Skeletal Muscle Cell Cultures Research Articles

Effects of thrombin on insulin signalling and glucose uptake in cultured human myotubes

BackgroundHyper-coagulability (elevated thrombin) is a feature of type 2 diabetes and contributes to an increased risk of thrombotic and vascular events. Skeletal muscle is the key peripheral tissue site of insulin resistance in type 2 diabetes. Cultured human skeletal muscle cells were used to explore the effects of thrombin on insulin signalling and glucose uptake. We hypothesized that thrombin affects insulin activity in human skeletal muscle cells which could link the hypercoagulability and insulin resistance in type 2 diabetes. MethodsHuman skeletal muscle cell cultures (myotubes) were treated with +/−5 units/ml thrombin for 6h. Insulin signalling pathway components and AMPK were examined by Western blotting. Real time PCR and glucose uptake assays were performed. ResultsThere was a significant decrease (p<0.01) in insulin mediated IRS-1 and Akt phosphorylation in response to thrombin in cultured myotubes. Diminished Akt phosphorylation was alleviated by treatment with a PKC inhibitor. Thrombin directly increased basal glucose uptake (p<0.05) that involved AMPK phosphorylation (p<0.01) and this was partly repressed by compound C (AMPK inhibitor). Thrombin also significantly increased the gene expression level of both GLUT1 and GLUT4 in cultured human skeletal muscle cells. ConclusionThrombin decreased insulin signalling in skeletal muscle cells through a PKC mediated mechanism, but did not affect the net action of insulin on glucose uptake. The direct stimulatory effect of thrombin on glucose uptake was mediated, at least in part, via an AMPK dependent mechanism. We conclude that thrombin activation results in multiple metabolic effects beyond increased thrombogenicity but does not include a decrease in insulin sensitivity (glucose uptake) in cultured human skeletal muscle cells.

Primary skeletal muscle myoblasts from chronic heart failure patients exhibit loss of anti-inflammatory and proliferative activity.

BackgroundPeripheral skeletal muscle wasting is a common finding with adverse effects in chronic heart failure (HF). Whereas its clinical relevance is beyond doubt, the underlying pathophysiological mechanisms are not yet fully elucidated. We aimed to introduce and characterize the primary culture of skeletal muscle cells from individual HF patients as a supportive model to study this muscle loss.Methods and resultsPrimary myoblast and myotubes cultures were successfully propagated from the m. vastus lateralis of 6 HF patients with reduced ejection fraction (HFrEF; LVEF <45 %) and 6 age and gender-matched healthy donors. HFrEF cultures were not different from healthy donors in terms of morphology, such as myoblast size, shape and actin microfilament. Differentiation and fusion indexes were identical between groups. Myoblast proliferation in logarithmic growth phase, however, was attenuated in the HFrEF group (p = 0.032). In addition, HFrEF myoblasts are characterized by a reduced TNFR2 expression and IL-6 secretion (p = 0.017 and p = 0.016; respectively).ConclusionBiopsy derived primary skeletal muscle myoblasts of HFrEF patients produce similar morphological and myogenic differentiation responses as myoblasts of healthy donors, though demonstrate loss of anti-inflammatory and proliferative activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12872-016-0278-3) contains supplementary material, which is available to authorized users.

Sodium nitrate decreases agrin-induced acetylcholine receptor clustering.

BackgroundHumans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined.MethodsC2C12 skeletal muscle cell cultures were maintained, myoblasts were fused into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance acetylcholine receptor (AChR) clustering. Untreated cultures were compared with cultures exposed to sodium nitrate at concentrations ranging from 10 ng/mL–100 μg/mL.ResultsThe results reported here demonstrate that 1 μg/mL sodium nitrate was sufficient to decrease the frequency of agrin-induced AChR clustering without affecting myotube formation. In addition, concentrations of sodium nitrate of 1 μg/mL or 100 μg/mL decreased gene expression of the myogenic transcription factor myogenin and AChR in correlation with the agrin-induced AChR clustering data.ConclusionsThese results reveal that sodium nitrate decreases the frequency of agrin-induced AChR clustering by a mechanism that includes myogenin and AChR gene expression. As a consequence sodium nitrate may pose a risk for skeletal muscle development and subsequent neuromuscular synapse formation in humans.

Human Skeletal Muscle Glyoxalase‐1 Protein Expression is Attenuated with Type 2 Diabetes

INTRODUCTIONIndividuals with type 2 diabetes mellitus (T2DM) present with increased levels of advanced glycation endproducts (AGEs), which have been associated with progression and complications of the disease. AGEs are known to contribute to the disruption of insulin signaling in skeletal muscle and are primarily produced in vivo by the damaging glycolytic byproduct, methylglyoxal (MG). MG is effectively detoxified by the enzyme, glyoxalase‐1 (GLO1), whose protein expression has been previously shown to be inversely related to MG‐mediated protein‐damage and AGE formation. Despite the role of skeletal muscle insulin resistance in the pathogenesis of T2DM, GLO1 protein expression has not been fully elucidated in skeletal muscle. Additionally, the metabolic effects of insulin stimulation on GLO1 protein in human skeletal muscle has not been described.PURPOSETherefore, the purpose of this study was to examine skeletal muscle GLO1 protein content in individuals with T2DM compared to lean, healthy controls (LHC). Further, we sought to examine the effects of insulin stimulation on GLO1 protein expression through in vivo and in vitro approaches that included the hyperinsulinemic‐euglycemic clamp and primary human skeletal muscle cell culture experiments, respectively.METHODSSkeletal muscle biopsies from the vastus lateralis were obtained from 7 individuals with T2DM (age: 55 ± 4 yrs, BMI: 35.6 ± 2.4 kg/m2) and 10 LHCs (age: 27 ± 1 yrs, BMI: 22.3 ± 0.8 kg/m2) during basal and insulin stimulated states of the hyperinsulinemic (40mU/m2/min)‐euglycemic (5 mM) clamp. To further investigate the effect of insulin, primary human skeletal muscle cells were incubated with 300 pM insulin (a concentration similar to that experienced during the hyperinsulinemic‐euglycemic clamp) for 0, 30, and 180 mins. GLO1 protein content in human tissue and cell culture was assessed via western blot.RESULTSIndividuals with T2DM displayed a 5.6 fold reduction (p&lt;0.0001) in GLO1 compared to LHC at baseline. Clamp‐induced hyperinsulinemia had no significant effect on GLO1 protein expression in either group (p&gt;0.05). In cell culture, 30 minutes of insulin stimulation increased GLO1 protein expression by 37 % (p=0.0093). Interestingly, continued insulin stimulation for 180 mins returned GLO1 protein expression to levels comparable to baseline (0 min vs 180 min, p&gt;0.05).CONCLUSIONThese data suggest that the skeletal muscle of T2DM may be susceptible to MG‐mediated protein damage and accumulation of AGEs. Further, prolonged exposure to hyperinsulinemia may attenuate early insulin mediated increases in GLO1 expression profiles. Further study is need to elucidate the exact mechanisms of these observations.

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation.

Ca2+ effects on glucose transport and fatty acid oxidation in L6 skeletal muscle cell cultures

We examined the effect of Ca2+ on skeletal muscle glucose transport and fatty acid oxidation using L6 cell cultures. Ca2+ stimulation of glucose transport is controversial. We found that caffeine (a Ca2+ secretagogue) stimulation of glucose transport was only evident in a two-part incubation protocol (“post-incubation”). Caffeine was present in the first incubation, the media removed, and labeled glucose added for the second. Caffeine elicited a rise in Ca2+ in the first incubation that was dissipated by the second. This post-incubation procedure was insensitive to glucose concentrations in the first incubation. With a single, direct incubation system (all components present together) caffeine caused a slight inhibition of glucose transport. This was likely due to caffeine induced inhibition of phosphatidylinositol 3-kinase (PI3K), since nanomolar concentrations of wortmannin, a selective PI3K inhibitor, also inhibited glucose transport, and previous investigators have also found this action.We did find a Ca2+ stimulation (using either caffeine or ionomycin) of fatty acid oxidation. This was observed in the absence (but not the presence) of added glucose. We conclude that Ca2+ stimulates fatty acid oxidation at a mitochondrial site, secondary to malonyl CoA inhibition (represented by the presence of glucose in our experiments). In summary, the experiments resolve a controversy on Ca2+ stimulation of glucose transport by skeletal muscle, introduce an important experimental consideration for the measurement of glucose transport, and uncover a new site of action for Ca2+ stimulation of fatty acid oxidation.

MRNA-seq reveals skeletal muscle atrophy in response to handling stress in a marine teleost, the red cusk-eel (Genypterus chilensis).

BackgroundFish reared under intensive conditions are repeatedly exposed to stress, which negatively impacts growth. Although most fish follow a conserved pattern of stress response, with increased concentrations of cortisol, each species presents specificities in the cell response and stress tolerance. Therefore, culturing new species requires a detailed knowledge of these specific responses. The red cusk-eel (Genypterus chilensis) is a new economically important marine species for the Chilean aquaculture industry. However, there is no information on the stress- and cortisol-induced mechanisms that decrease skeletal muscle growth in this teleost.ResultsUsing Illumina RNA-seq technology, skeletal muscle sequence reads for G. chilensis were generated under control and handling stress conditions. Reads were mapped onto a reference transcriptome, resulting in the in silico identification of 785 up-regulated and 167 down-regulated transcripts. Gene ontology enrichment analysis revealed a significant up-regulation of catabolic genes associated with skeletal muscle atrophy. These results were validated by RT-qPCR analysis for ten candidates genes involved in ubiquitin-mediated proteolysis, autophagy and skeletal muscle growth. Additionally, using a primary culture of fish skeletal muscle cells, the effect of cortisol was evaluated in relation to red cusk-eel skeletal muscle atrophy.ConclusionsThe present data demonstrated that handling stress promotes skeletal muscle atrophy in the marine teleost G. chilensis through the expression of components of the ubiquitin-proteasome and autophagy-lysosome systems. Furthermore, cortisol was a powerful inductor of skeletal muscle atrophy in fish myotubes. This study is an important step towards understanding the atrophy system in non-model teleost species and provides novel insights on the cellular and molecular mechanisms that control skeletal muscle growth in early vertebrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2232-7) contains supplementary material, which is available to authorized users.

Abstract 17608: Identification of a Tissue-specific Very Long-chain Acyl-CoA Synthetase Involved in the Inhibition of ATP-Citrate Lyase (ACL) by ETC-1002: A Novel Mechanism for Cholesterol Biosynthesis Inhibition in the Liver

ETC-1002 is a first-in-class oral small molecule that significantly lowers levels of low- density lipoprotein-cholesterol (LDL-C) by 30-40% in hypercholesterolemic patients with reduced potential for muscle-related adverse events. Our previous investigations into the mechanism of action demonstrated a requirement for acyl-CoA synthetase- (ACS) dependent coenzyme A (CoA) activation of ETC-1002 (ETC-1002-CoA) to inhibit its molecular target, ATP-citrate lyase (ACL), an enzyme upstream of HMG-CoA reductase in the cholesterol biosynthesis pathway. In the present study, we have elucidated a mechanistic basis for tissue-specific inhibition of ACL by providing evidence that ACSVL1 is the ACS isoform that catalyzes the CoA activation of ETC-1002. Genetic silencing of ACSVL1 in McArdle cells prevented the CoA activation of ETC-1002 and consequently its inhibition of de novo cholesterol synthesis. We demonstrate that ACSVL1 is highly expressed in primary human hepatocytes and human liver microsomes. Furthermore, we establish a statin-like regulatory link between ACL activity and cholesterol metabolism by showing that ACL siRNA silencing or pharmacological inhibition by ETC-1002-CoA, leads to increased LDLR expression and activity. Using primary human hepatocytes, we demonstrate the concentration-dependent inhibition of cholesterol synthesis by ETC-1002, an associated increase in mRNA expression of the sterol responsive genes srebp2, hmgr, pcsk9, and ldlr mRNA, and increased LDLR activity. In addition, we demonstrate that ACSVL1 is not expressed in L6 myotubes, primary human skeletal muscle cells, or human skeletal muscle microsomes. Unlike statins, ETC-1002 did not inhibit the de novo synthesis of sterols in skeletal muscle cell cultures, nor promote caspase 3/7 activation. Therefore, by establishing a regulatory link between ACL inhibition and LDLR up-regulation, and by identifying ACSVL1 as the ACS isoform that catalyzes the CoA activation of ETC-1002, we provide evidence for the tissue-specific inhibition of ACL by ETC-1002-CoA that results in LDL-C-lowering without activity in skeletal muscle or associated side effects.

Developmental MYH3 Myopathy Associated with Expression of Mutant Protein and Reduced Expression Levels of Embryonic MyHC

ObjectiveAn essential role for embryonic MyHC in foetal development has been found from its association with distal arthrogryposis syndromes, a heterogeneous group of disorders characterised by congenital contractions. The latter probably result from severe myopathy during foetal development. Lack of embryonic muscle biopsy material and suitable animal models has hindered study of the pathomechanisms linking mutations in MYH3 to prenatal myopathy.Methods and ResultsWe determined the pathomechanisms of developmental myopathy caused by recurrent p.Thr178Ile MYH3 heterozygosity, using patient-derived skeletal muscle cells in culture as an experimental disease model to emulate early embryonic development. These cultured cells were processed for discrimination and quantitative analysis of mutant and wild-type MYH3 alleles and MyHC transcripts, real-time RT-qPCR, sequence analysis, immunofluorescence microscopy, immunoblot, and proteomic assessments. Involvement of the ubiquitin proteasome system was investigated in patients with p.Thr178Ile mutations in MYH3 and MYH2. We found equal overall expression of mutant and wild-type MyHC mRNAs and proteins. Compared to the controls, however, expression of embryonic MyHC transcripts and proteins was reduced whereas expression of myosin-specific E3 ubiquitin ligase (MuRF1) was increased. We also found delayed myofibrillogenesis and atrophic myotubes but structured sarcomeres.ConclusionIn conclusion, this study suggests that developmental p.Thr178Ile MYH3 myopathy is associated with a combined pathomechanism of insufficient dosage of functional embryonic MyHC and production of mutant protein.

Low-Level Laser Therapy (LLLT) in Dystrophin-Deficient Muscle Cells: Effects on Regeneration Capacity, Inflammation Response and Oxidative Stress.

The present study evaluated low-level laser therapy (LLLT) effects on some physiological pathways that may lead to muscle damage or regeneration capacity in dystrophin-deficient muscle cells of mdx mice, the experimental model of Duchenne muscular dystrophy (DMD). Primary cultures of mdx skeletal muscle cells were irradiated only one time with laser and analyzed after 24 and 48 hours. The LLLT parameter used was 830 nm wavelengths at 5 J/cm² fluence. The following groups were set up: Ctrl (untreated C57BL/10 primary muscle cells), mdx (untreated mdx primary muscle cells), mdx LA 24 (mdx primary muscle cells - LLLT irradiated and analyzed after 24 h), and mdx LA 48 (mdx primary muscle cells - LLLT irradiated and analyzed after 48 h). The mdx LA 24 and mdx LA 48 groups showed significant increase in cell proliferation, higher diameter in muscle cells and decreased MyoD levels compared to the mdx group. The mdx LA 48 group showed significant increase in Myosin Heavy Chain levels compared to the untreated mdx and mdx LA 24 groups. The mdx LA 24 and mdx LA 48 groups showed significant increase in [Ca2+]i. The mdx group showed significant increase in H2O2 production and 4-HNE levels compared to the Ctrl group and LLLT treatment reduced this increase. GSH levels and GPx, GR and SOD activities increased in the mdx group. Laser treatment reduced the GSH levels and GR and SOD activities in dystrophic muscle cells. The mdx group showed significant increase in the TNF-α and NF-κB levels, which in turn was reduced by the LLLT treatment. Together, these results suggest that the laser treatment improved regenerative capacity and decreased inflammatory response and oxidative stress in dystrophic muscle cells, indicating that LLLT could be a helpful alternative therapy to be associated with other treatment for dystrophinopathies.

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

ObjectiveWhole-body protein metabolism is dysregulated with obesity. Our goal was to determine if activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity.MethodsWe utilized primary Human Skeletal Muscle cell (HSkM) cultures since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean (BMI ≤ 26.0 kg/m2) and 8 severely obese (BMI ≥ 39.0) women were examined basally and when stimulated to atrophy (serum and amino acid starvation).ResultsHSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin-proteasome system diverged according to obesity status, with a decrease in the lean and an increase in HSkM from obese subjects. HSkMC from the obese also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from lean and obese individuals under basal and starved conditions.ConclusionsOur data indicate that muscle cells of the lean and severely obese have innate differences in management of protein degradation, which may explain their metabolic differences.

Abstract 290: Lower Limb Revascularization of Patients with Peripheral Artery Disease Reduces Human Enterovirus Infection of the Gastrocnemius

Introduction: Peripheral artery disease (PAD), caused by obstruction of arteries supplying the lower extremities, is characterized by leg muscle degeneration. Recently, we showed that infectious human enterovirus (HEV), which has been implicated in other myodegenerative diseases, is present in ischemic muscle of PAD patients and that viral copy numbers correlate with disease severity. Hypothesis: We tested the hypothesis that prevalence of infectious HEV and viral copy number in the gastrocnemius of patients with PAD decrease in response to revascularization. Methods: Gastrocnemius biopsies were collected from controls (N=14) and from PAD patients (N=17), before and after revascularization. Biopsies were examined for the presence of HEV RNA, viral capsid protein, viral RNA copy number, and viral infectivity and for viral sub-genotype. Results: HEV RNA was detected in biopsies of 65% (11/17) of PAD patients and in none of the controls. After revascularization, the gastrocnemius of 5 patients who were HEV positive before surgery no longer harbored detectable virus. The prevalence of HEV infection was reduced from 65% (11/17 patients) before surgery to 35% (6/17 patients) after surgery. Positive-strand viral RNA copy numbers were decreased significantly after revascularization. Mean positive-strand copy number (average 10 2.61 copies/mg muscle wet weight) in post-surgical biopsies that were HEV-positive were lower (p&lt;0.001) compared to mean copy number (average 10 5.46 copies/mg muscle wet weight) in pre-surgical biopsies that were HEV-positive. Copy numbers of negative-strand (template) viral RNA were also decreased after revascularization. Viral replication was confirmed by detection of negative-strand viral RNA in all specimens positive for HEV. Cultures of HeLa and human skeletal muscle cells treated with muscle homogenates showed HEV replication and the presence of HEV capsid protein. By sequence analysis, HEV in PAD muscle exhibited 97% homology with Coxsackievirus B1. Conclusion: Our data introduce HEV infection as a potential mechanism for degeneration of ischemic muscle in PAD patients and demonstrate that revascularization of PAD patients reduces both prevalence of HEV infection and viral copy number in the gastrocnemius.

A High‐Fat Diet Activates Inflammatory Signaling in Human Skeletal Muscle Cultures from Severely Obese Women

The incidence of metabolic disease and severe obesity continues to rise; likely associated with consumption of a Western, high-fat diet. Several mechanisms have been postulated to account for comorbidities associated with severe obesity; one being inflammation. The objective of this study was to determine whether metabolic and/or inflammatory gene networks were differentially regulated in human skeletal muscle cell cultures (HSkMC) from lean and severely obese women as a response to lipid oversupply. RNA in HSkMC from lean and severely obese women were isolated after a 48hr lipid incubation (250µM oleate:palmitate), designed to imitate a high-fat diet. Genome-wide expression data was acquired using Illumina HumanHT-12 v4.0 Expression BeadChip. Network and pathway analysis was done using MetaCore software. The number of inflammatory response pathways associated with genes differentially regulated by the lipid treatment was 66, including the top responses, HMGB1/RAGE, TNF-R2, TSLP, and IL-17 signaling pathways, all of which are associated with pro-inflammatory cytokine production. In conclusion, the activation of inflammatory signaling due to the up-regulation of cytokine regulators in response to a high fat diet in the severely obese could be the link between obesity and metabolic dysfunction.

Chronic erythropoietin treatment improves diet-induced glucose intolerance in rats.

Erythropoietin (EPO) ameliorates glucose metabolism through mechanisms not fully understood. In this study, we investigated the effect of EPO on glucose metabolism and insulin signaling in skeletal muscle. A 2-week EPO treatment of rats fed with a high-fat diet (HFD) improved fasting glucose levels and glucose tolerance, without altering total body weight or retroperitoneal fat mass. Concomitantly, EPO partially rescued insulin-stimulated AKT activation, reduced markers of oxidative stress, and restored heat-shock protein 72 expression in soleus muscles from HFD-fed rats. Incubation of skeletal muscle cell cultures with EPO failed to induce AKT phosphorylation and had no effect on glucose uptake or glycogen synthesis. We found that the EPO receptor gene was expressed in myotubes, but was undetectable in soleus. Together, our results indicate that EPO treatment improves glucose tolerance but does not directly activate the phosphorylation of AKT in muscle cells. We propose that the reduced systemic inflammation or oxidative stress that we observed after treatment with EPO could contribute to the improvement of whole-body glucose metabolism.

Plastic Additives Decrease Agrin-Induced Acetylcholine Receptor Clusters and Myotube Formation in C2C12 Skeletal Muscle Cell Culture

Common additives in plastics such as bisphenol A (BPA) or phthalates like di-(2-ethylhexyl) phthalate (DEHP) are environmental estrogens that have been shown to be endocrine disruptors in some experimental animal models. This project used the C2C12 cell culture model to examine how exposure to BPA or DEHP affects two aspects of skeletal muscle development, the fusion of myoblasts into myotubes and agrin-induced clustering of acetylcholine receptors (AChRs). During myotube formation AChRs cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. For this project C2C12 cells were exposed to BPA or DEHP while myoblasts fused into myotubes. After exposure to 10 μM BPA or 100 μM DEHP the frequency of agrin-induced AChR clusters decreased. In addition, myotube formation decreased as a higher percentage of nuclei remained in myoblasts. Furthermore, BPA or DEHP reduced the amount of the myogenic regulatory factor myogenin. This suggests that BPA and DEHP decrease AChR clustering by reducing myogenin. Moreover, plastic additives like BPA and DEHP may pose a risk for skeletal muscle development in humans.

Low Levels of Lipopolysaccharide Modulate Mitochondrial Oxygen Consumption in Skeletal Muscle

ObjectiveWe have previously demonstrated that activation of toll-like receptor 4 (TLR4) in skeletal muscle results in an increased reliance on glucose as an energy source and a concomitant decrease in fatty acid oxidation under basal conditions. Herein, we examined the effects of lipopolysaccharide (LPS), the primary ligand for TLR4, on mitochondrial oxygen consumption in skeletal muscle cell culture and mitochondria isolated from rodent skeletal muscle. Materials/methodsSkeletal muscle cell cultures were exposed to LPS and oxygen consumption was assessed using a Seahorse Bioscience extracellular flux analyzer. Mice were also exposed to LPS and oxygen consumption was assessed in mitochondria isolated from skeletal muscle. ResultsAcute LPS exposure resulted in significant reductions in Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)-stimulated maximal respiration (state 3u) and increased oligomycin induced state 4 (state 4O) respiration in C2C12 and human primary myotubes. These findings were observed in conjunction with increased mRNA of uncoupling protein 3 (UCP3), superoxide dismutase 2 (SOD2), and pyruvate dehydrogenase activity. The LPS-mediated changes in substrate oxidation and maximal mitochondrial respiration were prevented in the presence of the antioxidants N-acetylcysteine and catalase, suggesting a potential role of reactive oxygen species in mediating these effects. Mitochondria isolated from red gastrocnemius and quadriceps femoris muscle from mice injected with LPS also demonstrated reduced respiratory control ratio (RCR), and ADP- and FCCP-stimulated respiration. ConclusionLPS exposure in skeletal muscle alters mitochondrial oxygen consumption and substrate preference, which is absent when antioxidants are present.

S143 Premature Ageing And Skeletal Muscle Dysfunction In Copd Patients: Development Of A Cell Culture Model

Introduction COPD is a disease of accelerated ageing, as increased cellular-ageing (senescence) occurs in the lungs of these patients. We aim at developing a primary skeletal muscle cell culture (Human skeletal muscle satellite cells [HSKMC]) model of muscle ageing to study the cascade of events that occur in muscle senescence in vitro and to explore the effect of inflammation (TNF alpha) and oxidative stress (H 2 O 2 ), two of the putative mechanisms related to muscle dysfunction and/wasting, on muscle differentiation and on protein loss in differentiated cells. Methods and results HSKMC were cultured to senescence when the cells stopped replicating. DNA was isolated from cells in serial passages of culture. Telomere length, a marker of biological ageing, was measured by qPCR and expressed as the ratio of telomere repeat copy number to single gene copy number in the experimental sample relative to a control sample (relative T/S ratio) (n = 3). Preliminary results show a progressive shortening of telomere length with cellular ageing when comparing early (passage 2, 1.433 ± 0.05 relative T/S ratio) with a later passage (passage 15, 0.340 ± 0.2 relative T/S ratio) (Fig 1). Conclusion We have developed a novel in vitro model of ageing skeletal muscle cells, which will help us to assess the role of accelerated ageing in muscle dysfunction and/wasting in COPD patients. Dr Lakhdar was funded by an LTERS fellowship grant.

P38 MAPK activation upregulates proinflammatory pathways in skeletal muscle cells from insulin-resistant type 2 diabetic patients

Skeletal muscle is the key site of peripheral insulin resistance in type 2 diabetes. Insulin-stimulated glucose uptake is decreased in differentiated diabetic cultured myotubes, which is in keeping with a retained genetic/epigenetic defect of insulin action. We investigated differences in gene expression during differentiation between diabetic and control muscle cell cultures. Microarray analysis was performed using skeletal muscle cell cultures established from type 2 diabetic patients with a family history of type 2 diabetes and clinical evidence of marked insulin resistance and nondiabetic control subjects with no family history of diabetes. Genes and pathways upregulated with differentiation in the diabetic cultures, compared with controls, were identified using Gene Spring and Gene Set Enrichment Analysis. Gene sets upregulated in diabetic myotubes were associated predominantly with inflammation. p38 MAPK was identified as a key regulator of the expression of these proinflammatory gene sets, and p38 MAPK activation was found to be increased in the diabetic vs. control myotubes. Although inhibition of p38 MAPK activity decreased cytokine gene expression from the cultured diabetic myotubes significantly, it did not improve insulin-stimulated glucose uptake. Increased cytokine expression driven by increased p38 MAPK activation is a key feature of cultured myotubes derived from insulin-resistant type 2 diabetic patients. p38 MAPK inhibition decreased cytokine expression but did not affect the retained defect of impaired insulin action in the diabetic muscle cells.

Effect of N-acetylcysteine plus deferoxamine on oxidative stress and inflammation in dystrophic muscle cells

ObjectivesOxidative stress and inflammatory process play an important role in the pathogenesis of Duchenne muscular dystrophy (DMD). We investigated whether deferoxamine (DFX) improves the antioxidant effects of N-acetylcysteine (NAC) on primary cultures of dystrophic muscle cells from mdx mice, the experimental model of DMD.MethodsPrimary cultures of skeletal muscle cells from mdx mice were treated with either NAC (10 mM), DFX (5 mM), or NAC plus DFX for 24 hours. The muscle cells of C57BL/10 mice were used as controls.ResultsProduction of hydrogen peroxide (H2O2) and levels of 4-hydroxynonenal (4-HNE), tumor necrosis factor alpha (TNF-α), and nuclear factor kappa-B (NF-κB) were significantly higher in mdx muscle cells than in C57BL/10 muscle cells. Treatment with NAC, DFX, or NAC plus DFX significantly decreased H2O2 production (24, 58, and 72%, respectively), and levels of 4-HNE-protein adducts (62, 33, and 71%, respectively), TNF-α (32, 29, and 31%, respectively), and NF-κB (34, 38, and 52%, respectively) on dystrophic muscle cells.DiscussionThis study demonstrates that mdx muscle cells are able to produce key oxidative stress and inflammatory markers, without the interference of inflammatory cells, and shows that NAC plus DFX reduced the inflammatory and oxidative stress indicators, mainly H2O2 production and NF-κB levels by dystrophic fibers.

Effects of rutaecarpine on inflammatory cytokines in insulin resistant primary skeletal muscle cells

It is now well established that inflammation plays an important role in the development of numerous chronic metabolic diseases including insulin resistance (IR) and type 2 diabetes (T2DM). Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Our pre- vious study has shown that rutaecarpine (Rut) can benefit blood lipid profile, mitigate inflammation, and improve kidney, liver, pan- creas pathology status of T2DM rats. However, the effects of Rut on inflammatory cytokines in the development of IR-skeletal muscle cells have not been studied. Thus, our objective was to investigate effects of Rut on inflammatory cytokines interleukiri (IL)-1, IL-6 and tumor necrosis factor (TNF)-α in insulin resistant primary skeletal muscle cells (IR-PSMC). Primary cultures of skeletal muscle cells were prepared from 5 neonate SD rats, and the primary rat skeletal muscle cells were identified by cell morphology, effect of ru- taecarpine on cell proliferation by MTT assay. IR-PSMC cells were induced by palmitic acid (PA), the glucose concentration was measured by glucose oxidase and peroxidase (GOD-POD) method. The effects of Rut on inflammatory cytokines IL-1, IL-6 and TNF-α in IR-PSMC cells were tested by enzyme-linked immunosorbent assay (ELISA) kit. The results show that the primary skeletal muscle cells from neonatal rat cultured for 2-4 days, parallel alignment regularly, and cultured for 7 days, cells fused and myotube formed. It was shown that Rut in concentration 0-180. 0 μmol x L(-1) possessed no cytotoxic effect towards cultured primary skeletal muscle cells. However, after 24 h exposure to 0.6 mmol x L(-1) PA, primary skeletal muscle cells were able to induce a state of insulin resistance. The results obtained indicated significant decrease (P < 0.05 to P < 0.001) IL-1, IL-6 and TNF-α production by cultured IR-PSMC cells when incubating 24 hours with Rut, beginning from 20 to 180.0 μmol x L(-1). IL-1, IL-6 and TNF-α in the Rut treated groups were dose-dependently decreased compared with that in the IR-PSMC control group. Our results demonstrated that the Rut promoted glucose consumption and improved insulin resistance possibly through suppression of inflammatory cytokines in the IR-PSMC cells.