacaine-induced sarcoplasmic/endoplasmic reticulum stress and apoptosis in primary human skeletal muscle cell.

  12. Role of the water extract from Coccinia indica stem on the stimulation of glucose transport in L8 myotubes

    Directory of Open Access Journals (Sweden)

    Chaweewan Jansakul

    2006-11-01

    Full Text Available Hypoglycemic effect of Coccinia indica used for treatment of diabetes in traditional remedies has known to relate with increased transport of glucose into peripheral tissues. However, the cellular mechanisms for this effect remain unclear. This present study reports that the water extract (WE of C. indica stem exhibited a dose-dependent induction of 2-deoxyglucose (2-DG uptake in rat L8 myotubes. Maximal uptake was observed with approximately 3-fold increase in 2-DG transport in 16 h treatment compared with the control. Effect of WE was stronger than that of 1 mM metformin. The effects of insulin and WE were additive. WE-induced glucose uptake was significantly inhibited by cycloheximide and partially reversed by SB203580. GLUT1 protein was markedly increased in response to WE. Conversely, WE had no effect on GLUT4 protein level. Redistribution of GLUT4 to the plasma membrane was demonstrated. Triterpenoids and carbohydrates were detected in WE. In conclusion, new GLUT1 protein synthesis is necessary for WEstimulated glucose transport while p38-MAPK-dependent activation of transporter intrinsic activity partly contributes to WE action. These results may explain and support the use of C. indica for the prevention and treatment of diabetes.

  13. Effects of DEHP and its metabolite MEHP on insulin signalling and proteins involved in GLUT4 translocation in cultured L6 myotubes.

    Science.gov (United States)

    Viswanathan, Mangala Priya; Mullainadhan, Vigneswari; Chinnaiyan, Mayilvanan; Karundevi, Balasubramanian

    2017-07-01

    Di-(2-ethyl hexyl) phthalate (DEHP) is the plasticizer used in variety of medical and consumer products to impart structural flexibility. DEHP and its primary metabolite mono-(2-ethyl hexyl)phthalate (MEHP) posed a considerable interest because of their contribution to insulin resistance, type-2 diabetes and obesity. Experimental and epidemiological data have shown that DEHP affects blood glucose homeostasis. However, direct effect of DEHP and its metabolite MEHP on insulin signal transduction and glucose transporter 4 (GLUT4) translocation remain obscure. The present study was delineated to decipher the direct effects of DEHP and MEHP on insulin signal transduction and proteins involved in GLUT4 translocation in cultured L6 myotubes, the rat skeletal muscle model. For this study we have exposed cells with 50 and 100μM DEHP and MEHP for 24h followed by insulin stimulation for 20min. GLUT4 level in both cytosol and plasma membrane fractions were analysed by western blot analysis and found to be significantly decreased. Further, DEHP and MEHP treatment significantly altered the insulin signalling molecules and proteins involved in GLUT4 translocation (Rab8A (Ras related proteins in skeletal muscle), insulin - regulated amino peptidase (IRAP), synaptosomal - associated protein 23 (SNAP23), Syntaxin4, Munc18c) from cytosol to plasma membrane. Impaired GLUT4 in the plasma membrane resulted in decreased 14 C-deoxy glucose uptake. 14 C-glucose oxidation and glycogen content were also significantly decreased in treated groups. In essence, the present study is first of its kind to show the direct adverse effects of DEHP and MEHP on insulin signal transduction and GLUT4 translocation in cultured L6 myotubes. Further, MEHP is found to be more effective than DEHP as a result of its differential structure and physico-chemical properties. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Skeletal Muscle Tissue Engineering: Methods to Form Skeletal Myotubes and Their Applications

    Science.gov (United States)

    Ostrovidov, Serge; Hosseini, Vahid; Ahadian, Samad; Fujie, Toshinori; Parthiban, Selvakumar Prakash; Ramalingam, Murugan; Bae, Hojae; Kaji, Hirokazu

    2014-01-01

    Skeletal muscle tissue engineering (SMTE) aims to repair or regenerate defective skeletal muscle tissue lost by traumatic injury, tumor ablation, or muscular disease. However, two decades after the introduction of SMTE, the engineering of functional skeletal muscle in the laboratory still remains a great challenge, and numerous techniques for growing functional muscle tissues are constantly being developed. This article reviews the recent findings regarding the methodology and various technical aspects of SMTE, including cell alignment and differentiation. We describe the structure and organization of muscle and discuss the methods for myoblast alignment cultured in vitro. To better understand muscle formation and to enhance the engineering of skeletal muscle, we also address the molecular basics of myogenesis and discuss different methods to induce myoblast differentiation into myotubes. We then provide an overview of different coculture systems involving skeletal muscle cells, and highlight major applications of engineered skeletal muscle tissues. Finally, potential challenges and future research directions for SMTE are outlined. PMID:24320971

  15. Substrate overload: Glucose oxidation in human myotubes conquers palmitate oxidation through anaplerosis

    DEFF Research Database (Denmark)

    Gaster, Michael

    2009-01-01

    by increasing concentrations of the other in human myotubes established from healthy, lean subjects exposed to acute stepwise increases in glucose and PA levels. At high substrate levels; PA oxidation was reduced while release of acid soluble metabolites was increased and, both glucose oxidation and release...... of citrate was increased which could be abolished by phenylacetic acid (inhibitor of pyruvate carboxylase (PC)). The present data challenges above preconceptions. Although they operate at low-moderate substrate levels additional two principles determine substrate oxidation at higher substrate concentrations......To date, two cardinal principles govern oxidation of glucose and fatty acids in skeletal muscle; exogenous fatty acid reduces glucose oxidation and glucose reduces fatty acid oxidation. Both glucose and palmitate (PA) oxidation was increased by increasing their concentration and inhibited...

  16. Liver X receptor antagonist reduces lipid formation and increases glucose metabolism in myotubes from lean, obese and type 2 diabetic individuals

    DEFF Research Database (Denmark)

    Kase, E T; Thoresen, G H; Westerlund, S

    2007-01-01

    with labelled precursors, and gene expression was analysed using real-time PCR. RESULTS: Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers...... lipogenesis and lipid formation was more pronounced in myotubes from type 2 diabetic patients than from lean individuals. 22-S-HC counteracted these effects and reduced de novo lipogenesis below baseline, while glucose uptake and oxidation were increased....

  17. Prosome cytodistribution relative to desmin and actin filaments in dividing C2.7 myoblasts and during myotube formation in vitro.

    Science.gov (United States)

    De Conto, F; Missorini, S; Arcangeletti, C; Pinardi, F; Montarras, D; Pinset, C; Vassy, J; Géraud, G; Chezzi, C; Scherrer, K

    1997-05-25

    Prosomes constitute the multicatalytic proteinase (MCP) core of the 26S proteasomes, but were first observed as subcomplexes of untranslated mRNP; this suggests that they play a putative role in the control of protein biosynthesis in addition to their catabolic enzymatic function. In previous investigations it was shown that some prosomes colocalize with the intermediate filaments (IF) of the cytoskeleton, of the cytokeratin type in epithelial cells, and of the vimentin type in fibroblasts. Studies on adult rat muscle carried out with prosome-specific monoclonal antibodies (p-mAbs) have shown, surprisingly, that specific types of prosomes predominantly occupy a particular zone in between the M and the Z lines of the sarcomeric structure. The data presented here show that the subunit composition of prosomes changes when the dividing C2.7 myoblasts fuse into myotubes. We show furthermore that, in dividing C2.7 myoblasts, prosomes colocalize with the desmin network as well as with that of actin, in a distribution that changes with the subunit pattern of the prosomes investigated by individual p-mAbs. Surprisingly, when myogenic fusion is induced, specific types of prosomes move first to the nuclei; later on, they reappear in the cytoplasm. There, superimposing initially onto the reorganizing desmin filaments that run from one pole of the prefusion myoblast to the other, prosomes gradually colocalize with the actin fibers in the fusing myotubes, finally forming a "pearl on a string" pattern. These results are discussed in relation to parallel observations of prosome distribution between the actin and IF networks not only in epithelial cells but also in fusing muscle satellite cells, which made it possible to monitor the complete buildup of the sarcomeric structure.

  18. Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Goh, Qingnian; Dearth, Christopher L.; Corbett, Jacob T. [Department of Kinesiology, The University of Toledo, Toledo, OH (United States); Pierre, Philippe [Centre d’Immunologie de Marseille-Luminy U2M, Aix-Marseille Université, Marseille (France); INSERM U631, Institut National de la Santé et Recherche Médicale, Marseille (France); CNRS UMR6102, Centre National de la Recherche Scientifique, Marseille (France); Chadee, Deborah N. [Department of Biological Sciences, The University of Toledo, Toledo, OH (United States); Pizza, Francis X., E-mail: Francis.Pizza@utoledo.edu [Department of Kinesiology, The University of Toledo, Toledo, OH (United States)

    2015-02-15

    We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast–myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube–myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube–myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle. - Highlights: • We examined mechanisms through which skeletal muscle cell expression of ICAM-1 facilitates events of in vitro myogenesis. • Expression of ICAM-1 by cultured myoblasts did not influence their ability to proliferate or differentiate. • Skeletal muscle cell expression of ICAM-1 augmented myoblast fusion, myotube alignment, myotube–myotube fusion, and myotube size. • ICAM-1 augmented myogenic processes through

  19. Myotube differentiation in clustered regularly interspaced short palindromic repeat/Cas9-mediated MyoD knockout quail myoblast cells

    Directory of Open Access Journals (Sweden)

    Si Won Kim

    2017-07-01

    Full Text Available Objective In the livestock industry, the regulatory mechanisms of muscle proliferation and differentiation can be applied to improve traits such as growth and meat production. We investigated the regulatory pathway of MyoD and its role in muscle differentiation in quail myoblast cells. Methods The MyoD gene was mutated by the clustered regularly interspaced short palindromic repeat (CRISPR/Cas9 technology and single cell-derived MyoD mutant sublines were identified to investigate the global regulatory mechanism responsible for muscle differentiation. Results The mutation efficiency was 73.3% in the mixed population, and from this population we were able to establish two QM7 MyoD knockout subline (MyoD KO QM7#4 through single cell pick-up and expansion. In the undifferentiated condition, paired box 7 expression in MyoD KO QM7#4 cells was not significantly different from regular QM7 (rQM7 cells. During differentiation, however, myotube formation was dramatically repressed in MyoD KO QM7#4 cells. Moreover, myogenic differentiation-specific transcripts and proteins were not expressed in MyoD KO QM7#4 cells even after an extended differentiation period. These results indicate that MyoD is critical for muscle differentiation. Furthermore, we analyzed the global regulatory interactions by RNA sequencing during muscle differentiation. Conclusion With CRISPR/Cas9-mediated genomic editing, single cell-derived sublines with a specific knockout gene can be adapted to various aspects of basic research as well as in functional genomics studies.

  20. Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

    DEFF Research Database (Denmark)

    Bunprajun, Tipwadee; Henriksen, Tora Ida; Scheele, Camilla

    2013-01-01

    , and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact...... derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy...... chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma...

  1. Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression.

    Directory of Open Access Journals (Sweden)

    Tipwadee Bunprajun

    Full Text Available Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs normally active or middle-aged (56.6 yrs individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.

  2. FA1 Induces Pro-Inflammatory and Anti-Adipogenic Pathways/Markers in Human Myotubes Established from Lean, Obese, and Type 2 Diabetic Subjects but Not Insulin Resistance

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Beck-Nielsen, Henning; Gaster, Michael

    2013-01-01

    established from lean, obese, and T2D subjects. Instead, chronic FA1 exposure induced pro-inflammatory cytokines expression (IL-6 and CCL2) in association with reducing adipogenic markers (ADD1, AP2, CD36, and PPARg2) in myotubes. Consistent with this observation, addition of FA1 to cultured myotubes was show...... stimulation of pro-inflammatory cytokines, which on turn inhibit adipogenesis in human myotubes....

  3. Fusion from myoblasts to myotubes is dependent on the rolling stone gene (rost) of Drosophila.

    Science.gov (United States)

    Paululat, A; Burchard, S; Renkawitz-Pohl, R

    1995-08-01

    The development and differentiation of the body wall musculature in Drosophila are accompanied by changes in gene expression and cellular architecture. We isolated a Drosophila gene, termed rolling stone (rost), which, when mutated, specifically blocks the fusion of mononucleated cells to myotubes in the body wall musculature. beta 3 tubulin, which is an early marker for the onset of mesoderm differentiation, is still expressed in these cells. Gastrulation and mesoderm formation, as well as the development of the epidermis and of the central and peripheral nervous systems, appear quite normal in homozygous rolling stone embryos. Embryonic development stops shortly before hatching in a P-element-induced mutant, as well as in 16 EMS-induced alleles. In mutant embryos, other mesodermal derivatives such as the visceral mesoderm and the dorsal vessel, develop fairly normally and defects are restricted to the body wall musculature. Myoblasts remain as single mononucleated cells, which express muscle myosin, showing that the developmental program of gene expression proceeds. These myoblasts occur at positions corresponding to the locations of dorsal, ventral and pleural muscles, showing that the gene rolling stone is involved in cell fusion, a process that is independent of cell migration in these mutants. This genetic analysis has set the stage for a molecular analysis to clarify where the rolling stone action is manifested in the fusion process and thus gives insight into the complex regulating network controlling the differentiation of the body wall musculature.

  4. Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu Qiuwei, E-mail: qiuwei_xu@merck.com; Vu, Heather; Liu Liping; Wang, Ting-Chuan; Schaefer, William H. [Merck Research Laboratories (United States)

    2011-04-15

    Mitochondrial toxicity has been a serious concern, not only in preclinical drug development but also in clinical trials. In mitochondria, there are several distinct metabolic processes including fatty acid {beta}-oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), and each process contains discrete but often intimately linked steps. Interruption in any one of those steps can cause mitochondrial dysfunction. Detection of inhibition to OXPHOS can be complicated in vivo because intermediate endogenous metabolites can be recycled in situ or circulated systemically for metabolism in other organs or tissues. Commonly used assays for evaluating mitochondrial function are often applied to ex vivo or in vitro samples; they include various enzymatic or protein assays, as well as functional assays such as measurement of oxygen consumption rate, membrane potential, or acidification rates. Metabolomics provides quantitative profiles of overall metabolic changes that can aid in the unraveling of explicit biochemical details of mitochondrial inhibition while providing a holistic view and heuristic understanding of cellular bioenergetics. In this paper, we showed the application of quantitative NMR metabolomics to in vitro myotube cells treated with mitochondrial toxicants, rotenone and antimycin A. The close coupling of the TCA cycle to the electron transfer chain (ETC) in OXPHOS enables specific diagnoses of inhibition to ETC complexes by discrete biochemical changes in the TCA cycle.

  5. Genome-wide examination of myoblast cell cycle withdrawal duringdifferentiation

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Xun; Collier, John Michael; Hlaing, Myint; Zhang, Leanne; Delshad, Elizabeth H.; Bristow, James; Bernstein, Harold S.

    2002-12-02

    Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40 percent fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly

  6. Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Bolado-Carrancio, A. [Department of Molecular Biology, University of Cantabria, IDIVAL, Santander (Spain); Riancho, J.A. [Department of Internal Medicine, Hospital U.M. Valdecilla-IDIVAL, University of Cantabria, RETICEF, Santander (Spain); Sainz, J. [Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC-University of Cantabria, Santander (Spain); Rodríguez-Rey, J.C., E-mail: rodriguj@unican.es [Department of Molecular Biology, University of Cantabria, IDIVAL, Santander (Spain)

    2014-04-04

    Highlights: • NR5A2 expression in C2C12 is associated with myotube differentiation. • DLPC induces an increase in GLUT4 levels and glucose uptake in C2C12 myotubes. • In high glucose conditions the activation of NR5A2 inhibits fatty acids oxidation. - Abstract: NR5A2 is a nuclear receptor which regulates the expression of genes involved in cholesterol metabolism, pluripotency maintenance and cell differentiation. It has been recently shown that DLPC, a NR5A2 ligand, prevents liver steatosis and improves insulin sensitivity in mouse models of insulin resistance, an effect that has been associated with changes in glucose and fatty acids metabolism in liver. Because skeletal muscle is a major tissue in clearing glucose from blood, we studied the effect of the activation of NR5A2 on muscle metabolism by using cultures of C2C12, a mouse-derived cell line widely used as a model of skeletal muscle. Treatment of C2C12 with DLPC resulted in increased levels of expression of GLUT4 and also of several genes related to glycolysis and glycogen metabolism. These changes were accompanied by an increased glucose uptake. In addition, the activation of NR5A2 produced a reduction in the oxidation of fatty acids, an effect which disappeared in low-glucose conditions. Our results suggest that NR5A2, mostly by enhancing glucose uptake, switches muscle cells into a state of glucose preference. The increased use of glucose by muscle might constitute another mechanism by which NR5A2 improves blood glucose levels and restores insulin sensitivity.

  7. Differentiation-Associated Downregulation of Poly(ADP-Ribose Polymerase-1 Expression in Myoblasts Serves to Increase Their Resistance to Oxidative Stress.

    Directory of Open Access Journals (Sweden)

    Gábor Oláh

    Full Text Available Poly(ADP-ribose polymerase 1 (PARP-1, the major isoform of the poly (ADP-ribose polymerase family, is a constitutive nuclear and mitochondrial protein with well-recognized roles in various essential cellular functions such as DNA repair, signal transduction, apoptosis, as well as in a variety of pathophysiological conditions including sepsis, diabetes and cancer. Activation of PARP-1 in response to oxidative stress catalyzes the covalent attachment of the poly (ADP-ribose (PAR groups on itself and other acceptor proteins, utilizing NAD+ as a substrate. Overactivation of PARP-1 depletes intracellular NAD+ influencing mitochondrial electron transport, cellular ATP generation and, if persistent, can result in necrotic cell death. Due to their high metabolic activity, skeletal muscle cells are particularly exposed to constant oxidative stress insults. In this study, we investigated the role of PARP-1 in a well-defined model of murine skeletal muscle differentiation (C2C12 and compare the responses to oxidative stress of undifferentiated myoblasts and differentiated myotubes. We observed a marked reduction of PARP-1 expression as myoblasts differentiated into myotubes. This alteration correlated with an increased resistance to oxidative stress of the myotubes, as measured by MTT and LDH assays. Mitochondrial function, assessed by measuring mitochondrial membrane potential, was preserved under oxidative stress in myotubes compared to myoblasts. Moreover, basal respiration, ATP synthesis, and the maximal respiratory capacity of mitochondria were higher in myotubes than in myoblasts. Inhibition of the catalytic activity of PARP-1 by PJ34 (a phenanthridinone PARP inhibitor exerted greater protective effects in undifferentiated myoblasts than in differentiated myotubes. The above observations in C2C12 cells were also confirmed in a rat-derived skeletal muscle cell line (L6. Forced overexpression of PARP1 in C2C12 myotubes sensitized the cells to oxidant

  8. Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes.

    Science.gov (United States)

    Sabourin, Jessica; Harisseh, Rania; Harnois, Thomas; Magaud, Christophe; Bourmeyster, Nicolas; Déliot, Nadine; Constantin, Bruno

    2012-12-01

    In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Antcin K, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects via Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in Muscles

    OpenAIRE

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching; Yang, Chang-Syun

    2016-01-01

    The purpose of this study was to screen firstly the potential effects of antcin K (AnK), the main constituent of the fruiting body of Antrodia camphorata, in vitro and further evaluate the activities and mechanisms in high-fat-diet- (HFD-) induced mice. Following 8-week HFD-induction, mice were treated with AnK, fenofibrate (Feno), metformin (Metf), or vehicle for 4 weeks afterward. In C2C12 myotube cells, the membrane GLUT4 and phospho-Akt expressions were higher in insulin and AnK-treated g...

  10. Testosterone treatment increases androgen receptor and aromatase gene expression in myotubes from patients with PCOS and controls, but does not induce insulin resistance.

    Science.gov (United States)

    Eriksen, Mette Brandt; Glintborg, Dorte; Nielsen, Michael Friberg Bruun; Jakobsen, Marianne Antonius; Brusgaard, Klaus; Tan, Qihua; Gaster, Michael

    2014-09-05

    Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7days testosterone treatment (100nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls. Testosterone treatment significantly increased aromatase and androgen receptor gene expression levels in patients and controls. Glucose transport in myotubes was comparable in patients with PCOS vs. controls and was unchanged by testosterone treatment (p=0.21 PCOS vs. controls). These results suggest that testosterone treatment of myotubes increases the aromatase and androgen receptor gene expression without affecting insulin sensitivity and if testosterone is implicated in muscular insulin resistance in PCOS, this is by and indirect mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: Effect on myotube fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roffe, Suzy [Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel); Hagai, Yosey [Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel); Institute of Animal Sciences, Volcani Center, Bet Dagan 50250 (Israel); Pines, Mark [Institute of Animal Sciences, Volcani Center, Bet Dagan 50250 (Israel); Halevy, Orna, E-mail: halevyo@agri.huji.ac.il [Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100 (Israel)

    2010-04-01

    Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of the phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.

  12. GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

    DEFF Research Database (Denmark)

    Ralston, E; Ploug, Thorkil

    1996-01-01

    There is little consensus on the nature of the storage compartment of the glucose transporter GLUT4, in non-stimulated cells of muscle and fat. More specifically, it is not known whether GLUT4 is localized to unique, specialized intracellular storage vesicles, or to vesicles that are part...... of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex...... (alpha mannosidase II and giantin), of the trans-Golgi network (TGN38), of lysosomes (lgp110), and of early and late endosomes (transferrin receptor and mannose-6-phosphate receptor, respectively), to define the position of their subcellular compartments. By immunofluorescence, GLUT4 appears concentrated...

  13. Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo-activated ERK1/2 is critical for the promyogenic function of Stim1.

    Science.gov (United States)

    Lee, Hye-Jin; Bae, Gyu-Un; Leem, Young-Eun; Choi, Hyun-Kyung; Kang, Tong Mook; Cho, Hana; Kim, Seong-Tae; Kang, Jong-Sun

    2012-04-01

    The promyogenic cell surface molecule Cdo is required for activation of extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells c3 (NFATc3) induced by netrin-2 in myogenic differentiation. However, the molecular mechanism leading to NFATc3 activation is unknown. Stromal interaction molecule 1 (Stim1), an internal calcium sensor of the endoplasmic reticulum store, promotes myogenesis via activation of NFATc3. In this study we investigated the functional interaction between Cdo and Stim1 in myogenic differentiation. Overexpression and depletion of Stim1 enhanced or decreased myotube formation, respectively. Of interest, Stim1 protein levels were decreased in Cdo-deficient perinatal hindlimb muscles or primary myoblasts; this correlates with defective NFATc3 activation in Cdo(-/-) myoblasts upon differentiation. Forced activation of NFATc3 by overexpression of calcineurin restored differentiation of Cdo-depleted C2C12 myoblasts. Furthermore, Cdo and Stim1 formed a complex in 293T cells or in differentiating C2C12 myoblasts. The netrin-2-mediated NFATc3 activation was coincident with robust interactions between Cdo and Stim1 in myoblasts and the ERK-mediated Stim1 phosphorylation at serine 575. The serine 575 phosphorylation was enhanced in C2C12 cells upon differentiation, and the alanine substitution of serine 575 failed to restore differentiation of Stim1-depleted myoblasts. Taken together, the results indicate that cell adhesion signaling triggered by netrin-2/Cdo induces Stim1 phosphorylation at serine 575 by ERK, which promotes myoblast differentiation.

  14. Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.

    Science.gov (United States)

    Dodds, E; Dunckley, M G; Naujoks, K; Michaelis, U; Dickson, G

    1998-04-01

    Cationic lipid-DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors. With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER. Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes. Reporter gene constructs expressing either E. coli beta-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively. Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully. However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin. This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

  15. Caffeine induces hyperacetylation of histones at the MEF2 site on the Glut4 promoter and increases MEF2A binding to the site via a CaMK-dependent mechanism.

    Science.gov (United States)

    Mukwevho, Emmanuel; Kohn, Tertius A; Lang, Dirk; Nyatia, Edward; Smith, James; Ojuka, Edward O

    2008-03-01

    This study was conducted to explore the mechanism by which caffeine increases GLUT4 expression in C(2)C(12) myotubes. Myoblasts were differentiated in DMEM containing 2% horse serum for 13 days and the resultant myotubes exposed to 10 mM caffeine in the presence or absence of 25 microM KN93 or 10 mM dantrolene for 2 h. After the treatment, cells were kept in serum-free medium and harvested between 0 and 6 h later, depending on the assay. Chromatin immunoprecipitation (ChIP) assays revealed that caffeine treatment caused hyperacetylation of histone H3 at the myocyte enhancer factor 2 (MEF2) site on the Glut4 promoter (P CaMK II), attenuated all the above caffeine-induced changes. These data indicate that caffeine increases GLUT4 expression by acetylating the MEF2 site to increase MEF2A binding via a mechanism that involves CaMK II.

  16. Transthyretin is a key regulator of myoblast differentiation.

    Directory of Open Access Journals (Sweden)

    Eun Ju Lee

    Full Text Available Transthyretin (TTR is a known carrier protein for thyroxine (T4 and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTRkd significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOGkd did not have any effect on TTR gene expression. Both TTRkd and MYOGkd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T4 uptake during myogenesis was observed in TTRkd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.

  17. The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control

    DEFF Research Database (Denmark)

    Minet, Ariane D; Gaster, Michael

    2011-01-01

    selects the mitochondria based on an antibody recognizing the mitochondrial outer membrane and not by size through gradient centrifugation. The dynamic equilibrium between ATP synthesis and ATP consumption is 35% lower in isolated mitochondria from myotubes established from type 2 diabetic subjects...... or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n=20 in each group), precultured under normophysiological...... compared to lean control. The ATP synthesis rate without ATP consumption was not different between groups and there were no significant gender differences. The mitochondrial dysfunction in type 2 diabetes in vivo is partly based on a primarily impaired ATP synthesis....

  18. Angiopoietin-like 4 mediates PPAR delta effect on lipoprotein lipase-dependent fatty acid uptake but not on beta-oxidation in myotubes.

    Directory of Open Access Journals (Sweden)

    Marius R Robciuc

    Full Text Available Peroxisome proliferator-activated receptor (PPAR delta is an important regulator of fatty acid (FA metabolism. Angiopoietin-like 4 (Angptl4, a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR, PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.

  19. Ecdysteroids: A novel class of anabolic agents?

    Directory of Open Access Journals (Sweden)

    MK Parr

    2015-05-01

    Full Text Available Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol, estradienedione (trenbolox, and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days. In C2C12 myotubes ecdysterone (1 μM induced a significant increase of the diameter comparable to dihydrotestosterone (1 μM and IGF 1 (1.3 nM. Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class “S1.2 Other Anabolic Agents” of the list of prohibited substances of the World Anti-Doping Agency.

  20. Potential Therapeutic Role of L-Carnitine in Skeletal Muscle Oxidative Stress and Atrophy Conditions

    Directory of Open Access Journals (Sweden)

    Anna Montesano

    2015-01-01

    Full Text Available The targeting of nutraceutical treatment to skeletal muscle damage is an emerging area of research, driven by the need for new therapies for a range of muscle-associated diseases. L-Carnitine (CARN is an essential nutrient and plays a key role in mitochondrial β-oxidation and in the ubiquitin-proteasome system regulation. As a dietary supplement to improve athletic performance, CARN has been studied for its potential to enhance β-oxidation. However, CARN effects on myogenesis, mitochondrial activity, and hypertrophy process are not completely elucidated. This in vitro study aims to investigate CARN role on skeletal muscle remodeling, differentiation process, and myotubes formation. We analyzed muscle differentiation and morphological features in C2C12 myoblasts exposed to 5 mM CARN. Our results showed that CARN was able to accelerate C2C12 myotubes formation and induce morphological changes, characterizing the start of hypertrophy process. In addition, CARN improved AKT activation and downstream cellular signaling pathways involved in skeletal muscle atrophy process prevention. Also, CARN positively regulated the pathways involved in oxidative stress defense. In this work, we provide an interesting novel mechanism of the potential therapeutic use of CARN to treat pathological conditions characterized by skeletal muscle morphological and functional impairment, oxidative stress production, and atrophy process in aging.

  1. A diacylglycerol kinase inhibitor, R59022, stimulates glucose transport through a MKK3/6-p38 signaling pathway in skeletal muscle cells.

    Science.gov (United States)

    Takahashi, Nobuhiko; Nagamine, Miho; Tanno, Satoshi; Motomura, Wataru; Kohgo, Yutaka; Okumura, Toshikatsu

    2007-08-17

    Diacylglycerol kinase (DGK) is one of lipid-regulating enzymes, catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Because skeletal muscle, a major insulin-target organ for glucose disposal, expresses DGK, we investigated in the present study a role of DGK on glucose transport in skeletal muscle cells. PCR study showed that C2C12 myotubes expressed DGKalpha, delta, epsilon, zeta, or theta isoform mRNA. R59022, a specific inhibitor of DGK, significantly increased glucose transport, p38 and MKK3/6 activation in C2C12 myotubes. The R59022-induced glucose transport was blocked by SB203580, a specific p38 inhibitor. In contrast, R59022 failed to stimulate both possible known mechanisms to enhance glucose transport, an IRS1-PI3K-Akt pathway, muscle contraction signaling or GLUT1 and 4 expression. All these results suggest that DGK may play a role in glucose transport in the skeletal muscle cells through modulating a MKK3/6-p38 signaling pathway.

  2. Modest Decrease in Pgc1α Results in TAG Accumulation but not in Insulin Resistance in L6 Myotubes

    Directory of Open Access Journals (Sweden)

    Bartlomiej Lukaszuk

    2015-03-01

    Full Text Available Background/Aims: PGC-1α is an important cellular protein (coactivator regulating myocyte mitochondria number and function, and therefore whole cellular energy status. The aim of this work was to investigate the effects of modest, temporary PGC-1α knock-down on L6 myotubes insulin resistance in a light of cellular lipid metabolism. Methods: Gas liquid chromatography was applied for assessing FAs content and composition. For the expression of mitochondrial enzymes, as well as FA and glucose transporters, Western Blot technique was adopted. Additionally, radiolabelled glucose and palmitic acid uptake was performed to estimate the nutrients cellular influx. Results: Modest (-24% PGC-1α protein ablation resulted in decreased mitochondrial activity in general (reduced Cyt C content and FAs oxidation in particular (diminished β-HAD expression without increased FAs cellular influx. The aforementioned intervention led to significantly increased TAG cellular level, but not DAG nor CER. Consequently, no changes in cellular insulin responsiveness were noticed. Conclusions: Modest (-24% PGC-1α protein depletion results in lipid accumulation, without causing insulin resistance. Importantly, it seems that this TAG loading is a result of decreased mitochondrial oxidative capacity and/or possibly increased lipid biosynthesis but not fatty acid cellular influx.

  3. Berberine promotes glucose consumption independently of AMP-activated protein kinase activation.

    Directory of Open Access Journals (Sweden)

    Miao Xu

    Full Text Available Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK pathway has been proposed as mechanism for berberine's action. This study aimed to examine whether AMPK activation was necessary for berberine's glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC phosphorylation were stimulated by 20 µmol/L berberine. Nevertheless, berberine was still effective on stimulating glucose utilization and lactate production, when the AMPK activation was blocked by (1 inhibition of AMPK activity by Compound C, (2 suppression of AMPKα expression by siRNA, and (3 blockade of AMPK pathway by adenoviruses containing dominant-negative forms of AMPKα1/α2. To test the effect of berberine on oxygen consumption, extracellular flux analysis was performed in Seahorse XF24 analyzer. The activity of respiratory chain complex I was almost fully blocked in C2C12 myotubes by berberine. Metformin, as a positive control, showed similar effects as berberine. These results suggest that berberine and metformin promote glucose metabolism by stimulating glycolysis, which probably results from inhibition of mitochondrial respiratory chain complex I, independent of AMPK activation.

  4. Long-Term Consumption of Platycodi Radix Ameliorates Obesity and Insulin Resistance via the Activation of AMPK Pathways

    Directory of Open Access Journals (Sweden)

    Chae Eun Lee

    2012-01-01

    Full Text Available This study was designed to evaluate the effects and mechanism of Platycodi radix, having white balloon flower (Platycodon grandiflorum for. albiflorum (Honda H. Hara on obesity and insulin resistance. The extracts of Platycodi radix with white balloon flower were tested in cultured cells and administered into mice on a high-fat diet. The Platycodi radix activated the AMPK/ACC phosphorylation in C2C12 myotubes and also suppressed adipocyte differentiation in 3T3-L1 cells. In experimental animal, it suppressed the weight gain of obese mice and ameliorated obesity-induced insulin resistance. It also reduced the elevated circulating mediators, including triglyceride (TG, T-CHO, leptin, resistin, and monocyte chemotactic protein (MCP-1 in obesity. As shown in C2C12 myotubes, the administration of Platycodi radix extracts also recovered the AMPK/ACC phosphorylation in the muscle of obese mice. These results suggest that Platycodi radix with white balloon flower ameliorates obesity and insulin resistance in obese mice via the activation of AMPK/ACC pathways and reductions of adipocyte differentiation.

  5. DHA at nutritional doses restores insulin sensitivity in skeletal muscle by preventing lipotoxicity and inflammation.

    Science.gov (United States)

    Capel, Frédéric; Acquaviva, Cécile; Pitois, Elodie; Laillet, Brigitte; Rigaudière, Jean-Paul; Jouve, Chrystèle; Pouyet, Corinne; Gladine, Cècile; Comte, Blandine; Vianey Saban, Christine; Morio, Bèatrice

    2015-09-01

    Skeletal muscle plays a major role in the control of whole body glucose disposal in response to insulin stimulus. Excessive supply of fatty acids to this tissue triggers cellular and molecular disturbances leading to lipotoxicity, inflammation, mitochondrial dysfunctions, impaired insulin response and decreased glucose uptake. This study was conducted to analyze the preventive effect of docosahexaenoic acid (DHA), a long-chain polyunsaturated n-3 fatty acid, against insulin resistance, lipotoxicity and inflammation in skeletal muscle at doses compatible with nutritional supplementation. DHA (30 μM) prevented insulin resistance in C2C12 myotubes exposed to palmitate (500 μM) by decreasing protein kinase C (PKC)-θ activation and restoring cellular acylcarnitine profile, insulin-dependent AKT phosphorylation and glucose uptake. Furthermore, DHA protected C2C12 myotubes from palmitate- or lipopolysaccharide-induced increase in Ptgs2, interleukin 6 and tumor necrosis factor-α mRNA level, probably through the inhibition of p38 MAP kinase and c-Jun amino-terminal kinase. In LDLR -/- mice fed a high-cholesterol-high-sucrose diet, supplementation with DHA reaching up to 2% of daily energy intake enhanced the insulin-dependent AKT phosphorylation and reduced the PKC-θ activation in skeletal muscle. Therefore, DHA used at physiological doses participates in the regulation of muscle lipid and glucose metabolisms by preventing lipotoxicity and inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. A Rhodiola rosea root extract protects skeletal muscle cells against chemically induced oxidative stress by modulating heat shock protein 70 (HSP70) expression.

    Science.gov (United States)

    Hernández-Santana, Aaron; Pérez-López, Verónica; Zubeldia, Jose María; Jiménez-del-Rio, Miguel

    2014-04-01

    Rhodiola rosea is a perennial plant in the Crassulaceae family, recently postulated to exert its adaptogenic functions partially by modulating the expression of molecular factors such as heat shock proteins (HSP). The aim of this study was to analyze the efficacy of a Rhodiola rosea extract (Rhodiolife) in protecting murine skeletal muscle cells (C2 C12 myotubes) from chemically induced oxidative stress and to establish whether modulation of HSP70 expression is observed. C2 C12 cells treated with Rhodiolife did not experience any loss of viability (p > 0.05) at concentrations of 1-100 µg/mL for up to 24 h. In control cultures, viability decreased 25% following exposure to 2 mM H2 O2 (1 h). However, no significant decrease in viability in cells pre-treated with extract at concentrations as low as 1 µg/mL was observed. HSP70 mRNA levels were up-regulated two-fold in cell cultures treated with Rhodiolife (10 µg/mL), and expression was further enhanced by exposure to H2 O2 (six-fold, p < 0.05). HSP70 protein levels were maintained in pre-treated cell cultures compared to controls but was significantly lower (-50%) in cells lacking treatment exposed to H2 O2 . The present results indicate that Rhodiolife protects C2 C12 myotubes against peroxide-induced oxidative stress through the modulation of the molecular chaperone HSP70. Copyright © 2013 John Wiley & Sons, Ltd.

  7. Role of vitamin D on the expression of glucose transporters in L6 myotubes

    OpenAIRE

    Bubblu Tamilselvan; Seshadri, Krishna G.; Ganesh Venkatraman

    2013-01-01

    Altered expression of glucose transporters is a major characteristic of diabetes. Vitamin D has evolved widespread interest in the pathogenesis and prevention of diabetes. The present study was designed to investigate the effect of vitamin D in the overall regulation of muscle cell glucose transporter expression. L6 cells were exposed to type 1 and type 2 diabetic conditions and the effect of calcitriol (1,25, dihydroxy cholicalciferol) on the expression of glucose transporters was studied by...

  8. Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary porcine myotubes

    DEFF Research Database (Denmark)

    Che, Brita Ngum; Oksbjerg, Niels; Hellgren, Lars

    2013-01-01

    ABSTRACT: BACKGROUND: Phytanic acid (PA) is a chlorophyll metabolite with potentials in regulating glucose metabolism, as it is a natural ligand of the peroxisome proliferator-activated receptor (PPAR) that is known to regulate hepatic glucose homeostasis. This study aimed to establish primary...... and tritiated 2-deoxyglucose (2-DOG) was used to measure glucose uptake, in relation to PA and 2-DOG exposure times and also in relation to PA and insulin concentrations. The MIXED procedure model of SAS was used for statistical analysis of data. RESULTS: PA increased glucose uptake by approximately 35...

  9. Role of vitamin D on the expression of glucose transporters in L6 myotubes

    Directory of Open Access Journals (Sweden)

    Bubblu Tamilselvan

    2013-01-01

    Full Text Available Altered expression of glucose transporters is a major characteristic of diabetes. Vitamin D has evolved widespread interest in the pathogenesis and prevention of diabetes. The present study was designed to investigate the effect of vitamin D in the overall regulation of muscle cell glucose transporter expression. L6 cells were exposed to type 1 and type 2 diabetic conditions and the effect of calcitriol (1,25, dihydroxy cholicalciferol on the expression of glucose transporters was studied by real time polymerase chain reaction (RT-PCR. There was a significant decrease in glucose transporter type 1 (GLUT1, GLUT4, vitamin D receptor (VDR, and IR expression in type 1 and 2 diabetic model compared to control group. Treatment of myoblasts with 10-7 M calcitriol for 24 h showed a significant increase in GLUT1, GLUT4, VDR, and insulin receptor (IR expression. The results indicate a potential antidiabetic function of vitamin D on GLUT1, GLUT4, VDR, and IR by improving receptor gene expression suggesting a role for vitamin D in regulation of expression of the glucose transporters in muscle cells.

  10. Testosterone treatment increases androgen receptor and aromatase gene expression in myotubes from patients with PCOS and controls, but does not induce insulin resistance

    DEFF Research Database (Denmark)

    Eriksen, Mette Brandt; Glintborg, Dorte; Nielsen, Michael Friberg Bruun

    2014-01-01

    Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conse......Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity...... is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown. We investigated the effect of 7days testosterone treatment (100nmol/l) on glucose transport and gene expression levels of hormone receptors and enzymes involved...... in the synthesis and conversion of testosterone (HSD17B1, HSD17B2, CYP19A1, SRD5A1-2, AR, ER-α, HSD17B6 and AKR1-3) in myotubes from ten patients with PCOS and ten matched controls. Testosterone treatment significantly increased aromatase and androgen receptor gene expression levels in patients and controls...

  11. Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes.

    Science.gov (United States)

    Allamand, Valérie; Bidou, Laure; Arakawa, Masayuki; Floquet, Célia; Shiozuka, Masataka; Paturneau-Jouas, Marion; Gartioux, Corine; Butler-Browne, Gillian S; Mouly, Vincent; Rousset, Jean-Pierre; Matsuda, Ryoichi; Ikeda, Daishiro; Guicheney, Pascale

    2008-02-01

    The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.

  12. Experiment list: DRX013455 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available erentiation from myoblast state was done by transferring to Dulbecco's modified Eag...q of Pol II-Ser5ph, C2C12 myotube sample_name=DRS013213 || sample comment=The sample was fixed with 0.5% formaldehyde for 5 min. Diff

  13. ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth

    DEFF Research Database (Denmark)

    Markljung, Ellen; Jiang, Lin; Jaffe, Jacob D

    2009-01-01

    and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (Ch...... is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth....

  14. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways ...

    Indian Academy of Sciences (India)

    The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA expression of myogenic ...

  15. Dexamethasone stimulates store-operated calcium entry and protein degradation in cultured L6 myotubes through a phospholipase A2-dependent mechanism

    Science.gov (United States)

    Itagaki, Kiyoshi; Menconi, Michael; Antoniu, Bozena; Zhang, Qin; Gonnella, Patricia; Soybel, David; Hauser, Carl

    2010-01-01

    Muscle wasting in various catabolic conditions is at least in part regulated by glucocorticoids. Increased calcium levels have been reported in atrophying muscle. Mechanisms regulating calcium homeostasis in muscle wasting, in particular the role of glucocorticoids, are poorly understood. Here we tested the hypothesis that glucocorticoids increase intracellular calcium concentrations in skeletal muscle and stimulate store-operated calcium entry (SOCE) and that these effects of glucocorticoids may at least in part be responsible for glucocorticoid-induced protein degradation. Treatment of cultured myotubes with dexamethasone, a frequently used in vitro model of muscle wasting, resulted in increased intracellular calcium concentrations determined by fura-2 AM fluorescence measurements. When SOCE was measured by using calcium “add-back” to muscle cells after depletion of intracellular calcium stores, results showed that SOCE was increased 15–25% by dexamethasone and that this response to dexamethasone was inhibited by the store-operated calcium channel blocker BTP2. Dexamethasone treatment stimulated the activity of calcium-independent phospholipase A2 (iPLA2), and dexamethasone-induced increase in SOCE was reduced by the iPLA2 inhibitor bromoenol lactone (BEL). In additional experiments, treatment of myotubes with the store-operated calcium channel inhibitor gadolinium ion or BEL reduced dexamethasone-induced increase in protein degradation. Taken together, the results suggest that glucocorticoids increase calcium concentrations in myocytes and stimulate iPLA2-dependent SOCE and that glucocorticoid-induced muscle protein degradation may at least in part be regulated by increased iPLA2 activity, SOCE, and cellular calcium levels. PMID:20107037

  16. Breaking sarcomeres by in vitro exercise.

    Science.gov (United States)

    Orfanos, Zacharias; Gödderz, Markus P O; Soroka, Ekaterina; Gödderz, Tobias; Rumyantseva, Anastasia; van der Ven, Peter F M; Hawke, Thomas J; Fürst, Dieter O

    2016-01-25

    Eccentric exercise leads to focal disruptions in the myofibrils, referred to as "lesions". These structures are thought to contribute to the post-exercise muscle weakness, and to represent areas of mechanical damage and/or remodelling. Lesions have been investigated in human biopsies and animal samples after exercise. However, this approach does not examine the mechanisms behind lesion formation, or their behaviour during contraction. To circumvent this, we used electrical pulse stimulation (EPS) to simulate exercise in C2C12 myotubes, combined with live microscopy. EPS application led to the formation of sarcomeric lesions in the myotubes, resembling those seen in exercised mice, increasing in number with the time of application or stimulation intensity. Furthermore, transfection with an EGFP-tagged version of the lesion and Z-disc marker filamin-C allowed us to observe the formation of lesions using live cell imaging. Finally, using the same technique we studied the behaviour of these structures during contraction, and observed them to be passively stretching. This passive behaviour supports the hypothesis that lesions contribute to the post-exercise muscle weakness, protecting against further damage. We conclude that EPS can be reliably used as a model for the induction and study of sarcomeric lesions in myotubes in vitro.

  17. Breaking sarcomeres by in vitro exercise

    Science.gov (United States)

    Orfanos, Zacharias; Gödderz, Markus P. O.; Soroka, Ekaterina; Gödderz, Tobias; Rumyantseva, Anastasia; van der Ven, Peter F. M.; Hawke, Thomas J.; Fürst, Dieter O.

    2016-01-01

    Eccentric exercise leads to focal disruptions in the myofibrils, referred to as “lesions”. These structures are thought to contribute to the post-exercise muscle weakness, and to represent areas of mechanical damage and/or remodelling. Lesions have been investigated in human biopsies and animal samples after exercise. However, this approach does not examine the mechanisms behind lesion formation, or their behaviour during contraction. To circumvent this, we used electrical pulse stimulation (EPS) to simulate exercise in C2C12 myotubes, combined with live microscopy. EPS application led to the formation of sarcomeric lesions in the myotubes, resembling those seen in exercised mice, increasing in number with the time of application or stimulation intensity. Furthermore, transfection with an EGFP-tagged version of the lesion and Z-disc marker filamin-C allowed us to observe the formation of lesions using live cell imaging. Finally, using the same technique we studied the behaviour of these structures during contraction, and observed them to be passively stretching. This passive behaviour supports the hypothesis that lesions contribute to the post-exercise muscle weakness, protecting against further damage. We conclude that EPS can be reliably used as a model for the induction and study of sarcomeric lesions in myotubes in vitro. PMID:26804343

  18. Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Kazuya, E-mail: asuno10k@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Wada, Eiji, E-mail: gacchu1@yahoo.co.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Zammit, Peter S., E-mail: peter.zammit@kcl.ac.uk [Randall Division of Cell and Molecular Biophysics, King' s College London, London SE1 1UL (United Kingdom); Shiozuka, Masataka, E-mail: cmuscle@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan); Matsuda, Ryoichi, E-mail: cmatsuda@mail.ecc.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo (Japan)

    2015-05-01

    Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then differentiate and fuse to form myofibers. Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. - Highlights: • Zinc has roles for promoting proliferation and inhibition differentiation of C2C12. • Zinc promotes activation of reserve cells. • Insulin and zinc synergize activation of reserve cells. • PI3K/Akt and ERK cascade affect zinc/insulin-mediated activation of reserve cells.

  19. Biocompatible, Biodegradable, and Electroactive Polyurethane-Urea Elastomers with Tunable Hydrophilicity for Skeletal Muscle Tissue Engineering.

    Science.gov (United States)

    Chen, Jing; Dong, Ruonan; Ge, Juan; Guo, Baolin; Ma, Peter X

    2015-12-30

    It remains a challenge to develop electroactive and elastic biomaterials to mimic the elasticity of soft tissue and to regulate the cell behavior during tissue regeneration. We designed and synthesized a series of novel electroactive and biodegradable polyurethane-urea (PUU) copolymers with elastomeric property by combining the properties of polyurethanes and conducting polymers. The electroactive PUU copolymers were synthesized from amine capped aniline trimer (ACAT), dimethylol propionic acid (DMPA), polylactide, and hexamethylene diisocyanate. The electroactivity of the PUU copolymers were studied by UV-vis spectroscopy and cyclic voltammetry. Elasticity and Young's modulus were tailored by the polylactide segment length and ACAT content. Hydrophilicity of the copolymer films was tuned by changing DMPA content and doping of the copolymer. Cytotoxicity of the PUU copolymers was evaluated by mouse C2C12 myoblast cells. The myogenic differentiation of C2C12 myoblasts on copolymer films was also studied by analyzing the morphology of myotubes and relative gene expression during myogenic differentiation. The chemical structure, thermal properties, surface morphology, and processability of the PUU copolymers were characterized by NMR, FT-IR, gel permeation chromatography (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM), and solubility testing, respectively. Those biodegradable electroactive elastic PUU copolymers are promising materials for repair of soft tissues such as skeletal muscle, cardiac muscle, and nerve.

  20. Metformin Treatment Prevents Sedentariness Related Damages in Mice

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    Pamela Senesi

    2016-01-01

    Full Text Available Metformin (METF, historical antihyperglycemic drug, is a likely candidate for lifespan extension, treatment and prevention of sedentariness damages, insulin resistance, and obesity. Skeletal muscle is a highly adaptable tissue, capable of hypertrophy response to resistance training and of regeneration after damage. Aims of this work were to investigate METF ability to prevent sedentariness damage and to enhance skeletal muscle function. Sedentary 12-week-old C57BL/6 mice were treated with METF (250 mg/kg per day, in drinking water for 60 days. METF role on skeletal muscle differentiation was studied in vitro using murine C2C12 myoblasts. Muscular performance evaluation revealed that METF enhanced mice physical performance (Estimated VO2max. Biochemical analyses of hepatic and muscular tissues indicated that in liver METF increased AMPK and CAMKII signaling. In contrast, METF inactivated ERKs, the principal kinases involved in hepatic stress. In skeletal muscle, METF activated AKT, key kinase in skeletal muscle mass maintenance. In in vitro studies, METF did not modify the C2C12 proliferation capacity, while it positively influenced the differentiation process and myotube maturation. In conclusion, our novel results suggest that METF has a positive action not only on the promotion of healthy aging but also on the prevention of sedentariness damages.

  1. APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffolding the TAK1-MKK3-p38 MAPK pathway

    Science.gov (United States)

    Xin, Xiaoban; Zhou, Lijun; Reyes, Caleb M.; Liu, Feng

    2011-01-01

    The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C2C12 cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C2C12 myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation. PMID:20978232

  2. Doxycycline Inhibits IL-17-Stimulated MMP-9 Expression by Downregulating ERK1/2 Activation: Implications in Myogenic Differentiation

    Science.gov (United States)

    Obradović, Hristina; Krstić, Jelena; Kukolj, Tamara; Đorđević, Ivana Okić; Jauković, Aleksandra; Jovčić, Gordana

    2016-01-01

    Interleukin 17 (IL-17) is a cytokine with pleiotropic effects associated with several inflammatory diseases. Although elevated levels of IL-17 have been described in inflammatory myopathies, its role in muscle remodeling and regeneration is still unknown. Excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting. In this study, the role of IL-17 on the expression of matrix metalloproteinase- (MMP-) 9 in myoblast cells was investigated. The expression of MMP-9 after IL-17 treatment was analyzed in mouse myoblasts C2C12 cell line. The increase in MMP-9 production by IL-17 was concomitant with its capacity to inhibit myogenic differentiation of C2C12 cells. Doxycycline (Doxy) treatment protected the myogenic capacity of myoblasts from IL-17 inhibition and, moreover, increased myotubes hypertrophy. Doxy blocked the capacity of IL-17 to stimulate MMP-9 production by regulating IL-17-induced ERK1/2 MAPK activation. Our results imply that MMP-9 mediates IL-17's capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that Doxy can modulate myoblast response to inflammatory induction by IL-17. PMID:28042204

  3. The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

    Science.gov (United States)

    Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

    Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

  4. Basal Lamina Mimetic Nanofibrous Peptide Networks for Skeletal Myogenesis

    Science.gov (United States)

    Yasa, I. Ceren; Gunduz, Nuray; Kilinc, Murat; Guler, Mustafa O.; Tekinay, Ayse B.

    2015-11-01

    Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells’ growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, “IKVAV”, and fibronectin-derived well known adhesive sequence, “RGD”, into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes.

  5. A Chemically Polymerized Electrically Conducting Composite of Polypyrrole Nanoparticles and Polyurethane for Tissue Engineering

    Science.gov (United States)

    Broda, Christopher R.; Lee, Jae Y.; Sirivisoot, Sirinrath; Schmidt, Christine E.; Harrison, Benjamin S.

    2011-01-01

    A variety of cell types respond to electrical stimuli, accordingly many conducting polymers (CPs) have been used as tissue engineering (TE) scaffolds, one such CP is polypyrrole (PPy). PPy is a well studied biomaterial with potential TE applications due to its electrical conductivity and many other beneficial properties. Combining its characteristics with an elastomeric material, such as polyurethane (PU), may yield a hybrid scaffold with electrical activity and significant mechanical resilience. Pyrrole was in situ polymerized within a PU emulsion mixture in weight ratios of 1:100, 1:20, 1:10 and 1:5, respectively. Morphology, electrical conductivity, mechanical properties and cytocompatibility with C2C12 myoblast cells were characterized. The polymerization resulted in a composite with a principle base of PU interspersed with an electrically percolating network of PPy nanoparticles. As the mass ratio of PPy to PU increased so did electrical conductivity of the composites. In addition, as the mass ratio of PPy to PU increased, stiffness of the composite increased while maximum elongation length decreased. Ultimate tensile strength was reduced by approximately 47% across all samples with the addition of PPy to the PU base. Cytocompatibility assay data indicated no significant cytotoxic effect from the composites. Static cellular seeding of C2C12 cells and subsequent differentiation showed myotube formation on the composite materials. PMID:21681943

  6. The Root Extract of Pueraria lobata and Its Main Compound, Puerarin, Prevent Obesity by Increasing the Energy Metabolism in Skeletal Muscle.

    Science.gov (United States)

    Jung, Hyo Won; Kang, An Na; Kang, Seok Yong; Park, Yong-Ki; Song, Mi Young

    2017-01-04

    Radix Pueraria lobata (RP) has been reported to prevent obesity and improve glucose metabolism; however, the mechanism responsible for these effects has not been elucidated. The mechanism underlying anti-obesity effect of RP was investigated in high-fat diet (HFD) induced obese mice and skeletal muscle cells (C2C12). Five-week-old C5BL/6 mice were fed a HFD containing or not containing RP (100 or 300 mg/kg) or metformin (250 mg/kg) for 16 weeks. RP reduced body weight gain, lipid accumulation in liver, and adipocyte and blood lipid levels. In addition, RP dose-dependently improved hyperglycemia, insulinemia, and glucose tolerance, and prevented the skeletal muscle atrophy induced by HFD. Furthermore, RP increased the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle tissues. RP and its main component, puerarin, increased mitochondrial biogenesis and myotube hypertrophy in C2C12 cells. The present study demonstrates that RP can prevent diet-induced obesity, glucose tolerance, and skeletal muscle atrophy in mouse models of obesity. The mechanism responsible for the effect of RP appears to be related to the upregulation of energy metabolism in skeletal muscle, which at the molecular level may be associated with PGC-1α and AMPK activation.

  7. Simvastatin induces insulin resistance in L6 skeletal muscle myotubes by suppressing insulin signaling, GLUT4 expression and GSK-3β phosphorylation.

    Science.gov (United States)

    Yaluri, Nagendra; Modi, Shalem; Kokkola, Tarja

    2016-11-11

    Simvastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor widely used for the treatment of hypercholesterolemia. Recent data indicates that simvastatin increases the risk of new-onset diabetes by impairing both insulin secretion and insulin sensitivity. However, systematic evaluation of mechanistic pathways is lacking. We aimed to explore the effects of simvastatin on glucose uptake and underlying mechanisms using L6 skeletal muscle myotubes. We performed our experiments at basal and insulin-stimulated conditions, at normal (5.5 mM) and high (16.7 mM) glucose. We showed that simvastatin inhibited glucose uptake at all conditions. We also found out that pravastatin, another widely used statin with different physicochemical properties, did not inhibit glucose uptake. The effect of simvastatin was reversed with geranylgeranyl pyrophosphate but not with farnesyl pyrophosphate, implying that reduced protein geranylgeranylation has a role in simvastatin-induced insulin resistance. Simvastatin also decreased phosphorylation of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), AKT and glycogen synthase kinase 3β (GSK-3β), and downregulated GLUT4. In conclusion, our data indicate that simvastatin decreased both basal and insulin-stimulated glucose uptake through inhibiting the critical steps in IR/IRS-1/AKT signaling cascade, and by hindering GLUT4 function and normal regulation of glycogen synthesis, contributing to insulin resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Magnolia officinalis Extract Contains Potent Inhibitors against PTP1B and Attenuates Hyperglycemia in db/db Mice

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    Jing Sun

    2015-01-01

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is an established therapeutic target for type 2 diabetes mellitus (T2DM and obesity. The aim of this study was to investigate the inhibitory activity of Magnolia officinalis extract (ME on PTP1B and its anti-T2DM effects. Inhibition assays and inhibition kinetics of ME were performed in vitro. 3T3-L1 adipocytes and C2C12 myotubes were stimulated with ME to explore its bioavailability in cell level. The in vivo studies were performed on db/db mice to probe its anti-T2DM effects. In the present study, ME inhibited PTP1B in a reversible competitive manner and displayed good selectivity against PTPs in vitro. Furthermore, ME enhanced tyrosine phosphorylation levels of cellular proteins, especially the insulin-induced tyrosine phosphorylations of insulin receptor β-subunit (IRβ and ERK1/2 in a dose-dependent manner in stimulated 3T3-L1 adipocytes and C2C12 myotubes. Meanwhile, ME enhanced insulin-stimulated GLUT4 translocation. More importantly, there was a significant decrease in fasting plasma glucose level of db/db diabetic mice treated orally with 0.5 g/kg ME for 4 weeks. These findings indicated that improvement of insulin sensitivity and hypoglycemic effects of ME may be attributed to the inhibition of PTP1B. Thereby, we pioneered the inhibitory potential of ME targeted on PTP1B as anti-T2DM drug discovery.

  9. Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line.

    Science.gov (United States)

    Varadi, G; Orlowski, J; Schwartz, A

    1989-07-03

    The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.

  10. Baicalin against obesity and insulin resistance through activation of AKT/AS160/GLUT4 pathway.

    Science.gov (United States)

    Fang, Penghua; Yu, Mei; Zhang, Lei; Wan, Dan; Shi, Mingyi; Zhu, Yan; Bo, Ping; Zhang, Zhenwen

    2017-06-15

    Obesity may cause several metabolic complications, including insulin resistance and type 2 diabetes mellitus. Despite great advances in medicine, people still keep exploring novel and effective drugs for treatment of obesity and insulin resistance. The aim of this study was to survey if baicalin might ameliorate obesity-induced insulin resistance and to explore its signal mechanisms in skeletal muscles of mice. Diet-induced obese (DIO) mice were given 50 mg/kg baicalin intraperitoneally (i.p.) once a day for 21 days, and C2C12 myotubes were treated with 100, 200, 400 μM baicalin for 12 h in this study. Then insulin resistance indexes and insulin signal protein levels in skeletal muscles were examined. We discovered that administration of baicalin decreased food intake, body weight, HOMA-IR and NT-PGC-1α levels, but enhanced GLUT4, PGC-1α, pP38MAPK, pAKT and pAS160 contents, as well as GLUT4 mRNA, PGC-1α mRNA, PPARγ mRNA, GLUT1 mRNA expression in skeletal muscles of obese mice and myotubes of C2C12 cells, and reversed high fat diet-induced glucose and insulin intolerance, hyperglycemia and insulin resistance in the mice. These results suggest that baicalin is a powerful and promising agent for treatment of obesity and insulin resistance via Akt/AS160/GLUT4 and P38MAPK/PGC1α/GLUT4 pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Alternative splicing factor ASF/SF2 is down regulated in inflamed muscle.

    Science.gov (United States)

    Xiong, Z; Shaibani, A; Li, Y-P; Yan, Y; Zhang, S; Yang, Y; Yang, F; Wang, H; Yang, X-F

    2006-08-01

    In our recent studies, alternative splicing has been shown to have a major role in inflammation and autoimmune muscle diseases. To examine the novel hypothesis that the expression of an essential alternative splicing factor, alternative splicing factor 2 (ASF/SF2), is modulated in muscle inflammation. ASF/SF2 expression in muscle biopsy samples from eight patients with inflammatory myopathy and six non-myositic controls was determined by using western blot with anti-ASF/SF2 antibodies. To further elucidate the mechanism of reduced ASF/SF2 expression in inflamed muscle, differentiated C2C12 myotubes were stimulated with proinflammatory cytokine tumour necrosis factor alpha (TNFalpha), followed by western blot analysis of ASF/SF2 expression. ASF/SF2 expression in the muscle biopsy samples from patients with inflammatory myopathy was found to be lower (mean of relative densitometric units 41.1 (2SD 20.7)) than that of the non-myositic controls (mean of relative densitometric units 76.7 (39.6); pASF/SF2 expression was seen to be significantly down regulated (sevenfold) in C2C12 myotubes compared with expression variations in the beta-actin control (0.62-fold; mean 1.22 (0.40); pASF/SF2 is down regulated in autoimmune inflammatory myositis-potentially via a TNFalpha-mediated pathway. The development of (1) novel autoantigen isoform microarrays for disease diagnosis and prognosis; (2) novel autoantigen-tolerising treatments for autoimmune diseases; and (3) novel splicing-redirection treatments can be facilitated by the ongoing study of alternative splicing of autoantigen transcripts.

  12. Imidacloprid Promotes High Fat Diet-Induced Adiposity in Female C57BL/6J Mice and Enhances Adipogenesis in 3T3-L1 Adipocytes via the AMPKα-Mediated Pathway.

    Science.gov (United States)

    Sun, Quancai; Qi, Weipeng; Xiao, Xiao; Yang, Szu-Hao; Kim, Daeyoung; Yoon, Kyong Sup; Clark, John M; Park, Yeonhwa

    2017-08-09

    Imidacloprid, a neonicotinoid insecticide, was previously reported to enhance adipogenesis and resulted in insulin resistance in cell culture models. It was also reported to promote high fat diet-induced obesity and insulin resistance in male C57BL/6J mice. Thus, the goal of the present study was to determine the effects of imidacloprid and dietary fat interaction on the development of adiposity and insulin resistance in female C57BL/6J mice. Mice were fed with a low (4% w/w) or high fat (20% w/w) diet containing imidacloprid (0.06, 0.6, or 6 mg/kg bw/day) for 12 weeks. Mice fed with imidacloprid (0.6 mg/kg bw/day) significantly enhanced high fat diet-induced weight gain and adiposity. Treatment with imidacloprid significantly increased serum insulin levels with high fat diet without effects on other markers of glucose homeostasis. AMPKα activation was significantly inhibited by 0.6 and 6 mg imidacloprid/kg bw/day in white adipose tissue. Moreover, AMPKα activation with 5-aminoimidazole-4-carboxamide ribonucleotide abolished the effects of imidacloprid (10 μM) on enhanced adipogenesis in 3T3-L1 adipocytes. N-Acetyl cysteine also partially reversed the effects of imidacloprid on reduced phosphorylation of protein kinase B (AKT) in C2C12 myotubes. These results indicate that imidacloprid may potentiate high fat diet-induced adiposity in female C57BL/6J mice and enhance adipogenesis in 3T3-L1 adipocytes via the AMPKα-mediated pathway. Imidacloprid might also influence glucose homeostasis partially by inducing cellular oxidative stress in C2C12 myotubes.

  13. Effect of Exercise Intensity on Isoform-Specific Expressions of NT-PGC-1α mRNA in Mouse Skeletal Muscle

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    Xingyuan Wen

    2014-01-01

    Full Text Available PGC-1α is an inducible transcriptional coactivator that regulates mitochondrial biogenesis and cellular energy metabolism in skeletal muscle. Recent studies have identified two additional PGC-1α transcripts that are derived from an alternative exon 1 (exon 1b and induced by exercise. Given that the PGC-1α gene also produces NT-PGC-1α transcript by alternative 3′ splicing between exon 6 and exon 7, we have investigated isoform-specific expression of NT-PGC-1α mRNA in mouse skeletal muscle during physical exercise with different intensities. We report here that NT-PGC-1α-a mRNA expression derived from a canonical exon 1 (exon 1a is increased by high-intensity exercise and AMPK activator AICAR in mouse skeletal muscle but not altered by low- and medium-intensity exercise and β2-adrenergic receptor agonist clenbuterol. In contrast, the alternative exon 1b-driven NT-PGC-1α-b (PGC-1α4 and NT-PGC-1α-c are highly induced by low-, medium-, and high-intensity exercise, AICAR, and clenbuterol. Ectopic expression of NT-PGC-1α-a in C2C12 myotube cells upregulates myosin heavy chain (MHC I, MHC II a and Glut4, which represent oxidative fibers, and promotes the expression of mitochondrial genes (Cyc1, COX5B, and ATP5B. In line with gene expression data, citrate synthase activity was significantly increased by NT-PGC-1α-a in C2C12 myotube cells. Our results indicate the regulatory role for NT-PGC-1α-a in mitochondrial biogenesis and adaptation of skeletal muscle to endurance exercise.

  14. Protein kinase D2 is an essential regulator of murine myoblast differentiation.

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    Alexander Kleger

    Full Text Available Muscle differentiation is a highly conserved process that occurs through the activation of quiescent satellite cells whose progeny proliferate, differentiate, and fuse to generate new myofibers. A defined pattern of myogenic transcription factors is orchestrated during this process and is regulated via distinct signaling cascades involving various intracellular signaling pathways, including members of the protein kinase C (PKC family. The protein kinase D (PKD isoenzymes PKD1, -2, and -3, are prominent downstream targets of PKCs and phospholipase D in various biological systems including mouse and could hence play a role in muscle differentiation. In the present study, we used a mouse myoblast cell line (C2C12 as an in vitro model to investigate the role of PKDs, in particular PKD2, in muscle stem cell differentiation. We show that C2C12 cells express all PKD isoforms with PKD2 being highly expressed. Furthermore, we demonstrate that PKD2 is specifically phosphorylated/activated during the initiation of mouse myoblast differentiation. Selective inhibition of PKCs or PKDs by pharmacological inhibitors blocked myotube formation. Depletion of PKD2 by shRNAs resulted in a marked inhibition of myoblast cell fusion. PKD2-depleted cells exhibit impaired regulation of muscle development-associated genes while the proliferative capacity remains unaltered. Vice versa forced expression of PKD2 increases myoblast differentiation. These findings were confirmed in primary mouse satellite cells where myotube fusion was also decreased upon inhibition of PKDs. Active PKD2 induced transcriptional activation of myocyte enhancer factor 2D and repression of Pax3 transcriptional activity. In conclusion, we identify PKDs, in particular PKD2, as a major mediator of muscle cell differentiation in vitro and thereby as a potential novel target for the modulation of muscle regeneration.

  15. Activation of type 2 cannabinoid receptors (CB2R) promotes fatty acid oxidation through the SIRT1/PGC-1α pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Xuqin [Department of Endocrinology, First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu Province 210029 (China); Sun, Tao [Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002 (China); Wang, Xiaodong, E-mail: xdwang666@hotmail.com [Department of Endocrinology, First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu Province 210029 (China)

    2013-07-05

    Highlights: •TC, a CB2R specific agonist, stimulates SIRT1 activity by PKA/CREB pathway. •TC promotes PGC-1α transcriptional activity by increasing its deacetylation. •TC increases the expression of genes linked to FAO and promotes the rate of FAO. •The effects of TC in FAO are dependent on CB2R. •Suggesting CB2R as a target to treat diseases with lipid dysregulation. -- Abstract: Abnormal fatty acid oxidation has been associated with obesity and type 2 diabetes. At the transcriptional level, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) has been reported to strongly increase the ability of hormone nuclear receptors PPARα and ERRα to drive transcription of fatty acid oxidation enzymes. In this study, we report that a specific agonist of the type 2 cannabinoid receptor (CB2R) can lead to fatty acid oxidation through the PGC-1α pathway. We have found that CB2R is expressed in differentiated C2C12 myotubes, and that use of the specific agonist trans-caryophyllene (TC) stimulates sirtuin 1 (SIRT1) deacetylase activity by increasing the phosphorylation of cAMP response element-binding protein (CREB), thus leading to increased levels of PGC-1α deacetylation. This use of TC treatment increases the expression of genes linked to the fatty acid oxidation pathway in a SIRT1/PGC-1α-dependent mechanism and also drastically accelerates the rate of complete fatty acid oxidation in C2C12 myotubes, neither of which occur when CB2R mRNA is knocked down using siRNA. These results reveal that activation of CB2R by a selective agonist promotes lipid oxidation through a signaling/transcriptional pathway. Our findings imply that pharmacological manipulation of CB2R may provide therapeutic possibilities to treat metabolic diseases associated with lipid dysregulation.

  16. Delta-like 1 homolog (dlk1: a marker for rhabdomyosarcomas implicated in skeletal muscle regeneration.

    Directory of Open Access Journals (Sweden)

    Louise H Jørgensen

    Full Text Available Dlk1, a member of the Epidermal Growth Factor family, is expressed in multiple tissues during development, and has been detected in carcinomas and neuroendocrine tumors. Dlk1 is paternally expressed and belongs to a group of imprinted genes associated with rhabdomyosarcomas but not with other primitive childhood tumors to date. Here, we investigate the possible roles of Dlk1 in skeletal muscle tumor formation. We analyzed tumors of different mesenchymal origin for expression of Dlk1 and various myogenic markers and found that Dlk1 was present consistently in myogenic tumors. The coincident observation of Dlk1 with a highly proliferative state in myogenic tumors led us to subsequently investigate the involvement of Dlk1 in the control of the adult myogenic programme. We performed an injury study in Dlk1 transgenic mice, ectopically expressing ovine Dlk1 (membrane bound C2 variant under control of the myosin light chain promotor, and detected an early, enhanced formation of myotubes in Dlk1 transgenic mice. We then stably transfected the mouse myoblast cell line, C2C12, with full-length Dlk1 (soluble A variant and detected an inhibition of myotube formation, which could be reversed by adding Dlk1 antibody to the culture supernatant. These results suggest that Dlk1 is involved in controlling the myogenic programme and that the various splice forms may exert different effects. Interestingly, both in the Dlk1 transgenic mice and the DLK1-C2C12 cells, we detected reduced myostatin expression, suggesting that the effect of Dlk1 on the myogenic programme might involve the myostatin signaling pathway. In support of a relationship between Dlk1 and myostatin we detected reciprocal expression of these two transcripts during different cell cycle stages of human myoblasts. Together our results suggest that Dlk1 is a candidate marker for skeletal muscle tumors and might be involved directly in skeletal muscle tumor formation through a modulatory effect on the

  17. Imidacloprid Promotes High Fat Diet-Induced Adiposity in Female C57BL/6J Mice and Enhances Adipogenesis in 3T3-L1 Adipocytes via the AMPKα-Mediated Pathway

    Science.gov (United States)

    2017-01-01

    Imidacloprid, a neonicotinoid insecticide, was previously reported to enhance adipogenesis and resulted in insulin resistance in cell culture models. It was also reported to promote high fat diet-induced obesity and insulin resistance in male C57BL/6J mice. Thus, the goal of the present study was to determine the effects of imidacloprid and dietary fat interaction on the development of adiposity and insulin resistance in female C57BL/6J mice. Mice were fed with a low (4% w/w) or high fat (20% w/w) diet containing imidacloprid (0.06, 0.6, or 6 mg/kg bw/day) for 12 weeks. Mice fed with imidacloprid (0.6 mg/kg bw/day) significantly enhanced high fat diet-induced weight gain and adiposity. Treatment with imidacloprid significantly increased serum insulin levels with high fat diet without effects on other markers of glucose homeostasis. AMPKα activation was significantly inhibited by 0.6 and 6 mg imidacloprid/kg bw/day in white adipose tissue. Moreover, AMPKα activation with 5-aminoimidazole-4-carboxamide ribonucleotide abolished the effects of imidacloprid (10 μM) on enhanced adipogenesis in 3T3-L1 adipocytes. N-Acetyl cysteine also partially reversed the effects of imidacloprid on reduced phosphorylation of protein kinase B (AKT) in C2C12 myotubes. These results indicate that imidacloprid may potentiate high fat diet-induced adiposity in female C57BL/6J mice and enhance adipogenesis in 3T3-L1 adipocytes via the AMPKα-mediated pathway. Imidacloprid might also influence glucose homeostasis partially by inducing cellular oxidative stress in C2C12 myotubes. PMID:28704996

  18. Tumor necrosis factor-α regulates distinct molecular pathways and gene networks in cultured skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Shephali Bhatnagar

    2010-10-01

    Full Text Available Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood.We have used microarray, quantitative real-time PCR (QRT-PCR, Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line. Microarray analyses of C2C12 myotubes treated with TNF-α (10 ng/ml for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-α. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-β signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes.TNF-α regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells

  19. Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca2+ release in rat skeletal myotubes.

    Science.gov (United States)

    Liberona, José Luis; Cárdenas, J César; Reyes, Roberto; Hidalgo, Jorge; Molgó, Jordi; Jaimovich, Enrique

    2008-09-01

    Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca2+ levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca2+ signals evoked by PbTx-3 were inhibited by blocking either IP3 receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP3 mass levels, dependent on extra-cellular Na+. A similar increase in IP3 mass was induced by high K+ depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP3 mass induced by either PbTx-3 or K+ was also detected in Ca2+-free medium. These results establish that the effect of the toxin on both intracellular Ca2+ and IP3 levels occurs via a membrane potential sensor instead of directly by Na+ flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP3-dependent calcium signal involved in regulation of gene expression.

  20. Transforming growth factor type-β inhibits Mas receptor expression in fibroblasts but not in myoblasts or differentiated myotubes; Relevance to fibrosis associated to muscular dystrophies.

    Science.gov (United States)

    Cofre, Catalina; Acuña, María José; Contreras, Osvaldo; Morales, María Gabriela; Riquelme, Cecilia; Cabello-Verrugio, Claudio; Brandan, Enrique

    2015-01-01

    Duchenne muscular dystrophy is a genetic disorder characterized by myofiber degeneration, muscle weakness, and increased fibrosis. Transforming growth factor type-β (TGF-β), a central mediator of fibrosis, is upregulated in fibrotic diseases. Angiotensin-(1-7) [Ang-(1-7)] is a peptide with actions that oppose those of angiotensin-II (Ang II). Ang-(1-7) effects are mediated by the Mas receptor. Treatment with Ang-(1-7) produce positive effects in the mdx mouse, normalizing skeletal muscle architecture, decreasing local fibrosis, and fibroblasts, and improving muscle function. Mdx mice deficient for the Mas receptor showed the opposite effects. To identify the cell type(s) responsible for Mas receptor expression, and to characterize whether profibrotic effectors had any effect on its expression, we determined the effect of profibrotic agents on Mas expression. TGF-β, but not connective tissue growth factor or Ang-II, reduced the expression of Mas receptor in fibroblasts isolated from skeletal muscle cells and fibroblasts from two established cell lines. In contrast, no effects were observed in myoblasts and differentiated myotubes. This inhibition was mediated by the Smad-dependent (canonical) and the PI3K and MEK1/2 (noncanonical) TGF-β signaling pathways. When both canonical and noncanonical inhibitors of the TGF-β-dependent pathways were added together, the inhibitory effect of TGF-β on Mas expression was lost. The decrease in Mas receptor induced by TGF-β in fibroblasts reduced the Ang-(1-7) mediated stimulation of phosphorylation of AKT pathway proteins. These results suggest that reduction of Mas receptor in fibroblasts, by TGF-β, could increase the fibrotic phenotype observed in dystrophic skeletal muscle decreasing the beneficial effect of Ang-(1-7). © 2015 International Union of Biochemistry and Molecular Biology.

  1. Stretchable degradable and electroactive shape memory copolymers with tunable recovery temperature enhance myogenic differentiation.

    Science.gov (United States)

    Deng, Zexing; Guo, Yi; Zhao, Xin; Li, Longchao; Dong, Ruonan; Guo, Baolin; Ma, Peter X

    2016-12-01

    Development of flexible degradable electroactive shape memory polymers (ESMPs) with tunable switching temperature (around body temperature) for tissue engineering is still a challenge. Here we designed and synthesized a series of shape memory copolymers with electroactivity, super stretchability and tunable recovery temperature based on poly(ε-caprolactone) (PCL) with different molecular weight and conductive amino capped aniline trimer, and demonstrated their potential to enhance myogenic differentiation from C2C12 myoblast cells. We characterized the copolymers by Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance ((1)H NMR), cyclic voltammetry (CV), ultraviolet-visible spectroscopy (UV-vis), differential scanning calorimetry (DSC), shape memory test, tensile test and in vitro enzymatic degradation study. The electroactive biodegradable shape memory copolymers showed great elasticity, tunable recovery temperature around 37°C, and good shape memory properties. Furthermore, proliferation and differentiation of C2C12 myoblasts were investigated on electroactive copolymers films, and they greatly enhanced the proliferation, myotube formation and related myogenic differentiation genes expression of C2C12 myoblasts compared to the pure PCL with molecular weight of 80,000. Our study suggests that these electroactive, highly stretchable, biodegradable shape memory polymers with tunable recovery temperature near the body temperature have great potential in skeletal muscle tissue engineering application. Conducting polymers can regulate cell behavior such cell adhesion, proliferation, and differentiation with or without electrical stimulation. Therefore, they have great potential for electrical signal sensitive tissue regeneration. Although conducting biomaterials with degradability have been developed, highly stretchable and electroactive degradable copolymers for soft tissue engineering have been rarely reported. On the other hand, shape

  2. The basal kinetic parameters of glycogen synthase in human myotube cultures are not affected by chronic high insulin exposure

    DEFF Research Database (Denmark)

    Gaster, M; Schrøder, H D; Handberg, A

    2001-01-01

    There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic pa...

  3. Regulation of REDD1 by Insulin-like Growth Factor-I in Skeletal Muscle and Myotubes

    OpenAIRE

    Frost, Robert A.; Huber, Danuta; Pruznak, Anne; Charles H Lang

    2009-01-01

    Insulin-like growth factor-I (IGF-I) is a major anabolic hormone for skeletal muscle and a potent stimulus for protein synthesis and translation initiation. Recent studies suggest that translation can be inhibited by over expression of the mammalian target of rapamycin (mTOR) repressor REDD1. The purpose of the present study was to determine whether IGF-I alters the expression of REDD1 and whether this is associated with a concomitant change in protein synthesis in vitro. Subcutaneous injecti...

  4. Creatine-induced activation of antioxidative defence in myotube cultures revealed by explorative NMR-based metabonomics and proteomics

    DEFF Research Database (Denmark)

    Young, Jette F; Larsen, Lotte B; Malmendal, Anders

    2010-01-01

    Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat......-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating...... to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress....

  5. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

    Directory of Open Access Journals (Sweden)

    Hitomi Maruta

    Full Text Available Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4 genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A, which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin.

  6. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

    Science.gov (United States)

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin.

  7. Upregulation of glucose uptake in L8 myotubes by the extract from Lagerstroemia speciosa: a possible mechanism of action

    Directory of Open Access Journals (Sweden)

    Juntipa Purintrapiban

    2009-12-01

    Full Text Available The leaf of Lagerstroemia speciosa L. is used as an anti-diabetic herbal remedy in many countries. In an attempt to discover mechanisms of action of the L. speciosa extract that stimulate glucose uptake, a cell-based radioactive assay of glucose uptake was performed using L8 muscle cells. In this study, the methanol fraction of L. speciosa leaves (LSE contained a high level of phenolic compounds and showed strong capability to stimulate glucose uptake in a dose-dependent manner. The LSE stimulation was slightly inhibited (8.8% by SB203580. The inhibitory effect (23.6% of wortmannin on LSE-stimulated glucose uptake was demonstrated, suggesting LSE action on glucose transporter translocation. LSE-induced glucose uptake was completely reversed by cycloheximide. In addition, an increased amount of total glucose-transporter-1 protein was observed indicating that new protein synthesis is necessary for elevated glucose transport. LSE also enhanced insulin-stimulated glucose transport. These results suggest that LSE action may be mediated primarily via the synthesis of new transporters and involve both insulin-dependent and independent pathways.

  8. Designing of a Si-MEMS device with an integrated skeletal muscle cell-based bio-actuator.

    Science.gov (United States)

    Fujita, Hideaki; Van Dau, Thanh; Shimizu, Kazunori; Hatsuda, Ranko; Sugiyama, Susumu; Nagamori, Eiji

    2011-02-01

    With the aim of designing a mechanical drug delivery system involving a bio-actuator, we fabricated a Micro Electro Mechanical Systems (MEMS) device that can be driven through contraction of skeletal muscle cells. The device is composed of a Si-MEMS with springs and ratchets, UV-crosslinked collagen film for cell attachment, and C2C12 muscle cells. The Si-MEMS device is 600 μm x 1000 μm in size and the width of the collagen film is 250 ~ 350 μm, which may allow the device to go through small blood vessels. To position the collagen film on the MEMS device, a thermo-sensitive polymer was used as the sacrifice-layer which was selectively removed with O₂ plasma at the positions where the collagen film was glued. The C2C12 myoblasts were seeded on the collagen film, where they proliferated and formed myotubes after induction of differentiation. When C2C12 myotubes were stimulated with electric pulses, contraction of the collagen film-C2C12 myotube complex was observed. When the edge of the Si-MEMS device was observed, displacement of ~8 μm was observed, demonstrating the possibility of locomotive movement when the device is placed on a track of adequate width. Here, we propose that the C2C12-collagen film complex is a new generation actuator for MEMS devices that utilize glucose as fuel, which will be useful in environments in which glucose is abundant such as inside a blood vessel.

  9. A comprehensive study of novel microcapsules incorporating gliclazide and a permeation enhancing bile acid: hypoglycemic effect in an animal model of Type-1 diabetes.

    Science.gov (United States)

    Mathavan, Sangeetha; Chen-Tan, Nigel; Arfuso, Frank; Al-Salami, Hani

    2016-10-01

    Gliclazide (G) is a commonly prescribed drug for Type 2 diabetes (T2D). In a recent study, we found that when G was combined with a primary bile acid, and gavaged to an animal model of Type 1 diabetes (T1D), it exerted a hypoglycemic effect. We hypothesized this to be due to metabolic activation of the primary bile acid into a secondary or a tertiary bile acid, which enhanced G solubility and absorption. The tertiary bile acid, taurocholic acid (TCA), has shown strong permeation-enhancing effects in vivo. Thus, we aimed to design, characterize, and test microcapsules incorporating G and TCA in an animal model of T1D. Microcapsules were prepared using the polymer sodium alginate (SA). G-SA microcapsules (control) and G-TCA-SA microcapsules (test) were extensively examined (in-vitro) at different pH and temperatures. The microcapsules were gavaged to diabetic rats, and blood glucose and G concentrations in serum were examined. Ex-vivo studies were also performed using a muscle cell line (C2C12), and cell viability and glucose intake post-treatment were examined. G-TCA-SA microcapsules showed good stability, uniformity, and thermal and chemical excipient compatibilities. TCA did not change the size or the shape of the microcapsules, but it enhanced their mechanical resistance and reduced their swelling properties. G-TCA-SA enhanced the viability of C2C12 cells over 24 hours, and exerted a hypoglycemic effect in alloxan-induced type-1 diabetic rats. The incorporation of TCA into G-microcapsules resulted in functionally improved microcapsules with a positive effect on cell viability and glycemic control in Type-1 diabetic animals.

  10. An extract of Urtica dioica L. mitigates obesity induced insulin resistance in mice skeletal muscle via protein phosphatase 2A (PP2A).

    Science.gov (United States)

    Obanda, Diana N; Ribnicky, David; Yu, Yongmei; Stephens, Jacqueline; Cefalu, William T

    2016-02-26

    The leaf extract of Urtica dioica L. (UT) has been reported to improve glucose homeostasis in vivo, but definitive studies on efficacy and mechanism of action are lacking. We investigated the effects of UT on obesity- induced insulin resistance in skeletal muscle. Male C57BL/6J mice were divided into three groups: low-fat diet (LFD), high-fat diet (HFD) and HFD supplemented with UT. Body weight, body composition, plasma glucose and plasma insulin were monitored. Skeletal muscle (gastrocnemius) was analyzed for insulin sensitivity, ceramide accumulation and the post translational modification and activity of protein phosphatase 2A (PP2A). PP2A is activated by ceramides and dephosphorylates Akt. C2C12 myotubes exposed to excess free fatty acids with or without UT were also evaluated for insulin signaling and modulation of PP2A. The HFD induced insulin resistance, increased fasting plasma glucose, enhanced ceramide accumulation and PP2A activity in skeletal muscle. Supplementation with UT improved plasma glucose homeostasis and enhanced skeletal muscle insulin sensitivity without affecting body weight and body composition. In myotubes, UT attenuated the ability of FFAs to induce insulin resistance and PP2A hyperactivity without affecting ceramide accumulation and PP2A expression. UT decreased PP2A activity through posttranslational modification that was accompanied by a reduction in Akt dephosphorylation.

  11. Evaluation of the hypoglycemic effects of flavonoids and extracts from Jatropha gossypifolia L.

    Science.gov (United States)

    Granados, Sergio; Balcázar, Norman; Guillén, Alis; Echeverri, Fernando

    2015-04-09

    Jatropha gossypifolia L. (Euphorbiaceae) is a plant widely used in the treatment of type 2 diabetes mellitus (T2DM), but there are few scientific reports validating its activity in this area. In this work and through a bioguided assay, a crude extract stimulated glucose uptake in C2C12 myotubes up to 30%, thereby reducing insulin resistance induced by fatty acids compared to the basal control. A chromatographic fraction applied intraperitoneally (IP) in mice reduced glucose by 42% in a mouse model of T2DM, after administration of 10 doses during 20 days. A flavanone was purified from this active fraction and its structure was assigned by 1H- and 13C-NMR (1D and 2D) and MS. This compound retains the previously reported activity, stimulating in vitro the glucose uptake in a concentration-dependent manner. This study indicates that Jatropha gossypifolia L. extracts enhance glucose uptake in cultured myotubes and adipocytes and also improving glucose tolerance in an in vivo model.

  12. Regulation of mitochondrial biogenesis during myogenesis

    OpenAIRE

    Remels, A.H.V.; Langen, R.C.J.; Schrauwen, P.; Schaart, G.; Schols, A.M.W.J.; Gosker, H. R.

    2009-01-01

    Abstract Pathways involved in mitochondrial biogenesis associated with myogenic differentiation are poorly defined. Therefore, C2C12 myoblasts were differentiated into multi-nucleated myotubes and parameters/regulators of mitochondrial biogenesis were investigated. Mitochondrial respiration, citrate synthase- and ?-hydroxyacyl-CoA dehydrogenase activity as well as protein content of complexes I, II, III and V of the mitochondrial respiratory chain increased 4-8 fold during differen...

  13. Skeletal muscle differentiation evokes endogenous XIAP to restrict the apoptotic pathway.

    Science.gov (United States)

    Smith, Michelle I; Huang, Yolanda Y; Deshmukh, Mohanish

    2009-01-01

    Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells) are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells) and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP). Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms.

  14. Skeletal muscle differentiation evokes endogenous XIAP to restrict the apoptotic pathway.

    Directory of Open Access Journals (Sweden)

    Michelle I Smith

    Full Text Available Myotube apoptosis occurs normally during muscle development and aging but it can lead to destruction of skeletal muscle in neuromuscular diseases. Therefore, understanding how myotube apoptosis is regulated is important for developing novel strategies for treatment of muscle loss. We investigated the regulation of apoptosis in skeletal muscle and report a striking increase in resistance to apoptosis following differentiation. We find mitotic C2C12 cells (myoblast-like cells are sensitive to cytosolic cytochrome c microinjection. However, differentiated C2C12 cells (myotube-like cells and primary myotubes are markedly resistant. This resistance is due to endogenous X-linked inhibitor of apoptotic protein (XIAP. Importantly, the selective difference in the ability of XIAP to block myotube but not myoblast apoptosis is not due to a change in XIAP but rather a decrease in Apaf-1 expression. This decrease in Apaf-1 links XIAP to caspase activation and death. Our findings suggest that in order for myotubes to die, they may degrade XIAP, functionally inactivate XIAP or upregulate Apaf-1. Importantly, we identify a role for endogenous Smac in overcoming XIAP to allow myotube death. However, in postmitotic cardiomyocytes, where XIAP also restricts apoptosis, endogenous Smac was not capable of overcoming XIAP to cause death. These results show that as skeletal muscle differentiate, they become resistant to apoptosis because of the ability of XIAP to regulate caspase activation. The increased restriction of apoptosis in myotubes is presumably important to ensure the long term survival of these postmitotic cells as they play a vital role in the physiology of organisms.

  15. Angiotensin-(1-7 Prevents Skeletal Muscle Atrophy Induced by Transforming Growth Factor Type Beta (TGF-β via Mas Receptor Activation

    Directory of Open Access Journals (Sweden)

    Johanna Ábrigo

    2016-11-01

    Full Text Available Background: Transforming growth factor type beta 1 (TGF-β1 produces skeletal muscle atrophy. Angiotensin-(1-7 (Ang-(1-7, through the Mas receptor, prevents the skeletal muscle atrophy induced by sepsis, immobilization, or angiotensin II (Ang-II. However, the effect of Ang-(1-7 on muscle wasting induced by TGF-β1 is unknown. Aim: To evaluate whether Ang-(1-7/Mas receptor axis could prevent the skeletal muscle atrophy induced by TGF-β1. Methods: This study assessed the atrophic effect of TGF-β1 in C2C12 myotubes and mice in absence or presence of Ang-(1-7, and the receptor participation using A779, an antagonist of the Mas receptor. The levels of myosin heavy chain (MHC, polyubiquitination, and MuRF-1 were detected by western blot. Myotube diameter was also evaluated. In vivo analysis included the muscle strength, fibre diameter, MHC and MuRF-1 levels by western blot, and ROS levels by DCF probe detection. Results: The results showed that Ang-(1-7 prevented the increase in MuRF-1 and polyubiquitined protein levels, the decrease of MHC levels, the myotubes/fibre diameter diminution, and the increased production of reactive oxygen species (ROS induced by TGF-β1. Utilizing A779 inhibited the anti-atrophic effect of Ang-(1-7. Conclusion: The preventive effect of Ang-(1-7 on skeletal muscle atrophy induced by TGF-β1 is produced through inhibition of ROS production and proteasomal degradation of MHC.

  16. Loss of catalytically inactive lipid phosphatase myotubularin-related protein 12 impairs myotubularin stability and promotes centronuclear myopathy in zebrafish.

    Directory of Open Access Journals (Sweden)

    Vandana A Gupta

    2013-06-01

    Full Text Available X-linked myotubular myopathy (XLMTM is a congenital disorder caused by mutations of the myotubularin gene, MTM1. Myotubularin belongs to a large family of conserved lipid phosphatases that include both catalytically active and inactive myotubularin-related proteins (i.e., "MTMRs". Biochemically, catalytically inactive MTMRs have been shown to form heteroligomers with active members within the myotubularin family through protein-protein interactions. However, the pathophysiological significance of catalytically inactive MTMRs remains unknown in muscle. By in vitro as well as in vivo studies, we have identified that catalytically inactive myotubularin-related protein 12 (MTMR12 binds to myotubularin in skeletal muscle. Knockdown of the mtmr12 gene in zebrafish resulted in skeletal muscle defects and impaired motor function. Analysis of mtmr12 morphant fish showed pathological changes with central nucleation, disorganized Triads, myofiber hypotrophy and whorled membrane structures similar to those seen in X-linked myotubular myopathy. Biochemical studies showed that deficiency of MTMR12 results in reduced levels of myotubularin protein in zebrafish and mammalian C2C12 cells. Loss of myotubularin also resulted in reduction of MTMR12 protein in C2C12 cells, mice and humans. Moreover, XLMTM mutations within the myotubularin interaction domain disrupted binding to MTMR12 in cell culture. Analysis of human XLMTM patient myotubes showed that mutations that disrupt the interaction between myotubularin and MTMR12 proteins result in reduction of both myotubularin and MTMR12. These studies strongly support the concept that interactions between myotubularin and MTMR12 are required for the stability of their functional protein complex in normal skeletal muscles. This work highlights an important physiological function of catalytically inactive phosphatases in the pathophysiology of myotubular myopathy and suggests a novel therapeutic approach through

  17. Urotensin II inhibits skeletal muscle glucose transport signaling pathways via the NADPH oxidase pathway.

    Directory of Open Access Journals (Sweden)

    Hong-Xia Wang

    Full Text Available Our previous studies have demonstrated that the urotensin (UII and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM, but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.

  18. Cinnamomum cassia Prevents High-Fat Diet-Induced Obesity in Mice through the Increase of Muscle Energy.

    Science.gov (United States)

    Song, Mi Young; Kang, Seok Yong; Kang, Anna; Hwang, Ji Hye; Park, Yong-Ki; Jung, Hyo Won

    2017-01-01

    The cortex of Cinnamomum cassia Presl (Cinnamomi Cortex: CC) has commonly been used for weight control in traditional medicines, but without a scientific basis. Therefore, this study was undertaken to investigate the anti-obesity effect of CC extract in a high-fat diet (HFD)-induced obese mouse model and in C2C12 mouse skeletal muscle cells. Male C57BL/6 mice were fed a normal diet or a HFD for 16 consecutive weeks, and orally administered CC extract (100 or 300[Formula: see text]mg/kg) or metformin (250[Formula: see text]mg/kg; positive control) daily for 16 weeks. CC extract administration significantly decreased body weights, food intakes, and serum levels of glucose, insulin, total cholesterol and ALT levels, prevented oral glucose tolerance and insulin resistance, inhibited the protein expressions of MyHC and PGC1[Formula: see text] and the phosphorylation of AMPK, suppressed lipid accumulation in liver, decreased adipocyte size and increased muscle mass in obese mice. For this in vitro study, C2C12 myoblasts were differentiated into the myotubes for five days, and then treated with CC extract (0.1 or 0.2[Formula: see text]mg/ml) for 24[Formula: see text]h. CC extract significantly increased ATP levels by increasing the mRNA expressions of mitochondrial biogenesis-related factors, such as, PGC1[Formula: see text], NRF-1, and Tfam, and the phosphorylations of AMPK and ACC. Our results suggest CC extract controls weight gain in obese mice by inhibiting lipid accumulation and increasing energy expenditure, and that its action mechanism involves the up-regulation of mitochondrial biogenesis in skeletal muscle cells.

  19. Short-term starvation is a strategy to unravel the cellular capacity of oxidizing specific exogenous/endogenous substrates in mitochondria.

    Science.gov (United States)

    Zeidler, Julianna D; Fernandes-Siqueira, Lorena O; Carvalho, Ana S; Cararo-Lopes, Eduardo; Dias, Matheus H; Ketzer, Luisa A; Galina, Antonio; Da Poian, Andrea T

    2017-08-25

    Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. In vitro studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most in vitro experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles. We found that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. In vitro anti-diabetic activity of Sclerocarya birrea and Ziziphus mucronata.

    Science.gov (United States)

    Mousinho, Nuno M H Da Costa; van Tonder, Jacob J; Steenkamp, Vanessa

    2013-09-01

    Type 2 diabetes mellitus is on the increase worldwide. Current treatments possess undesirable side-effects and therefore investigations into alternative remedies, which may be cost-effective and devoid of such side-effects, are on-going. Aqueous and methanol extracts of the bark of Sclerocarya birrea (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro (Anacardiaceae) and Ziziphus mucronata Willd. subsp. mucronata (Rhamnaceae) were subjected to testing in order to assess their in vitro anti-diabetic activity. Total polyphenolic content and antioxidant activity, as well as the effect on alpha-amylase, alpha-glucosidase and cytotoxicity, were measured. Furthermore, glucose uptake was evaluated in C2C12 myotubes, 3T3-L1 adipocytes and HepG2 hepatocarcinoma cells, and insulin secretion in RIN-m5F rat pancreatic beta-cells. The extracts of both plants inhibited the activities of alpha-amylase and alpha-glucosidase in a concentration dependent manner, with results being comparable with the positive control, acarbose. All the extracts contained antioxidant activity, with the methanol extracts of S. birrea displaying the strongest free radical scavenging capacity (IC50 = 2.16 microg/mL). Most of the extracts were not cytotoxic to the cell lines with IC50 values > 100 microg/mL. Extracts of both plants significantly increased glucose uptake in C2C12, 3T3-L1 and HepG2 cells. However, insulin secretion from RIN-m5F cells was not altered. This study provides evidence that the plants possess in vitro anti-diabetic properties.

  1. β-agonists selectively modulate proinflammatory gene expression in skeletal muscle cells via non-canonical nuclear crosstalk mechanisms.

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    Krzysztof Kolmus

    Full Text Available The proinflammatory cytokine Tumour Necrosis Factor (TNF-α is implicated in a variety of skeletal muscle pathologies. Here, we have investigated how in vitro cotreatment of skeletal muscle C2C12 cells with β-agonists modulates the TNF-α-induced inflammatory program. We observed that C2C12 myotubes express functional TNF receptor 1 (TNF-R1 and β2-adrenoreceptors (β2-ARs. TNF-α activated the canonical Nuclear Factor-κB (NF-κB pathway and Mitogen-Activated Protein Kinases (MAPKs, culminating in potent induction of NF-κB-dependent proinflammatory genes. Cotreatment with the β-agonist isoproterenol potentiated the expression of inflammatory mediators, including Interleukin-6 (IL-6 and several chemokines. The enhanced production of chemotactic factors upon TNF-α/isoproterenol cotreatment was also suggested by the results from migrational analysis. Whereas we could not explain our observations by cytoplasmic crosstalk, we found that TNF-R1-and β2-AR-induced signalling cascades cooperate in the nucleus. Using the IL-6 promoter as a model, we demonstrated that TNF-α/isoproterenol cotreatment provoked phosphorylation of histone H3 at serine 10, concomitant with enhanced promoter accessibility and recruitment of the NF-κB p65 subunit, cAMP-response element-binding protein (CREB, CREB-binding protein (CBP and RNA polymerase II. In summary, we show that β-agonists potentiate TNF-α action, via nuclear crosstalk, that promotes chromatin relaxation at selected gene promoters. Our data warrant further study into the mode of action of β-agonists and urge for caution in their use as therapeutic agents for muscular disorders.

  2. Integrated strain array for cellular mechanobiology studies

    Science.gov (United States)

    Simmons, C. S.; Sim, J. Y.; Baechtold, P.; Gonzalez, A.; Chung, C.; Borghi, N.; Pruitt, B. L.

    2011-05-01

    We have developed an integrated strain array for cell culture enabling high-throughput mechano-transduction studies. Biocompatible cell culture chambers were integrated with an acrylic pneumatic compartment and microprocessor-based control system. Each element of the array consists of a deformable membrane supported by a cylindrical pillar within a well. For user-prescribed waveforms, the annular region of the deformable membrane is pulled into the well around the pillar under vacuum, causing the pillar-supported region with cultured cells to be stretched biaxially. The optically clear device and pillar-based mechanism of operation enables imaging on standard laboratory microscopes. Straightforward fabrication utilizes off-the-shelf components, soft lithography techniques in polydimethylsiloxane and laser ablation of acrylic sheets. Proof of compatibility with basic biological assays and standard imaging equipment were accomplished by straining C2C12 skeletal myoblasts on the device for 6 h. At higher strains, cells and actin stress fibers realign with a circumferential preference.

  3. Salidroside alleviates cachexia symptoms in mouse models of cancer cachexia via activating mTOR signalling.

    Science.gov (United States)

    Chen, Xiangzheng; Wu, Yangping; Yang, Tinghan; Wei, Mingtian; Wang, Yuxi; Deng, Xiangbing; Shen, Congcong; Li, Wenting; Zhang, Hang; Xu, Weiyong; Gou, Lantu; Zeng, Yong; Zhang, Yonghui; Wang, Ziqiang; Yang, Jinliang

    2016-05-01

    Cachexia has a devastating impact on survival and quality of life for many cancer patients and contributes to nearly one-third of all cancer deaths; also, it is associated with poor responses to chemotherapy and survival. A better understanding of the underlying mechanisms of cancer-associated cachexia (CAC), coupled with effective therapeutic approaches, will improve management of progressive functional impairment in cancer patients. Salidroside, a phenylpropanoid glycoside in Rhodiola rosea L, has been reported to possess potential anti-fatigue, anti-ageing, and anti-Alzheimer's disease properties. It is widely consumed as a nutritional supplement, but its effects on CAC and the possible mechanism remain a mystery. In the murine models of cachexia induced by CT-26 and Lewis lung carcinoma (LLC) tumour, respectively, main features of CAC were determined after treatment of salidroside or chemotherapy. In vitro experiments were performed using murine C2C12 myotubes, which were treated by tumour necrosis factor-α. Levels of several critical muscle-related signal proteins such as mammalian target of rapamycin (mTOR), p-mTOR, and myosin heavy chain (MyHC) were examined using western blot both in vitro and in vivo. In the present study, we showed the exciting effect of salidroside on the treatment of CAC. In CT-26 and LLC models, respectively, salidroside treatment could effectively preserve the tumour-free body weight, decrease loss of adipose and gastrocnemius muscles, alleviate tumour burden, and prolong their survival time. Additionally, in combined chemotherapy, salidroside could synergistically enhance the anti-tumour activity of cisplatin, especially decreased or eliminated chemotherapy-induced cachexia. Further analysis demonstrated that salidroside could significantly increase expression of mTOR, p-mTOR, and MyHC in gastrocnemius muscle. Also, results in vitro showed that salidroside could not only obviously increase mTOR, p-mTOR, and MyHC expression in C2C12

  4. Analysis of MicroRNA Expression Profiles in Weaned Pig Skeletal Muscle after Lipopolysaccharide Challenge

    Directory of Open Access Journals (Sweden)

    Jing Zhang

    2015-09-01

    Full Text Available MicroRNAs (miRNAs constitute a class of non-coding RNAs that play a crucial regulatory role in skeletal muscle development and disease. Several acute inflammation conditions including sepsis and cancer are characterized by a loss of skeletal muscle due primarily to excessive muscle catabolism. As a well-known inducer of acute inflammation, a lipopolysaccharide (LPS challenge can cause serious skeletal muscle wasting. However, knowledge of the role of miRNAs in the course of inflammatory muscle catabolism is still very limited. In this study, RNA extracted from the skeletal muscle of pigs injected with LPS or saline was subjected to small RNA deep sequencing. We identified 304 conserved and 114 novel candidate miRNAs in the pig. Of these, four were significantly increased in the LPS-challenged samples and five were decreased. The expression of five miRNAs (ssc-miR-146a-5p, ssc-miR-221-5p, ssc-miR-148b-3p, ssc-miR-215 and ssc-miR-192 were selected for validation by quantitative polymerase chain reaction (qPCR, which found that ssc-miR-146a-5p and ssc-miR-221-5p were significantly upregulated in LPS-challenged pig skeletal muscle. Moreover, we treated mouse C2C12 myotubes with 1000 ng/mL LPS as an acute inflammation cell model. Expression of TNF-α, IL-6, muscle atrophy F-box (MAFbx and muscle RING finger 1 (MuRF1 mRNA was strongly induced by LPS. Importantly, miR-146a-5p and miR-221-5p also showed markedly increased expression in LPS-treated C2C12 myotubes, suggesting the two miRNAs may be involved in muscle catabolism systems in response to acute inflammation caused by a LPS challenge. To our knowledge, this study is the first to examine miRNA expression profiles in weaned pig skeletal muscle challenged with LPS, and furthers our understanding of miRNA function in the regulation of inflammatory muscle catabolism.

  5. Proteomic profiling of glucocorticoid-exposed myogenic cells: Time series assessment of protein translocation and transcription of inactive mRNAs

    Directory of Open Access Journals (Sweden)

    Hoffman Eric P

    2009-07-01

    Full Text Available Abstract Background Prednisone, one of the most highly prescribed drugs, has well characterized effects on gene transcription mediated by the glucocorticoid receptor. These effects are typically occurring on the scale of hours. Prednisone also has a number of non-transcriptional effects (occurring on minutes scale on protein signaling, yet these are less well studied. We sought to expand the understanding of acute effects of prednisone action on cell signaling using a combination of SILAC strategy and subcellular fractionations from C2C12 myotubes. Results De novo translation of proteins was inhibited in both SILAC labeled and unlabeled C2C12 myotubes. Unlabeled cells were exposed to prednisone while SILAC labeled cells remained untreated. After 0, 5, 15, and 30 minutes of prednisone exposure, labeled and unlabeled cells were mixed at 1:1 ratios and fractionated into cytosolic and nuclear fractions. A total of 534 proteins in the cytosol and 626 proteins in the nucleus were identified and quantitated, using 3 or more peptides per protein with peptide based probability ≤ 0.001. We identified significant increases (1.7- to 3.1- fold in cytoplasmic abundance of 11 ribosomal proteins within 5 minutes of exposure, all of which returned to baseline by 30 min. We hypothesized that these drug-induced acute changes in the subcellular localization of the cell's protein translational machinery could lead to altered translation of quiescent RNAs. To test this, de novo protein synthesis was assayed after 15 minutes of drug exposure. Quantitative fluorography identified 16 2D gel spots showing rapid changes in translation; five of these were identified by MS/MS (pyruvate kinase, annexin A6 isoform A and isoform B, nasopharyngeal epithelium specific protein 1, and isoform 2 of Replication factor C subunit 1, and all showed the 5' terminal oligopyrimidine motifs associated with mRNA sequestration to and from inactive mRNA pools. Conclusion We describe novel

  6. Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23 Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

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    Yoshiaki Shimoda

    Full Text Available Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23 is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

  7. In vitro toxicity studies of polymer-coated gold nanorods

    Energy Technology Data Exchange (ETDEWEB)

    Rayavarapu, Raja G; Petersen, Wilma; Manohar, Srirang; Van Leeuwen, Ton G [Biomedical Photonic Imaging Group, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, PO Box 217, 7500AE Enschede (Netherlands); Hartsuiker, Liesbeth; Otto, Cees [Medical Cell Biophysics, MIRA Institute for Biomedical Technology and Technical Medicine, Faculty of Science and Technology, University of Twente, PO Box 217, 7500AE Enschede (Netherlands); Chin, Patrick; Van Leeuwen, Fijs W B [Division of Diagnostic Oncology, Netherlands Cancer Institute, 1066 CX Amsterdam (Netherlands); Janssen, Hans, E-mail: S.Manohar@utwente.nl [Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam (Netherlands)

    2010-04-09

    We evaluated cellular responses to polymer-treated gold nanorods, which were synthesized using the standard wet-chemistry method that utilizes hexadecyltrimethylammonium bromide (CTAB). The nanorod dispersions were coated with either polystyrene sulfonate (PSS) or polyethylene glycol (PEG). Two sizes of nanorods were tested, with optical responses peaking at 628 and 773 nm. The cells were from mammary adenocarcinoma (SKBR3), Chinese Hamster Ovary (CHO), mouse myoblast (C2C12) and Human Leukemia (HL60) cell lines. Their mitochondrial function following exposure to the nanorods were assessed using the MTS assay. We found PEGylated particles to have superior biocompatibility compared with PSS-coated nanorods, which showed substantial cytotoxicity. Electron microscopy showed no cellular uptake of PEGylated particles compared with their PSS counterparts. PEGylated gold nanorods also exhibited better dispersion stability in the presence of cell growth medium; PSS-coated rods tended to flocculate or cluster. In the case of the PSS particles, toxicity correlated with surface area across the two sizes of nanorods studied.

  8. Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system.

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    Xiaodong Mu

    2011-02-01

    Full Text Available Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied.In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells.These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.

  9. Nilotinib impairs skeletal myogenesis by increasing myoblast proliferation

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    Osvaldo Contreras

    2018-02-01

    Full Text Available Abstract Background Tyrosine kinase inhibitors (TKIs are effective therapies with demonstrated antineoplastic activity. Nilotinib is a second-generation FDA-approved TKI designed to overcome Imatinib resistance and intolerance in patients with chronic myelogenous leukemia (CML. Interestingly, TKIs have also been shown to be an efficient treatment for several non-malignant disorders such fibrotic diseases, including those affecting skeletal muscles. Methods We investigated the role of Nilotinib on skeletal myogenesis using the well-established C2C12 myoblast cell line. We evaluated the impact of Nilotinib during the time course of skeletal myogenesis. We compared the effect of Nilotinib with the well-known p38 MAPK inhibitor SB203580. MEK1/2 UO126 and PI3K/AKT LY294002 inhibitors were used to identify the signaling pathways involved in Nilotinib-related effects on myoblast. Adult primary myoblasts were also used to corroborate the inhibition of myoblasts fusion and myotube-nuclei positioning by Nilotinib. Results We found that Nilotinib inhibited myogenic differentiation, reducing the number of myogenin-positive myoblasts and decreasing myogenin and MyoD expression. Furthermore, Nilotinib-mediated anti-myogenic effects impair myotube formation, myosin heavy chain expression, and compromise myotube-nuclei positioning. In addition, we found that p38 MAPK is a new off-target protein of Nilotinib, which causes inhibition of p38 phosphorylation in a similar manner as the well-characterized p38 inhibitor SB203580. Nilotinib induces the activation of ERK1/2 and AKT on myoblasts but not in myotubes. We also found that Nilotinib stimulates myoblast proliferation, a process dependent on ERK1/2 and AKT activation. Conclusions Our findings suggest that Nilotinib may have important negative effects on muscle homeostasis, inhibiting myogenic differentiation but stimulating myoblasts proliferation. Additionally, we found that Nilotinib stimulates the activation

  10. Análise dos genes diferencialmente expressos durante a osteodiferenciação induzida por proteínas morfogenéticas de osso (BMP2 e BMP7) em células C2C12 e super-expressão de rhBMP2 e rhBMP7 em células de mamíferos

    OpenAIRE

    Juan Carlos Bustos Valenzuela

    2008-01-01

    As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-β (Transforming Growth Factor β ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto \\"in vitro\\" como \\"in vivo,\\" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia ...

  11. Bioactive Components from Flowers of Sambucus nigra L. Increase Glucose Uptake in Primary Porcine Myotube Cultures and Reduce Fat Accumulation in Caenorhabditis elegans

    DEFF Research Database (Denmark)

    Bhattacharya, Sumangala; B. Christensen, Kathrine; C. B. Olsen, Louise

    2013-01-01

    elegans. Bioassay guided chromatographic fractionations of extracts and fractions resulted in the identification of naringenin and 5-O- caffeoylquinic acid exhibiting a significant increase in GU. In addition, phenolic compounds related to those found in elderflowers were also tested, and among these......, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, and isorhamnetin-3-O-glucoside and the related phenolic compounds kaempferol and ferulic acid. The study indicates that elderflower extracts contain bioactive compounds capable of modulating glucose and lipid metabolism, suitable for nutraceutical...

  12. Functional evaluation of artificial skeletal muscle tissue constructs fabricated by a magnetic force-based tissue engineering technique.

    Science.gov (United States)

    Yamamoto, Yasunori; Ito, Akira; Fujita, Hideaki; Nagamori, Eiji; Kawabe, Yoshinori; Kamihira, Masamichi

    2011-01-01

    Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2 μN [1.06 mN/mm2]). Rheobase and chronaxie of the tissue were determined as 4.45 V and 0.72 ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies.

  13. Cellular Trafficking of Phospholamban and Formation of Functional Sarcoplasmic Reticulum During Myocyte DIfferentiation

    Energy Technology Data Exchange (ETDEWEB)

    Stenoien, David L.; Knyushko, Tatyana V.; Londono, Monica P.; Opresko, Lee; Mayer, M. Uljana; Brady, Scott T.; Squier, Thomas C.; Bigelow, Diana J.

    2007-06-01

    The sarco/endoplasmic reticulum Ca-ATPase (SERCA) family members are transmembrane proteins that play an essential role in regulating intracellular calcium levels. Phospholamban (PLB), a 52 amino acid phosphoprotein, regulates SERCA activity in adult heart and skeletal muscle. Using the C2C12 myocyte cell line, we find endogenous PLB constitutively expressed in both myoblasts and myotubes, whereas SERCA expression coincides with activation of the differentiation program. PLB has a punctuate distribution in myoblasts changing to a reticular distribution in myotubes where it colocalizes with SERCAs. To examine the distribution and dynamics of PLB and SERCA, we expressed fluorescent fusion proteins (GFP, CFP, and YFP) of PLB and SERCA in myoblasts. Coexpressed PLB and SERCA localize to distinct cellular compartments in myoblasts but begin to colocalize as cells differentiate. Fluorescence Recovery After Photobleaching (FRAP) studies show different recovery patterns for each protein in myoblasts confirming their localization to distinct compartments. To extend these studies, we created stable cell lines expressing O6-alkylguanine-DNA alkyltransferase (AGT) fusions with PLB or SERCA to track their localization as myocytes differentiate. These experiments demonstrate that PLB localizes to punctate vesicles in myoblasts and adopts a reticular distribution that coincides with SERCA distribution after differentiation. Colocalization experiments indicate that a subset of PLB in myoblasts colocalizes with endosomes, Golgi, and the plasma membrane however PLB also localizes to other, as yet unidentified vesicles. Our results indicate that differentiation plays a critical role in regulating PLB distribution to ensure its colocalization within the same cellular compartment as SERCA in differentiated cells. The presence and altered distribution of PLB in undifferentiated myoblasts raises the possibility that this protein has additional functions distinct from SERCA regulation.

  14. Association between expression of FABPpm in skeletal muscle and insulin sensitivity in intramyocellular lipid-accumulated nonobese men.

    Science.gov (United States)

    Kawaguchi, Minako; Tamura, Yoshifumi; Kakehi, Saori; Takeno, Kageumi; Sakurai, Yuko; Watanabe, Takahiro; Funayama, Takashi; Sato, Fumihiko; Ikeda, Shinichi; Ogura, Yuji; Saga, Norio; Naito, Hisashi; Fujitani, Yoshio; Kanazawa, Akio; Kawamori, Ryuzo; Watada, Hirotaka

    2014-09-01

    Intramyocellular lipid (IMCL) accumulation is observed in both insulin-resistant subjects and insulin-sensitive endurance athletes (athlete's paradox). We hypothesized that the expression pattern of fatty acid transporters may influence oxidative capacity and determine the association between IMCL and insulin resistance. The objective of the study was to investigate the muscle expression of fatty acid transporters and their function related to insulin sensitivity in IMCL-accumulated subjects. The study subjects were 36 nonobese healthy men. Their IMCL levels were measured by (1)H-magnetic resonance spectroscopy, and their insulin sensitivity was evaluated by steady-state glucose infusion rate (GIR) during a euglycemic-hyperinsulinemic clamp. Gene expression levels in the vastus lateralis were evaluated by quantitative RT-PCR. We compared the clinical phenotypes and the expression levels of genes involved in lipid metabolism in skeletal muscle between IMCL-accumulated high-GIR (H-GIR) subjects (n = 8) and low-GIR subjects (n = 9). The functions of candidate fatty acid transporters were determined by in vitro analyses. Compared with the low-GIR group, body fat was lower and maximum oxygen uptake was higher in the H-GIR group. Several lipid oxidation genes in muscle were up-regulated in the H-GIR group, and this was associated with increased expression of higher plasma membrane-associated fatty acid-binding protein (FABPpm) and decreased expression of fatty acid transport protein (FATP)-1. Overexpression of FABPpm in C2C12 myotubes increased fatty acid oxidation coupled with the elevated expression of genes related to fatty acid oxidation. These changes were not observed in FATP1-overexpressed myotubes. Differences in the gene expression of fatty acid transporters may, at least in part, affect insulin sensitivity in IMCL-accumulated nonobese men.

  15. Effect of Phenolic Compounds from Elderflowers on Glucose- and Fatty Acid Uptake in Human Myotubes and HepG2-Cells

    Directory of Open Access Journals (Sweden)

    Giang Thanh Thi Ho

    2017-01-01

    Full Text Available Type 2 diabetes (T2D is manifested by progressive metabolic impairments in tissues such as skeletal muscle and liver, and these tissues become less responsive to insulin, leading to hyperglycemia. In the present study, stimulation of glucose and oleic acid uptake by elderflower extracts, constituents and metabolites were tested in vitro using the HepG2 hepatocellular liver carcinoma cell line and human skeletal muscle cells. Among the crude extracts, the 96% EtOH extract showed the highest increase in glucose and oleic acid uptake in human skeletal muscle cells and HepG2-cells. The flavonoids and phenolic acids contained therein were potent stimulators of glucose and fatty acid uptake in a dose-dependent manner. Most of the phenolic constituents and several of the metabolites showed high antioxidant activity and showed considerably higher α-amylase and α-glucosidase inhibition than acarbose. Elderflower might therefore be valuable as a functional food against diabetes.

  16. Sustained elevation of circulating growth and differentiation factor-15 and a dynamic imbalance in mediators of muscle homeostasis are associated with the development of acute muscle wasting following cardiac surgery.

    Science.gov (United States)

    Bloch, Susannah A A; Lee, Jen Y; Wort, S John; Polkey, Michael I; Kemp, Paul R; Griffiths, Mark J D

    2013-04-01

    Acute muscle wasting in the critically ill is common and causes significant morbidity. In a novel human model of acute muscle wasting following cardiac surgery, known or potential circulating modulators of muscle mass--insulin-like growth factor-1, myostatin, and growth and differentiation factor-15--were measured over a week. It was hypothesized that patients who developed acute muscle wasting would show distinct patterns of change in these mediators. A prospective longitudinal observational study of high-risk elective cardiac surgical patients identifying, by ultrasound, those developing muscle wasting. Tertiary cardiothoracic referral center: Royal Brompton Hospital, London, UK. Forty-two patients undergoing elective high-risk cardiothoracic surgery. Circulating insulin-like growth factor-1, myostatin, and growth and differentiation factor-15 were assayed preoperatively and over the first week postoperatively. The ability of growth and differentiation factor-15 to cause muscle wasting in vitro was determined in C2C12 myotubes. Of the 42 patients, 23 (55%) developed quadriceps atrophy. There was an acute decrease in insulin-like growth factor-1 and unexpectedly myostatin, known mediators of muscle hypertrophy and atrophy, respectively. By contrast, plasma growth and differentiation factor-15 concentrations increased in all patients. This increase in growth and differentiation factor-15 was sustained at day 7 in those who developed muscle wasting (day 7 compared with baseline, p0.05). Insulin-like growth factor-1 did not recover in those who developed muscle wasting (day 7 compared with baseline, p0.05). Finally, we demonstrated that growth and differentiation factor-15 caused atrophy of myotubes in vitro. These data support the hypothesis that acute muscle loss occurs as a result of an imbalance between drivers of muscle atrophy and hypertrophy. Growth and differentiation factor-15 is a potential novel factor associated with muscle atrophy, which may become a

  17. Fine-tuning of substrate architecture and surface chemistry promotes muscle tissue development.

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    Guex, A G; Kocher, F M; Fortunato, G; Körner, E; Hegemann, D; Carrel, T P; Tevaearai, H T; Giraud, M N

    2012-04-01

    Tissue engineering has been increasingly brought to the scientific spotlight in response to the tremendous demand for regeneration, restoration or substitution of skeletal or cardiac muscle after traumatic injury, tumour ablation or myocardial infarction. In vitro generation of a highly organized and contractile muscle tissue, however, crucially depends on an appropriate design of the cell culture substrate. The present work evaluated the impact of substrate properties, in particular morphology, chemical surface composition and mechanical properties, on muscle cell fate. To this end, aligned and randomly oriented micron (3.3±0.8 μm) or nano (237±98 nm) scaled fibrous poly(ε-caprolactone) non-wovens were processed by electrospinning. A nanometer-thick oxygen functional hydrocarbon coating was deposited by a radio frequency plasma process. C2C12 muscle cells were grown on pure and as-functionalized substrates and analysed for viability, proliferation, spatial orientation, differentiation and contractility. Cell orientation has been shown to depend strongly on substrate architecture, being most pronounced on micron-scaled parallel-oriented fibres. Oxygen functional hydrocarbons, representing stable, non-immunogenic surface groups, were identified as strong triggers for myotube differentiation. Accordingly, the highest myotube density (28±15% of total substrate area), sarcomeric striation and contractility were found on plasma-coated substrates. The current study highlights the manifold material characteristics to be addressed during the substrate design process and provides insight into processes to improve bio-interfaces. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  18. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

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    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  19. Pyrrolidine Dithiocarbamate (PDTC Attenuates Cancer Cachexia by Affecting Muscle Atrophy and Fat Lipolysis

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    Chunxiao Miao

    2017-12-01

    Full Text Available Cancer cachexia is a kind of whole body metabolic disorder syndrome accompanied with severe wasting of muscle and adipose tissue. NF-κB signaling plays an important role during skeletal muscle atrophy and fat lipolysis. As an inhibitor of NF-κB signaling, Pyrrolidine dithiocarbamate (PDTC was reported to relieve cancer cachexia; however, its mechanism remains largely unknown. In our study, we showed that PDTC attenuated cancer cachexia symptom in C26 tumor bearing mice models in vivo without influencing tumor volume. What’s more, PDTC inhibited muscle atrophy and lipolysis in cells models in vitro induced by TNFα and C26 tumor medium. PDTC suppressed atrophy of myotubes differentiated from C2C12 by reducing MyoD and upregulating MuRF1, and preserving the expression of perilipin as well as blocking the activation of HSL in 3T3-L1 mature adipocytes. Meaningfully, we observed that PDTC also inhibited p38 MAPK signaling besides the NF-κB signaling in cancer cachexia in vitro models. In addition, PDTC also influenced the protein synthesis of skeletal muscle by activating AKT signaling and regulated fat energy metabolism by inhibiting AMPK signaling. Therefore, PDTC primarily influenced different pathways in different tissues. The study not only established a simple and reliable screening drugs model of cancer cachexia in vitro but also provided new theoretical basis for future treatment of cancer cachexia.

  20. Study of in Vitro and in Vivo Bone Formation in Composite Cryogels and the Influence of Electrical Stimulation.

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    Mishra, Ruchi; Raina, Deepak Bushan; Pelkonen, Mea; Lidgren, Lars; Tägil, Magnus; Kumar, Ashok

    2015-01-01

    This work studies osteoinduction and bone conduction in polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogels along with the synergistic effect of electrical stimulation. In vitro osteoinduction of C2C12 myoblast towards osteogenic lineage is demonstrated through alkaline phosphatase assay, scanning electron microscopy and energy dispersive X-ray spectroscopy. These results were followed by in vivo implantation studies of PTAC biocomposite cryogel scaffolds in the bone conduction chamber model depicting bone formation after 24 days based on immunohistological staining for osteogenic markers, i.e., collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP). Further, osteogenic differentiation of murine mesenchymal stem cells was studied with and without electrical stimulation. The q-PCR analysis shows that the electrically stimulated cryogels exhibit ~ 6 folds higher collagen type I and ~ 10 folds higher osteopontin mRNA level, in comparison to the unstimulated cryogels. Thus, PTAC biocomposite cryogels present osteoinductive and osteoconductive properties during in vitro and in vivo studies and support osteogenic differentiation of mesenchymal stem cells under the influence of electrical stimulation.

  1. Muscle wasting and impaired myogenesis in tumor bearing mice are prevented by ERK inhibition.

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    Fabio Penna

    Full Text Available BACKGROUND: The onset of cachexia is a frequent feature in cancer patients. Prominent characteristic of this syndrome is the loss of body and muscle weight, this latter being mainly supported by increased protein breakdown rates. While the signaling pathways dependent on IGF-1 or myostatin were causally involved in muscle atrophy, the role of the Mitogen-Activated-Protein-Kinases is still largely debated. The present study investigated this point on mice bearing the C26 colon adenocarcinoma. METHODOLOGY/PRINCIPAL FINDINGS: C26-bearing mice display a marked loss of body weight and muscle mass, this latter associated with increased phosphorylated (p-ERK. Administration of the ERK inhibitor PD98059 to tumor bearers attenuates muscle depletion and weakness, while restoring normal atrogin-1 expression. In C26 hosts, muscle wasting is also associated with increased Pax7 expression and reduced myogenin levels. Such pattern, suggestive of impaired myogenesis, is reversed by PD98059. Increased p-ERK and reduced myosin heavy chain content can be observed in TNFα-treated C2C12 myotubes, while decreased myogenin and MyoD levels occur in differentiating myoblasts exposed to the cytokine. All these changes are prevented by PD98059. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that ERK is involved in the pathogenesis of muscle wasting in cancer cachexia and could thus be proposed as a therapeutic target.

  2. Upregulation of skeletal muscle PGC-1α through the elevation of cyclic AMP levels by Cyanidin-3-glucoside enhances exercise performance.

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    Matsukawa, Toshiya; Motojima, Hideko; Sato, Yuki; Takahashi, Shinya; Villareal, Myra O; Isoda, Hiroko

    2017-03-20

    Regular exercise and physical training enhance physiological capacity and improve metabolic diseases. Skeletal muscles require peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) in the process of their adaptation to exercise owing to PGC-1α's ability to regulate mitochondrial biogenesis, angiogenesis, and oxidative metabolism. Cyanidin-3-glucoside (Cy3G) is a natural polyphenol and a nutraceutical factor, which has several beneficial effects on human health. Here, the effect of Cy3G on exercise performance and the underlying mechanisms involved were investigated. ICR mice were given Cy3G (1 mg/kg, orally) everyday and made to perform weight-loaded swimming exercise for 15 days. The endurance of mice orally administered with Cy3G was improved, enabling them to swim longer (time) and while the levels of exercise-induced lactate and fatigue markers (urea nitrogen, creatinine and total ketone bodies) were reduced. Additionally, the expression of lactate metabolism-related genes (lactate dehydrogenase B and monocarboxylate transporter 1) in gastrocnemius and biceps femoris muscles was increased in response to Cy3G-induced PGC-1α upregulation. In vitro, using C2C12 myotubes, Cy3G-induced elevation of intracellular cyclic AMP levels increased PGC-1α expression via the Ca(2+)/calmodulin-dependent protein kinase kinase pathway. This study demonstrates that Cy3G enhances exercise performance by activating lactate metabolism through skeletal muscle PGC-1α upregulation.

  3. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) activates promyogenic signaling pathways, thereby promoting myoblast differentiation

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    Lee, Sang-Jin; Go, Ga-Yeon; Yoo, Miran; Kim, Yong Kee [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Seo, Dong-Wan [College of Pharmacy, Dankook University, Cheonan 330-714 (Korea, Republic of); Kang, Jong-Sun [Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Samsung Biomedical Research Institute, Suwon 440-746 (Korea, Republic of); Bae, Gyu-Un, E-mail: gbae@sookmyung.ac.kr [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2016-01-29

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) regulates postnatal myogenesis by alleviating myostatin activity, but the molecular mechanisms by which it regulates myogenesis are not fully understood. In this study, we investigate molecular mechanisms of PPARβ/δ in myoblast differentiation. C2C12 myoblasts treated with a PPARβ/δ agonist, GW0742 exhibit enhanced myotube formation and muscle-specific gene expression. GW0742 treatment dramatically activates promyogenic kinases, p38MAPK and Akt, in a dose-dependent manner. GW0742-stimulated myoblast differentiation is mediated by p38MAPK and Akt, since it failed to restore myoblast differentiation repressed by inhibition of p38MAPK and Akt. In addition, GW0742 treatment enhances MyoD-reporter activities. Consistently, overexpression of PPARβ/δ enhances myoblast differentiation accompanied by elevated activation of p38MAPK and Akt. Collectively, these results suggest that PPARβ/δ enhances myoblast differentiation through activation of promyogenic signaling pathways. - Highlights: • A PPARβ/δ agonist, GW0742 promotes myoblast differentiation. • GW0742 activates both p38MAPK and Akt activation in myogenic differentiation. • GW0742 enhances MyoD activity for myogenic differentiation. • Overexpression of PPARβ/δ enhances myoblast differentiation via activating promyogenic signaling pathways. • This is the first finding for agonistic mechanism of PPARβ/δ in myogenesis.

  4. Amla Enhances Mitochondrial Spare Respiratory Capacity by Increasing Mitochondrial Biogenesis and Antioxidant Systems in a Murine Skeletal Muscle Cell Line

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    Hirotaka Yamamoto

    2016-01-01

    Full Text Available Amla is one of the most important plants in Indian traditional medicine and has been shown to improve various age-related disorders while decreasing oxidative stress. Mitochondrial dysfunction is a proposed cause of aging through elevated oxidative stress. In this study, we investigated the effects of Amla on mitochondrial function in C2C12 myotubes, a murine skeletal muscle cell model with abundant mitochondria. Based on cell flux analysis, treatment with an extract of Amla fruit enhanced mitochondrial spare respiratory capacity, which enables cells to overcome various stresses. To further explore the mechanisms underlying these effects on mitochondrial function, we analyzed mitochondrial biogenesis and antioxidant systems, both proposed regulators of mitochondrial spare respiratory capacity. We found that Amla treatment stimulated both systems accompanied by AMPK and Nrf2 activation. Furthermore, we found that Amla treatment exhibited cytoprotective effects and lowered reactive oxygen species (ROS levels in cells subjected to t-BHP-induced oxidative stress. These effects were accompanied by increased oxygen consumption, suggesting that Amla protected cells against oxidative stress by using enhanced spare respiratory capacity to produce more energy. Thus we identified protective effects of Amla, involving activation of mitochondrial function, which potentially explain its various effects on age-related disorders.

  5. Production of IL-6 by human myoblasts stimulated with Abeta: relevance in the pathogenesis of IBM.

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    Baron, P; Galimberti, D; Meda, L; Scarpini, E; Conti, G; Cogiamanian, F; Scarlato, G

    2001-11-13

    To determine whether amyloid-beta protein (Abeta) can induce the production of proinflammatory cytokines by cultured normal muscle cells. Sporadic inclusion body myositis (IBM) is characterized by the presence of rimmed vacuoles and fibrillary inclusions of Abeta in muscle fibers, and often inflammatory cells. Endomysial expression of proinflammatory molecules has suggested an ongoing immune process, but the site of sensitization and the mechanisms that trigger an inflammatory reaction is unknown. The authors used Northern blot analysis and specific immunoassays to study the expression and secretion in cell-free supernatants of tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) by purified human myoblasts and C2C12 mouse skeletal muscle cells incubated with Abeta[1-42] or Abeta[25-35] peptides. Nonstimulated muscle cells produced detectable IL-6, whereas secretion of IL-1beta and TNFalpha was absent. Incubation with Abeta peptides increased IL-6 production, whereas TNFalpha and IL-1beta levels remained undetectable. Northern blot analysis of Abeta-stimulated human myoblasts revealed an increase in IL-6 mRNA expression. Cultured muscle cells increase the constitutive production of IL-6 in response to local deposition of Abeta in sporadic IBM. IL-6 could be a CD8(+) proliferation and differentiation agent, an autocrine proteolysis-inducing factor of damaged myotubes, and a proliferation-stimulating agent for satellite cells to replace the destroyed myofibers in IBM.

  6. L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF- Members Are Candidates to Developmentally Control Their Fiber Formation

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    Stefania Rossi

    2010-01-01

    Full Text Available This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs and follistatin as well as the highly related TGF- members Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF- ligands involved in developmental regulation of fiber size.

  7. Clones of ectopic stem cells in the regeneration of muscle defects in vivo.

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    Rujing Yang

    2010-10-01

    Full Text Available Little is known about whether clones of ectopic, non-muscle stem cells contribute to muscle regeneration. Stem/progenitor cells that are isolated for experimental research or therapeutics are typically heterogeneous. Non-myogenic lineages in a heterogeneous population conceptually may compromise tissue repair. In this study, we discovered that clones of mononucleated stem cells of human tooth pulp fused into multinucleated myotubes that robustly expressed myosin heavy chain in vitro with or without co-culture with mouse skeletal myoblasts (C2C12 cells. Cloned cells were sustainably Oct4+, Nanog+ and Stro1+. The fusion indices of myogenic clones were approximately 16-17 folds greater than their parent, heterogeneous stem cells. Upon infusion into cardio-toxin induced tibialis anterior muscle defects, undifferentiated clonal progenies not only engrafted and colonized host muscle, but also expressed human dystrophin and myosin heavy chain more efficaciously than their parent heterogeneous stem cell populations. Strikingly, clonal progenies yielded ∼9 times more human myosin heavy chain mRNA in regenerating muscles than those infused with their parent, heterogeneous stem cells. The number of human dystrophin positive cells in regenerating muscles infused with clonal progenies was more than ∼3 times greater than muscles infused with heterogeneous stem cells from which clonal progenies were derived. These findings suggest the therapeutic potential of ectopic myogenic clones in muscle regeneration.

  8. Application of cell co-culture system to study fat and muscle cells.

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    Pandurangan, Muthuraman; Hwang, Inho

    2014-09-01

    Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

  9. Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve.

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    Rachelle N Palchesko

    Full Text Available Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line and C2C12 (muscle cell line were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.

  10. Myokines (muscle-derived cytokines and chemokines) including ciliary neurotrophic factor (CNTF) inhibit osteoblast differentiation.

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    Johnson, Rachelle W; White, Jason D; Walker, Emma C; Martin, T John; Sims, Natalie A

    2014-07-01

    Muscle and bone are intimately linked by bi-directional signals regulating both muscle and bone cell gene expression and proliferation. It is generally accepted that muscle cells secrete factors (myokines) that influence adjacent bone cells, but these myokines are yet to be identified. We have previously shown that osteocyte-specific deletion of the co-receptor subunit utilized by IL-6 family cytokines, glycoprotein 130 (gp130), resulted in impaired bone formation in the trabecular bone, but enhanced periosteal expansion, suggesting a gp130-dependent periosteum-specific inhibition of osteoblast function, potentially induced by the local muscle fibres. We report here that differentiated primary calvarial osteoblasts cultured in myotube-conditioned media (CM) from myogenic C2C12 cells show reduced mRNA levels of genes associated with osteoblast differentiation. Alkaline phosphatase protein activity and all mRNA markers of osteoblast differentiation in the tested panel (runx2, osterix, alkaline phosphatase, parathyroid hormone (PTH) receptor, osteoprotegerin, osteocalcin, sclerostin) were reduced following culture with myotube CM. The exception was RANKL, which was significantly elevated in differentiated primary osteoblast cultures expressing osteocytic genes. A cytokine array of the C2C12 myotube-conditioned media identified TIMP-1 and MCP-1 as the most abundant myokines, but treatment with recombinant TIMP-1 or MCP-1 did not inhibit osteoblast gene expression. Rather, the IL-6 family cytokine ciliary neurotrophic factor (CNTF), which we found abundantly expressed by mouse muscle at the transcript and protein level, reduced osteoblast gene expression, although not to the same extent as the myotube-conditioned media. These data indicate that muscle cells secrete abundant TIMP-1, MCP-1, and CNTF, and that of these, only CNTF has the ability to suppress osteoblast function and gene expression in a similar manner to myotube-conditioned medium. This suggests that CNTF is

  11. Fibrodysplasia ossificans progressiva-related activated activin-like kinase signaling enhances osteoclast formation during heterotopic ossification in muscle tissues.

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    Yano, Masato; Kawao, Naoyuki; Okumoto, Katsumi; Tamura, Yukinori; Okada, Kiyotaka; Kaji, Hiroshi

    2014-06-13

    Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. CD36 is required for myoblast fusion during myogenic differentiation

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    Park, Seung-Yoon [Department of Biochemistry, College of Medicine, Dongguk University and Medical Institute of Dongguk University, Gyeongju 780-714 (Korea, Republic of); Yun, Youngeun [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Kim, In-San, E-mail: iskim@knu.ac.kr [Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422 (Korea, Republic of); Biomedical Research Institute, Korea Institute Science and Technology, Seoul (Korea, Republic of)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer CD36 expression was induced during myogenic differentiation. Black-Right-Pointing-Pointer CD36 expression was localized in multinucleated myotubes. Black-Right-Pointing-Pointer The expression of myogenic markers is attenuated in CD36 knockdown C2C12 cells. Black-Right-Pointing-Pointer Knockdown of CD36 significantly inhibited myotube formation during differentiation. -- Abstract: Recently, CD36 has been found to be involved in the cytokine-induced fusion of macrophage. Myoblast fusion to form multinucleated myotubes is required for myogenesis and muscle regeneration. Because a search of gene expression database revealed the attenuation of CD36 expression in the muscles of muscular dystrophy patients, the possibility that CD36 could be required for myoblast fusion was investigated. CD36 expression was markedly up-regulated during myoblast differentiation and localized in multinucleated myotubes. Knockdown of endogenous CD36 significantly decreased the expression of myogenic markers as well as myotube formation. These results support the notion that CD36 plays an important role in cell fusion during myogenic differentiation. Our finding will aid the elucidation of the common mechanism governing cell-to-cell fusion in various fusion models.

  13. CLP-1 associates with MyoD and HDAC to restore skeletal muscle cell regeneration.

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    Galatioto, Josephine; Mascareno, Eduardo; Siddiqui, M A Q

    2010-11-01

    Emerging evidence suggests that eukaryotic gene transcription is regulated primarily at the elongation stage by association and dissociation of the inhibitory protein cardiac lineage protein 1 (CLP-1/HEXIM1) from the positive transcription elongation factor b (P-TEFb) complex. It was reported recently that P-TEFb interacts with skeletal muscle-specific regulatory factor, MyoD, suggesting a linkage between CLP-1-mediated control of transcription and skeletal myogenesis. To examine this, we produced CLP-1 knockdown skeletal muscle C2C12 cells by homologous recombination, and demonstrated that the C2C12 CLP-1 +/- cells failed to differentiate when challenged by low serum in the medium. We also showed that CLP-1 interacts with both MyoD and histone deacetylases (HDACs) maximally at the early stage of differentiation of C2C12 cells. This led us to hypothesize that the association might be crucial to inhibition of MyoD-target proliferative genes. Chromatin immunoprecipitation analysis revealed that the CLP-1/MyoD/HDAC complex binds to the promoter of the cyclin D1 gene, which is downregulated in differentiated muscle cells. These findings suggest a novel transcriptional paradigm whereby CLP-1, in conjunction with MyoD and HDAC, acts to inhibit growth-related gene expression, a requirement for myoblasts to exit the cell cycle and transit to myotubes.

  14. Integrated analysis of chronic lipotoxicity on muscle metabolism and stress and its reversal by antioxidants.

    Science.gov (United States)

    Verma, Mahesh Kumar; Yateesh, Aggunda Nagaraju; Smitha, Rachapalli; Neelima, Korrapati; Pallavi, Puttrevana M; Reddy, Madhusudhan; Poornima, Jayaram; Oommen, Anup M; Jagannath, Madanahalli R; Somesh, Baggavalli P

    2014-01-01

    Apart from elevated glucose, triglyceride and cholesterol, elevated levels of serum free-fatty acid (FFA) are observed in diabetic patients. Increased FFA load can cause multiple dysregulation which are collectively known as lipotoxicity. Impacts of FFA induced lipotoxicity were evaluated on various cellular responses of metabolism and stress in skeletal muscle myotubes. Under lipotoxicity, oxidative capacity of C2C12 myotubes was reduced and decreased levels ATP and NAD were observed. Lipotoxicity augmented non-oxidative disposal of metabolites in terms of lactate release, IMTG and ceramide synthesis. Concomitantly, insulin resistance was also observed. These impacts were in conjunction with increased cellular stress, inflammation, proteolysis and apoptosis. Quenching of lipotoxicity mediated oxidative stress by antioxidant reverted its deleterious impacts and restored insulin stimulated glucose uptake. In conclusion, the in vitro lipotoxicity makes a system which resembles in vivo pathology of muscle as seen in diabetic patients and represents an integrated perspective of lipotoxicity on various parameters of metabolism and stress.

  15. Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells

    Science.gov (United States)

    Gorman, Jennifer L.; Liu, Sammy T. K.; Slopack, Dara; Shariati, Khashayar; Hasanee, Adam; Olenich, Sara; Olfert, I. Mark; Haas, Tara L.

    2014-01-01

    Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload

  16. IL-6 regulation on skeletal muscle mitochondrial remodeling during cancer cachexia in the ApcMin/+ mouse

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    White James P

    2012-07-01

    Full Text Available Abstract Background Muscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood. The purposes of this study were to 1 determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and 2 to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia. Methods ApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts. Results Mitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2 protein expression was repressed. With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1 was induced. IL-6 receptor antibody administration after the onset of cachexia improved mitochondrial content, PGC-1α, Mfn1/Mfn2 and FIS1 protein expression. IL-6 over-expression in pre-cachectic mice accelerated body weight loss and muscle wasting, without reducing mitochondrial content, while PGC-1α and Mfn1/Mfn2 protein expression was suppressed and FIS1 protein expression

  17. Specification of skeletal muscle differentiation by repressor element-1 silencing transcription factor (REST)-regulated Kv7.4 potassium channels.

    Science.gov (United States)

    Iannotti, Fabio Arturo; Barrese, Vincenzo; Formisano, Luigi; Miceli, Francesco; Taglialatela, Maurizio

    2013-02-01

    Changes in the expression of potassium (K(+)) channels is a pivotal event during skeletal muscle differentiation. In mouse C(2)C(12) cells, similarly to human skeletal muscle cells, myotube formation increased the expression of K(v)7.1, K(v)7.3, and K(v)7.4, the last showing the highest degree of regulation. In C(2)C(12) cells, K(v)7.4 silencing by RNA interference reduced the expression levels of differentiation markers (myogenin, myosin heavy chain, troponinT-1, and Pax3) and impaired myotube formation and multinucleation. In K(v)7.4-silenced cells, the differentiation-promoting effect of the K(v)7 activator N-(2-amino-4-(4-fluorobenzylamino)-phenyl)-carbamic acid ethyl ester (retigabine) was abrogated. Expression levels for the repressor element-1 silencing transcription factor (REST) declined during myotube formation. Transcript levels for K(v)7.4, as well as for myogenin, troponinT-1, and Pax3, were reduced by REST overexpression and enhanced upon REST suppression by RNA interference. Four regions containing potential REST-binding sites in the 5' untranslated region and in the first intron of the K(v)7.4 gene were identified by bioinformatic analysis. Chromatin immunoprecipitation assays showed that REST binds to these regions, exhibiting a higher efficiency in myoblasts than in myotubes. These data suggest that K(v)7.4 plays a permissive role in skeletal muscle differentiation and highlight REST as a crucial transcriptional regulator for this K(+) channel subunit.

  18. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Muratore, Massimo, E-mail: M.Muratore@ed.ac.uk [Institute of Integrated Micro and Nano System, School of Engineering, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Mitchell, Steve [Institute of Molecular Plant Science, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Waterfall, Martin [Institute of Immunology and Infection Research, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JT (United Kingdom)

    2013-09-06

    Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy.

  19. Valproic acid attenuates skeletal muscle wasting by inhibiting C/EBPβ-regulated atrogin1 expression in cancer cachexia.

    Science.gov (United States)

    Sun, Rulin; Zhang, Santao; Hu, Wenjun; Lu, Xing; Lou, Ning; Yang, Zhende; Chen, Shaoyong; Zhang, Xiaoping; Yang, Hongmei

    2016-07-01

    Muscle wasting is the hallmark of cancer cachexia and is associated with poor quality of life and increased mortality. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has important biological effects in the treatment of muscular dystrophy. To verify whether VPA could ameliorate muscle wasting induced by cancer cachexia, we explored the role of VPA in two cancer cachectic mouse models [induced by colon-26 (C26) adenocarcinoma or Lewis lung carcinoma (LLC)] and atrophied C2C12 myotubes [induced by C26 cell conditioned medium (CCM) or LLC cell conditioned medium (LCM)]. Our data demonstrated that treatment with VPA increased the mass and cross-sectional area of skeletal muscles in tumor-bearing mice. Furthermore, treatment with VPA also increased the diameter of myotubes cultured in conditioned medium. The skeletal muscles in cachectic mice or atrophied myotubes treated with VPA exhibited reduced levels of CCAAT/enhancer binding protein beta (C/EBPβ), resulting in atrogin1 downregulation and the eventual alleviation of muscle wasting and myotube atrophy. Moreover, atrogin1 promoter activity in myotubes was stimulated by CCM via activating the C/EBPβ-responsive cis-element and subsequently inhibited by VPA. In contrast to the effect of VPA on the levels of C/EBPβ, the levels of inactivating forkhead box O3 (FoxO3a) were unaffected. In summary, VPA attenuated muscle wasting and myotube atrophy and reduced C/EBPβ binding to atrogin1 promoter locus in the myotubes. Our discoveries indicate that HDAC inhibition by VPA might be a promising new approach for the preservation of skeletal muscle in cancer cachexia. Copyright © 2016 the American Physiological Society.

  20. Citrus junos Tanaka Peel Extract Exerts Antidiabetic Effects via AMPK and PPAR-γ both In Vitro and In Vivo in Mice Fed a High-Fat Diet

    Directory of Open Access Journals (Sweden)

    Sung Hee Kim

    2013-01-01

    Full Text Available The antidiabetic effect of the Citrus junos Tanaka (also known as yuja or yuzu was examined. Ethanol extract of yuja peel (YPEE significantly stimulated 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-ylamino]-2-deoxy-D-glucose (2-NBDG uptake in C2C12 myotubes. However, ethanol extract of yuja pulp (YpEE and water extract of yuja peel (YPWE or pulp (YpWE did not stimulate glucose uptake. In addition, peroxisome proliferator-activated receptor gamma (PPAR-γ and AMP-activated protein kinase (AMPK activities were increased by YPEE in a dose-dependent manner. Pretreatment of AMPK inhibitor decreased the glucose uptake stimulated by YPEE in C2C12 myotubes. We confirmed the anti-diabetic effect of YPEE in mice fed a high fat-diet (HFD. Compared with control mice on a normal diet (ND, these mice showed increased body weight, liver fat, insulin resistance, triacylglycerol (TG, and total cholesterol content. Addition of 5% YPEE significantly reduced the weight gain and rise in liver fat content, serum triacylglycerol (TG, total cholesterol, and insulin resistance found in mice fed a high-fat diet (HFD. Moreover, YPEE reduced the secretion of HFD-induced adipocytokines such as leptin and resistin. YPEE also resulted in increased phosphorylation of AMPK in muscle tissues. These results suggest that ethanol extract of yuja peel exerts anti-diabetic effects via AMPK and PPAR-γ in both cell culture and mouse models.

  1. ADP-ribosylation factor-like GTPase 15 enhances insulin-induced AKT phosphorylation in the IR/IRS1/AKT pathway by interacting with ASAP2 and regulating PDPK1 activity.

    Science.gov (United States)

    Zhao, Jie; Wang, Min; Deng, Wuquan; Zhong, Daping; Jiang, Youzhao; Liao, Yong; Chen, Bing; Zhang, Xiaoli

    2017-05-13

    Decreased phosphorylation in the insulin signalling pathway is a hallmark of insulin resistance. The causes of this phenomenon are complicated and multifactorial. Recently, genomic analyses have identified ARL15 as a new candidate gene related to diabetes. However, the ARL15 protein function remains unclear. Here, we show that ARL15 is upregulated by insulin stimulation. This effect was impaired in insulin-resistant pathophysiology in TNF-α-treated C2C12 myotubes and in the skeletal muscles of leptin knockout mice. In addition, ARL15 localized to the cytoplasm in the resting state and accumulated in the Golgi apparatus around the nucleus upon insulin stimulation. ARL15 overexpression can enhance the phosphorylation of the key insulin signalling pathway molecules IR, IRS1 and AKT in C2C12 myotubes. Moreover, ARL15 knockdown can also specifically inhibit the phosphorylation of PDPK1 Ser241, thereby reducing PDPK1 activity and its downstream phosphorylation of AKT Thr308. Co-immunoprecipitation assays identified ASAP2 as an ARL15-interacting protein. In conclusion, we have identified that ARL15 acts as an insulin-sensitizing effector molecule to upregulate the phosphorylation of members of the canonical IR/IRS1/PDPK1/AKT insulin pathway by interacting with its GAP ASAP2 and activating PDPK1. This research may provide new insights into GTPase-mediated insulin signalling regulation and facilitate the development of new pharmacotherapeutic targets for insulin sensitization. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Salamanders on the bench - A biocompatibility study of salamander skin secretions in cell cultures.

    Science.gov (United States)

    von Byern, Janek; Mebs, Dietrich; Heiss, Egon; Dicke, Ursula; Wetjen, Oliver; Bakkegard, Kristin; Grunwald, Ingo; Wolbank, Susanne; Mühleder, Severin; Gugerell, Alfred; Fuchs, Heidemarie; Nürnberger, Sylvia

    2017-09-01

    Salamanders have evolved a wide variety of antipredator mechanisms and behavior patterns, including toxins and noxious or adhesive skin secretions. The high bonding strength of the natural bioadhesives makes these substances interesting for biomimetic research and applications in industrial and medical sectors. Secretions of toxic species may help to understand the direct effect of harmful substances on the cellular level. In the present study, the biocompatibility of adhesive secretions from four salamander species (Plethodon shermani, Plethodon glutinosus, Ambystoma maculatum, Ambystoma opacum) were analyzed using the MTT assay in cell culture and evaluated against toxic secretions of Pleurodeles waltl, Triturus carnifex, Pseudotriton ruber, Tylototriton verrucosus, and Salamandra salamandra. Their effect on cells was tested in direct contact (direct culture) or under the influence of the extract (indirect exposure) in accordance with the protocol of the international standard norm ISO 10993-5. Human dermal fibroblasts (NHDF), umbilical vein endothelial cells (HUVEC), and articular chondrocytes (HAC), as well as the cell lines C2C12 and L929 were used in both culture types. While the adhesive secretions from Plethodon shermani are cytocompatible and those of Ambystoma opacum are even advantageous, those of Plethodon glutinosus and Ambystoma maculatum appear to be cytotoxic to NDHF and HUVEC. Toxic secretions from Salamandra salamandra exhibited harmful effects on all cell types. Pseudotriton ruber and Triturus carnifex secretions affected certain cell types marginally; those from Pleurodeles waltl and Tylototriton verrucosus were generally well tolerated. The study shows for the first time the effect of salamander secretions on the viability of different cell types in culture. Two adhesive secretions appeared to be cell compatible and are therefore promising candidates for future investigations in the field of medical bioadhesives. Among the toxic secretions

  3. A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

    Directory of Open Access Journals (Sweden)

    Jakob D Wikstrom

    Full Text Available The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

  4. Cellular cardiomyoplasty into infracted swine's hearts by retrograde infusion through the venous coronary sinus: An experimental study

    Energy Technology Data Exchange (ETDEWEB)

    Prifti, Edvin, E-mail: edvinprifti@hotmail.com [Division of Cardiac Surgery, University Hospital Center of Tirana (Albania); Di Lascio, Gabriella [Anesthesiology and Intensive Care Section, Department of Health Sciences, University of Florence, Florence (Italy); Harmelin, Guy [Cardiac Surgery, Department of Experimental and Clinical Medicine, University of Florence, Florence (Italy); Bani, Daniele [Research Unit of Histology & Embryology, Departments of Clinical & Experimental Medicine, University of Florence, Florence (Italy); Briganti, Vittorio [Unit of Nuclear Medicine, Careggi Hospital, Florence (Italy); Veshti, Altin [Division of Cardiac Surgery, University Hospital Center of Tirana (Albania); Bonacchi, Massimo [Cardiac Surgery, Department of Experimental and Clinical Medicine, University of Florence, Florence (Italy)

    2016-06-15

    Objectives: The aim was to create a model of myocardial infarction with a borderline myocardial impairment which would enable evaluation of the retrograde cellular cardiomyoplasty through the venous coronary sinus in a large animal model. Materials and methods: Fifteen (study group) and 10 juvenile farm pigs (control group) underwent distal left anterior descending artery ligation. One month later the study group animals underwent sternotomy and a murine myoblastic line C2-C12 was injected at a constant pressure of 30 mmHg, into the coronary sinus. Thirty days later all animals that survived from both groups underwent transthoracic echocardiography and 99Tc scintigraphy and were later euthanized and specimens were taken for microscopic evaluation. Results: Cardiac output decreased significantly after ligation (p < 0.001) and increased significantly after cardiomyoplasty (p < 0.001). In all animals, the surgical induction of myocardial infarction caused a marked decline in the echocardiographic values of cardiac function; however, the cardiac function and dimensions were significantly improved in the study group after cardiomyoplasty versus the control group. All animals undergoing cardiomyoplasty demonstrated a significant reduction of the perfusion deficit in the left anterior descending artery territory, instead such data remained unchanged in the control group. The histological examination demonstrated the engrafted myoblasts could be distinguished from the activated fibroblasts in the scar tissue because they never showed any signs of collagen secretion and fiber buildup. Conclusions: In conclusion, the venous retrograde delivery route through the coronary sinus is safe and effective, providing a significant improvement in function and viability.

  5. A Key Role for Leukemia Inhibitory Factor in C26 Cancer Cachexia.

    Science.gov (United States)

    Seto, Danielle N; Kandarian, Susan C; Jackman, Robert W

    2015-08-07

    Cachexia is an exacerbating event in many types of cancer that is strongly associated with a poor prognosis. We have identified cytokine, signaling, and transcription factors that are required for cachexia in the mouse C26 colon carcinoma model of cancer. C2C12 myotubes treated with conditioned medium from C26 cancer cells induced atrophy and activated a STAT-dependent reporter gene but not reporter genes dependent on SMAD, FOXO, C/EBP, NF-κB, or AP-1. Of the gp130 family members IL-11, IL-6, oncostatin M (OSM), and leukemia inhibitory factor (LIF), only OSM and LIF were sufficient to activate the STAT reporter in myotubes. LIF was elevated in C26 conditioned medium (CM), but IL-6, OSM, TNFα, and myostatin were not. A LIF-blocking antibody abolished C26 CM-induced STAT reporter activation, STAT3 phosphorylation, and myotube atrophy but blocking antibodies to IL-6 or OSM did not. JAK2 inhibitors also blocked C26 CM-induced STAT reporter activation, STAT3 phosphorylation, and atrophy in myotubes. LIF at levels found in the C26 CM was sufficient for STAT reporter activation and atrophy in myotubes. In vivo, an increase in serum LIF preceded the increase in IL-6 in mice with C26 tumors. Overexpression of a dominant negative Stat3Cβ-EGFP gene in myotubes and in mouse muscle blocked the atrophy caused by C26 CM or C26 tumors, respectively. Taken together, these data support an important role of LIF-JAK2-STAT3 in C26 cachexia and point to a therapeutic approach for at least some types of cancer cachexia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Stimulatory Effects of Balanced Deep Sea Water on Mitochondrial Biogenesis and Function.

    Directory of Open Access Journals (Sweden)

    Byung Geun Ha

    Full Text Available The worldwide prevalence of metabolic diseases, including obesity and diabetes, is increasing. Mitochondrial dysfunction is recognized as a core feature of these diseases. Emerging evidence also suggests that defects in mitochondrial biogenesis, number, morphology, fusion, and fission, contribute to the development and progression of metabolic diseases. Our previous studies revealed that balanced deep-sea water (BDSW has potential as a treatment for diabetes and obesity. In this study, we aimed to investigate the mechanism by which BDSW regulates diabetes and obesity by studying its effects on mitochondrial metabolism. To determine whether BDSW regulates mitochondrial biogenesis and function, we investigated its effects on mitochondrial DNA (mtDNA content, mitochondrial enzyme activity, and the expression of transcription factors and mitochondria specific genes, as well as on the phosphorylation of signaling molecules associated with mitochondria biogenesis and its function in C2C12 myotubes. BDSW increased mitochondrial biogenesis in a time and dose-dependent manner. Quantitative real-time PCR revealed that BDSW enhances gene expression of PGC-1α, NRF1, and TFAM for mitochondrial transcription; MFN1/2 and DRP1 for mitochondrial fusion; OPA1 for mitochondrial fission; TOMM40 and TIMM44 for mitochondrial protein import; CPT-1α and MCAD for fatty acid oxidation; CYTC for oxidative phosphorylation. Upregulation of these genes was validated by increased mitochondria staining, CS activity, CytC oxidase activity, NAD+ to NADH ratio, and the phosphorylation of signaling molecules such as AMPK and SIRT1. Moreover, drinking BDSW remarkably improved mtDNA content in the muscles of HFD-induced obese mice. Taken together, these results suggest that the stimulatory effect of BDSW on mitochondrial biogenesis and function may provide further insights into the regulatory mechanism of BDSW-induced anti-diabetic and anti-obesity action.

  7. Pregnancy-associated Plasma Protein-A Regulates Myoblast Proliferation and Differentiation through an Insulin-like Growth Factor-dependent Mechanism*

    OpenAIRE

    Kumar, Ashok; Mohan, Subburaman; Newton, Jacqueline; Rehage, Mark; Tran, Kiet; Baylink, David J.; Qin, Xuezhong

    2005-01-01

    Pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, is an important regulator of mammalian growth and development. However, the role of PAPP-A and its mechanism of action in various cellular processes remain unknown. In this study, we have investigated the role of PAPP-A in skeletal myogenesis using C2C12 myoblasts. Recombinant PAPP-A was purified from the conditioned medium of HT1080 cells overexpressing PAPP-A. Treatment of C2C12 myoblasts with PAP...

  8. Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor

    OpenAIRE

    John, Scott A.; Ottolia, Michela; Weiss, James N.; Ribalet, Bernard

    2007-01-01

    To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600µM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (τ =5 to 15 s) in COS and C2C12 cells and slow (τ =20 to 40 s) in HEK and CHO cells. Upon ...

  9. Sialylation of Glycosylphosphatidylinositol (GPI) Anchors of Mammalian Prions Is Regulated in a Host-, Tissue-, and Cell-specific Manner*

    Science.gov (United States)

    Katorcha, Elizaveta; Srivastava, Saurabh; Klimova, Nina; Baskakov, Ilia V.

    2016-01-01

    Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrPC. PrPC is posttranslationally modified with N-linked glycans and a sialylated glycosylphosphatidylinositol (GPI) anchor. Conformational conversion of PrPC gives rise to glycosylated and GPI-anchored PrPSc. The question of the sialylation status of GPIs within PrPSc has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrPSc, with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrPC with sialylo-GPIs could be recruited into PrPSc, whereas PrPC with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed the sialylation status of GPIs within PrPSc generated in the brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrPC with both sialo- and asialo-GPIs is a common feature of PrPSc. The mixtures of sialo- and asialo-GPIs were observed in PrPSc universally regardless of prion strain as well as host, tissue, or type of cells that produced PrPSc. Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue, and cell type but not prion strain. In summary, this study found no strain-specific preferences for selecting PrPC with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrPSc is regulated in a cell-, tissue-, or host-specific manner and is likely to be determined by the specifics of GPI biosynthesis. PMID:27317661

  10. Hydroxy monounsaturated fatty acids as agonists for peroxisome proliferator-activated receptors.

    Science.gov (United States)

    Yokoi, Hiroshi; Mizukami, Hajime; Nagatsu, Akito; Tanabe, Hiroki; Inoue, Makoto

    2010-01-01

    The physiological and pathological role of oxidized polyunsaturated fatty acids (PUFAs) has been extensively studied, whereas those of hydroxy monounsaturated fatty acids (MUFAs) are not well understood. This study demonstrated that 11-hydroxy-(9Z)-octadecenoic acid ((9Z)-11-HOE), which was isolated from adlay seeds (Coix lacryma-jobi L. var. ma-yuen STAF.), can activate peroxisome proliferator-activated receptor (PPAR)alpha, delta and gamma in luciferase reporter assays more efficiently than (9Z)-octadecenoic acid (oleic acid), and to the same degree as linoleic acid. (9Z)-11-HOE increased the mRNA levels of UCP2 and CD36 in C2C12 myotubes and THP- 1 cells, respectively, and these effects were blocked by the PPARdelta- and gamma-specific antagonists GSK0660 and T0070907, respectively. Evaluation of the structure.activity relationship between hydroxy MUFAs and PPAR activation revealed that (9E)-11-HOE, the geometrical isomer of (9Z)-11-HOE, activated PPARs more potently than (9Z)-11-HOE, and that PPAR activation by hydroxyl MUFAs was not markedly influenced by the position of the hydroxy group or the double bond, although PPARdelta seemed to possess ligand specificity different to that of PPARalpha or gamma . Additionally, the finding that 11-hydroxy octadecanoic acid, the hydrogenated product of (9E)-11- HOE, was also capable of activating PPARs to a similar extent as (9E)-11-HOE indicates that the double bond in hydroxy MUFAs is not essential for PPAR activation. In conclusion, (9Z)-11-HOE derived from alday seeds and hydroxy MUFAs with a chain length of 16 or 18 acted as PPAR agonists. Hydroxylation of MUFAs may change these compounds from silent PPAR ligands to active PPAR agonists.

  11. Insulin resistance is associated with MCP1-mediated macrophage accumulation in skeletal muscle in mice and humans.

    Directory of Open Access Journals (Sweden)

    David Patsouris

    Full Text Available Inflammation is now recognized as a major factor contributing to type 2 diabetes (T2D. However, while the mechanisms and consequences associated with white adipose tissue inflammation are well described, very little is known concerning the situation in skeletal muscle. The aim of this study was to investigate, in vitro and in vivo, how skeletal muscle inflammation develops and how in turn it modulates local and systemic insulin sensitivity in different mice models of T2D and in humans, focusing on the role of the chemokine MCP1. Here, we found that skeletal muscle inflammation and macrophage markers are increased and associated with insulin resistance in mice models and humans. In addition, we demonstrated that intra-muscular TNFα expression is exclusively restricted to the population of intramuscular leukocytes and that the chemokine MCP1 was associated with skeletal muscle inflammatory markers in these models. Furthermore, we demonstrated that exposure of C2C12 myotubes to palmitate elevated the production of the chemokine MCP1 and that the muscle-specific overexpression of MCP1 in transgenic mice induced the local recruitment of macrophages and altered local insulin sensitivity. Overall our study demonstrates that skeletal muscle inflammation is clearly increased in the context of T2D in each one of the models we investigated, which is likely consecutive to the lipotoxic environment generated by peripheral insulin resistance, further increasing MCP1 expression in muscle. Consequently, our results suggest that MCP1-mediated skeletal muscle macrophages recruitment plays a role in the etiology of T2D.

  12. Ca(2+) calmodulin kinase and calcineurin mediate IGF-1-induced skeletal muscle dihydropyridine receptor alpha(1S) transcription.

    Science.gov (United States)

    Zheng, Z; Wang, Z M; Delbono, O

    2004-01-15

    The skeletal muscle L-type Ca(2+) channel or dihydropyridine(DHP)-sensitive receptor is a key molecule involved in membrane voltage-sensing, sarcoplasmic reticulum Ca(2+) release, and muscle contraction. Previous work from our laboratory has shown that the insulin-like growth factor-1 (IGF-1) increases skeletal muscle L-type Ca(2+) channel or dihydropyridine-sensitive receptor DHPRalpha(1S) transcriptional activity by acting on the cyclic AMP response element binding protein (CREB) element of the promoter region; however, the cellular signaling mediating this process is not known. In this study, we investigated the signaling pathway whereby IGF-1 enhances the expression of DHPRalpha(1S) in C2C12 myotubes, using a molecular, pharmacological and electrophysiological approach. We found that inhibition of the Ca(2+)/Calmodulin (CaM)-dependent protein kinase or calcineurin, influenced IGF-1-induced increase in DHPRalpha(1S) expression, as detected by recording the luminescence of the DHPRalpha(1S) promoter-luciferase fusion construct and by immunoblot analysis of the DHPR alpha1 subunit. IGF-1 significantly increased CaM kinase and calcineurin activity and the cellular levels of phosphorylated CREB in a time-dependent manner. The role of CaM kinase and calcineurin in DHPRalpha(1S) expression was confirmed by functional recording of the effects of the inhibition of the kinase and phosphatase on IGF-1-mediated enhancement of charge movement. These results support the conclusion that IGF-1 controls CREB phosphorylation by activating a phosphorylation and dephosphorylation cascade, which ultimately modulates the DHPRalpha(1S) gene transcription.

  13. Antcin K, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects via Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in Muscles

    Directory of Open Access Journals (Sweden)

    Yueh-Hsiung Kuo

    2016-01-01

    Full Text Available The purpose of this study was to screen firstly the potential effects of antcin K (AnK, the main constituent of the fruiting body of Antrodia camphorata, in vitro and further evaluate the activities and mechanisms in high-fat-diet- (HFD- induced mice. Following 8-week HFD-induction, mice were treated with AnK, fenofibrate (Feno, metformin (Metf, or vehicle for 4 weeks afterward. In C2C12 myotube cells, the membrane GLUT4 and phospho-Akt expressions were higher in insulin and AnK-treated groups than in the control group. It was observed that AnK-treated mice significantly lowered blood glucose, triglyceride, total cholesterol, and leptin levels in AnK-treated groups. Of interest, AnK at 40 mg/kg/day dosage displayed both antihyperglycemic effect comparable to Metf (300 mg/kg/day and antihypertriglyceridemic effect comparable to Feno (250 mg/kg/day. The combination of significantly increased skeletal muscular membrane expression levels of glucose transporter 4 (GLUT4 but decreased hepatic glucose-6-phosphatase (G6 Pase mRNA levels by AnK thus contributed to a decrease in blood glucose levels. Furthermore, AnK enhanced phosphorylation of AMP-activated protein kinase (phospho-AMPK expressions in the muscle and liver. Moreover, AnK treatment exhibited inhibition of hepatic fatty acid synthase (FAS but enhancement of fatty acid oxidation peroxisome proliferator-activated receptor α (PPARα expression coincident with reduced sterol response element binding protein-1c (SREBP-1c mRNA levels in the liver may contribute to decreased plasma triglycerides, hepatic steatosis, and total cholesterol levels. The present findings indicate that AnK displays an advantageous therapeutic potential for the management of type 2 diabetes and hyperlipidemia.

  14. Antcin K, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects via Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in Muscles.

    Science.gov (United States)

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching; Yang, Chang-Syun

    2016-01-01

    The purpose of this study was to screen firstly the potential effects of antcin K (AnK), the main constituent of the fruiting body of Antrodia camphorata, in vitro and further evaluate the activities and mechanisms in high-fat-diet- (HFD-) induced mice. Following 8-week HFD-induction, mice were treated with AnK, fenofibrate (Feno), metformin (Metf), or vehicle for 4 weeks afterward. In C2C12 myotube cells, the membrane GLUT4 and phospho-Akt expressions were higher in insulin and AnK-treated groups than in the control group. It was observed that AnK-treated mice significantly lowered blood glucose, triglyceride, total cholesterol, and leptin levels in AnK-treated groups. Of interest, AnK at 40 mg/kg/day dosage displayed both antihyperglycemic effect comparable to Metf (300 mg/kg/day) and antihypertriglyceridemic effect comparable to Feno (250 mg/kg/day). The combination of significantly increased skeletal muscular membrane expression levels of glucose transporter 4 (GLUT4) but decreased hepatic glucose-6-phosphatase (G6 Pase) mRNA levels by AnK thus contributed to a decrease in blood glucose levels. Furthermore, AnK enhanced phosphorylation of AMP-activated protein kinase (phospho-AMPK) expressions in the muscle and liver. Moreover, AnK treatment exhibited inhibition of hepatic fatty acid synthase (FAS) but enhancement of fatty acid oxidation peroxisome proliferator-activated receptor α (PPARα) expression coincident with reduced sterol response element binding protein-1c (SREBP-1c) mRNA levels in the liver may contribute to decreased plasma triglycerides, hepatic steatosis, and total cholesterol levels. The present findings indicate that AnK displays an advantageous therapeutic potential for the management of type 2 diabetes and hyperlipidemia.

  15. Intake of a Ketone Ester Drink during Recovery from Exercise Promotes mTORC1 Signaling but Not Glycogen Resynthesis in Human Muscle.

    Science.gov (United States)

    Vandoorne, Tijs; De Smet, Stefan; Ramaekers, Monique; Van Thienen, Ruud; De Bock, Katrien; Clarke, Kieran; Hespel, Peter

    2017-01-01

    Purpose: Ketone bodies are energy substrates produced by the liver during prolonged fasting or low-carbohydrate diet. The ingestion of a ketone ester (KE) rapidly increases blood ketone levels independent of nutritional status. KE has recently been shown to improve exercise performance, but whether it can also promote post-exercise muscle protein or glycogen synthesis is unknown. Methods: Eight healthy trained males participated in a randomized double-blind placebo-controlled crossover study. In each session, subjects undertook a bout of intense one-leg glycogen-depleting exercise followed by a 5-h recovery period during which they ingested a protein/carbohydrate mixture. Additionally, subjects ingested a ketone ester (KE) or an isocaloric placebo (PL). Results: KE intake did not affect muscle glycogen resynthesis, but more rapidly lowered post-exercise AMPK phosphorylation and resulted in higher mTORC1 activation, as evidenced by the higher phosphorylation of its main downstream targets S6K1 and 4E-BP1. As enhanced mTORC1 activation following KE suggests higher protein synthesis rates, we used myogenic C2C12 cells to further confirm that ketone bodies increase both leucine-mediated mTORC1 activation and protein synthesis in muscle cells. Conclusion: Our results indicate that adding KE to a standard post-exercise recovery beverage enhances the post-exercise activation of mTORC1 but does not affect muscle glycogen resynthesis in young healthy volunteers. In vitro, we confirmed that ketone bodies potentiate the increase in mTORC1 activation and protein synthesis in leucine-stimulated myotubes. Whether, chronic oral KE intake during recovery from exercise can facilitate training-induced muscular adaptation and remodeling need to be further investigated.

  16. Involvement of mTOR in Type 2 CRF Receptor Inhibition of Insulin Signaling in Muscle Cells.

    Science.gov (United States)

    Chao, Hongxia; Li, Haochen; Grande, Rebecca; Lira, Vitor; Yan, Zhen; Harris, Thurl E; Li, Chien

    2015-06-01

    Type 2 corticotropin-releasing factor receptor (CRFR2) is expressed in skeletal muscle and stimulation of the receptor has been shown to inhibit the effect of insulin on glucose uptake in muscle cells. Currently, little is known about the mechanisms underlying this process. In this study, we first showed that both in vivo and in vitro CRFR2 expression in muscle was closely correlated with insulin sensitivity, with elevated receptor levels observed in insulin resistant muscle cells. Stimulation of CRFR2 by urocortin 2 (Ucn 2), a CRFR2-selective ligand, in C2C12 myotubes greatly attenuated insulin-induced glucose uptake. The inhibitory effect of CRFR2 signaling required cAMP production and is involved the mammalian target of rapamycine pathway, as rapamycin reversed the inhibitory effect of CRFR2 stimulation on insulin-induced glucose uptake. Moreover, stimulation of CRFR2 failed to inhibit glucose uptake in muscle cells induced by platelet-derived growth factor, which, similar to insulin, signals through Akt-mediated pathway but is independently of insulin receptor substrate (IRS) proteins to promote glucose uptake. This result argues that CRFR2 signaling modulates insulin's action likely at the levels of IRS. Consistent with this notion, Ucn 2 reduced insulin-induced tyrosine phosphorylation of IRS-1, and treatment with rapamycin reversed the inhibitory effect of Ucn 2 on IRS-1 and Akt phosphorylation. In conclusion, the inhibitory effect of CRFR2 signaling on insulin action is mediated by cAMP in a mammalian target of rapamycine-dependent manner, and IRS-1 is a key nodal point where CRFR2 signaling modulates insulin-stimulated glucose uptake in muscle cells.

  17. Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells.

    Science.gov (United States)

    Kobayashi, Tatsuo; Akiyama, Yuko; Akiyama, Nobuteru; Katoh, Hideaki; Yamamoto, Sachiko; Funatsuki, Kenzo; Yanagimoto, Toru; Notoya, Mitsuru; Asakura, Kenji; Shinosaki, Toshihiro; Hanasaki, Kohji

    2010-12-15

    Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulin-stimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Comparative study of the cytolytic activity of snake venoms from African spitting cobras (Naja spp., Elapidae) and its neutralization by a polyspecific antivenom.

    Science.gov (United States)

    Méndez, Ileana; Gutiérrez, José María; Angulo, Yamileth; Calvete, Juan J; Lomonte, Bruno

    2011-11-01

    Venoms of several Naja species found in Sub-Saharan Africa, and commonly known as "spitting cobras", induce a predominantly cytotoxic pattern of envenomings that may evolve into tissue necrosis and gangrene. Cytotoxic components of their venoms have been identified as members of the three-finger toxin and phospholipase A(2) protein families. In this study, an in vitro assay using the myogenic cell line C2C12, was utilized to compare the cytolytic activities of venoms from five species of spitting cobras: Naja nigricollis, Naja katiensis, Naja pallida, Naja nubiae, and Naja mossambica. These venoms were strongly cytotoxic, causing a 50% effect at ~1.5 μg/well (15 μg/ml), except for N. katiensis venom, which required nearly twice this amount. Using the cell-based assay, the ability of an equine polyspecific antivenom (EchiTab-Plus-ICP) to neutralize cytotoxicity was assessed. The antivenom completely inhibited the cytotoxic activity of all five venoms, although high antivenom/venom ratios were needed. Neutralization curves displayed the following decreasing order of efficiency: N. nubiae > N. pallida > N. mossambica > N. nigricollis > N. katiensis. Results indicate that neutralizing antibodies toward toxins responsible for this particular effect are present in the antivenom, albeit in low titers. Fucoidan, a natural sulfated polysaccharide known to inhibit the toxic effects of some basic snake venom components, was unable to reduce cytotoxicity of Naja venoms. Results emphasize the need of enhancing the immunogenicity of low molecular mass toxins during antivenom production, as well as to search for useful toxin inhibitors which could complement antivenom therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesis.

    Directory of Open Access Journals (Sweden)

    Xingxing Kong

    2010-07-01

    Full Text Available Sirtuin 3 (SIRT3 is one of the seven mammalian sirtuins, which are homologs of the yeast Sir2 gene. SIRT3 is the only sirtuin with a reported association with the human life span. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. Knockdown of PGC-1alpha led to decreased Sirt3 gene expression. PGC-1alpha activated the mouse SIRT3 promoter, which was mediated by an estrogen-related receptor (ERR binding element (ERRE (-407/-399 mapped to the promoter region. Chromatin immunoprecipitation and electrophoretic mobility shift assays confirmed that ERRalpha bound to the identified ERRE and PGC-1alpha co-localized with ERRalpha in the mSirt3 promoter. Knockdown of ERRalpha reduced the induction of Sirt3 by PGC-1alpha in C(2C(12 myotubes. Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2C(12 myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2C(12 myotubes.Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy

  20. Neonatal Satellite Cells Form Small Myotubes in Vitro

    NARCIS (Netherlands)

    Carvajal Monroy, P.L.; Grefte, S.; Kuijpers-Jagtman, A.M.; Den Hoff, Von J.W.; Wagener, F.A.D.T.G.

    2017-01-01

    Although palatal muscle reconstruction in patients with cleft palate takes place during early childhood, normal speech development is often not achieved. We hypothesized that the intrinsic properties of head satellite cells (SCs) and the young age of these patients contribute to the poor muscle

  1. Nonionizing radiation as a noninvasive strategy in regenerative medicine: the effect of Ca(2+)-ICR on mouse skeletal muscle cell growth and differentiation.

    Science.gov (United States)

    De Carlo, Flavia; Ledda, Mario; Pozzi, Deleana; Pierimarchi, Pasquale; Zonfrillo, Manuela; Giuliani, Livio; D'Emilia, Enrico; Foletti, Alberto; Scorretti, Riccardo; Grimaldi, Settimio; Lisi, Antonella

    2012-11-01

    Controlling cell differentiation and proliferation with minimal manipulation is one of the most important goals for cell therapy in clinical applications. In this work, we evaluated the hypothesis that the exposure of myoblast cells (C2C12) to nonionizing radiation (tuned at an extremely low-frequency electromagnetic field at calcium-ion cyclotron frequency of 13.75 Hz) may drive their differentiation toward a myogenic phenotype. C2C12 cells exposed to calcium-ion cyclotron resonance (Ca(2+)-ICR) showed a decrease in cellular growth and an increase in the G(0)/G(1) phase. Severe modifications in the shape and morphology and a change in the actin distribution were revealed by the phalloidin fluorescence analysis. A significant upregulation at transcriptional and translational levels of muscle differentiation markers such as myogenin (MYOG), muscle creatine kinase (MCK), and alpha skeletal muscle actin (ASMA) was observed in exposed C2C12 cells. Moreover, the pretreatment with nifedipine (an L-type voltage-gated Ca(2+) channel blocker) led to a reduction of the Ca(2+)-ICR effect. Consequently, it induced a downregulation of the MYOG, MCK, and ASMA mRNA expression affecting adversely the differentiation process. Therefore, our data suggest that Ca(2+)-ICR exposure can upregulate C2C12 differentiation. Although further studies are needed, these results may have important implications in myodegenerative pathology therapies.

  2. The parafibromin tumor suppressor protein interacts with actin-binding proteins actinin-2 and actinin-3

    Directory of Open Access Journals (Sweden)

    Marx Stephen J

    2008-08-01

    Full Text Available Abstract Background Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait. Results Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3, but not with non-muscle alpha-actinins (actinin-1 and actinin-4. The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells, but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment. Conclusion These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin participate in sequestering parafibromin in the cytoplasmic compartment.

  3. Regulation of myoblast differentiation by metabolic perturbations induced by metformin.

    Directory of Open Access Journals (Sweden)

    Theodora Pavlidou

    Full Text Available The metabolic perturbation caused by calorie restriction enhances muscle repair by playing a critical role in regulating satellite cell availability and activity in the muscles of young and old mice. To clarify the underlying mechanisms we asked whether myoblast replication and differentiation are affected by metformin, a calorie restriction-mimicking drug. C2C12, a mouse myoblast cell line, readily differentiate in vitro and fuse to form myotubes. However, when incubated with metformin, C2C12 slow their replication and do not differentiate. Interestingly, lower doses of metformin promote myogenic differentiation. We observe that metformin treatment modulates the expression of cyclins and cyclin inhibitors thereby inducing a cell cycle perturbation that causes a delay in the G2/M transition. The effect of metformin treatment is reversible since after drug withdrawal, myoblasts can re-enter the cell cycle and/or differentiate, depending on culture conditions. Myoblasts cultured under metformin treatment fail to up-regulate MyoD and p21cip1, a key step in cell cycle exit and terminal differentiation. Although the details of the molecular mechanisms underlying the effect of the drug on myoblasts still need to be clarified, we propose that metformin negatively affects myogenic differentiation by inhibiting irreversible exit from the cell cycle through reduction of MyoD and p21cip1 levels.

  4. Liquid Crystalline Networks toward Regenerative Medicine and Tissue Repair.

    Science.gov (United States)

    Martella, Daniele; Paoli, Paolo; Pioner, Josè M; Sacconi, Leonardo; Coppini, Raffaele; Santini, Lorenzo; Lulli, Matteo; Cerbai, Elisabetta; Wiersma, Diederik S; Poggesi, Corrado; Ferrantini, Cecilia; Parmeggiani, Camilla

    2017-12-01

    The communication reports the use of liquid crystalline networks (LCNs) for engineering tissue cultures with human cells. Their ability as cell scaffolds for different cell lines is demonstrated. Preliminary assessments of the material biocompatibility are performed on human dermal fibroblasts and murine muscle cells (C2C12), demonstrating that coatings or other treatments are not needed to use the acrylate-based materials as support. Moreover, it is found that adherent C2C12 cells undergo differentiation, forming multinucleated myotubes, which show the typical elongated shape, and contain bundles of stress fibers. Once biocompatibility is demonstrated, the same LCN films are used as a substrate for culturing human induced pluripotent stem cell-derived cardiomyocites (hiPSC-CMs) proving that LCNs are capable to develop adult-like dimensions and a more mature cell function in a short period of culture in respect to standard supports. The demonstrated biocompatibility together with the extraordinary features of LCNs opens to preparation of complex cell scaffolds, both patterned and stimulated, for dynamic cell culturing. The ability of these materials to improve cell maturation and differentiation will be developed toward engineered heart and skeletal muscular tissues exploring regenerative medicine toward bioartificial muscles for injured sites replacement. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Control of Cell Alignment and Morphology by Redesigning ECM-Mimetic Nanotopography on Multilayer Membranes.

    Science.gov (United States)

    Sousa, Maria P; Caridade, Sofia G; Mano, João F

    2017-08-01

    Inspired by native extracellular matrix (ECM) together with the multilevel architecture observed in nature, a material which topography recapitulates topographic features of the ECM and the internal architecture mimics the biological materials organization is engineered. The nanopatterned design along the XY plane is combined with a nanostructured organization along the Z axis on freestanding membranes prepared by layer-by-layer deposition of chitosan and chondroitin sulfate. Cellular behavior is monitored using two different mammalian cell lines, fibroblasts (L929) and myoblasts (C2C12), in order to perceive the response to topography. Viability, proliferation, and morphology of L929 are sensitively controlled by topography; also differentiation of C2C12 into myotubes is influenced by the presence of nanogrooves. This kind of nanopatterned structure has also been associated with strong cellular alignment. To the best of the knowledge, it is the first time that such a straightforward and inexpensive strategy is proposed to produce nanopatterned freestanding multilayer membranes. Controlling cellular alignment plays a critical role in many human tissues, such as muscles, nerves, or blood vessels, so these membranes can be potentially useful in specific tissue regeneration strategies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Comparisons of ELISA and Western blot assays for detection of autophagy flux.

    Science.gov (United States)

    Oh, Sung-Hee; Choi, Yong-Bok; Kim, June-Hyun; Weihl, Conrad C; Ju, Jeong-Sun

    2017-08-01

    We analyzed autophagy/mitophagy flux in vitro (C2C12 myotubes) and in vivo (mouse skeletal muscle) following the treatments of autophagy inducers (starvation, rapamycin) and a mitophagy inducer (carbonyl cyanide m-chlorophenylhydrazone, CCCP) using two immunodetection methods, ELISA and Western blotting, and compared their working range, accuracy, and reliability. The ELISAs showed a broader working range than that of the LC3 Western blots (Table 1). Table 2 showed that data value distribution was tighter and the average standard error from the ELISA was much smaller than those of the Western blot, directly relating to the accuracy of the assay. Test-retest reliability analysis showed good reliability for three individual ELISAs (interclass correlation, ≥ 0.7), but poor reliability for three individual Western blots (interclass correlation, ≤ 0.4) (Table 3).

  7. Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

    Science.gov (United States)

    Whitson, Peggy A.; Stuart, Charles A.; Huls, M. H.; Sams, Clarence F.; Cintron, Nitza M.

    1989-01-01

    The effect of dexamethasone on the activity of creatine kinase (CK) and the insulin-like growth factor I (IGF-I) binding were investigated using skeletal- and cardiac-muscle-derived cultured cell lines (mouse, C2C12; rat, L6 and H9c2). It was found that, in skeletal muscle cells, dexamethasone treatment during differentiation of skeletal-muscle cells caused dose-dependent increases in CK activity and increases in the degree of myotube formation, whereas cardiac cells (H9c2) exhibited very low CK activity during culture or dexamethasone treatment. Results for IGF-I binding were similar in all three cell lines. The IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr on the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities.

  8. Sub-cellular localisation of fukutin related protein in different cell lines and in the muscle of patients with MDC1C and LGMD2I

    DEFF Research Database (Denmark)

    Torelli, Silvia; Brown, Susan C; Brockington, Martin

    2005-01-01

    MDC1C and LGMD2I are two allelic forms of muscular dystrophies caused by mutations in the gene encoding for fukutin related protein (FKRP). FKRP encodes for a putative glycosyltransferase, the precise function of which is unknown. However, the marked reduction of alpha-dystroglycan glycosylation...... in the muscle of MDC1C and LGMD2I patients suggests a role for FKRP in dystroglycan processing. Using a polyclonal antibody raised against FKRP we now show that endogenous FKRP locates to the Golgi apparatus of neuronal, oligodendroglial, and the cardiac muscle cell line H9c2. In differentiated C2C12 myotubes...... and in transverse sections of normal skeletal and cardiac muscle, endogenous FKRP surrounded the myonuclei. This localisation was unaffected in the skeletal muscle of patients with MDC1C and LGMD2I carrying various FKRP mutations. These observations imply a specific role for FKRP during striated muscle, neuronal...

  9. The transcription coactivator ASC-1 is a regulator of skeletal myogenesis, and its deficiency causes a novel form of congenital muscle disease.

    Science.gov (United States)

    Davignon, Laurianne; Chauveau, Claire; Julien, Cédric; Dill, Corinne; Duband-Goulet, Isabelle; Cabet, Eva; Buendia, Brigitte; Lilienbaum, Alain; Rendu, John; Minot, Marie Christine; Guichet, Agnès; Allamand, Valérie; Vadrot, Nathalie; Fauré, Julien; Odent, Sylvie; Lazaro, Leïla; Leroy, Jean Paul; Marcorelles, Pascale; Dubourg, Odile; Ferreiro, Ana

    2016-04-15

    Despite recent progress in the genetic characterization of congenital muscle diseases, the genes responsible for a significant proportion of cases remain unknown. We analysed two branches of a large consanguineous family in which four patients presented with a severe new phenotype, clinically marked by neonatal-onset muscle weakness predominantly involving axial muscles, life-threatening respiratory failure, skin abnormalities and joint hyperlaxity without contractures. Muscle biopsies showed the unreported association of multi-minicores, caps and dystrophic lesions. Genome-wide linkage analysis followed by gene and exome sequencing in patients identified a homozygous nonsense mutation in TRIP4 encoding Activating Signal Cointegrator-1 (ASC-1), a poorly characterized transcription coactivator never associated with muscle or with human inherited disease. This mutation resulted in TRIP4 mRNA decay to around 10% of control levels and absence of detectable protein in patient cells. ASC-1 levels were higher in axial than in limb muscles in mouse, and increased during differentiation in C2C12 myogenic cells. Depletion of ASC-1 in cultured muscle cells from a patient and in Trip4 knocked-down C2C12 led to a significant reduction in myotube diameter ex vivo and in vitro, without changes in fusion index or markers of initial myogenic differentiation. This work reports the first TRIP4 mutation and defines a novel form of congenital muscle disease, expanding their histological, clinical and molecular spectrum. We establish the importance of ASC-1 in human skeletal muscle, identify transcriptional co-regulation as novel pathophysiological pathway, define ASC-1 as a regulator of late myogenic differentiation and suggest defects in myotube growth as a novel myopathic mechanism. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. YB-1 gene expression is kept constant during myocyte differentiation through replacement of different transcription factors and then falls gradually under the control of neural activity.

    Science.gov (United States)

    Kobayashi, Shunsuke; Tanaka, Toru; Moue, Masamitsu; Ohashi, Sachiyo; Nishikawa, Taishi

    2015-11-01

    We have previously reported that translation of acetylcholine receptor α-subunit (AChR α) mRNA in skeletal muscle cells is regulated by Y-box binding protein 1 (YB-1) in response to neural activity, and that in the postnatal mouse developmental changes in the amount of YB-1 mRNA are similar to those of AChR α mRNA, which is known to be regulated by myogenic transcription factors. Here, we examined transcriptional regulation of the YB-1 gene in mouse skeletal muscle and differentiating C2C12 myocytes. Although neither YB-1 nor AChR α was detected at either the mRNA or protein level in adult hind limb muscle, YB-1 expression was transiently activated in response to denervation of the sciatic nerve and completely paralleled that of AChR α, suggesting that these genes are regulated by the same transcription factors. However, during differentiation of C2C12 cells to myotubes, the level of YB-1 remained constant even though the level of AChR α increased markedly. Reporter gene, gel mobility shift and ChIP assays revealed that in the initial stage of myocyte differentiation, transcription of the YB-1 gene was regulated by E2F1 and Sp1, and was then gradually replaced under the control of both MyoD and myogenin through an E-box sequence in the proximal region of the YB-1 gene promoter. These results suggest that transcription factors for the YB-1 gene are exchanged during skeletal muscle cell differentiation, perhaps playing a role in translational control of mRNAs by YB-1 in both myotube formation and the response of skeletal muscle tissues to neural stimulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Muscle-specific AMPK β1β2-null mice display a myopathy due to loss of capillary density in nonpostural muscles

    Science.gov (United States)

    Thomas, Melissa M.; Wang, David C.; D'Souza, Donna M.; Krause, Matthew P.; Layne, Andrew S.; Criswell, David S.; O'Neill, Hayley M.; Connor, Michael K.; Anderson, Judy E.; Kemp, Bruce E.; Steinberg, Gregory R.; Hawke, Thomas J.

    2014-01-01

    AMP-activated protein kinase (AMPK) is a master regulator of metabolism. While muscle-specific AMPK β1β2 double-knockout (β1β2M-KO) mice display alterations in metabolic and mitochondrial capacity, their severe exercise intolerance suggested a secondary contributor to the observed phenotype. We find that tibialis anterior (TA), but not soleus, muscles of sedentary β1β2M-KO mice display a significant myopathy (decreased myofiber areas, increased split and necrotic myofibers, and increased centrally nucleated myofibers. A mitochondrial- and fiber-type-specific etiology to the myopathy was ruled out. However, β1β2M-KO TA muscles displayed significant (Pmuscle resulted from impaired AMPK-nNOSμ signaling, causing increased platelet aggregation, impaired vasodilation, and, ultimately, ischemic injury. Consistent with this hypothesis, AMPK-specific phosphorylation (Ser1446) of nNOSμ was decreased in β1β2M-KO compared to wild-type (WT) mice. The AMPK-nNOSμ relationship was further demonstrated by administration of 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) to β1β2-MKO muscles and C2C12 myotubes. AICAR significantly increased nNOSμ phosphorylation and nitric oxide production (Pmuscles and C2C12 myotubes but not in β1β2M-KO muscles. These findings highlight the importance of the AMPK-nNOSμ pathway in resting skeletal muscle.—Thomas, M. M., Wang, D. C., D'Souza, D. M., Krause, M. P., Layne, A. S., Criswell, D. S., O'Neill, H. M., Connor, M. K., Anderson, J. E., Kemp, B. E., Steinberg, G. R., and Hawke, T. J. Muscle-specific AMPK β1β2-null mice display a myopathy due to loss of capillary density in nonpostural muscles. PMID:24522207

  12. Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism.

    Science.gov (United States)

    Uruno, Akira; Yagishita, Yoko; Katsuoka, Fumiki; Kitajima, Yasuo; Nunomiya, Aki; Nagatomi, Ryoichi; Pi, Jingbo; Biswal, Shyam S; Yamamoto, Masayuki

    2016-06-01

    Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Endothelial protective genes induced by statin are mimicked by ERK5 activation as triggered by a drug combination of FTI-277 and GGTI-298.

    Science.gov (United States)

    Chu, Uyen B; Duellman, Tyler; Weaver, Sara J; Tao, Yunting; Yang, Jay

    2015-07-01

    Statins are potent inhibitors of cholesterol biosynthesis and are clinically beneficial in preventing cardiovascular diseases, however, the therapeutic utility of these drugs is limited by myotoxicity. Here, we explored the mechanism of statin-mediated activation of ERK5 in the human endothelium with the goal of identifying compounds that confer endothelial protection but are nontoxic to muscle. An ERK5-one hybrid luciferase reporter transfected into COS-7 cells with pharmacological and molecular manipulations dissected the signaling pathway leading to statin activation of ERK5. qRT-PCR of HUVEC cells documented the transcriptional activation of endothelial-protective genes. Lastly, morphological and cellular ATP analysis, and induction of atrogin-1 in C2C12 myotubes were used to assess statin-induced myopathy. Statin activation of ERK5 is dependent on the cellular reduction of GGPPs. Furthermore, we found that the combination of FTI-277 (inhibitor of farnesyl transferase) and GGTI-298 (inhibitor of geranylgeranyl transferase I) mimicked the statin-mediated activation of ERK5. FTI-277 and GGTI-298 together recapitulated the beneficial effects of statins by transcriptionally upregulating anti-inflammatory mediators such as eNOS, THBD, and KLF2. Finally, C2C12 skeletal myotubes treated with both FTI-277 and GGTI-298 evoked less morphological and cellular changes recognized as biomarkers of statin-associated myopathy. Statin-induced endothelial protection and myopathy are mediated by distinct metabolic intermediates and co-inhibition of farnesyl transferase and geranylgeranyl transferase I confer endothelial protection without myopathy. The combinatorial FTI-277 and GGTI-298 drug regimen provides a promising alternative avenue for endothelial protection without myopathy. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Endothelial protective genes induced by statin is mimicked by FTI-277 and GGTI-298 drug combination-mediated ERK5 activation

    Science.gov (United States)

    Chu, Uyen B.; Duellman, Tyler; Weaver, Sara J.; Tao, Yunting; Yang, Jay

    2015-01-01

    Background Statins are potent inhibitors of cholesterol biosynthesis and are clinically beneficial in preventing cardiovascular diseases, however, the therapeutic utility of these drugs is limited by myotoxicity. Here, we explored the mechanism of statin-mediated activation of ERK5 in the human endothelium with the goal of identifying compounds that confer endothelial protection but are nontoxic to muscle. Methods An ERK5-one hybrid luciferase reporter transfected into COS-7 cells with pharmacological and molecular manipulations dissected the signaling pathway leading to statin activation of ERK5. qRT-PCR of HUVEC cells documented the transcriptional activation of endothelial-protective genes. Lastly, morphological and cellular ATP analysis, and induction of atrogin-1 in C2C12 myotubes were used to assess statin-induced myopathy. Results Statin activation of ERK5 is dependent on the cellular reduction of GGPPs. Furthermore, we found that the combination of FTI-277 (inhibitor of farnesyl transferase) and GGTI-298 (inhibitor of geranylgeranyl transferase I) mimicked the statin-mediated activation of ERK5. FTI-277 and GGTI-298 together recapitulated the beneficial effects of statins by transcriptionally upregulating anti-inflammatory mediators such as eNOS, THBD, and KLF2. Finally, C2C12 skeletal myotubes treated with both FTI-277 and GGTI-298 evoked less morphological and cellular changes recognized as biomarkers of statin-associated myopathy. Conclusions Statin-induced endothelial protection and myopathy are mediated by distinct metabolic intermediates and co-inhibition of farnesyl transferase and geranylgeranyl transferase I confer endothelial protection without myopathy. General Significance The combinatorial FTI-277 and GGTI-298 drug regimen provides a promising alternative avenue for endothelial protection without myopathy. PMID:25829196

  15. Distinct effects of oleic acid and its trans-isomer elaidic acid on the expression of myokines and adipokines in cell models.

    Science.gov (United States)

    Granados, Nuria; Amengual, Jaume; Ribot, Joan; Palou, Andreu; Bonet, M Luisa

    2011-04-01

    Trans-fatty acids (TFA) and cis-monounsaturated fat appear to exert detrimental and beneficial effects, respectively, on glucose metabolism and insulin sensitivity. Adipose tissue and skeletal muscle are a source of signalling proteins (adipokines and myokines), some of which have been related to the control of insulin sensitivity. Here, we investigated the possible differential effects of elaidic acid (EA; trans-9-18 : 1) - the major component in industrially produced TFA - and oleic acid (OA; cis-9-18 : 1) - its cis-isomer naturally present in food - on cellular glucose uptake and the expression of selected myokines and adipokines using cell models. Differentiated C2C12 myotubes and 3T3-L1 adipocytes were pretreated with the vehicle (control cells) or fatty acids for 24 h, after which basal and insulin-stimulated 2-deoxyglucose uptake and the expression of selected signalling proteins were measured. In C2C12 myotubes, pretreatment with OA, but not with EA, led to increased insulin-stimulated 2-deoxyglucose uptake and IL-6 expression levels, while pretreatment with EA, but not with OA, led to reduced IL-15 mRNA levels and increased TNF-α expression levels. In 3T3-L1 adipocytes, exposure to OA, but not to EA, resulted in reduced resistin gene expression and increased adiponectin gene expression. The results show evidence of distinct, direct effects of OA and EA on muscle glucose uptake and the expression of target myokines and adipokines, thus suggesting novel mechanisms by which cis- and trans-monounsaturated fat may differentially affect systemic functions.

  16. L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism.

    Directory of Open Access Journals (Sweden)

    Daniel J Ham

    Full Text Available Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF or growth factors and nutrients (HEPES buffered saline; HBS. Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control or L-alanine (negative control and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37% and myotube diameter (HBS: +18%, SF: +29%. L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%. The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS and oxidative stress (H2O2 induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.

  17. Sosiho‑tang ameliorates cachexia‑related symptoms in mice bearing colon 26 adenocarcinoma by reducing systemic inflammation and muscle loss.

    Science.gov (United States)

    Kim, Aeyung; Im, Minju; Ma, Jin Yeul

    2016-03-01

    affecting myoblast proliferation and differentiation, and C2C12 myotube wasting. Taken together, these results suggest that SO is a safe and useful anti‑cachectic therapy for cancer patients with severe weight loss.

  18. Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration

    Science.gov (United States)

    Lee, Bora; Lee, Seung-Min; Bahn, Young Jae; Lee, Kwang-Pyo; Kang, Moonkyung; Kim, Yeon-Soo; Woo, Sun-Hee; Lim, Jae-Young; Kim, Eunhee; Kwon, Ki-Sun

    2016-01-01

    Skeletal muscle mass and power decrease with age, leading to impairment of mobility and metabolism in the elderly. Ca2+ signaling is crucial for myoblast differentiation as well as muscle contraction through activation of transcription factors and Ca2+-dependent kinases and phosphatases. Ca2+ channels, such as dihydropyridine receptor (DHPR), two-pore channel (TPC) and inositol 1,4,5-triphosphate receptor (ITPR), function to maintain Ca2+ homeostasis in myoblasts. Here, we observed a significant decrease in expression of type 1 IP3 receptor (ITPR1), but not types 2 and 3, in aged mice skeletal muscle and isolated myoblasts, compared with those of young mice. ITPR1 knockdown using shRNA-expressing viruses in C2C12 myoblasts and tibialis anterior muscle of mice inhibited myotube formation and muscle regeneration after injury, respectively, a typical phenotype of aged muscle. This aging phenotype was associated with repression of muscle-specific genes and activation of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. ERK inhibition by U0126 not only induced recovery of myotube formation in old myoblasts but also facilitated muscle regeneration after injury in aged muscle. The conserved decline in ITPR1 expression in aged human skeletal muscle suggests utility as a potential therapeutic target for sarcopenia, which can be treated using ERK inhibition strategies. PMID:27658230

  19. Age-associated repression of type 1 inositol 1, 4, 5-triphosphate receptor impairs muscle regeneration.

    Science.gov (United States)

    Choi, Jeong Yi; Hwang, Chae Young; Lee, Bora; Lee, Seung-Min; Bahn, Young Jae; Lee, Kwang-Pyo; Kang, Moonkyung; Kim, Yeon-Soo; Woo, Sun-Hee; Lim, Jae-Young; Kim, Eunhee; Kwon, Ki-Sun

    2016-09-21

    Skeletal muscle mass and power decrease with age, leading to impairment of mobility and metabolism in the elderly. Ca(2+) signaling is crucial for myoblast differentiation as well as muscle contraction through activation of transcription factors and Ca(2+)-dependent kinases and phosphatases. Ca(2+) channels, such as dihydropyridine receptor (DHPR), two-pore channel (TPC) and inositol 1,4,5-triphosphate receptor (ITPR), function to maintain Ca(2+) homeostasis in myoblasts. Here, we observed a significant decrease in expression of type 1 IP3 receptor (ITPR1), but not types 2 and 3, in aged mice skeletal muscle and isolated myoblasts, compared with those of young mice. ITPR1 knockdown using shRNA-expressing viruses in C2C12 myoblasts and tibialis anterior muscle of mice inhibited myotube formation and muscle regeneration after injury, respectively, a typical phenotype of aged muscle. This aging phenotype was associated with repression of muscle-specific genes and activation of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. ERK inhibition by U0126 not only induced recovery of myotube formation in old myoblasts but also facilitated muscle regeneration after injury in aged muscle. The conserved decline in ITPR1 expression in aged human skeletal muscle suggests utility as a potential therapeutic target for sarcopenia, which can be treated using ERK inhibition strategies.

  20. Alix Protein Is Substrate of Ozz-E3 Ligase and Modulates Actin Remodeling in Skeletal Muscle*

    Science.gov (United States)

    Bongiovanni, Antonella; Romancino, Daniele P.; Campos, Yvan; Paterniti, Gaetano; Qiu, Xiaohui; Moshiach, Simon; Di Felice, Valentina; Vergani, Naja; Ustek, Duran; d'Azzo, Alessandra

    2012-01-01

    Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz−/− myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling. PMID:22334701

  1. Alix protein is substrate of Ozz-E3 ligase and modulates actin remodeling in skeletal muscle.

    Science.gov (United States)

    Bongiovanni, Antonella; Romancino, Daniele P; Campos, Yvan; Paterniti, Gaetano; Qiu, Xiaohui; Moshiach, Simon; Di Felice, Valentina; Vergani, Naja; Ustek, Duran; d'Azzo, Alessandra

    2012-04-06

    Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz(-/-) myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling.

  2. Effect of zinc-alpha2-glycoprotein (ZAG) on expression of uncoupling proteins in skeletal muscle and adipose tissue.

    Science.gov (United States)

    Sanders, Paul M; Tisdale, Michael J

    2004-08-20

    The plasma protein zinc-alpha2-glycoprotein (ZAG) has been shown to be identical with a lipid mobilizing factor capable of inducing loss of adipose tissue in cancer cachexia through an increased lipid mobilization and utilization. The ability of ZAG to induce uncoupling protein (UCP) expression has been determined using in vitro models of adipose tissue and skeletal muscle. ZAG induced a concentration-dependent increase in the expression of UCP-1 in primary cultures of brown, but not white, adipose tissue, and this effect was attenuated by the beta3-adrenergic receptor (beta3-AR) antagonist SR59230A. A 6.5-fold increase in UCP-1 expression was found in brown adipose tissue after incubation with 0.58 microM ZAG. ZAG also increased UCP-2 expression 3.5-fold in C2C12 murine myotubes, and this effect was also attenuated by SR59230A and potentiated by isobutylmethylxanthine, suggesting a cyclic AMP-mediated process through interaction with a beta3-AR. ZAG also produced a dose-dependent increase in UCP-3 in murine myotubes with a 2.5-fold increase at 0.58 microM ZAG. This effect was not mediated through the beta3-AR, but instead appeared to require mitogen activated protein kinase. These results confirm the ability of ZAG to directly influence UCP expression, which may play an important role in lipid utilization during cancer cachexia.

  3. Sodium nitrate decreases agrin-induced acetylcholine receptor clustering.

    Science.gov (United States)

    Jarosz, Jess; White, Cullen; Grow, Wade A

    2016-05-01

    Humans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined. C2C12 skeletal muscle cell cultures were maintained, myoblasts were fused into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance acetylcholine receptor (AChR) clustering. Untreated cultures were compared with cultures exposed to sodium nitrate at concentrations ranging from 10 ng/mL-100 μg/mL. The results reported here demonstrate that 1 μg/mL sodium nitrate was sufficient to decrease the frequency of agrin-induced AChR clustering without affecting myotube formation. In addition, concentrations of sodium nitrate of 1 μg/mL or 100 μg/mL decreased gene expression of the myogenic transcription factor myogenin and AChR in correlation with the agrin-induced AChR clustering data. These results reveal that sodium nitrate decreases the frequency of agrin-induced AChR clustering by a mechanism that includes myogenin and AChR gene expression. As a consequence sodium nitrate may pose a risk for skeletal muscle development and subsequent neuromuscular synapse formation in humans.

  4. Hyperammonaemia‐induced skeletal muscle mitochondrial dysfunction results in cataplerosis and oxidative stress

    Science.gov (United States)

    Davuluri, Gangarao; Allawy, Allawy; Thapaliya, Samjhana; Rennison, Julie H.; Singh, Dharmvir; Kumar, Avinash; Sandlers, Yana; Van Wagoner, David R.; Flask, Chris A.; Hoppel, Charles; Kasumov, Takhar

    2016-01-01

    Key points Hyperammonaemia occurs in hepatic, cardiac and pulmonary diseases with increased muscle concentration of ammonia.We found that ammonia results in reduced skeletal muscle mitochondrial respiration, electron transport chain complex I dysfunction, as well as lower NAD+/NADH ratio and ATP content.During hyperammonaemia, leak of electrons from complex III results in oxidative modification of proteins and lipids.Tricarboxylic acid cycle intermediates are decreased during hyperammonaemia, and providing a cell‐permeable ester of αKG reversed the lower TCA cycle intermediate concentrations and increased ATP content.Our observations have high clinical relevance given the potential for novel approaches to reverse skeletal muscle ammonia toxicity by targeting the TCA cycle intermediates and mitochondrial ROS. Abstract Ammonia is a cytotoxic metabolite that is removed primarily by hepatic ureagenesis in humans. Hyperammonaemia occurs in advanced hepatic, cardiac and pulmonary disease, and in urea cycle enzyme deficiencies. Increased skeletal muscle ammonia uptake and metabolism are the major mechanism of non‐hepatic ammonia disposal. Non‐hepatic ammonia disposal occurs in the mitochondria via glutamate synthesis from α‐ketoglutarate resulting in cataplerosis. We show skeletal muscle mitochondrial dysfunction during hyperammonaemia in a comprehensive array of human, rodent and cellular models. ATP synthesis, oxygen consumption, generation of reactive oxygen species with oxidative stress, and tricarboxylic acid (TCA) cycle intermediates were quantified. ATP content was lower in the skeletal muscle from cirrhotic patients, hyperammonaemic portacaval anastomosis rat, and C2C12 myotubes compared to appropriate controls. Hyperammonaemia in C2C12 myotubes resulted in impaired intact cell respiration, reduced complex I/NADH oxidase activity and electron leak occurring at complex III of the electron transport chain. Consistently, lower NAD+/NADH ratio was observed

  5. STAT3 Activation in Skeletal Muscle Links Muscle Wasting and the Acute Phase Response in Cancer Cachexia

    Science.gov (United States)

    Kunzevitzky, Noelia; Guttridge, Denis C.; Khuri, Sawsan; Koniaris, Leonidas G.; Zimmers, Teresa A.

    2011-01-01

    Background Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia. Methodology/Principal Findings Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer. Conclusions/Significance These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such

  6. IgG-specific cell-based assay detects potentially pathogenic MuSK-Abs in seronegative MG.

    Science.gov (United States)

    Huda, Saif; Waters, Patrick; Woodhall, Mark; Leite, Maria Isabel; Jacobson, Leslie; De Rosa, Anna; Maestri, Michelangelo; Ricciardi, Roberta; Heckmann, Jeannine M; Maniaol, Angelina; Evoli, Amelia; Cossins, Judy; Hilton-Jones, David; Vincent, Angela

    2017-07-01

    To increase the detection of MuSK-Abs using a CBA and test their pathogenicity. Sera from 69 MuSK-RIA-positive patients with myasthenia gravis (MG) (Definite MuSK-MG), 169 patients negative for MuSK-RIA and AChR-RIA (seronegative MG, SNMG), 35 healthy individuals (healthy controls, HCs), and 16 NMDA receptor-Ab-positive (NMDAR-Ab) disease controls were tested for binding to MuSK on a CBA using different secondary antibodies. Initially, in addition to 18% of SNMG sera, 11% of HC and 19% of NMDAR-Ab sera showed positive binding to MuSK-transfected cells; this low specificity was due to anti-IgG(H+L) detection of IgM bound nonspecifically to MuSK. Using an IgG Fc gamma-specific secondary antibody, MuSK-Abs were detected by CBA in 68/69 (99%) of Definite MuSK-MG, 0/35 HCs, 0/16 NMDAR-Ab, and 14/169 (8%) of SNMG sera, providing increased sensitivity with high specificity. The RIA-negative, CBA-positive MuSK-IgG sera, but not IgM-MuSK-binding sera, reduced agrin-induced AChR clustering in C2C12 myotubes, qualitatively similar to RIA-positive MuSK-Abs. An IgG-specific MuSK-CBA can reliably detect IgG MuSK-Abs and increase sensitivity. In the MuSK-CBA, IgG specificity is essential. The positive sera demonstrated pathogenic potential in the in vitro AChR-clustering assay, although less effective than Definite MuSK-MG sera, and the patients had less severe clinical disease. Use of IgG-specific secondary antibodies may improve the results of other antibody tests. This study provides Class III evidence that an IgG-specific MuSK-CBA identifies patients with MG.

  7. Enhanced release of bone morphogenetic proteins from demineralized bone matrix by gamma irradiation

    Science.gov (United States)

    Sung, Nak-Yun; Choi, Jong-il

    2015-06-01

    Gamma irradiation is a useful method for sterilizing demineralized bone matrix (DBM), but its effect on the osteoinductivity of DBM is still controversial. In this study, the osteoinductive activity of gamma-irradiated DBM was examined using a mouse myoblastic cell line (C2C12). DBM was extracted from adult bovine bone and was irradiated at a dose of 25 kGy using a 60cobalt gamma-irradiator. Cell proliferation with DBM was not affected by gamma-irradiation, but alkaline phosphatase and osteocalcin productions were significantly increased in C2C12 cell groups treated with gamma-irradiated DBM. It was reasoned that bone morphogenetic proteins were more efficiently released from gamma-irradiated DBM than from the non-irradiated control. This result suggests the effectiveness of radiation sterilization of bone implants

  8. Inhibition of in vitro myogenic differentiation by cellular transcription factor E2F1

    DEFF Research Database (Denmark)

    Wang, J; Helin, K; Jin, P

    1995-01-01

    Terminal differentiation of cultured myocytes requires withdrawal of the cells from the cell cycle. Constitutive overexpression of several oncogenes in myoblasts can inhibit in vitro myogenesis. Here we studied the role of the cellular transcription factor E2F1 on myogenic differentiation. E2F1...... expression is irreversibly down-regulated during differentiation of C2C12 myocytes. Furthermore, deregulated E2F1 expression in C2C12 cells prevented myogenic differentiation. This inhibition of myogenesis was associated with the repression of myogenin expression and an elevated cyclin D1 expression....... Moreover, E2F1-overexpressing myocytes failed to exit the cell cycle under differentiation conditions. These results are consistent with the notion that E2F1 can function as an oncogene and further suggest that E2F1 down-regulation is required for myogenic differentiation....

  9. Assessment of the antidiabetic potential of selected medicinal plants using in vitro bioassays of muscle glucose transport and liver glucose production

    DEFF Research Database (Denmark)

    Beidokhti, M N; Sanchez Villavicencio, M L; Eid, H M

    2016-01-01

    by skeletal muscle cells are the principal contributors to the associated hyperglycemic state. The aim of this study was to assess the antidiabetic potential of five medicinal plant extracts using in vitro cell based assays targeting glucose uptake in C2C12 skeletal muscle cells [1] and glucose-6-phosphatase...... activity (G6Pase) [2] in rat hepatoma H4IIE. Cells were treated for 18h with maximal non-toxic concentrations (50 µg/mL) of the ethanolic extract of Psidium guajava (leaf and bark), Phyllanthus niruri (aerial parts), Eugenia jambolana (dried fruit) and Rhizophora mucronata (bark), which were determined...... by the lactate dehydrogenase (LDH) cytotoxicity assay. None of the extracts were able to reduce G6Pase activity. In contrast, one plant extract (P. guajava leaf extract) was found to significantly increase deoxyglucose uptake in C2C12 muscle cells (161%, p ≤0.001), to levels higher that of the positive control...

  10. Oligonol, a Low-Molecular Weight Polyphenol Derived from Lychee, Alleviates Muscle Loss in Diabetes by Suppressing Atrogin-1 and MuRF1

    Directory of Open Access Journals (Sweden)

    Hung-Wen Liu

    2017-09-01

    Full Text Available Stimulation of the ubiquitin-proteasome pathway—especially E3 ubiquitin ligases Atrogin-1 and MuRF1—is associated with muscle loss in diabetes. Elevated lipid metabolites impair myogenesis. Oligonol, a low molecular weight polyphenol derived from lychee, exhibited anti-diabetic and anti-obesity properties, suggesting it could be a proper supplement for attenuating muscle loss. Dietary (10 weeks oligonol supplementation (20 or 200 mg/kg diet on the skeletal muscle loss was investigated in diabetic db/db mice. Transcription factors NF-κB and FoxO3a involved in regulation of Atrogin-1 and MuRF1 were also investigated. Attenuation of muscle loss by oligonol (both doses was associated with down-regulation of Atrogin-1 and MuRF1 gene expression. Oligonol supplementation decreased NF-κB expression in the nuclear fraction compared with db/db mice without oligonol supplement. Upregulation of sirtuin1 (SIRT1 expression prevented FoxO3a nuclear localization in db/db mice supplemented with oligonol. Marked increases in AMPKα activity and Ppara mRNA expression leading to lower lipid accumulation by oligonol provided additional benefits for attenuating muscle loss. Oligonol limited palmitate-induced senescent phenotype and cell cycle arrest and suppressed Atrogin-1 and MuRF1 mRNA expression in palmitate-treated C2C12 muscle cells, thus contributing to improving the impaired myotube formation. In conclusion, oligonol-mediated downregulation of Atrogin-1 and MuRF1 gene expression alleviates muscle loss and improves the impaired myotube formation, indicating that oligonol supplementation may be useful for the attenuation of myotube loss.

  11. Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

    Science.gov (United States)

    Pournourmohammadi, Shirin; Grimaldi, Mariagrazia; Stridh, Malin H; Lavallard, Vanessa; Waagepetersen, Helle S; Wollheim, Claes B; Maechler, Pierre

    2017-07-01

    Glucose homeostasis is determined by insulin secretion from the ß-cells in pancreatic islets and by glucose uptake in skeletal muscle and other insulin target tissues. While glutamate dehydrogenase (GDH) senses mitochondrial energy supply and regulates insulin secretion, its role in the muscle has not been elucidated. Here we investigated the possible interplay between GDH and the cytosolic energy sensing enzyme 5'-AMP kinase (AMPK), in both isolated islets and myotubes from mice and humans. The green tea polyphenol epigallocatechin-3-gallate (EGCG) was used to inhibit GDH. Insulin secretion was reduced by EGCG upon glucose stimulation and blocked in response to glutamine combined with the allosteric GDH activator BCH (2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid). Insulin secretion was similarly decreased in islets of mice with ß-cell-targeted deletion of GDH (ßGlud1 -/- ). EGCG did not further reduce insulin secretion in the mutant islets, validating its specificity. In human islets, EGCG attenuated both basal and nutrient-stimulated insulin secretion. Glutamine/BCH-induced lowering of AMPK phosphorylation did not operate in ßGlud1 -/- islets and was similarly prevented by EGCG in control islets, while high glucose systematically inactivated AMPK. In mouse C2C12 myotubes, like in islets, the inhibition of AMPK following GDH activation with glutamine/BCH was reversed by EGCG. Stimulation of GDH in primary human myotubes caused lowering of insulin-induced 2-deoxy-glucose uptake, partially counteracted by EGCG. Thus, mitochondrial energy provision through anaplerotic input via GDH influences the activity of the cytosolic energy sensor AMPK. EGCG may be useful in obesity by resensitizing insulin-resistant muscle while blunting hypersecretion of insulin in hypermetabolic states. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Astragalus Polysaccharide Suppresses Skeletal Muscle Myostatin Expression in Diabetes: Involvement of ROS-ERK and NF-κB Pathways

    Directory of Open Access Journals (Sweden)

    Min Liu

    2013-01-01

    Full Text Available Objective. The antidiabetes drug astragalus polysaccharide (APS is capable of increasing insulin sensitivity in skeletal muscle and improving whole-body glucose homeostasis. Recent studies suggest that skeletal muscle secreted growth factor myostatin plays an important role in regulating insulin signaling and insulin resistance. We hypothesized that regulation of skeletal muscle myostatin expression may be involved in the improvement of insulin sensitivity by APS. Methods. APS was administered to 13-week-old diabetic KKAy and nondiabetic C57BL/6J mice for 8 weeks. Complementary studies examined APS effects on the saturated acid palmitate-induced insulin resistance and myostatin expression in C2C12 cells. Results. APS treatment ameliorated hyperglycemia, hyperlipidemia, and insulin resistance and decreased the elevation of myostatin expression and malondialdehyde production in skeletal muscle of noninsulin-dependent diabetic KKAy mice. In C2C12 cells in vitro, saturated acid palmitate-induced impaired glucose uptake, overproduction of ROS, activation of extracellular regulated protein kinases (ERK, and NF-κB were partially restored by APS treatment. The protective effects of APS were mimicked by ERK and NF-κB inhibitors, respectively. Conclusion. Our study demonstrates elevated myostatin expression in skeletal muscle of type 2 diabetic KKAy mice and in cultured C2C12 cells exposed to palmitate. APS is capable of improving insulin sensitivity and decreasing myostatin expression in skeletal muscle through downregulating ROS-ERK-NF-κB pathway.

  13. Reactive oxygen species are important mediators of taurine release from skeletal muscle cells

    DEFF Research Database (Denmark)

    Ørtenblad, Niels; Feveile Young, Jette; Oksbjerg, Niels

    2003-01-01

    C2C12, calcium, cell volume regulation, 5-lipoxygenase, melittin, anoxia, secretory phospholipase A2......C2C12, calcium, cell volume regulation, 5-lipoxygenase, melittin, anoxia, secretory phospholipase A2...

  14. ErbB3 binding protein-1 (Ebp1) controls proliferation and myogenic differentiation of muscle stem cells.

    Science.gov (United States)

    Figeac, Nicolas; Serralbo, Olivier; Marcelle, Christophe; Zammit, Peter S

    2014-02-01

    Satellite cells are resident stem cells of skeletal muscle, supplying myoblasts for post-natal muscle growth, hypertrophy and repair. Many regulatory networks control satellite cell function, which includes EGF signalling via the ErbB family of receptors. Here we investigated the role of ErbB3 binding protein-1 (Ebp1) in regulation of myogenic stem cell proliferation and differentiation. Ebp1 is a well-conserved DNA/RNA binding protein that is implicated in cell growth, apoptosis and differentiation in many cell types. Of the two main Ebp1 isoforms, only p48 was expressed in satellite cells and C2C12 myoblasts. Although not present in quiescent satellite cells, p48 was strongly induced during activation, remaining at high levels during proliferation and differentiation. While retroviral-mediated over-expression of Ebp1 had only minor effects, siRNA-mediated Ebp1 knockdown inhibited both proliferation and differentiation of satellite cells and C2C12 myoblasts, with a clear failure of myotube formation. Ebp1-knockdown significantly reduced ErbB3 receptor levels, yet over-expression of ErbB3 in Ebp1 knockdown cells did not rescue differentiation. Ebp1 was also expressed by muscle cells during developmental myogenesis in mouse. Since Ebp1 is well-conserved between mouse and chick, we switched to chick to examine its role in muscle formation. In chick embryo, Ebp1 was expressed in the dermomyotome, and myogenic differentiation of muscle progenitors was inhibited by specific Ebp1 down-regulation using shRNA electroporation. These observations demonstrate a conserved function of Ebp1 in the regulation of embryonic muscle progenitors and adult muscle stem cells, which likely operates independently of ErbB3 signaling. © 2013 Published by Elsevier Inc.

  15. Emergent behavior of cells on microfabricated soft polymeric substrates

    Science.gov (United States)

    Anand, Sandeep Venkit

    ) with a single cell scale (micro scale) cross section. We plate C2C12 cells on the platform and characterize their migration, proliferation, and differentiation patterns in contrast to 2D culture. We find that the cells land on the 2D surface, and then migrate to the filament only when the 2D surface has become nearly confluent. Individual and isolated cells randomly approaching the filament always retract away towards the 2D surface. Once on the filament, their differentiation to myotubes is expedited compared to that on 2D substrate. The myotubes generate periodic twitching forces that deform the filament producing more than 17 um displacement at the tip. Such flagellar motion can be used to develop autonomous micro scale bio-bots. Finally, the design and fabrication of a polymeric micro-pillar based force sensor capable of measuring cellular focal-adhesion forces under externally applied stretch is discussed. The force sensor consists of arrays of uniformly spaced PDMS micro-pillars of 1-2 um diameter and 2-3 um spacing on a macroscale PDMS substrate. The tips of the micro-pillars are selectively patterned with fluorescently labeled ECM proteins using micro-contact printing to promote cell adhesion while simultaneously acting as markers for strain measurements. Cells adhere and spread on top of the pillars causing them to deform. When stretched, the cells reorganize their internal structure and modulate their traction forces in response to the applied stretch. The dynamically varying cellular forces in response to the stretch are computed by measuring the cell induced displacements estimated by isolating the displacements caused by the applied stretch from the net displacements of the tips.

  16. Melengestrol acetate alters muscle cell proliferation in heifers and steers.

    Science.gov (United States)

    Sissom, E K; Reinhardt, C D; Johnson, B J

    2006-11-01

    In vitro experiments were performed to investigate the effects of melengestrol acetate (MGA) or progesterone (P4) on bovine muscle satellite cells and C2C12 myoblasts. Addition of MGA at physiological and supraphysiological concentrations resulted in a dose-dependent decrease (P < 0.05) in DNA synthesis as measured by [3H]-thymidine incorporation (TI). Similarly, P4 addition (0.01 nM) reduced (P < 0.05) TI. Addition of MGA (10 nM) increased (P < 0.05) IGF-I mRNA abundance but did not affect myogenin mRNA. Progesterone addition (10 nM) increased myogenin mRNA abundance (P < 0.05). In C2C12 cultures, P4 addition resulted in a dose-dependent decrease in TI. The antiprogestin RU486, in combination with MGA or P4, also resulted in reduced (P < 0.05) TI. Treatment with RU486 alone had a negative effect (P < 0.05) on TI that was similar to the progestins. Treatment of C2C12 myoblasts with MGA (100 nM) resulted in an increase (P < 0.05) in myogenin mRNA. These studies suggest that progestins may reduce satellite cell proliferation, ultimately affecting carcass composition.

  17. miR-145a-5p Promotes Myoblast Differentiation

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    Jingjing Du

    2016-01-01

    Full Text Available MicroRNAs are a class of 18–22-nucleotide noncoding RNAs that posttranscriptionally regulate gene expression and have been shown to play an important role during myoblast differentiation. In this study, we found that the expression of miR-145a-5p was gradually increased during C2C12 myoblast differentiation, and miR-145a-5p inhibitors or mimics significantly suppressed or promoted the relative expression of specific myogenesis related marker genes. Moreover, overexpression or inhibition of miR-145a-5p enhanced or repressed the expression of some special genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicated that miR-145a-5p might be considered as a new myogenic differentiation-associated microRNA that can promote C2C12 myoblast differentiation by enhancing genes related to myoblasts differentiation.

  18. Characterization of the Methylation Status of and Myogenic Regulator Factors in Cell Myogenic Differentiation

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    Zhe Chao

    2016-07-01

    Full Text Available Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

  19. MuSK myasthenia gravis IgG4 disrupts the interaction of LRP4 with MuSK but both IgG4 and IgG1-3 can disperse preformed agrin-independent AChR clusters.

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    Inga Koneczny

    Full Text Available A variable proportion of patients with generalized myasthenia gravis (MG have autoantibodies to muscle specific tyrosine kinase (MuSK. During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4, which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a agrin, reflecting neuromuscular development, and