Primary Human Myotubes Research Articles

Effects of Inflammation on Arginine Transport and Metabolism in Human Primary Myotubes

RationaleSkeletal muscle atrophy among older adults increases the risk for falls and fractures, loss of mobility and independence, physical disabilities, and even mortality. Development of efficacious therapies to attenuate skeletal muscle loss in the older population is necessary. Metabolomics research from our laboratory indicates that skeletal muscle mass (measured as appendicular lean mass divided by height squared) in older adults is associated with arginine metabolism. Arginine supplementation, as part of a multi‐nutrient supplement, has been given to individuals to attenuate skeletal muscle wasting induced by diseases with heightened inflammation (e.g., HIV and cancer cachexia). Older adults in general have chronic increased circulating and intramuscular levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) that is associated with skeletal muscle atrophy. The effect of TNFα on skeletal muscle arginine metabolism is largely unknown.ObjectiveTo investigate the effects of age and TNFα on skeletal muscle arginine metabolism.MethodsSkeletal muscle biopsies were collected from 11 younger (21–39 years old) and 5 older (68–80 years old) males and females (YM, YF, OM, OF) after an overnight fast. To assess inflammatory signaling and arginine metabolism, the gene expression of TNFα, signal transducer and activator of transcription 3 (STAT3), interleukin‐6 (IL‐6), the IL‐6 receptors (IL‐6R and IL‐6ST), and cationic amino acid transporter 1 and 2 (CAT‐1 and CAT‐2) in whole tissue lysate was measured using quantitative PCR. To investigate the effects of TNFα specifically, primary skeletal muscle progenitor cells from 6 YM were cultured in growth media for 7 days, switched to differentiation media (DM) for 3 days, then incubated in DM containing 10 ng/mL TNFα for 2 days. Gene expression of the arginine transporters and metabolic enzymes in the TNFα treated primary human myotubes was measured using quantitative PCR.ResultsThere was no difference between young and old adults for CAT‐1 or CAT‐2 gene expression in whole skeletal muscle tissue lysate. There was also no difference in the gene expression of TNFα, STAT3, IL‐6, or the IL‐6 receptors. Incubation with TNFα decreased CAT‐1 gene expression slightly (−1.1 fold change, p=0.045) and increased CAT‐2 gene expression (11.2‐fold change, p=0.002) in primary human myotubes.ConclusionsThe proinflammatory cytokine TNFα increased gene expression of the arginine transporter CAT‐2 in primary human myotubes, indicating that older adults with elevated TNFα may have altered arginine transport, and potentially altered metabolism, in skeletal muscle. Ongoing research in our laboratory is assessing the effects of increased arginine availability in the skeletal myotubes in a proinflammatory culture. Future research is needed to determine if altering dietary arginine consumption in older adults can attenuate the atrophic effects of TNFα and thus, be a possible therapy to reduce skeletal muscle atrophy.Support or Funding InformationCornell Start‐up

Paraoxonase 1 (PON1) and pomegranate influence circadian gene expression and period length

ABSTRACTThe circadian timing system regulates key aspects of mammalian physiology. Here, we analyzed the effect of the endogenous antioxidant paraoxonase 1 (PON1), a high-density lipoprotein-associated lipolactonase that hydrolyses lipid peroxides and attenuates atherogenesis, on circadian gene expression in C57BL/6J and PON1KO mice fed a normal chow diet or a high-fat diet (HFD). Expression levels of core-clock transcripts Nr1d1, Per2, Cry2 and Bmal1 were altered in skeletal muscle in PON1-deficient mice in response to HFD. These findings were supported by circadian bioluminescence reporter assessments in mouse C2C12 and human primary myotubes, synchronized in vitro, where administration of PON1 or pomegranate juice modulated circadian period length.

Myokine Expression in Muscle and Myotubes in Response to Exercise Stimulation.

Myokines have been shown to affect muscle physiology and exert systemic effects. We endeavored to investigate a panel of myokine mRNA expression after a single exercise bout (studies 1 and 2) to measure myokine mRNA in primary human myotubes in an in vitro exercise model (study 2). Vastus lateralis muscle biopsies were obtained from 20 healthy males (age, 24.0 ± 4.5 yr; BMI, 23.6 ± 1.8 kg·m)(-2) before and after a single exercise bout (650 kcal at 50% V˙O2max). Primary myotubes from active and sedentary male donors were treated with a pharmacological cocktail (palmitate, forskolin, and ionomycin (PFI)) to mimic exercise-stimulated contractions in vitro. Interleukin 6 and 8 (IL-6 and IL-8), leukocyte-inducing factor, and connective tissue growth factor (CTGF) mRNA levels increased approximately 10-fold after a single exercise bout (all P < 0.001), whereas myostatin levels decreased (P < 0.05). Key correlations between myokine expression and parameters of muscle and whole-body physiology were found: myostatin versus skeletal muscle citrate synthase activity (r = -0.69, P < 0.001), V˙O2max (r = -0.64, P = 0.002) and the percentage of Type I fibers (r = -0.55, P = 0.01); IL-6 versus the RER (r = 0.45, P = 0.04), homeostatic model assessment of insulin resistance (r = 0.44, P = 0.05), and serum lactate (r = 0.50, P = 0.02). Myokine expressions in myotubes from sedentary donors for CTGF and myostatin decreased, whereas IL-6 and IL-8 increased after PFI treatment. In myotubes from active donors, myokine expression increased for IL-6, CTGF, and myostatin but decreased for IL-8 after PFI treatment. These data offer insight into the differences in regulation of myokine expression and their possible physiologic relationships.

Tumor necrosis factor-α impairs adiponectin signalling, mitochondrial biogenesis, and myogenesis in primary human myotubes cultures.

Skeletal muscle metabolic changes are common in patients with chronic heart failure (HF). Previously, we demonstrated a functional skeletal muscle adiponectin resistance in HF patients with reduced left ventricular ejection fraction (HFrEF). We aimed to examine the impact of adiponectin receptor 1 (AdipoR1) deficiency and TNF-α treatment on adiponectin signaling, proliferative capacity, myogenic differentiation, and mitochondrial biogenesis in primary human skeletal muscle cells. Primary cultures of myoblasts and myotubes were initiated from the musculus vastus lateralis of 10 HFrEF patients (left ventricular ejection fraction; 31.30 ± 2.89%) and 10 age- and gender-matched healthy controls. Healthy control cultures were transfected with siAdipoR1 and/or exposed to TNF-α (10 ng/ml; 72 h). Primary cultures from HFrEF patients preserved the features of adiponectin resistance in vivo. AdipoR1 mRNA was negatively correlated with time to reach maximal cell index (r = -0.7319, P = 0.003). SiRNA-mediated AdipoR1 silencing reduced pAMPK (P < 0.01), AMPK activation (P = 0.046), and myoblast proliferation rate (xCELLigence Real-Time Cellular Analysis; P < 0.0001). Moreover, TNF-α decreased the mRNA expression of genes involved in glucose (APPL1, P = 0.0002; AMPK, P = 0.021), lipid (PPARα, P = 0.025; ACADM, P = 0.003), and mitochondrial (FOXO3, P = 0.018) metabolism, impaired myogenesis (MyoD1, P = 0.053; myogenin, P = 0.048) and polarized cytokine secretion toward a growth-promoting phenotype (IL-10, IL-1β, IFN-γ, P < 0.05 for all; Meso Scale Discovery Technology). Major features of adiponectin resistance are retained in primary cultures from the skeletal muscle of HFrEF patients. In addition, our results suggest that an increased inflammatory constitution contributes to adiponectin resistance and confers alterations in skeletal muscle differentiation, growth, and function.

CX3CL1--a macrophage chemoattractant induced by a single bout of exercise in human skeletal muscle.

Monocytes/macrophages (MOs/MΦs) are suggested to be crucial for skeletal muscle repair and remodeling. This has been attributed to their proangiogenic potential, secretion of growth factors, and clearance of tissue debris. Skeletal muscle injury increases the number of MΦs in the tissue, and their importance for muscle regeneration has been supported by studies demonstrating that depletion of MOs/MΦs greatly impairs repair after muscle injury. Whether noninjurious exercise leads to induced expression of chemoattractants for MOs/MΦs is poorly investigated. To this end, we analyzed the expression of CX3CL1 (fractalkine), CCL2 (MCP-1), and CCL22 (MDC) in human skeletal muscle after a bout of exercise, all of which are established MO/MΦ chemotactic factors that are expressed by human myoblasts. Muscle biopsies from the musculus vastus lateralis were obtained up to 24 h after 1 h of cycle exercise in healthy individuals and in age-matched nonexercised controls. CX3CL1 increased at both the mRNA and protein level in human skeletal muscle after one bout of exercise. It was not possible to distinguish changes in CCL2 or CCL22 mRNA levels between biopsy vs. exercise effects, and the expression of CCL22 was very low. CX3CL1 mainly localized to the skeletal muscle endothelium, and it increased in human umbilical vein endothelial cells stimulated with tissue fluid from exercised muscle. CX3CL1 increased the expression of proinflammatory and proangiogenic factors in THP-1 monocytes (a human acute monocytic leukemia cell line) and in human primary myoblasts and myotubes. Altogether, this suggests that CX3CL1 participates in cross-talk mechanisms between endothelium and other muscle tissue cells and may promote a shift in the microenvironment toward a more regenerative milieu.

Effect of serial cell passaging in the retention of fiber type and mitochondrial content in primary human myotubes.

The purpose of the study was to determine the effects of passaging on retention of donor phenotypic characteristics in primary human myotubes. Primary muscle cultures and serial passaged myotubes from physically active, sedentary lean, and individuals with type 2 diabetes were established. Maximal ATP synthesis capacity (ATPmax) and resting ATP flux (ATPase) in vivo were measured by (31) P magnetic resonance spectroscopy, type-I fibers and intramyocelluar lipid (IMCL) in vastus lateralis tissue were determined using immunohistochemistry techniques, and oxidative phosphorylation complexes (OXPHOS) were measured by Western immunoblotting. Similar in vitro measures for lipid and type-I fibers were made in myotubes, along with mitochondrial content measured by MitoTracker. Passage 4 and 5 measures for myotubes correlated positively with in vivo measurements for percent type-I fibers (P4: R(2) = 0.39, p = 0.02; P5: R(2) = 0.48, p = 0.01), ATPmax (P4: R(2) = 0.30, p = 0.03; P5: R(2) = 0.22, p = 0.05), and OXPHOS (P4: R(2) = 0.44, p = 0.04; P5: R(2) = 0.59, p = 0.006). No correlations were observed for IMCL. However, passage 4 measures for myotubes correlated with passage 5 measures for percent type-I fibers (R(2) = 0.49, p = 0.01), IMCL (R(2) = 0.80, p < 0.001), and mitochondrial content (R(2) = 0.26, p = 0.03). Myotubes through the first two passages following immunopurification (referred to as passage 4 and 5) reflect the mitochondrial and type-I fiber content in vivo phenotype of the donor.

High-intensity sprint training inhibits mitochondrial respiration through aconitase inactivation.

Intense exercise training is a powerful stimulus that activates mitochondrial biogenesis pathways and thus increases mitochondrial density and oxidative capacity. Moderate levels of reactive oxygen species (ROS) during exercise are considered vital in the adaptive response, but high ROS production is a serious threat to cellular homeostasis. Although biochemical markers of the transition from adaptive to maladaptive ROS stress are lacking, it is likely mediated by redox sensitive enzymes involved in oxidative metabolism. One potential enzyme mediating such redox sensitivity is the citric acid cycle enzyme aconitase. In this study, we examined biopsy specimens of vastus lateralis and triceps brachii in healthy volunteers, together with primary human myotubes. An intense exercise regimen inactivated aconitase by 55-72%, resulting in inhibition of mitochondrial respiration by 50-65%. In the vastus, the mitochondrial dysfunction was compensated for by a 15-72% increase in mitochondrial proteins, whereas H2O2 emission was unchanged. In parallel with the inactivation of aconitase, the intermediary metabolite citrate accumulated and played an integral part in cellular protection against oxidative stress. In contrast, the triceps failed to increase mitochondrial density, and citrate did not accumulate. Instead, mitochondrial H2O2 emission was decreased to 40% of the pretraining levels, together with a 6-fold increase in protein abundance of catalase. In this study, a novel mitochondrial stress response was highlighted where accumulation of citrate acted to preserve the redox status of the cell during periods of intense exercise.

Adipogenic progenitors from obese human skeletal muscle give rise to functional white adipocytes that contribute to insulin resistance.

Recent reports indicate that inter/intramuscular adipose tissue (IMAT), composed by adipocytes underneath the deep fascia of the muscles, is positively correlated with aging, obesity and insulin resistance in humans. However, no molecular/cellular evidence is available to support these interactions. The current study aimed to better characterize human skeletal muscle-derived adipogenic progenitors obtained from obese volunteers and investigate the impact of derived adipocytes on insulin action in primary skeletal muscle cells. Primary cultured stroma-vascular fraction (SVF) obtained from vastus lateralis muscle biopsies of middle-aged obese subjects was immunoseparated (magnetic beads or flow cytometry). The characteristics and/or metabolic phenotype of CD56(+), CD56(-) and CD56(-)CD15(+) cellular fractions were investigated by complementary approaches (flow cytometry, cytology, quantitative PCR and metabolic assays). The effects of conditioned media from CD56(-)CD15(+) cells differentiated into adipocytes on insulin action and signaling in human primary myotubes was also examined. Our data indicate that CD56(+) and CD56(-) cellular fractions isolated from cultured SVF of human muscle contain two distinct committed progenitors: CD56(+) cells (that is, satellite cells) as myogenic progenitors and CD15(+) cells as adipogenic progenitors, respectively. CD56(-)CD15(+)-derived adipocytes display the phenotype and metabolic properties of white adipocytes. Secretions of CD56(-)CD15(+) cells differentiated into functional white adipocytes reduced insulin-mediated non-oxidative glucose disposal (P=0.0002) and insulin signaling. Using in-vitro models, we show for the first time that secretions of skeletal muscle adipocytes are able to impair insulin action and signaling of muscle fibers. This paracrine effect could explain, at least in part, the negative association between high levels of IMAT and insulin sensitivity in obesity and aging.

Defective Natriuretic Peptide Receptor Signaling in Skeletal Muscle Links Obesity to Type 2 Diabetes.

Circulating natriuretic peptide (NP) levels are reduced in obesity and predict the risk of type 2 diabetes (T2D). Since skeletal muscle was recently shown as a key target tissue of NP, we aimed to investigate muscle NP receptor (NPR) expression in the context of obesity and T2D. Muscle NPRA correlated positively with whole-body insulin sensitivity in humans and was strikingly downregulated in obese subjects and recovered in response to diet-induced weight loss. In addition, muscle NP clearance receptor (NPRC) increased in individuals with impaired glucose tolerance and T2D. Similar results were found in obese diabetic mice. Although no acute effect of brain NP (BNP) on insulin sensitivity was observed in lean mice, chronic BNP infusion improved blood glucose control and insulin sensitivity in skeletal muscle of obese and diabetic mice. This occurred in parallel with a reduced lipotoxic pressure in skeletal muscle due to an upregulation of lipid oxidative capacity. In addition, chronic NP treatment in human primary myotubes increased lipid oxidation in a PGC1α-dependent manner and reduced palmitate-induced lipotoxicity. Collectively, our data show that activation of NPRA signaling in skeletal muscle is important for the maintenance of long-term insulin sensitivity and has the potential to treat obesity-related metabolic disorders.

Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion

ObjectiveCircadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion.MethodsWe established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock.ResultsBioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption.ConclusionsOur study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

143. Off-Target Analysis of a rAAV-U7snRNA Vector Used for the Treatment of Duchenne Patients By Exon Skipping

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1 male in 5000 and caused by mutations in the gene encoding for the Dystrophin protein. So far, there are no strongly effective treatments but gene-based therapies are currently being developed. Among them, the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in shorter in-frame transcripts that are translated into functionally active Dystrophin. Exon skipping can be achieved using modified U7-small nuclear RNA (snRNA) in which a therapeutic antisense oligonucleotide (AO) is cloned. Such optimized U7snRNA, called U7.OPT, can be packaged in recombinant vectors derived from Adeno-Associated Virus (rAAV), allowing a long-lasting repair of the dystrophin messenger after one single administration.We recently published the high ability of a rAAV8.U7.OPT vector to transduce the skeletal muscle but also the liver and restore the open reading frame of the dystrophin transcript in GRMD dogs muscles injected in one forelimb. We are now moving forward to a phase I/II clinical trial in DMD patients treatable by skipping of the exon 53 of the dystrophin messenger.Because an abundant presence of the therapeutic U7.OPT RNA can in theory lead to partial hybridization to non-targeted messenger(s) with unwanted side effects, we looked for possible off-target events. Primary human myotubes and hepatocytes, untransduced or transduced with our clinical rAAV.U7.OPT vector, served as our in vitro model system. The skipping of exon 53 of the human dystrophin messenger upon vector transduction was validated. In silico analysis using the miRanda algorithm identified some gene candidates expressed in liver and muscle, i.e. messengers which could be the target of our dystrophin-specific AO. Three of them were analyzed by RT-PCR and RTqPCR and showed not detectable exon skipping events or differential expression. Other candidates are currently analyzed. Simultaneously, further investigation included a full transcriptomic analysis by RNA sequencing comparing the samples obtained from untransduced vs transduced primary cells. Preliminary results revealed differential gene expression patterns between the two types of samples. We are now exploring the respective impact of the rAAV transduction itself vs the AO effect per se to finally identity potential off-target events.The complete data will be presented and discussed in the context of our future clinical trial in DMD patients.This project is supported by AFM (Association Francaise contre les Myopathies) and ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Merieux and supported by research and innovation aid from the French public agency, BpiFrance.

Intramyocellular Triacylglycerol is Associated with Peroxisomal Biogenesis in Skeletal Muscle from Lean and Obese Humans

Obesity is associated with elevated levels of lipids (intramyocellular triacylglycerols; IMTG) and reductions in mitochondrial fatty acid oxidation in skeletal muscle, both which are associated with insulin resistance. Known predominately for their actions in liver, peroxisomes are subcellular compartments essential for lipid disposal by chain‐shortening very long‐ and long‐chain fatty acids to acyl‐carnitines, permitting CPT‐1 independent entry into the mitochondria for oxidation. We hypothesized that peroxisome proliferation in human skeletal muscle occurs as an adaptive response to elevated lipid supply and accumulation (IMTG) which could facilitate mitochondrial fatty acid oxidation. Assays for the peroxisomal biogenesis marker PEX19 protein content and IMTG were assessed using vastus lateralis from obese and lean humans. Peroxisomal membrane protein 70 (PMP70) was measured after lipid incubation of human skeletal muscle primary myotubes. Results: 1. A significant relationship between tissue IMTG and PEX19 was identified in human skeletal muscle tissue (P&lt;0.05) but not % body fat or BMI 2) PEX19 protein content was elevated (P&lt;0.05) in tissues from obese individuals with high IMTG and 3) An increase in peroxisomal PMP70 was observed (P&lt;0.05) with high fat incubation of human primary myotubes.ConclusionThis is the first report associating lipid oversupply, IMTG accumulation, and peroxisomal biogenesis in human skeletal muscle suggesting a functional‐adaptive role for peroxisomes in mitochondrial lipid oxidation and obesity.

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.

Increased pyruvate dehydrogenase kinase expression in cultured myotubes from obese and diabetic individuals.

To investigate the mechanisms of impairments in oxidative metabolism in obese and diabetic (T2DM) skeletal muscle, this study analysed the adaptive expression of genes involved in fatty acid (FA) oxidation and mitochondrial biogenesis in primary myotubes treated with elevated FAs. Muscle samples from obese or obese T2DM donors were stored or processed into human primary skeletal muscle myotubes, which were treated for 6h with a saturated (palmitic acid) or a monounsaturated (oleic acid) FA with or without a polyunsaturated FA (eicosapentaenoic acid: EPA). Real-time PCR analysis was used to determine mRNA expression. Basal pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression in whole muscle samples from obese and T2DM subjects was increased compared to lean (P<0.05; n=13-20/group). In obese- and T2DM-derived myotubes, oleic acid treatment alone and in combination with EPA increased PDK4 mRNA expression compared to control (P<0.05; n=7/group), whereas palmitic acid alone and in combination with EPA only increased PDK4 mRNA in T2DM-derived myotubes compared to control (P<0.05; n=7/group). EPA alone did not alter mRNA expression of PDK4. These findings show that FAs induce the expression of PDK4 mRNA, which was increased in myotubes cultured from obese and T2DM donors. This persistent difference in PDK4 expression, present after culturing, suggests a fundamental alteration in the FA-mediated gene expression. This may in turn translate to differences in the regulation of oxidative substrate flux to impact on insulin sensitivity.

Involvement of oxidative stress, Nuclear Factor kappa B and the Ubiquitin proteasomal pathway in dysferlinopathy

AimsDysferlinopathies are autosomal recessive neuromuscular disorders arising from mutations of the protein dysferlin that preferentially affect the limbs which waste and weaken. The pathomechanisms of the diseases are not known and effective treatment is not available. Although free radicals and upstream signaling by the redox sensitive transcription factor, NF-κB, in activation of the ubiquitin pathway are shown to occur in several muscle wasting disorders, their involvement in dysferlinopathy is not known. This study analyzed the role of oxidative stress, NF-κB and the ubiquitin pathway in dysferlinopathic muscle and in dysferlin knockdown human myoblasts and myotubes. Main methodsFourteen dysferlinopathic muscle biopsies and 8 healthy control muscle biopsies were analyzed for oxidative stress, NF-κB activation and protein ubiquitinylation and human primary myoblasts and myotubes knocked down for dysferlin were studied for their state of oxidative stress. Key findingsDysferlinopathic muscle biopsies showed NF-κB p65 signaling induced protein ubiquitinylation in response to oxidative stress. Dysferlin knock down primary muscle cell cultures confirmed that oxidative stress is induced in the absence of dysferlin in muscle. SignificanceAnti-oxidants that also inhibit nitrosative stress and NF-κB activation, might prove to be of therapeutic benefit in slowing the progression of muscle wasting that occurs with dysferlinopathy.

Dietary nitrate reduces resting metabolic rate: a randomized, crossover study in humans

Nitrate, which is an inorganic anion abundant in vegetables, increases the efficiency of isolated human mitochondria. Such an effect might be reflected in changes in the resting metabolic rate (RMR) and formation of reactive oxygen species. The bioactivation of nitrate involves its active accumulation in saliva followed by a sequential reduction to nitrite, nitric oxide, and other reactive nitrogen species. We studied effects of inorganic nitrate, in amounts that represented a diet rich in vegetables, on the RMR in healthy volunteers. In a randomized, double-blind, crossover study, we measured the RMR by using indirect calorimetry in 13 healthy volunteers after a 3-d dietary intervention with sodium nitrate (NaNO₃) or a placebo (NaCl). The nitrate dose (0.1 mmol · kg⁻¹ · d⁻¹) corresponded to the amount in 200-300 g spinach, beetroot, lettuce, or other vegetable that was rich in nitrate. Effects of direct nitrite exposure on cell respiration were studied in cultured human primary myotubes. The RMR was 4.2% lower after nitrate compared with placebo administration, and the change correlated strongly to the degree of nitrate accumulation in saliva (r² = 0.71). The thyroid hormone status, insulin sensitivity, glucose uptake, plasma concentration of isoprostanes, and total antioxidant capacity were unaffected by nitrate. The administration of nitrite to human primary myotubes acutely inhibited respiration. Dietary inorganic nitrate reduces the RMR. This effect may have implications for the regulation of metabolic function in health and disease.

BRCA1 is a novel regulator of metabolic function in skeletal muscle

Breast cancer type 1 (BRCA1) susceptibility protein is expressed across multiple tissues including skeletal muscle. The overall objective of this investigation was to define a functional role for BRCA1 in skeletal muscle using a translational approach. For the first time in both mice and humans, we identified the presence of multiple isoforms of BRCA1 in skeletal muscle. In response to an acute bout of exercise, we found increases in the interaction between the native forms of BRCA1 and the phosphorylated form of acetyl-CoA carboxylase. Decreasing BRCA1 content using a shRNA approach in cultured primary human myotubes resulted in decreased oxygen consumption by the mitochondria and increased reactive oxygen species production. The decreased BRCA1 content also resulted in increased storage of intracellular lipid and reduced insulin signaling. These results indicate that BRCA1 plays a critical role in the regulation of metabolic function in skeletal muscle. Collectively, these data reveal BRCA1 as a novel target to consider in our understanding of metabolic function and risk for development of metabolic-based diseases.

Exercise-mimicking treatment fails to increase Fndc5 mRNA & irisin secretion in primary human myotubes

Irisin, myokine secreted by skeletal muscle, was suggested to mediate some of exercise health benefits via “browning” of white adipose tissue. However, mounting evidence contradicts the regulatory role of exercise for muscle irisin production/secretion in humans. Thus, we explored the direct effect of exercise-mimicking treatment on irisin in human primary muscle cells in vitro. Human primary muscle cell cultures were established from lean, obese prediabetic and type-2-diabetic individuals. Complex metabolic phenotyping included assessment of insulin sensitivity (euglycemic hyperinsulinemic clamp) and adiposity content&distribution (MRI&MRS). In vitro exercise-mimicking treatment (forskolin+ionomycin) was delivered in 1-h pulse/day during differentiation. Fndc5 mRNA (qRT-PCR) and secreted irisin (ELISA) were determined in cells and media. Exercise-mimicking treatment more than doubled Pgc1α mRNA in differentiated muscle cells. Nevertheless, Fndc5 mRNA was reduced by 18% and irisin in media by 20%. Moreover, Fncd5 mRNA was increased in myotubes derived from individuals with type-2-diabetes, independent on exercise-mimicking treatment. Fndc5 mRNA in cells was positively related to fasting glycemia (p=0.0001) and negatively to whole-body insulin sensitivity (p<0.05). Collectively, our data do not support the role of exercise-related signaling pathways in irisin regulation in human skeletal muscle and confirm our previous observations on increased Fndc5 expression in muscle cells from individuals with type-2-diabetes.

Skeletal Muscle Perilipin 3 and Coatomer Proteins Are Increased following Exercise and Are Associated with Fat Oxidation

Lipid droplet-associated proteins such as perilipin 3 (PLIN3) and coatomer GTPase proteins (GBF1, ARF1, Sec23a, and ARFRP1) are expressed in skeletal muscle but little is known so far as to their regulation of lipolysis. We aimed here to explore the effects of lipolytic stimulation in vitro in primary human myotubes as well as in vivo following an acute exercise bout. In vitro lipolytic stimulation by epinephrine (100 μM) or by a lipolytic cocktail (30 μM palmitate, 4 μM forskolin, and 0.5 μM ionomycin, PFI) resulted in increases in PLIN3 protein content. Coatomer GTPases such as GBF1, ARF1, Sec23a, and ARFRP1 also increased in response to lipolytic stimuli. Furthermore, a long duration endurance exercise bout (20 males; age 24.0±4.5 y; BMI 23.6±1.8 kg/m2) increased PLIN3 protein in human skeletal muscle (p = 0.03) in proportion to ex vivo palmitate oxidation (r = 0.45, p = 0.04) and whole body in vivo fat oxidation (r = 0.52, p = 0.03). Protein content of ARF1 was increased (p = 0.04) while mRNA expression was increased for several other coatomers (GBF1, ARF1, and Sec23a, all p<0.05). These data provide novel observational insight into the possible relationships between lipolysis and PLIN3 along with these coatomoer GTPase proteins in human skeletal muscle.

Defects in TLR3 expression and RNase L activation lead to decreased MnSOD expression and insulin resistance in muscle cells of obese people

Obesity is associated with chronic low-grade inflammation and oxidative stress that blunt insulin response in its target tissues, leading to insulin resistance (IR). IR is a characteristic feature of type 2 diabetes. 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. Interestingly, some obese people stay insulin-sensitive and metabolically healthy. With the aim of understanding this difference and identifying the mechanisms responsible for insulin sensitivity maintenance/IR development during obesity, we explored the role of the latent endoribonuclease (RNase L) in skeletal muscle cells. RNase L is a regulator of innate immunity, of double-stranded RNA sensors and of toll-like receptor (TLR) 4 signaling. It is regulated during inflammation by interferons and its activity is dependent on its binding to 2-5A, an oligoadenylate synthesized by oligoadenylate synthetases (OAS). Increased expression of RNase L or downregulation of its inhibitor (RLI) improved insulin response in mouse myogenic C2C12 cells and in primary human myotubes from normal-weight subjects treated with palmitate, a saturated free fatty acid (FFA) known to induce inflammation and oxidative stress via TLR4 activation. While RNase L and RLI levels remained unchanged, OAS level was decreased in primary myotubes from insulin-resistant obese subjects (OB-IR) compared with myotubes from insulin-sensitive obese subjects (OB-IS). TLR3 and mitochondrial manganese superoxide dismutase (MnSOD) were also underexpressed in OB-IR myotubes. Activation of RNase L by 2-5A transfection allowed to restore insulin response, OAS, MnSOD and TLR3 expression in OB-IR myotubes. Due to low expression of OAS, OB-IR myotubes present a defect in RNase L activation and TLR3 regulation. Consequently, MnSOD level is low and insulin sensitivity is reduced. These results support that RNase L activity limits FFA/obesity-induced impairment of insulin response in muscle cells via TLR3 and MnSOD expression.