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Journal articles on the topic 'Myotubes humains'
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Jacquemin, V., A. Bigot, D. Furling, G. Butler-Browne, and V. Mouly. "L'IGF-1 induit une augmentation de la taille et du contenu en myosine des myotubes humains." Science & Sports 20, no. 4 (August 2005): 199–201. http://dx.doi.org/10.1016/j.scispo.2005.01.011.
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Henrotin, Y., C. Lambert, A. Florin, J. Zappia, P. Centonze, and C. Sanchez. "Évaluation de l’activité des urolithines A et B sur le muscle : modulation transcriptomique sur les myotubes primaires humains." Revue du Rhumatisme 89 (December 2022): A49—A50. http://dx.doi.org/10.1016/j.rhum.2022.10.061.
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Thrush, A. Brianne, Rui Zhang, William Chen, Erin L. Seifert, Jessica K. Quizi, Ruth McPherson, Robert Dent, and Mary-Ellen Harper. "Lower Mitochondrial Proton Leak and Decreased Glutathione Redox in Primary Muscle Cells of Obese Diet-Resistant Versus Diet-Sensitive Humans." Journal of Clinical Endocrinology & Metabolism 99, no. 11 (November 1, 2014): 4223–30. http://dx.doi.org/10.1210/jc.2014-1726.
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Context: Weight loss success in response to energy restriction is highly variable. This may be due in part to differences in mitochondrial function and oxidative stress. Objective: The objective of the study was to determine whether mitochondrial function, content, and oxidative stress differ in well-matched obese individuals in the upper [obese diet sensitive (ODS)] vs lower quintiles [obese diet resistant (ODR)] for rate of weight loss. Design: Primary myotubes derived from muscle biopsies of individuals identified as ODS or ODR were studied. Setting: Compliant ODS and ODR females who completed in the Ottawa Hospital Weight Management Program and identified as ODS and ODR participated in this study. Patients or Other Participants: Eleven ODS and nine ODR weight-stable females matched for age, body mass, and body mass index participated in this study. Intervention: Vastus lateralis muscle biopsies were obtained and processed for muscle satellite cell isolation. Main Outcome Measures: Mitochondrial respiration, content, reactive oxygen species, and glutathione redox ratios were measured in the myotubes of ODS and ODR individuals. Results: Mitochondrial proton leak was increased in myotubes of ODS compared with ODR (P < .05). Reduced and oxidized glutathione was decreased in the myotubes of ODR vs ODS (P < .05), indicating a more oxidized glutathione redox state. There were no differences in myotube mitochondrial content, uncoupling protein 3, or adenine nucleotide translocase levels. Conclusions: Lower rate of mitochondrial proton leak in muscle is a cell autonomous phenomenon in ODR vs ODS individuals, and this is associated with a more oxidized glutathione redox state in ODR vs ODS myotubes. The muscle of ODR subjects may thus have a lower capacity to adapt to oxidative stress as compared with ODS.
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Woo, Jin Seok, Ji-Hye Hwang, Jae-Kyun Ko, Noah Weisleder, Do Han Kim, Jianjie Ma, and Eun Hui Lee. "S165F mutation of junctophilin 2 affects Ca2+ signalling in skeletal muscle." Biochemical Journal 427, no. 1 (March 15, 2010): 125–34. http://dx.doi.org/10.1042/bj20091225.
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JPs (junctophilins) contribute to the formation of junctional membrane complexes in muscle cells by physically linking the t-tubule (transverse-tubule) and SR (sarcoplasmic reticulum) membranes. In humans with HCM (hypertrophic cardiomyopathy), mutations in JP2 are linked to altered Ca2+ signalling in cardiomyocytes; however, the effects of these mutations on skeletal muscle function have not been examined. In the present study, we investigated the role of the dominant-negative JP2-S165F mutation (which is associated with human HCM) in skeletal muscle. Consistent with the hypertrophy observed in human cardiac muscle, overexpression of JP2-S165F in primary mouse skeletal myotubes led to a significant increase in myotube diameter and resting cytosolic Ca2+ concentration. Single myotube Ca2+ imaging experiments showed reductions in both the excitation–contraction coupling gain and RyR (ryanodine receptor) 1-mediated Ca2+ release from the SR. Immunoprecipitation assays revealed defects in the PKC (protein kinase C)-mediated phosphorylation of the JP2-S165F mutant protein at Ser165 and in binding of JP2-S165F to the Ca2+ channel TRPC3 (transient receptor potential cation canonical-type channel 3) on the t-tubule membrane. Therefore both the hypertrophy and altered intracellular Ca2+ signalling in the JP2-S165F-expressing skeletal myotubes can be linked to altered phosphorylation of JP2 and/or altered cross-talk among Ca2+ channels on the t-tubule and SR membranes.
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Pasternak, C., S. Wong, and E. L. Elson. "Mechanical function of dystrophin in muscle cells." Journal of Cell Biology 128, no. 3 (February 1, 1995): 355–61. http://dx.doi.org/10.1083/jcb.128.3.355.
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We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.
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Morissette, Michael R., Stuart A. Cook, Cattleya Buranasombati, Michael A. Rosenberg, and Anthony Rosenzweig. "Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt." American Journal of Physiology-Cell Physiology 297, no. 5 (November 2009): 1124–32. http://dx.doi.org/10.1152/ajpcell.00043.2009.
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Myostatin is a highly conserved negative regulator of skeletal muscle growth. Loss of functional myostatin in cattle, mice, sheep, dogs, and humans results in increased muscle mass. The molecular mechanisms responsible for this increase in muscle growth are not fully understood. Previously, we have reported that phenylephrine-induced cardiac muscle growth and Akt activation are enhanced in myostatin knockout mice compared with controls. Here we report that skeletal muscle from myostatin knockout mice show increased Akt protein expression and overall activity at baseline secondary to an increase in Akt mRNA. We examined the functional role of myostatin modulation of Akt in C2C12 myotubes, a well-established in vitro model of skeletal muscle hypertrophy. Adenoviral overexpression of myostatin attenuated the insulin-like growth factor-I (IGF-I)-mediated increase in myotube diameter, as well as IGF-I-stimulated Akt phosphorylation. Inhibition of myostatin by overexpression of the NH2-terminal portion of myostatin was sufficient to increase myotube diameter and Akt phosphorylation. Coexpression of myostatin and constitutively active Akt (myr-Akt) restored the increase in myotube diameter. Conversely, expression of dominant negative Akt (dn-Akt) with the inhibitory myostatin propeptide blocked the increase in myotube diameter. Of note, ribosomal protein S6 phosphorylation and atrogin-1/muscle atrophy F box mRNA were increased in skeletal muscle from myostain knockout mice. Together, these data suggest myostatin regulates muscle growth at least in part through regulation of Akt.
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Baquero-Perez, Belinda, Suresh V. Kuchipudi, Jemima Ho, Sujith Sebastian, Anita Puranik, Wendy Howard, Sharon M. Brookes, Ian H. Brown, and Kin-Chow Chang. "Chicken and Duck Myotubes Are Highly Susceptible and Permissive to Influenza Virus Infection." Journal of Virology 89, no. 5 (December 24, 2014): 2494–506. http://dx.doi.org/10.1128/jvi.03421-14.
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ABSTRACTSkeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-β) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection.IMPORTANCEInfection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.
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Gundersen, Anders E., Benjamin A. Kugler, Paul M. McDonald, Alexey Veraksa, Joseph A. Houmard, and Kai Zou. "Altered mitochondrial network morphology and regulatory proteins in mitochondrial quality control in myotubes from severely obese humans with or without type 2 diabetes." Applied Physiology, Nutrition, and Metabolism 45, no. 3 (March 2020): 283–93. http://dx.doi.org/10.1139/apnm-2019-0208.
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Healthy mitochondrial networks are maintained via balanced integration of mitochondrial quality control processes (biogenesis, fusion, fission, and mitophagy). The purpose of this study was to investigate the effects of severe obesity and type 2 diabetes (T2D) on mitochondrial network morphology and expression of proteins regulating mitochondrial quality control processes in cultured human myotubes. Primary human skeletal muscle cells were isolated from biopsies from lean, severely obese nondiabetic individuals and severely obese type 2 diabetic individuals (n = 8–9/group) and were differentiated to myotubes. Mitochondrial network morphology was determined in live cells via confocal microscopy and protein markers of mitochondrial quality control were measured by immunoblotting. Myotubes from severely obese nondiabetic and type 2 diabetic humans exhibited fragmented mitochondrial networks (P < 0.05). Mitochondrial fission protein Drp1 (Ser616) phosphorylation was higher in myotubes from severely obese nondiabetic humans when compared with the lean controls (P < 0.05), while mitophagy protein Parkin expression was lower in myotubes from severely obese individuals with T2D in comparison to the other groups (P < 0.05). These data suggest that regulatory proteins in mitochondrial quality control processes, specifically mitochondrial fission protein Drp1 (Ser616) phosphorylation and mitophagy protein Parkin, are intrinsically dysregulated at cellular level in skeletal muscle from severely obese nondiabetic and type 2 diabetic humans, respectively. These differentially expressed mitochondrial quality control proteins may play a role in mitochondrial fragmentation evident in skeletal muscle from severely obese and type 2 diabetic humans. Novelty Mitochondrial network morphology and mitochondrial quality control proteins are intrinsically dysregulated in skeletal muscle cells from severely obese humans with or without T2D.
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Garza, M. Carmen, Sang-Gyun Kang, Chiye Kim, Eva Monleón, Jacques van der van der Merwe, David A. Kramer, Richard Fahlman, et al. "In Vitro and In Vivo Evidence towards Fibronectin’s Protective Effects against Prion Infection." International Journal of Molecular Sciences 24, no. 24 (December 15, 2023): 17525. http://dx.doi.org/10.3390/ijms242417525.
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A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix, mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich extracellular matrix while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues.
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Gallagher, Harrison, Hanzla Naeem, Nathanael Wood, Hélène N. Daou, Marcelo G. Pereira, Peter V. Giannoudis, Lee D. Roberts, Anthony Howard, and T. Scott Bowen. "LOCAL AND SYSTEMIC MEDIATORS OF SKELETAL MUSCLE WASTING IN HUMANS FOLLOWING ACUTE TRAUMA." Orthopaedic Proceedings 105-B, SUPP_16 (November 17, 2023): 18. http://dx.doi.org/10.1302/1358-992x.2023.16.018.
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AbstractIntroductionSkeletal muscle wasting is an important clinical issue following acute traumatic injury, and can delay recovery and cause permanent functional disability particularly in the elderly. However, the fundamental mechanisms involved in trauma-induced muscle wasting remain poorly defined and therapeutic interventions are limited.ObjectivesTo characterise local and systemic mediators of skeletal muscle wasting in elderly patients following acute trauma.MethodsExperiments were approved by a local NHS Research Ethics Committee and all participants provided written informed consent. Vastus lateralis biopsies and serum samples were taken from human male and female patients shortly after acute trauma injury in lower limbs (n=6; mean age 78.7±4.4 y) and compared to age-matched controls (n=6; mean age 72.6±6.3 y). Atrogenes and upstream regulators (MuRF1; MAFbx; IL6, TNFα, PGC-1α) mRNA expression was assessed in muscle samples via RT-qPCR. Serum profiling of inflammatory markers (e.g. IL6, TNFα, IL1β) was further performed via multiplex assays. To determine whether systemic factors induced by trauma directly affect muscle phenotype, differentiated primary human myotubes were treated in vitro with serum from controls or trauma patients (pooled; n=3 each) in the final 24 hours of differentiation. Cells were then fixed, stained for myogenin and imaged to determine minimum ferret diameter. Statistical significance was determined at P<0.05.ResultsThere was an increase in skeletal muscle mRNA expression for E3 ligase MAFbx and inflammatory cytokine IL-6 (4.6 and 21.5-fold respectively; P<0.05) in trauma patients compared to controls. Expression of myogenic determination factor MyoD and regulator of mitochondrial biogenesis PGC-1α was lower in muscle of trauma patients vs controls (0.5 and 0.39-fold respectively; P<0.05). In serum, trauma patients showed increased concentrations of circulating pro-inflammatory cytokines IL-6 (14.5 vs. 0.3 pg/ml; P<0.05) and IL-16 (182.7 vs. 85.2 pg/ml; P<0.05) compared to controls. Primary myotube experiments revealed serum from trauma patients induced atrophy (32% decrease in diameter) compared to control serum-treated cells (P<0.001).ConclusionSkeletal muscle from patients following acute trauma injury showed greater expression of atrophy and inflammatory markers. Trauma patient serum exhibited higher circulating pro-inflammatory cytokine concentrations. Primary human myotubes treated with serum from trauma patients showed significant atrophy compared to healthy serum-treated controls. We speculate a mechanism(s) acting via circulating factors may contribute to skeletal muscle pathology following acute trauma.Declaration of Interest(b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.
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Steinberg, Gregory R., Andrew J. McAinch, Michael B. Chen, Paul E. O’Brien, John B. Dixon, David Cameron-Smith, and Bruce E. Kemp. "The Suppressor of Cytokine Signaling 3 Inhibits Leptin Activation of AMP-Kinase in Cultured Skeletal Muscle of Obese Humans." Journal of Clinical Endocrinology & Metabolism 91, no. 9 (September 1, 2006): 3592–97. http://dx.doi.org/10.1210/jc.2006-0638.
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Abstract Context: Leptin is thought to regulate whole-body adiposity and insulin sensitivity, at least in part, by stimulating fatty acid metabolism via activation of AMP-kinase (AMPK) in skeletal muscle. Human obesity is associated with leptin resistance, and recent studies have demonstrated that hypothalamic expression of the suppressors of cytokine signaling 3 (SOCS3) regulates leptin sensitivity in rodents. Objective: The objective of the study was to investigate the effects of leptin on fatty acid oxidation and AMPK signaling in primary myotubes derived from lean and obese skeletal muscle and evaluate the contribution of SOCS3 to leptin resistance and AMPK signaling in obese humans. Results: We demonstrate that leptin stimulates AMPK activity and increases AMPK Thr172 and acetyl-CoA carboxylase-β Ser222 phosphorylation and fatty acid oxidation in lean myotubes but that in obese subjects leptin-dependent AMPK signaling and fatty acid oxidation are suppressed. Reduced activation of AMPK was associated with elevated expression of IL-6 (∼3.5-fold) and SOCS3 mRNA (∼2.5-fold) in myotubes of obese subjects. Overexpression of SOCS3 via adenovirus-mediated infection in lean myotubes to a similar degree as observed in obese myotubes prevented leptin but not AICAR (5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside) activation of AMPK signaling. Conclusions: These data demonstrate that SOCS3 inhibits leptin activation of AMPK. These data suggest that this impairment of leptin signaling in skeletal muscle may contribute to the aberrant regulation of fatty acid metabolism observed in obesity and that pharmacological activation of AMPK may be an effective therapy to bypass SOCS3-mediated skeletal muscle leptin resistance for the treatment of obesity-related disorders.
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Girgis, Christian M., Roderick J. Clifton-Bligh, Nancy Mokbel, Kim Cheng, and Jenny E. Gunton. "Vitamin D Signaling Regulates Proliferation, Differentiation, and Myotube Size in C2C12 Skeletal Muscle Cells." Endocrinology 155, no. 2 (February 1, 2014): 347–57. http://dx.doi.org/10.1210/en.2013-1205.
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Vitamin D deficiency is linked to a range of muscle disorders including myalgia, muscle weakness, and falls. Humans with severe vitamin D deficiency and mice with transgenic vitamin D receptor (VDR) ablation have muscle fiber atrophy. However, molecular mechanisms by which vitamin D influences muscle function and fiber size remain unclear. A central question is whether VDR is expressed in skeletal muscle and is able to regulate transcription at this site. To address this, we examined key molecular and morphologic changes in C2C12 cells treated with 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25(OH)2D). As well as stimulating VDR expression, 25(OH)D and 1,25(OH)2D dose-dependently increased expression of the classic vitamin D target cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1), demonstrating the presence of an autoregulatory vitamin D-endocrine system in these cells. Luciferase reporter studies demonstrated that cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1) was functional in these cells. Both 25OHD and 1,25(OH)2D altered C2C12 proliferation and differentiation. These effects were related to the increased expression of genes involved in G0/G1 arrest (retinoblastoma protein [Rb], 1.3-fold; ATM, 1.5-fold, both P < .05), downregulation of mRNAs involved in G1/S transition, including myc and cyclin-D1 (0.7- and 0.8-fold, both P < .05) and reduced phosphorylation of Rb protein (0.3-fold, P < .005). After serum depletion, 1,25(OH)2D (100nM) suppressed myotube formation with decreased mRNAs for key myogenic regulatory factors (myogenin, 0.5-fold; myf5, 0.4-fold, P < .005) but led to a 1.8-fold increase in cross-sectional size of individual myotubes associated with markedly decreased myostatin expression (0.2-fold, P < .005). These data show that vitamin D signaling alters gene expression in C2C12 cells, with effects on proliferation, differentiation, and myotube size.
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Rajesh, P., and K. Balasubramanian. "Di(2-ethylhexyl)phthalate exposure impairs insulin receptor and glucose transporter 4 gene expression in L6 myotubes." Human & Experimental Toxicology 33, no. 7 (October 15, 2013): 685–700. http://dx.doi.org/10.1177/0960327113506238.
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Di(2-ethyl hexyl)-phthalate (DEHP) is an endocrine disrupter and is the most abundantly used phthalate derivative, which is suspected to be an inevitable environmental exposure contributing to the increasing incidence of type-2 diabetes in humans. Therefore, the present study was designed to address the dose-dependent effects of DEHP on insulin signaling molecules in L6 myotubes. L6 myotubes were exposed to different concentrations (25, 50, and 100 μM) of DEHP for 24 h. At the end of exposure, cells were utilized for assessing various parameters. Insulin receptor and glucose transporter4 (GLUT4) gene expression, insulin receptor protein concentration, glucose uptake and oxidation, and enzymatic and nonenzymatic antioxidants were significantly reduced, but glutamine fructose-6-phosphate amidotransferase, nitric oxide, lipid peroxidation, and reactive oxygen species levels were elevated in a dose-dependent manner in L6 myotubes exposed to DEHP. The present study in turn shows the direct adverse effect of DEHP on the expression of insulin receptor and GLUT4 gene, glucose uptake, and oxidation in L6 myotubes suggesting that DEHP exposure may have a negative influence on insulin signaling.
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Boyle, K. E., D. Zheng, E. J. Anderson, P. D. Neufer, and J. A. Houmard. "Mitochondrial lipid oxidation is impaired in cultured myotubes from obese humans." International Journal of Obesity 36, no. 8 (October 25, 2011): 1025–31. http://dx.doi.org/10.1038/ijo.2011.201.
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Dlakic, Wendy M., Eric Grigg, and Richard A. Bessen. "Prion Infection of Muscle Cells In Vitro." Journal of Virology 81, no. 9 (February 21, 2007): 4615–24. http://dx.doi.org/10.1128/jvi.02628-06.
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ABSTRACT The prion agent has been detected in skeletal muscle of humans and animals with prion diseases. Here we report scrapie infection of murine C2C12 myoblasts and myotubes in vitro following coculture with a scrapie-infected murine neuroblastoma (N2A) cell line but not following incubation with a scrapie-infected nonneuronal cell line or a scrapie brain homogenate. Terminal differentiation of scrapie-infected C2C12 myoblasts into myotubes resulted in an increase in the expression of the disease-specific prion protein, PrPSc. The amount of scrapie infectivity or PrPSc in C2C12 myotubes was comparable to the levels found in scrapie-infected N2A cells, indicating that a high level of infection was established in muscle cells. Subclones of scrapie-infected C2C12 cells produced high levels of PrPSc in myotubes, and the C-terminal C2 polypeptide fragment of PrPSc was found based on deglycosylation and PrPSc-specific immunoprecipitation of cell lysates. This is the first report of a stable prion infection in muscle cells in vitro and of a long-term prion infection in a nondividing, differentiated peripheral cell type in culture. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.
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Zou, Kai, J. Matthew Hinkley, Sanghee Park, Donghai Zheng, Terry E. Jones, Walter J. Pories, Pamela J. Hornby, James Lenhard, G. Lynis Dohm, and Joseph A. Houmard. "Altered tricarboxylic acid cycle flux in primary myotubes from severely obese humans." International Journal of Obesity 43, no. 4 (June 11, 2018): 895–905. http://dx.doi.org/10.1038/s41366-018-0137-7.
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Liu, Xuhui, Steven Garcia, Hubert Kim, Robert Raffai, Brian Feeley, and Michael Davies. "Poster 155: Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes that Treat Rotator Cuff Muscle Degeneration in Mice and are Identified in Humans by Single-Cell RNA Sequencing." Orthopaedic Journal of Sports Medicine 10, no. 7_suppl5 (July 1, 2022): 2325967121S0071. http://dx.doi.org/10.1177/2325967121s00716.
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Objectives: Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white- and beige adipose tissue (WAT; BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in a variety of injury models. The purpose of this study was to characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their pro-myogenic effect is mediated via exosomes, and analyze human FAPs for an analogous pro-myogenic, exosome-rich subpopulation. Given their non-myogenic differentiation capacity, it was hypothesized that FAP-BAT exerts a pro-myogenic effect on the injured muscle environment through the secretion of exosomes. Methods: FAPs from UCP1-reporter mice were isolated via FACS and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1-), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNAseq was performed on UCP1-, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ (Exo-FB) and UCP1- (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and MEFs in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat WT C57B/L6J mice that had undergone massive rotator cuff tear. At 6 weeks mice were sacrificed and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing (scRNAseq) was then performed on FAPs isolated from human rotator cuff muscle that were treated with mirabegron or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans. Results: Flow cytometry analysis of sorted UCP1-reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with three distinct transcriptomic profiles characterized with bulk RNAseq. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers (Table 1). UCP1- cells were marked by fibrogenic markers and UCP1+ cells were characterized differential enrichment of WAT markers (Table 1). Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, with a myotube fusion index of 33.3 ± 6.3% compared to 7.1 ± 3.2% with Exo-nFB (p <0.001) and 8.8 ± 3.3% with exosome-free media (p = 0.0014), while treatment of MEFs resulted in transdifferentiation into myotubes (Fig. 1). Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and FI compared to treatment with Exo-nFB or PBS (Fig. 2). ScRNAseq of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with differential expression of mitochondrial genes, certain BAT markers, and exosome surface markers noted within one of the subpopulations (Fig. 3). Conclusions: FAP-BAT cells comprise a subpopulation of FAPs with upregulated mitochondrial gene expression and exosome production that mediates promyogenic effects in vitro and in vivo, and are present as a transcriptomically similar subpopulation of FAPs in humans. [Table: see text][Figure: see text][Figure: see text]
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Volonte, Daniela, Aaron J. Peoples, and Ferruccio Galbiati. "Modulation of Myoblast Fusion by Caveolin-3 in Dystrophic Skeletal Muscle Cells: Implications for Duchenne Muscular Dystrophy and Limb-Girdle Muscular Dystrophy-1C." Molecular Biology of the Cell 14, no. 10 (October 2003): 4075–88. http://dx.doi.org/10.1091/mbc.e03-03-0161.
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Caveolae are vesicular invaginations of the plasma membrane. Caveolin-3 is the principal structural component of caveolae in skeletal muscle cells in vivo. We have recently generated caveolin-3 transgenic mice and demonstrated that overexpression of wild-type caveolin-3 in skeletal muscle fibers is sufficient to induce a Duchenne-like muscular dystrophy phenotype. In addition, we have shown that caveolin-3 null mice display mild muscle fiber degeneration and T-tubule system abnormalities. These data are consistent with the mild phenotype observed in Limb-girdle muscular dystrophy-1C (LGMD-1C) in humans, characterized by a ∼95% reduction of caveolin-3 expression. Thus, caveolin-3 transgenic and null mice represent valid mouse models to study Duchenne muscular dystrophy (DMD) and LGMD-1C, respectively, in humans. Here, we derived conditionally immortalized precursor skeletal muscle cells from caveolin-3 transgenic and null mice. We show that overexpression of caveolin-3 inhibits myoblast fusion to multinucleated myotubes and lack of caveolin-3 enhances the fusion process. M-cadherin and microtubules have been proposed to mediate the fusion of myoblasts to myotubes. Interestingly, we show that M-cadherin is downregulated in caveolin-3 transgenic cells and upregulated in caveolin-3 null cells. For the first time, variations of M-cadherin expression have been linked to a muscular dystrophy phenotype. In addition, we demonstrate that microtubules are disorganized in caveolin-3 null myotubes, indicating the importance of the cytoskeleton network in mediating the phenotype observed in these cells. Taken together, these results propose caveolin-3 as a key player in myoblast fusion and suggest that defects of the fusion process may represent additional molecular mechanisms underlying the pathogenesis of DMD and LGMD-1C in humans.
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Schiffer, Tomas A., Maria Peleli, Michaela L. Sundqvist, Björn Ekblom, Jon O. Lundberg, Eddie Weitzberg, and Filip J. Larsen. "Control of human energy expenditure by cytochrome c oxidase subunit IV-2." American Journal of Physiology-Cell Physiology 311, no. 3 (September 1, 2016): C452—C461. http://dx.doi.org/10.1152/ajpcell.00099.2016.
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Resting metabolic rate (RMR) in humans shows pronounced individual variations, but the underlying molecular mechanism remains elusive. Cytochrome c oxidase (COX) plays a key role in control of metabolic rate, and recent studies of the subunit 4 isoform 2 (COX IV-2) indicate involvement in the cellular response to hypoxia and oxidative stress. We evaluated whether the COX subunit IV isoform composition may explain the pronounced individual variations in resting metabolic rate (RMR). RMR was determined in healthy humans by indirect calorimetry and correlated to levels of COX IV-2 and COX IV-1 in vastus lateralis. Overexpression and knock down of the COX IV isoforms were performed in primary myotubes followed by evaluation of the cell respiration and production of reactive oxygen species. Here we show that COX IV-2 protein is constitutively expressed in human skeletal muscle and strongly correlated to RMR. Primary human myotubes overexpressing COX IV-2 displayed markedly (>60%) lower respiration, reduced (>50%) cellular H2O2 production, higher resistance toward both oxidative stress, and severe hypoxia compared with control cells. These results suggest an important role of isoform COX IV-2 in the control of energy expenditure, hypoxic tolerance, and mitochondrial ROS homeostasis in humans.
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Tamura, Yuki, Karina Kouzaki, Takaya Kotani, and Koichi Nakazato. "Electrically stimulated contractile activity-induced transcriptomic responses and metabolic remodeling in C2C12 myotubes: twitch vs. tetanic contractions." American Journal of Physiology-Cell Physiology 319, no. 6 (December 1, 2020): C1029—C1044. http://dx.doi.org/10.1152/ajpcell.00494.2019.
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The contraction of myotubes using electrical pulse stimulation is a research tool used to mimic muscle contractile activity and exercise in rodents and humans. Most protocols employed in previous work used low-frequency twitch contractions. However, high-frequency tetanus contractions that are more physiologically relevant to muscle contractions in vivo are poorly characterized. In this report, the similarities and differences in acute responses and chronic adaptations with different contractile modes using twitches (2 Hz, continuous, 3 h) and tetanus (66 Hz, on: 5 s/off: 5 s, 3 h) were investigated. RNA sequencing-based transcriptome analysis and subsequent bioinformatics analysis suggest that tetanus may promote bioenergetic remodeling rather than twitch. Based on in silico analyses, metabolic remodeling after three contractile sessions of twitch and tetanus were investigated. Although twitch and tetanus had no significant effect on glycolysis, both types of contraction upregulated glucose oxidation capacity. Both twitch and tetanus qualitatively caused mitochondrial adaptations (increased content, respiratory chain enzyme activity, and respiratory function). The magnitude of adaptation was much greater under tetanus conditions. Our findings indicate that the contraction of myotubes by tetanus may be a useful experimental model, especially in the study of metabolic adaptations in C2C12 myotubes.
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Brook, M. S., D. J. Wilkinson, W. K. Mitchell, J. L. Lund, B. E. Phillips, N. J. Szewczyk, H. Kainulainen, et al. "A novel D2O tracer method to quantify RNA turnover as a biomarker of de novo ribosomal biogenesis, in vitro, in animal models, and in human skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 313, no. 6 (December 1, 2017): E681—E689. http://dx.doi.org/10.1152/ajpendo.00157.2017.
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Current methods to quantify in vivo RNA dynamics are limited. Here, we developed a novel stable isotope (D2O) methodology to quantify RNA synthesis (i.e., ribosomal biogenesis) in cells, animal models, and humans. First, proliferating C2C12 cells were incubated in D2O-enriched media and myotubes ±50 ng/ml IGF-I. Second, rat quadriceps (untrained, n = 9; 7-wk interval-“like” training, n = 13) were collected after ~3-wk D2O (70 atom %) administration, with body-water enrichment monitored via blood sampling. Finally, 10 (23 ± 1 yr) men consumed 150-ml D2O followed by 50 ml/wk and undertook 6-wk resistance exercise (6 × 8 repetitions, 75% 1-repetition maximum 3/wk) with body-water enrichment monitored by saliva sampling and muscle biopsies (for determination of RNA synthesis) at 0, 3, and 6 wk. Ribose mole percent excess (r-MPE) from purine nucleotides was analyzed via GC-MS/MS. Proliferating C2C12 cell r-MPE exhibited a rise to plateau, whereas IGF-I increased myotube RNA from 76 ± 3 to 123 ± 3 ng/μl and r-MPE by 0.39 ± 0.1% (both P < 0.01). After 3 wk, rat quadriceps r-MPE had increased to 0.25 ± 0.01% ( P < 0.01) and was greater with running exercise (0.36 ± 0.02%; P < 0.01). Human muscle r-MPE increased to 0.06 ± 0.01 and 0.13 ± 0.02% at 3/6 wk, respectively, equating to synthesis rates of ~0.8%/day, increasing with resistance exercise to 1.7 ± 0.3%/day ( P < 0.01) and 1.2 ± 0.1%/day ( P < 0.05) at 3/6 wk, respectively. Therefore, we have developed and physiologically validated a novel technique to explore ribosomal biogenesis in a multimodal fashion.
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Mason, Rachael R., Ruth C. R. Meex, Robert Lee-Young, Benedict J. Canny, and Matthew J. Watt. "Phosphorylation of adipose triglyceride lipase Ser404 is not related to 5′-AMPK activation during moderate-intensity exercise in humans." American Journal of Physiology-Endocrinology and Metabolism 303, no. 4 (August 15, 2012): E534—E541. http://dx.doi.org/10.1152/ajpendo.00082.2012.
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Intramyocellular triacylglycerol provides fatty acid substrate for ATP generation in contracting muscle. The protein adipose triglyceride lipase (ATGL) is a key regulator of triacylglycerol lipolysis and whole body energy metabolism at rest and during exercise, and ATGL activity is reported to be enhanced by 5′-AMP-activated protein kinase (AMPK)-mediated phosphorylation at Ser406 in mice. This is a curious observation, because AMPK activation reduces lipolysis in several cell types. We investigated whether the phosphorylation of ATGL Ser404 (corresponding to murine Ser406) was increased during exercise in human skeletal muscle and with pharmacological AMPK activation in myotubes in vitro. In human experiments, skeletal muscle and venous blood samples were obtained from recreationally active male subjects before and at 5 and 60 min during exercise. ATGL Ser404 phosphorylation was not increased from rest during exercise, but ATGL Ser404 phosphorylation correlated with myosin heavy chain 1 expression, suggesting a possible fiber type dependency. ATGL Ser404 phosphorylation was not related to increases in AMPK activity, and immunoprecipitation experiments indicated no interaction between AMPK and ATGL. Rather, ATGL Ser404 phosphorylation was associated with protein kinase A (PKA) signaling. ATGL Ser406 phosphorylation in C2C12 myotubes was unaffected by 5-aminoimidazole-4-carboxaminde-1-β-d-ribofuranoside, an AMPK activator, and the PKA activator forskolin. Our results demonstrate that ATGL Ser404 phosphorylation is not increased in mixed skeletal muscle during moderate-intensity exercise and that AMPK does not appear to be an activating kinase for ATGL Ser404/406 in skeletal muscle.
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Richards, Nicholas J., Ali Alqallaf, Robert D. Mitchell, Andrew Parnell, Husain Bin Haidar, José R. Almeida, Jarred Williams, et al. "Indian Ornamental Tarantula (Poecilotheria regalis) Venom Affects Myoblast Function and Causes Skeletal Muscle Damage." Cells 12, no. 16 (August 15, 2023): 2074. http://dx.doi.org/10.3390/cells12162074.
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Envenomation by the Indian ornamental tarantula (Poecilotheria regalis) is medically relevant to humans, both in its native India and worldwide, where they are kept as pets. Muscle-related symptoms such as cramps and pain are commonly reported in humans following envenomation by this species. There is no specific treatment, including antivenom, for its envenomation. Moreover, the scientific knowledge of the impact of this venom on skeletal muscle function is highly limited. Therefore, we carried out this study to better understand the myotoxic properties of Poecilotheria regalis venom by determining its effects in cultured myoblasts and in the tibialis anterior muscle in mice. While there was no effect found on undifferentiated myoblasts, the venom affected differentiated multinucleated myotubes resulting in the reduction of fusion and atrophy of myotubes. Similarly, intramuscular administration of this venom in the tibialis anterior muscle in mice resulted in extensive muscle damage on day 5. However, by day 10, the regeneration was evident, and the regeneration process continued until day 20. Nevertheless, some tissue abnormalities including reduced dystrophin expression and microthrombi presence were observed on day 20. Overall, this study demonstrates the ability of this venom to induce significant muscle damage and affect its regeneration in the early stages. These data provide novel mechanistic insights into this venom-induced muscle damage and guide future studies to isolate and characterise individual toxic component(s) that induce muscle damage and their significance in developing better therapeutics.
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Deng, Wenqian, Benjamin A. Kugler, Bergomi Francois, Meaghan Nasta, Joseph Houmard, and Kai Zou. "Effects Of Electrical Pulse Stimulation On Mitochondrial Dynamics In Myotubes From Lean And Severely Obese Humans." Medicine & Science in Sports & Exercise 52, no. 7S (July 2020): 927–28. http://dx.doi.org/10.1249/01.mss.0000685628.20238.6a.
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Houzelle, Alexandre, Dennis Dahlmans, Emmani B. M. Nascimento, Gert Schaart, Johanna A. Jörgensen, Esther Moonen‐Kornips, Sander Kersten, Xu Wang, and Joris Hoeks. "MicroRNA‐204‐5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans." Journal of Cellular Physiology 235, no. 12 (May 26, 2020): 9851–63. http://dx.doi.org/10.1002/jcp.29797.
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Sun, Jingyu, Yajuan Su, Jiajia Chen, Duran Qin, Yaning Xu, Hang Chu, Tianfeng Lu, Jingmei Dong, Lili Qin, and Weida Li. "Differential Roles of CD36 in Regulating Muscle Insulin Response Depend on Palmitic Acid Load." Biomedicines 11, no. 3 (February 28, 2023): 729. http://dx.doi.org/10.3390/biomedicines11030729.
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The possible role of fatty acid translocase (CD36) in the treatment of obesity has gained increasing research interest since researchers recognized its coordinated function in fatty acid uptake and oxidation. However, the effect of CD36 deficiency on intracellular insulin signaling is complex and its impact may depend on different nutritional stresses. Therefore, we investigated the various effects of CD36 deletion on insulin signaling in C2C12 myotubes with or without palmitic acid (PA) overload. In the present work, we reported the upregulated expression levels of CD36 in the skeletal muscle tissues of obese humans and mice as well as in C2C12 myotubes with PA stimulation. CD36 knockdown using RNA interference showed that insulin signaling was impaired in CD36-deficient C2C12 cells in the absence of PA loading, suggesting that CD36 is essential for the maintenance of insulin action, possibly resulting from increased mitochondrial dysfunction and endoplasmic reticulum (ER) stress; however, CD36 deletion improved insulin signaling in the presence of PA overload due to a reduction in lipid overaccumulation. In conclusion, we identified differential roles of CD36 in regulating muscle insulin response under conditions with and without PA overload, which provides supportive evidence for further research into therapeutic approaches to diabetes.
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Noipha, Kusumarn, and Putrada Ninla-Aesong. "Antidiabetic Activity of Zingiber officinale Roscoe Rhizome Extract: an In Vitro Study." HAYATI Journal of Biosciences 25, no. 4 (December 4, 2018): 160. http://dx.doi.org/10.4308/hjb.25.4.160.
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The potential roles of Zingiber officinale Roscoe (ginger) for treating and preventing diabetes have been investigated in both humans and experimental animals. However, the mode of its action has not yet been elucidated. This study aimed to investigate the effects of ginger extract on glucose uptake activity and its activation pathway in L6 myotubes. Cells were co-cultured for 24 h with a variable concentration of either ginger extract or 2 mM metformin or 200 nM insulin or 20 μM Troglitazone (TGZ), followed by a 10-min 2-[3H]-deoxy-D-glucose (2-DG) uptake. The levels of glucose transporters 1 (GLUT1) and GLUT4 protein and mRNA expression were determined. Ginger extract at 400 μg/ml significantly enhanced glucose uptake in L6 myotubes (208.03 ± 10.65% above basal value, p<0.05) after co-culture for 24 h. The ginger-enhancement of glucose uptake was inhibited by 3.5 μM cycloheximide, a protein synthesis inhibitor, 1 μM wortmannin (Phosphatidylinositol 3-Kinase (PI3 kinase) inhibitor) and 15 nM rapamycin (mammalian target of rapamycin (mTOR) inhibitor). The enhancement of glucose transport by ginger extract at 400 μg/ml was accompanied with the increased expression of GLUT1 protein (1.60 ± 0.20, 2.03 ± 0.19, and 2.25 ± 0.35 folds of basal at 4, 8, and 24 h, respectively p<0.05) and mRNA (1.22 ± 0.96, 1.45 ± 0.93, 1.91 ± 0.75, 2.32±0.92, and 2.20 ± 0.64 folds of basal at 1, 2, 4, 8, and 24 h, respectively p<0.05) in a time-dependent manner. Z. officinale Roscoe rhizome extract increase glucose transport activity of L6 myotubes by enhancing GLUT1 expression, the results of PI3-Kinase and 5’-AMP-activated kinase (AMPK) stimulation.
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Poulsen, K. A., S. F. Pedersen, M. Kolko, and I. H. Lambert. "Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants." American Journal of Physiology-Cell Physiology 293, no. 5 (November 2007): C1605—C1615. http://dx.doi.org/10.1152/ajpcell.00012.2007.
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The involvement of group VI Ca2+-independent PLA2s (iPLA2-VI) in in vitro ischemia [oxygen and glucose deprivation (OGD)] in mouse C2C12 myotubes was investigated. OGD induced a time-dependent (0–6 h) increase in bromoenol lactone (BEL)-sensitive iPLA2 activity, which was suppressed by specific short interfering (si)RNA knockdown of iPLA2-VIA. OGD was associated with an increase in iPLA2-VIA protein levels, whereas mRNA levels were unchanged. The levels of iPLA2-VIB mRNA and protein were not increased by OGD. RT-PCR and Western blot analysis identified a mouse iPLA2-VIA homolog to catalytically inactive 50-kDa iPLA2-VIA-ankyrin variants previously identified in humans. Both the mRNA and protein levels of this ∼50-kDa variant were reduced significantly within 1 h following OGD. In C2C12 myoblasts, iPLA2-VIA seemed to predominantly reside at the endoplasmatic reticulum, where it accumulated further during OGD. A time-dependent reduction in cell viability during the early OGD period (3 h) was partially prevented by iPLA2-VIA knockdown or pharmacological inhibition (10 μM BEL), whereas iPLA2-VIA overexpression had no effect on cell viability. Taken together, these data demonstrate that OGD in C2C12 myotubes is associated with an increase in iPLA2-VIA activity that decreases cell viability. iPLA2-VIA activation may be modulated by changes in the levels of active and inactive iPLA2-VIA isoforms.
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Watt, Matthew J., Anna G. Holmes, Srijan K. Pinnamaneni, Andrew P. Garnham, Gregory R. Steinberg, Bruce E. Kemp, and Mark A. Febbraio. "Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue." American Journal of Physiology-Endocrinology and Metabolism 290, no. 3 (March 2006): E500—E508. http://dx.doi.org/10.1152/ajpendo.00361.2005.
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Hormone-sensitive lipase (HSL) is important for the degradation of triacylglycerol in adipose and muscle tissue, but the tissue-specific regulation of this enzyme is not fully understood. We investigated the effects of adrenergic stimulation and AMPK activation in vitro and in circumstances where AMPK activity and catecholamines are physiologically elevated in humans in vivo (during physical exercise) on HSL activity and phosphorylation at Ser563 and Ser660, the PKA regulatory sites, and Ser565, the AMPK regulatory site. In human experiments, skeletal muscle, subcutaneous adipose and venous blood samples were obtained before, at 15 and 90 min during, and 120 min after exercise. Skeletal muscle HSL activity was increased by ∼80% at 15 min compared with rest and returned to resting rates at the cessation of and 120 min after exercise. Consistent with changes in plasma epinephrine, skeletal muscle HSL Ser563 and Ser660 phosphorylation were increased by 27% at 15 min ( P < 0.05), remained elevated at 90 min, and returned to preexercise values postexercise. Skeletal muscle HSL Ser565 phosphorylation and AMPK signaling were increased at 90 min during, and after, exercise. Phosphorylation of adipose tissue HSL paralleled changes in skeletal muscle in vivo, except HSL Ser660 was elevated 80% in adipose compared with 35% in skeletal muscle during exercise. Studies in L6 myotubes and 3T3-L1 adipocytes revealed important tissue differences in the regulation of HSL. AMPK inhibited epinephrine-induced HSL activity in L6 myotubes and was associated with reduced HSL Ser660 but not Ser563 phosphorylation. HSL activity was reduced in L6 myotubes expressing constitutively active AMPK, confirming the inhibitory effects of AMPK on HSL activity. Conversely, in 3T3-L1 adipocytes, AMPK activation after epinephrine stimulation did not prevent HSL activity or glycerol release, which coincided with maintenance of HSL Ser660 phosphorylation. Taken together, these data indicate that HSL activity is maintained in the face of AMPK activation as a result of elevated HSL Ser660 phosphorylation in adipose tissue but not skeletal muscle.
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Benegiamo, Giorgia, Maroun Bou Sleiman, Martin Wohlwend, Sandra Rodríguez-López, Ludger J. E. Goeminne, Pirkka-Pekka Laurila, Marie Klevjer, et al. "COX7A2L genetic variants determine cardiorespiratory fitness in mice and human." Nature Metabolism 4, no. 10 (October 17, 2022): 1336–51. http://dx.doi.org/10.1038/s42255-022-00655-0.
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AbstractMitochondrial respiratory complexes form superassembled structures called supercomplexes. COX7A2L is a supercomplex-specific assembly factor in mammals, although its implication for supercomplex formation and cellular metabolism remains controversial. Here we identify a role for COX7A2L for mitochondrial supercomplex formation in humans. By using human cis-expression quantitative trait loci data, we highlight genetic variants in the COX7A2L gene that affect its skeletal muscle expression specifically. The most significant cis-expression quantitative trait locus is a 10-bp insertion in the COX7A2L 3′ untranslated region that increases messenger RNA stability and expression. Human myotubes harboring this insertion have more supercomplexes and increased respiration. Notably, increased COX7A2L expression in the muscle is associated with lower body fat and improved cardiorespiratory fitness in humans. Accordingly, specific reconstitution of Cox7a2l expression in C57BL/6J mice leads to higher maximal oxygen consumption, increased lean mass and increased energy expenditure. Furthermore, Cox7a2l expression in mice is induced specifically in the muscle upon exercise. These findings elucidate the genetic basis of mitochondrial supercomplex formation and function in humans and show that COX7A2L plays an important role in cardiorespiratory fitness, which could have broad therapeutic implications in reducing cardiovascular mortality.
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Costford, Sheila R., Sudip Bajpeyi, Magdalena Pasarica, Diana C. Albarado, Shantele C. Thomas, Hui Xie, Timothy S. Church, Sharon A. Jubrias, Kevin E. Conley, and Steven R. Smith. "Skeletal muscle NAMPT is induced by exercise in humans." American Journal of Physiology-Endocrinology and Metabolism 298, no. 1 (January 2010): E117—E126. http://dx.doi.org/10.1152/ajpendo.00318.2009.
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In mammals, nicotinamide phosphoribosyltransferase (NAMPT) is responsible for the first and rate-limiting step in the conversion of nicotinamide to nicotinamide adenine dinucleotide (NAD+). NAD+ is an obligate cosubstrate for mammalian sirtuin-1 (SIRT1), a deacetylase that activates peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), which in turn can activate mitochondrial biogenesis. Given that mitochondrial biogenesis is activated by exercise, we hypothesized that exercise would increase NAMPT expression, as a potential mechanism leading to increased mitochondrial content in muscle. A cross-sectional analysis of human subjects showed that athletes had about a twofold higher skeletal muscle NAMPT protein expression compared with sedentary obese, nonobese, and type 2 diabetic subjects ( P < 0.05). NAMPT protein correlated with mitochondrial content as estimated by complex III protein content ( R 2 = 0.28, P < 0.01), MRS-measured maximal ATP synthesis ( R 2 = 0.37, P = 0.002), and V̇o2max ( R 2 = 0.63, P < 0.0001). In an exercise intervention study, NAMPT protein increased by 127% in sedentary nonobese subjects after 3 wk of exercise training ( P < 0.01). Treatment of primary human myotubes with forskolin, a cAMP signaling pathway activator, resulted in an ∼2.5-fold increase in NAMPT protein expression, whereas treatment with ionomycin had no effect. Activation of AMPK via AICAR resulted in an ∼3.4-fold increase in NAMPT mRNA ( P < 0.05) as well as modest increases in NAMPT protein ( P < 0.05) and mitochondrial content ( P < 0.05). These results demonstrate that exercise increases skeletal muscle NAMPT expression and that NAMPT correlates with mitochondrial content. Further studies are necessary to elucidate the pathways regulating NAMPT as well as its downstream effects.
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Girgis, Christian M., Nancy Mokbel, Kuan Minn Cha, Peter J. Houweling, Myriam Abboud, David R. Fraser, Rebecca S. Mason, Roderick J. Clifton-Bligh, and Jenny E. Gunton. "The Vitamin D Receptor (VDR) Is Expressed in Skeletal Muscle of Male Mice and Modulates 25-Hydroxyvitamin D (25OHD) Uptake in Myofibers." Endocrinology 155, no. 9 (September 1, 2014): 3227–37. http://dx.doi.org/10.1210/en.2014-1016.
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Abstract Vitamin D deficiency is associated with a range of muscle disorders, including myalgia, muscle weakness, and falls. In humans, polymorphisms of the vitamin D receptor (VDR) gene are associated with variations in muscle strength, and in mice, genetic ablation of VDR results in muscle fiber atrophy and motor deficits. However, mechanisms by which VDR regulates muscle function and morphology remain unclear. A crucial question is whether VDR is expressed in skeletal muscle and directly alters muscle physiology. Using PCR, Western blotting, and immunohistochemistry (VDR-D6 antibody), we detected VDR in murine quadriceps muscle. Detection by Western blotting was dependent on the use of hyperosmolar lysis buffer. Levels of VDR in muscle were low compared with duodenum and dropped progressively with age. Two in vitro models, C2C12 and primary myotubes, displayed dose- and time-dependent increases in expression of both VDR and its target gene CYP24A1 after 1,25(OH)2D (1,25 dihydroxyvitamin D) treatment. Primary myotubes also expressed functional CYP27B1 as demonstrated by luciferase reporter studies, supporting an autoregulatory vitamin D-endocrine system in muscle. Myofibers isolated from mice retained tritiated 25-hydroxyvitamin D3, and this increased after 3 hours of pretreatment with 1,25(OH)2D (0.1nM). No such response was seen in myofibers from VDR knockout mice. In summary, VDR is expressed in skeletal muscle, and vitamin D regulates gene expression and modulates ligand-dependent uptake of 25-hydroxyvitamin D3 in primary myofibers.
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Mahlapuu, Margit, Carina Johansson, Kerstin Lindgren, Göran Hjälm, Brian R. Barnes, Anna Krook, Juleen R. Zierath, Leif Andersson, and Stefan Marklund. "Expression profiling of the γ-subunit isoforms of AMP-activated protein kinase suggests a major role for γ3 in white skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 286, no. 2 (February 2004): E194—E200. http://dx.doi.org/10.1152/ajpendo.00147.2003.
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Expression patterns of the three isoforms of the regulatory γ-subunit of AMP-activated protein kinase (AMPK) were determined in various tissues from adult humans, mice, and rats, as well as in human primary muscle cells. Real-time PCR-based quantification of mRNA showed similar expression patterns in the three species and a good correlation with protein expression in mice and rats. The γ3-isoform appeared highly specific to skeletal muscle, whereas γ1 and γ2 showed broad tissue distributions. Moreover, the proportion of white, type IIb fibers in the mouse and rat muscle samples, as indicated by real-time PCR quantification of Atp1b2 mRNA, showed a strong positive correlation with the expression of γ3. In samples of white skeletal muscle, γ3 clearly appeared to be the most abundant γ-isoform. Differentiation of human primary muscle cells from myoblasts into multinucleated myotubes was accompanied by upregulation of γ3 mRNA expression, whereas levels of γ1 and γ2 remained largely unchanged. However, even in these cultured myotubes, γ2 was the most highly expressed isoform, indicating a considerable difference compared with adult skeletal muscle. Immunoblot analysis of mouse gastrocnemius and quadriceps muscle extracts precipitated with a γ3-specific antibody showed that γ3 was exclusively associated with the α2- and β2-subunit isoforms. The observation that the AMPKγ3 isoform is expressed primarily in white skeletal muscle, in which it is the predominant γ-isoform, strongly suggests that γ3 has a key role in this tissue.
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Kong, JY, HK Kim, HJ Lee, SC Yeon, JK Park, KS Jeong, and IH Hong. "Embryonal rhabdomyosarcoma in a Siberian chipmunk (Tamias sibiricus): a case report." Veterinární Medicína 62, No. 9 (September 20, 2017): 527–31. http://dx.doi.org/10.17221/173/2016-vetmed.
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A 2-year-old female Siberian chipmunk (Tamias sibiricus) was presented to the veterinary clinic for swelling, pain and lameness of the left rear leg. Radiologically, an invasive tumour around the distal femur was suspected, and the leg was surgically amputated and submitted for histopathological diagnosis. Microscopically, the mass was densely packed with multinucleated strap cells that had round-to-oval, or elongated nuclei with prominent nucleoli. These neoplastic cells occasionally formed myotubes with cross-striations and were immunohistochemically positive for muscle markers including desmin and myogenin. Consequently, embryonal rhabdomyosaroma myotubular variant of the leg with metastasis to the femur was diagnosed. Spontaneous rhabdomyosaromas are rare tumours in animals and humans, and this is the first report of its occurrence in a Siberian chipmunk.
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Yang, Tianzhong, Joyce Riehl, Eric Esteve, Klaus I. Matthaei, Samuel Goth, Paul D. Allen, Isaac N. Pessah, and José R. Lopez. "Pharmacologic and Functional Characterization of Malignant Hyperthermia in the R163C RyR1 Knock-in Mouse." Anesthesiology 105, no. 6 (December 1, 2006): 1164–75. http://dx.doi.org/10.1097/00000542-200612000-00016.
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Background Malignant hyperthermia is a pharmacogenetic disorder affecting humans, dogs, pigs, and horses. In the majority of human cases and all cases in animals, malignant hyperthermia has been associated with missense mutations in the skeletal ryanodine receptor (RyR1). Methods The authors used a "knock-in" targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation. Results Validation of this new mouse model of human malignant hyperthermia susceptibility includes (1) proof of transcription of the R163C allele and expression of ryanodine receptor protein in R163C heterozygous and R163C homozygous animals; (2) fulminant malignant hyperthermia episodes in R163C heterozygous mice after exposure to 1.25-1.75% halothane or an ambient temperature of 42 degrees C characterized by increased rectal temperature, respiratory rate, and inspiratory effort, with significant blood biochemical changes indicating metabolic acidosis, ending in death and hyperacute rigor mortis; (3) intraperitoneal pretreatment with dantrolene provided 100% protection from the halothane-triggered fulminant malignant hyperthermia episode; (4) significantly increased sensitivity (decreased effective concentration causing 50% of the maximal response) of R163C heterozygous and homozygous myotubes to caffeine, 4-chloro-m-cresol, and K-induced depolarization; (5) R163C heterozygous and homozygous myotubes have a significantly increased resting intracellular Ca concentration compared with wild type; (6) R163C heterozygous sarcoplasmic reticulum membranes have a twofold higher affinity (Kd = 35.4 nm) for [H]ryanodine binding compared with wild type (Kd = 80.1 nm) and a diminished inhibitory regulation by Mg. Conclusions Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.
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Romero, Matthew A., Petey W. Mumford, Paul A. Roberson, Shelby C. Osburn, Hailey A. Parry, Andreas N. Kavazis, L. Bruce Gladden, et al. "Five months of voluntary wheel running downregulates skeletal muscle LINE-1 gene expression in rats." American Journal of Physiology-Cell Physiology 317, no. 6 (December 1, 2019): C1313—C1323. http://dx.doi.org/10.1152/ajpcell.00301.2019.
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Transposable elements (TEs) are mobile DNA and constitute approximately half of the human genome. LINE-1 (L1) is the only active autonomous TE in the mammalian genome and has been implicated in a number of diseases as well as aging. We have previously reported that skeletal muscle L1 expression is lower following acute and chronic exercise training in humans. Herein, we used a rodent model of voluntary wheel running to determine whether long-term exercise training affects markers of skeletal muscle L1 regulation. Selectively bred high-running female Wistar rats ( n = 11 per group) were either given access to a running wheel (EX) or not (SED) at 5 wk of age, and these conditions were maintained until 27 wk of age. Thereafter, mixed gastrocnemius tissue was harvested and analyzed for L1 mRNA expression and DNA content along with other L1 regulation markers. We observed significantly ( P < 0.05) lower L1 mRNA expression, higher L1 DNA methylation, and less L1 DNA in accessible chromatin regions in EX versus SED rats. We followed these experiments with 3-h in vitro drug treatments in L6 myotubes to mimic transient exercise-specific signaling events. The AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 4 mM) significantly decreased L1 mRNA expression in L6 myotubes. However, this effect was not facilitated through increased L1 DNA methylation. Collectively, these data suggest that long-term voluntary wheel running downregulates skeletal muscle L1 mRNA, and this may occur through chromatin modifications. Enhanced AMPK signaling with repetitive exercise bouts may also decrease L1 mRNA expression, although the mechanism of action remains unknown.
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Ludlow, Andrew T., Laila C. J. Lima, Jenny Wang, Erik D. Hanson, Lisa M. Guth, Espen E. Spangenburg, and Stephen M. Roth. "Exercise alters mRNA expression of telomere-repeat binding factor 1 in skeletal muscle via p38 MAPK." Journal of Applied Physiology 113, no. 11 (December 1, 2012): 1737–46. http://dx.doi.org/10.1152/japplphysiol.00200.2012.
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Telomeres protect chromosome ends and shorten with age in most tissues. Integral to the maintenance of telomeres is the protein complex shelterin. The gene expression regulation of shelterin proteins to physiological stressors is not understood in vivo. We have recently reported increased telomere-repeat binding factor 1 (TRF1) protein expression and longer telomere length in skeletal muscle of sedentary compared with chronically active mice. These provocative observations led us to examine the effects of acute physiological stress on shelterin expression in vivo in mice and to further define potential mechanisms associated with gene regulation of shelterin. Three groups of female C57Bl/6 mice were studied: one control group and two groups that underwent a 30-min treadmill running bout and were killed either immediately following or 1-h after the exercise. Following the exercise bout, mRNA expression of Trf1 was significantly reduced in the plantaris muscle, and this reduction was paralleled by significant increases in p38 MAPK phosphorylation. To determine if p38 mediated the decreases in Trf1 mRNA expression, C2C12 myotubes were treated with the calcium ionophore, A23187. In response to the A23187, Trf1 gene expression was significantly reduced, coupled with significant increases in p38 phosphorylation, similar to in vivo data. C2C12 myotubes pretreated with a p38 inhibitor (SB-202190) prevented the A23187-induced decrease in Trf1 mRNA expression, indicating a link between Trf1 gene expression and p38 MAPK activation. While it is too early to definitively report the effect of exercise on telomere biology in rodents or humans, these data provide important mechanistic insights into the paradoxical telomere shortening that occurs in skeletal muscle in response to chronic exercise in mice.
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Grozovsky, Renata, Scott Ribich, Matthew L. Rosene, Michelle A. Mulcahey, Stephen A. Huang, Mary Elizabeth Patti, Antonio C. Bianco, and Brian W. Kim. "Type 2 Deiodinase Expression Is Induced by Peroxisomal Proliferator-Activated Receptor-γ Agonists in Skeletal Myocytes." Endocrinology 150, no. 4 (November 26, 2008): 1976–83. http://dx.doi.org/10.1210/en.2008-0938.
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The thyroid hormone activating type 2 deiodinase (D2) is known to play a role in brown adipose tissue-mediated adaptive thermogenesis in rodents, but the finding of D2 in skeletal muscle raises the possibility of a broader metabolic role. In the current study, we examined the regulation of the D2 pathway in primary skeletal muscle myoblasts taken from both humans and mice. We found that pioglitazone treatment led to a 1.6- to 1.9-fold increase in primary human skeletal myocyte D2 activity; this effect was seen with other peroxisomal proliferator-activated receptor-γ agonists. D2 activity in primary murine skeletal myotubes increased 2.8-fold in response to 5 μm pioglitazone and 1.6-fold in response to 5 nm insulin and increased in a dose-dependent manner in response to lithocholic acid (maximum response at 25 μm was ∼3.8-fold). We compared Akt phosphorylation in primary myotubes derived from wild-type and D2 knockout (D2KO) mice: phospho-Akt was reduced by 50% in the D2KO muscle after 1 nm insulin exposure. Expression of T3-responsive muscle genes via quantitative RT-PCR suggests that D2KO cells have decreased thyroid hormone signaling, which could contribute to the abnormalities in insulin signaling. D2 activity in skeletal muscle fragments from both murine and human sources was low, on the order of about 0.01 fmol/min · mg of muscle protein. The phenotypic changes seen with D2KO cells support a metabolic role for D2 in muscle, hinting at a D2-mediated linkage between thyroid hormone and insulin signaling, but the low activity calls into question whether skeletal muscle D2 is a major source of plasma T3.
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Norrbom, J., E. K. Sällstedt, H. Fischer, C. J. Sundberg, H. Rundqvist, and T. Gustafsson. "Alternative splice variant PGC-1α-b is strongly induced by exercise in human skeletal muscle." American Journal of Physiology-Endocrinology and Metabolism 301, no. 6 (December 2011): E1092—E1098. http://dx.doi.org/10.1152/ajpendo.00119.2011.
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The present study investigated whether exercise induces the expression of PGC-1α splice variants in human skeletal muscle and the possible influence of metabolic perturbation on this response. The subjects exercised one leg for 45 min with restricted blood flow (R-leg), followed by 45 min of exercise using the other leg at the same absolute workload but with normal blood flow (NR-leg). This ischemic model (R-leg) has been shown previously to induce a greater metabolic perturbation and enhance the expression of PGC-1α beyond that observed in the NR-leg. Cultured human myotubes were used to test suggested exercise-induced regulatory stimuli of PGC-1α. We showed, for the first time, that transcripts from both the canonical promoter (PGC-1α-a) and the proposed upstream-located promoter (PGC-1α-b) are present in human skeletal muscle. Both transcripts were upregulated after exercise in the R-leg, but the fold change increase of PGC-1α-b was much greater than that of PGC-1α-a. No differences were observed between the two conditions regarding the marker for calcineurin activation, MCIP1, or p38 phosphorylation. AMPK phosphorylation increased to a greater extent in the R-leg, and AICAR stimulation of cultured human myotubes induced the expression of PGC-1α-a and PGC-1α-b. AICAR combined with norepinephrine yielded an additive effect on the PGC-1α-b expression only. Our results indicate clearly that exercise can activate an upstream promoter in humans and support AMPK as a major regulator of transcripts from the canonical PGC-1α promoter and the involvement of β-adrenergic stimulation in combination with AMPK in the regulation of PGC-1α-b.
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Kugler, Benjamin A., Anders E. Gundersen, Junhan Li, Wenqian Deng, Nancy Eugene, Philimon N. Gona, Joseph A. Houmard, and Kai Zou. "Roux-en-Y gastric bypass surgery restores insulin-mediated glucose partitioning and mitochondrial dynamics in primary myotubes from severely obese humans." International Journal of Obesity 44, no. 3 (October 17, 2019): 684–96. http://dx.doi.org/10.1038/s41366-019-0469-y.
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Hinkley, J. Matthew, Kai Zou, Sanghee Park, Donghai Zheng, G. Lynis Dohm, and Joseph A. Houmard. "Differential acute and chronic responses in insulin action in cultured myotubes following from nondiabetic severely obese humans following gastric bypass surgery." Surgery for Obesity and Related Diseases 13, no. 11 (November 2017): 1853–62. http://dx.doi.org/10.1016/j.soard.2017.05.019.
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Lendoye, E., B. Sibille, A.-S. Rousseau, J. Murdaca, P. A. Grimaldi, and P. Lopez. "PPARβ Activation Induces Rapid Changes of Both AMPK Subunit Expression and AMPK Activation in Mouse Skeletal Muscle." Molecular Endocrinology 25, no. 9 (September 1, 2011): 1487–98. http://dx.doi.org/10.1210/me.2010-0504.
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AMP-activated protein kinases (AMPK) are heterotrimeric, αβγ, serine/threonine kinases. The γ3-AMPK subunit is particularly interesting in muscle physiology because 1) it is specifically expressed in skeletal muscle, 2) α2β2γ3 is the AMPK heterotrimer activated during exercise in humans, and 3) it is down-regulated in humans after a training period. However, mechanisms underlying this decrease of γ3-AMPK expression remained unknown. We investigated whether the expression of AMPK subunits and particularly that of γ3-AMPK are regulated by the PPARβ pathway. We report that PPARβ activation with GW0742 induces a rapid (2 h) and sustained down-regulation of γ3-AMPK expression both in mouse skeletal muscles and in culture myotubes. Concomitantly, phosphorylation levels of both AMPK and acetyl-coenzyme A carboxylase are rapidly modified. The γ3-AMPK down-regulation is also observed in muscles from young and adult transgenic mice with muscle-specific overexpression of peroxisome proliferator-activated receptor β (PPARβ). We showed that γ3-AMPK down-regulation is a rapid physiological muscle response observed in mouse after running exercise or fasting, two situations leading to PPARβ activation. Finally, using C2C12, we demonstrated that dose and time-dependent down-regulation of γ3-AMPK expression upon GW0742 treatment, is due to decrease γ3-AMPK promoter activity.
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Huang, Tai-Yu, Donghai Zheng, Joseph A. Houmard, Jeffrey J. Brault, Robert C. Hickner, and Ronald N. Cortright. "Overexpression of PGC-1α increases peroxisomal activity and mitochondrial fatty acid oxidation in human primary myotubes." American Journal of Physiology-Endocrinology and Metabolism 312, no. 4 (April 1, 2017): E253—E263. http://dx.doi.org/10.1152/ajpendo.00331.2016.
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Peroxisomes are indispensable organelles for lipid metabolism in humans, and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI = 24.0 ± 0.6 kg/m2; n = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (peroxins) and genes ( PEXs) responsible for proliferation and functions were assessed by Western blotting and real-time qRT-PCR, respectively. [1-14C]palmitic acid and [1-14C]lignoceric acid (exclusive peroxisomal-specific substrate) were used to assess mitochondrial oxidation of peroxisomal-derived metabolites. After overexpression of PGC-1α, 1) peroxisomal membrane protein 70 kDa (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated ( P < 0.05), 2) PGC-1α, PMP70, key PEXs, and peroxisomal β-oxidation mRNA expression levels were significantly upregulated ( P < 0.05), and 3) a concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed ( P < 0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomal activity and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation, as observed in HSkM cells.
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Mestareehi, Aktham, Xiangmin Zhang, Berhane Seyoum, Zaher Msallaty, Abdullah Mallisho, Kyle Jon Burghardt, Anjaneyulu Kowluru, and Zhengping Yi. "Metformin Increases Protein Phosphatase 2A Activity in Primary Human Skeletal Muscle Cells Derived from Lean Healthy Participants." Journal of Diabetes Research 2021 (July 28, 2021): 1–6. http://dx.doi.org/10.1155/2021/9979234.
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Context. Skeletal muscle insulin resistance is one of the primary contributors of type 2 diabetes (T2D). Metformin is the first-line drug for the treatment of T2D. The primary effects of metformin include decreasing glucose production in the liver and decreasing insulin resistance in the skeletal muscle. However, the molecular mechanism of metformin’s action in skeletal muscle is not well understood. Protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, plays a pivotal role in cellular processes, such as signal transduction, cell proliferation, and apoptosis, and acts through dephosphorylating key signaling molecules such as AKT and AMPK. However, whether PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells remains to be elucidated. Objective. To investigate if PP2A plays a role in metformin-induced insulin sensitivity improvement in human skeletal muscle cells. Participants. Eight lean insulin-sensitive nondiabetic participants (4 females and 4 males; age: 21.0 ± 1.0 years; BMI: 22.0 ± 0.7 kg / m 2 ; 2-hour OGTT: 97.0 ± 6.0 mg / dl ; HbA1c: 5.3 ± 0.1 % ; fasting plasma glucose: 87.0 ± 2.0 mg / dl ; M value; 11.0 ± 1.0 mg / kgBW / min ). Design. A hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity in human subjects, and skeletal muscle biopsy samples were obtained. Primary human skeletal muscle cells (shown to retain metabolic characteristics of donors) were cultured from these muscle biopsies that included 8 lean insulin-sensitive participants. Cultured cells were expanded, differentiated into myotubes, and treated with 50 μM metformin for 24 hours before harvesting. PP2Ac activity was measured by a phosphatase activity assay kit (Millipore) according to the manufacturer’s protocol. Results. The results indicated that metformin significantly increased the activity of PP2A in the myotubes for all 8 lean insulin-sensitive nondiabetic participants, and the average fold increase is 1.54 ± 0.11 ( P < 0.001 ). Conclusions. These results provided the first evidence that metformin can activate PP2A in human skeletal muscle cells derived from lean healthy insulin-sensitive participants and may help to understand metformin’s action in skeletal muscle in humans.
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Hayashiji, Nozomi, Genri Kawahara, Xing Xu, Tomohiko Fukuda, Aurelien Kerever, Jianguo Gu, Yukiko K. Hayashi, and Eri Arikawa-Hirasawa. "α-1,6-Fucosyltransferase Is Essential for Myogenesis in Zebrafish." Cells 12, no. 1 (December 29, 2022): 144. http://dx.doi.org/10.3390/cells12010144.
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Glycosylation is an important mechanism regulating various biological processes, including intercellular signaling and adhesion. α-1,6-fucosyltransferase (Fut8) belongs to a family of enzymes that determine the terminal structure of glycans. Fut8 is widely conserved from Caenorhabditis elegans to humans, and its mutants have been reported in humans, mice, and zebrafish. Although mutants show various symptoms, such as spinal deformity and growth retardation, its effects on skeletal muscles are unknown. We aimed to elucidate the function of Fut8 in skeletal muscle using zebrafish and C2C12 cells for evaluation. We observed that most fut8a morphants died at 2 days post-fertilization (dpf) or in earlier developmental stages even at low concentrations of morpholino oligonucleotides (MOs). Mutant juveniles also had small body sizes, and abnormal myocepta and sarcomere structures, suggesting that Fut8a plays important roles in myogenesis. Moreover, treatment of C2C12 cells with 2-fluorofucose (2FF), a fucosylation inhibitor, during cell differentiation dramatically reduced the expression of myogenic genes, such as Myomaker and other myogenic fusion genes, and inhibited myotube formation. These results indicate that Fut8 is an important factor in myogenesis, and myofusion in particular.
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Barnes, Tyler, Katie M. Di Sebastiano, Filip Vlavcheski, Joe Quadrilatero, Evangelia Litsa Tsiani, and Marina Mourtzakis. "Glutamate increases glucose uptake in L6 myotubes in a concentration- and time-dependent manner that is mediated by AMPK." Applied Physiology, Nutrition, and Metabolism 43, no. 12 (December 2018): 1307–13. http://dx.doi.org/10.1139/apnm-2018-0174.
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Various in vivo studies have investigated the insulin response that is elicited when glutamate is elevated in circulation or in a given tissue; fewer studies have investigated the effects of glutamate on glucose uptake and handling. Glutamate ingestion in humans can attenuate rises in blood glucose following a carbohydrate load in the absence of increases in serum insulin concentrations. However, the underlying mechanisms have yet to be investigated. To elucidate the effects of glutamate on glucose handling in skeletal muscle tissue, differentiated rat L6 myocytes were treated with glutamate, and glucose uptake was assessed with the use of 2-[3H]-deoxy-d-glucose ([3H]-2-DG). Cells treated with 2 mmol/L glutamate experienced the greatest increase in [3H]-2-DG uptake relative to the control condition (177% ± 2% of control, P < 0.001) and the uptake was similar to that of metformin (184% ± 4%, P < 0.001). In line with these findings, differentiated glucose transporter 4 (GLUT4)-overexpressing myotubes treated with 2 mmol/L glutamate displayed significantly increased GLUT4 translocation when compared with the control condition (159% ± 8% of control, P < 0.001) and to an extent similar to that of insulin and metformin (181% ± 7% and 159% ± 12%, respectively). An AMP-activated protein kinase (AMPK) inhibitor (Compound C) abolished the glutamate-stimulated glucose uptake (98% ± 12% of control), and Western blotting revealed significantly elevated AMPK phosphorylation (278% ± 17% of control, P < 0.001) by glutamate. Our findings suggest that when muscle cells are exposed to increased glutamate concentrations, glucose uptake into these cells is augmented through AMPK activation, through mechanisms distinct from those of insulin and leucine.
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Hurel, Stevn, Francis Ofei, Ann Wells, José Newkirk, Mark Sopwith, Douglas Turnbull, and Roy Taylor. "O-48: TNFα and insulin sensitivity in humans: effects in vivo of antibody blockade in obese NIDDM patients and in vitro upon human cultured myotubes." Experimental and Clinical Endocrinology & Diabetes 104, S 02 (July 15, 2009): 59–60. http://dx.doi.org/10.1055/s-0029-1211539.
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Krützfeldt, Jan, and Alexandra Fahrner. "OR24-1 Paracrine Growth Factor Signaling Regulates Muscle Regeneration in Aged Mice via microRNA 501." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A351. http://dx.doi.org/10.1210/jendso/bvac150.729.
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Abstract Background Skeletal muscle is one of to the most dynamic and plastic tissues. The capacity to regenerate arises from adult muscle stem cells termed myogenic progenitors (MPs) that are activated in response to injury and differentiate to replenish damaged muscle fibers. The age-related loss of muscle mass is reflected in the reduced regenerative potential of MPs. The underlying mechanisms are still unclear and insights into alterations in adult muscle stem cells during ageing are urgently needed. We have previously identified a novel muscle-specific microRNA, miR-501-3p, that is enriched in activated MPs. Pharmacological inhibition of miR-501 during muscle regeneration promoted small-diameter neofibers. Here, we provide evidence for the regulation of miR-501 in skeletal muscle during aging and its effect on newly formed myofibers using genetic deletion in mice. Methods We analysed the regulation and upstream signalling pathways of miR-501 in primary myoblasts from mice and humans and in skeletal muscle from young and aged mice. We generated a novel Cre-loxP mouse model, which allowed for global and MP-specific deletion of miR-501. Skeletal muscle regeneration was induced using cardiotoxin (CTX) and assessed using immunofluorescence and transcriptomic analysis. Results miR-501 was upregulated in muscle cells by the paracrine growth factors platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) through the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription-3 (STAT3) signalling axis. Expression levels of miR-501 as well as its upstream regulators Pdgf and Vegf were significantly reduced in skeletal muscle from aged mice (22 months) compared to young mice (3 months) by 67.8% (p<0.001), 40.0% (p<0.01) and 36.3% (p<0.05), respectively. Genetic deletion of miR-501 in mice resulted in muscle fibers with decreased size in adult skeletal muscle and in newly formed myofibers during muscle regeneration. RNA-seq of regenerating muscle annotated a critical role for miR-501 in the organisation of muscle filaments. We identified a miR-501-dependent sarcomeric gene signature that involves genes such as myosin heavy chains, titin and tubulin polymerization-promoting protein. This was confirmed in vitro in myotubes from mice and humans in which miR-501 was deleted either genetically or pharmacologically using antagomirs. Altered sarcomeric gene expression correlated with an increased sarcomere length in myotubes lacking miR-501 (2.49 ± 0.08μm vs 3.22 ± 0.15μm, p<0.01). Conclusion Our data indicate that paracrine growth factor signalling contributes to muscle fiber formation via miR-501 in myogenic progenitor cells and its effect on the sarcomere. Restoring the Pdgf+Vegf/miR-501 axis might have the potential to improve muscle formation during aging. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.
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Sheppard, Ryan L., Espen E. Spangenburg, Eva R. Chin, and Stephen M. Roth. "Androgen receptor polyglutamine repeat length affects receptor activity and C2C12 cell development." Physiological Genomics 43, no. 20 (October 2011): 1135–43. http://dx.doi.org/10.1152/physiolgenomics.00049.2011.
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Testosterone (T) has an anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. The effects of AR glutamine repeat length on skeletal muscle are unknown. In this study we examined the effect of AR CAG repeat length on AR function in C2C12 cells. AR expression vectors harboring 14, 24, and 33 CAG repeats were used to assess AR transcriptional activity. C2C12 cell proliferation, differentiation, gene expression, myotube formation, and myonuclear fusion index were assessed. Transcriptional activity increased with increasing repeat length and in response to testosterone (AR14 = 3.91 ± 0.26, AR24 = 25.21 ± 1.72, AR33 = 36.08 ± 3.22 relative light units; P < 0.001). Ligand activation was increased for AR33 (2.10 ± 0.04) compared with AR14 (1.54 ± 0.09) and AR24 (1.57 ± 0.05, P < 0.001). AR mRNA expression was elevated in each stably transfected line. AR33 cell proliferation (20,512.3 ± 1,024.0) was decreased vs. AR14 (27,604.17 ± 1,425.3; P < 0.001) after 72 h. Decreased CK activity in AR14 cells (54.9 ± 2.9 units/μg protein) in comparison to AR33 (70.8 ± 8.1) ( P < 0.05) was noted. The myonuclear fusion index was lower for AR14 (15.21 ± 3.24%) and AR33 (9.97 ± 3.14%) in comparison to WT (35.07 ± 5.60%, P < 0.001). AR14 and AR33 cells also displayed atypical myotube morphology. RT-PCR revealed genotype differences in myostatin and myogenin expression. We conclude that AR polyglutamine repeat length is directly associated with transcriptional activity and alters the growth and development of C2C12 cells. This polymorphism may contribute to the heritability of muscle mass in humans.
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Owens, Daniel J., Adam P. Sharples, Ioanna Polydorou, Nura Alwan, Timothy Donovan, Jonathan Tang, William D. Fraser, et al. "A systems-based investigation into vitamin D and skeletal muscle repair, regeneration, and hypertrophy." American Journal of Physiology-Endocrinology and Metabolism 309, no. 12 (December 15, 2015): E1019—E1031. http://dx.doi.org/10.1152/ajpendo.00375.2015.
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Skeletal muscle is a direct target for vitamin D. Observational studies suggest that low 25[OH]D correlates with functional recovery of skeletal muscle following eccentric contractions in humans and crush injury in rats. However, a definitive association is yet to be established. To address this gap in knowledge in relation to damage repair, a randomised, placebo-controlled trial was performed in 20 males with insufficient concentrations of serum 25(OH)D (45 ± 25 nmol/l). Prior to and following 6 wk of supplemental vitamin D3 (4,000 IU/day) or placebo (50 mg of cellulose), participants performed 20 × 10 damaging eccentric contractions of the knee extensors, with peak torque measured over the following 7 days of recovery. Parallel experimentation using isolated human skeletal muscle-derived myoblast cells from biopsies of 14 males with low serum 25(OH)D (37 ± 11 nmol/l) were subjected to mechanical wound injury, which enabled corresponding in vitro studies of muscle repair, regeneration, and hypertrophy in the presence and absence of 10 or 100 nmol 1α,25(OH)2D3. Supplemental vitamin D3 increased serum 25(OH)D and improved recovery of peak torque at 48 h and 7 days postexercise. In vitro, 10 nmol 1α,25(OH)2D3 improved muscle cell migration dynamics and resulted in improved myotube fusion/differentiation at the biochemical, morphological, and molecular level together with increased myotube hypertrophy at 7 and 10 days postdamage. Together, these preliminary data are the first to characterize a role for vitamin D in human skeletal muscle regeneration and suggest that maintaining serum 25(OH)D may be beneficial for enhancing reparative processes and potentially for facilitating subsequent hypertrophy.