Human Skeletal Muscle Myotubes Research Articles

Identifying novel small molecule integrin agonists to mimic the favourable effects of the RGD domain of IGFBP-1 to treat cardiometabolic disease

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): British Heart Foundation (BHF) 4-Year PhD Programme Background Insulin-like growth factor binding protein (IGFBP)-1 has previously been shown to be associated with reduced type 2 diabetes (T2D) progression and preferable cardiovascular profiles in patients. Our laboratory previously demonstrated that IGFBP-1 is an insulin sensitiser in mouse models, in addition to having favourable vascular effects, independently of IGFs. IGFBP-1 has an Arg-Gly-Asp (RGD) domain which is thought to facilitate the direct interaction with the heterodimeric cell-surface receptor, integrin α5β1, inducing downstream cell signalling. We then showed the positive effects of IGFBP-1 could be mimicked with an RGD-containing linear peptide, GRGDTP, however this peptide is not a suitable therapeutic as it is rapidly metabolised in vivo. To our knowledge, RGD-based therapeutics have not been explored for cardiometabolic disease. Purpose There are currently few therapeutics which have been specifically designed to address both insulin resistance and the high risk of macrovascular disease in T2D. Therefore, exploring RGD-based integrin agonists will be essential in developing new insulin sensitising therapeutics which prevent cardiovascular complications. Methods The ROCS high speed ligand-based virtual screening tool was used to screen commercially available small molecule libraries mimicking the chemical structure of the RGD sequence of IGFBP-1. Additionally, we performed in silico modelling to predict the binding interactions of these molecules with the integrin α5β1 headpiece to select candidates. We then used a LIVE/DEAD cell viability assay to initially profile any cytotoxic effects of our selected compounds in integrin-expressing human umbilical vein endothelial cells (HUVECs) and C2C12 skeletal muscle myotubes. We further assessed the effects of compounds on relevant HUVEC functions using a high-throughput wound closure assay. Results The structure of the RGD domain of IGFBP-1 can be mimicked with small molecules containing amino acid moieties. These molecules are predicted to bind with micromolar affinity to the integrin, promoting beneficial interactions for integrin agonism. We selected 17 structurally diverse small molecules with the best predicted binding interactions. Initial in vitro profiling demonstrates that the molecules are not cytotoxic. In preliminary analyses, three of the selected compounds show a trend towards inhibition of HUVEC migration and proliferation, while six show a trend towards increased migration and proliferation. Conclusion The RGD domain of IGFBP-1 can be mimicked with novel small molecules, of which we have selected 17 ligands. Our compounds do not affect cell viability in relevant cell types, so can be further profiled. Our initial findings indicate that the compounds have different functional effects on HUVECs which allows us to prioritise hits having favourable effects in the context of T2D, such as enhancing angiogenic properties of endothelial cells.

Sex-specific increases in myostatin and SMAD3 contribute to obesity-related insulin resistance in human skeletal muscle and primary human myotubes.

The purpose of the present study was to determine the effects of obesity and biological sex on myostatin expression in humans and to examine the direct effects of myostatin, SMAD2, and SMAD3 on insulin signaling in primary human skeletal muscle cells (HSkMCs). For cohort 1, 15 lean [body mass index (BMI): 22.1 ± 0.5 kg/m2; n = 8 males; n = 7 females] and 14 obese (BMI: 40.6 ± 1.4 kg/m2; n = 7 males; n = 7 females) individuals underwent skeletal muscle biopsies and an oral glucose tolerance test. For cohort 2, 14 young lean (BMI: 22.4 ± 1.9 kg/m2; n = 6 males; n = 8 females) and 14 obese (BMI: 39.3 ± 7.9 kg/m2; n = 6 males; n = 8 females) individuals underwent muscle biopsies for primary HSkMC experiments. Plasma mature myostatin (P = 0.041), skeletal muscle precursor myostatin (P = 0.048), and skeletal muscle SMAD3 (P = 0.029) were elevated in obese females compared to lean females, and plasma mature myostatin (r = 0.58, P = 0.029) and skeletal muscle SMAD3 (r = 0.56, P = 0.037) were associated with insulin resistance in females but not males. Twenty-four hours of myostatin treatment impaired insulin signaling in primary HSkMCs derived from females (P < 0.024) but not males. Overexpression of SMAD3, but not SMAD2, impaired insulin-stimulated AS160 phosphorylation in HSkMCs derived from lean females (-27%, P = 0.040), whereas silencing SMAD3 improved insulin-stimulated AS160 phosphorylation and insulin-stimulated glucose uptake (25%, P < 0.014) in HSkMCs derived from obese females. These results suggest for the first time that myostatin-induced impairments in skeletal muscle insulin signaling are sex specific and that increased body fat in females is associated with detrimental elevations in myostatin and SMAD3, which contribute to obesity-related insulin resistance.NEW & NOTEWORTHY Obesity is considered a main risk factor for the development of insulin resistance and type 2 diabetes. The present study utilizes in vivo and in vitro experiments in human skeletal muscle to demonstrate for the first time that females are inherently more susceptible to myostatin-induced insulin resistance, which is further enhanced with obesity due to increased myostatin and SMAD3 expression.

Human HDL subclasses modulate energy metabolism in skeletal muscle cells

In addition to its antiatherogenic role, HDL reportedly modulates energy metabolism at the whole-body level. HDL functionality is associated with its structure and composition, and functional activities can differ between HDL subclasses. Therefore, we studied if HDL2 and HDL3, the two major HDL subclasses, are able to modulate energy metabolism of skeletal muscle cells. Differentiated mouse and primary human skeletal muscle myotubes were used to investigate the influences of human HDL2 and HDL3 on glucose and fatty uptake and oxidation. HDL-induced changes in lipid distribution and mRNA expression of genes related to energy substrate metabolism, mitochondrial function, and HDL receptors were studied with human myotubes. Additionally, we examined the effects of apoA-I and discoidal, reconstituted HDL particles on substrate metabolism. In mouse myotubes, HDL subclasses strongly enhanced glycolysis upon high and low glucose concentrations. HDL3 caused a minor increase in ATP-linked respiration upon glucose conditioning but HDL2 improved complex I–mediated mitochondrial respiration upon fatty acid treatment. In human myotubes, glucose metabolism was attenuated but fatty acid uptake and oxidation were markedly increased by both HDL subclasses, which also increased mRNA expression of genes related to fatty acid metabolism and HDL receptors. Finally, both HDL subclasses induced incorporation of oleic acid into different lipid classes. These results, demonstrating that HDL subclasses enhance fatty acid oxidation in human myotubes but improve anaerobic metabolism in mouse myotubes, support the role of HDL as a circulating modulator of energy metabolism. Exact mechanisms and components of HDL causing the change, require further investigation.

Cancer-Cachexia-Induced Human Skeletal Muscle Myotube Degeneration Is Prevented via Cannabinoid Receptor 2 Agonism In Vitro.

Cachexia syndrome, leading to reduced skeletal muscle and fat mass, is highly prevalent in cancer patients, resulting in further negative implications for these patients. To date, there is no approved therapy for cachexia syndrome. The objective of this study was to establish an in vitro model of cancer cachexia in mature human skeletal muscle myotubes, with the intention of exploiting the cell model to assess potential cachexia therapeutics, specifically cannabinoid related drugs. Having cultured and differentiated primary human muscle myoblasts to mature myotubes, we successfully established two cancer cachexia models using conditioned media (CM) from human colon adenocarcinoma (SW480) and from non-small-cell lung carcinoma (H1299) cultured cells. The cancer-CM-induced extensive myotube degeneration, demonstrated by a significant reduction in mature myotube diameter, which progressed over the period studied. Myotube degeneration is a characteristic feature of cancer cachexia and was used in this study as an index of cachexia. Expression of cannabinoid 1 and 2 receptors (CB1R and CB2R) was confirmed in the mature human skeletal muscle myotubes. Subsequently, the effect of cannabinoid compounds on this myotube degeneration were assessed. Tetrahydrocannabinol (THC), a partial CB1R/CB2R agonist, and JWH133, a selective CB2R agonist, proved efficacious in protecting mature human myotubes from the deleterious effects of both (SW480 and H1299) cancer cachexia conditions. ART27.13, a full, peripherally selective CB1R/CB2R agonist, currently being trialled in cancer cachexia (IRAS ID 278450, REC 20/NE/0198), was also significantly protective against myotube degeneration in both (SW480 and H1299) cancer cachexia conditions. Furthermore, the addition of the CB2R antagonist AM630, but not the CB1R antagonist Rimonabant, abolished the protective effect of ART27.13. In short, we have established a convenient and robust in vitro model of cancer-induced human skeletal muscle cachexia. The data obtained using the model demonstrate the therapeutic potential of ART27.13 in cancer-induced cachexia prevention and provides evidence indicating that this effect is via CB2R, and not CB1R.

The role and mechanism of myeloperoxidase in dermatomyositis

ObjectiveDermatomyositis (DM) is the best known subtype of idiopathic inflammatory myopathies. The hallmarks of DM muscle pathology including microangiopathy, inflammatory infiltration, and perifascicular atrophy. Recent findings have revealed pathogenetic effects of myeloperoxidase (MPO) by causing oxidative damage and regulating abnormal immunity in multiple disease conditions. In this study, we aimed to explore the role of MPO in the pathogenesis of DM. MethodsThe peripheral blood mononuclear cell (PBMC) mRNA expression and DNA methylation of MPO were verified using real-time qPCR and bisulfite pyrosequencing, respectively. Plasma MPO levels were measured with enzyme-linked immunosorbent assay, and their relationships with clinical characteristics were analyzed. The expression and distribution of MPO in muscle were tested by immunofluorescence. Purified human native MPO protein was used to stimulate human dermal microvascular endothelial cells (HDMECs) and skeletal muscle myotubes. The cell viability, tube forming capacity, permeability, adhesion molecule expressions in HDMECs, and atrophy and programmed cell death pathways in myotubes were then observed. ResultsMPO gene methylation was decreased, while mRNA expression and plasma levels were increased in DM. Plasma MPO of DM patients was positively correlated with serum creatine kinase (CK). MPO mainly distributed around endomysia capillaries and perifascicular atrophy in DM muscle biopsies, and was co-localized with CD4+, CD8+ T cells and CD19+ B cells. MPO not only could influence the cell viability, tube forming capacity, permeability and expression of adhesion molecules (including ICAM 1, VCAM 1 and E-selectin) of HDMECs, but also could cause atrophy of myotubes. ConclusionsOur study disclosed, for the first time, that MPO plays an important role in promoting inflammatory infiltration and inducing muscle damage in DM patients. MPO may be a potential biomarker for DM muscle involvement and MPO targeted drugs may be promising in DM treatment.

Antidiabetic effects and mechanisms of action of γ-conglutin from lupin seeds

The glucose modulating properties of lupin have been attributed to its seed protein γ-conglutin. Here we explored the antidiabetic potential of γ-conglutin purified from lupin seeds in-vitro. To mimic the effects of an orally administered supplement, purified γ-conglutin was hydrolysed by gastrointestinal proteolytic enzymes and the resulting peptides evaluated for their antidiabetic effects in pancreatic β-cells and primary human skeletal muscle myotubes. γ-conglutin peptides did not promote insulin secretion in β-cells but elicited a potent insulin-mimetic action by activating insulin signalling pathways responsible for glycogen, protein synthesis, and glucose transport into myotubes. Additionally, the peptides potently suppressed the activity of DPP4 indicating their potential to increase the half-life of incretin hormones in circulation. These results substantiate the health benefits of consuming lupin seeds as part of a healthy diet and can drive the current market for lupins from primarily stockfeed, towards value-added lupin-based food products for human consumption.

Associations Among Adipose Tissue Immunology, Inflammation, Exosomes and Insulin Sensitivity in People With Obesity and Nonalcoholic Fatty Liver Disease

Insulin resistance is a key factor in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We evaluated the importance of subcutaneous abdominal adipose tissue (SAAT) inflammation and both plasma and SAAT-derived exosomes in regulating insulin sensitivity in people with obesity and NAFLD. Adipose tissue inflammation (macrophage and T-cell content and expression of proinflammatory cytokines), liver and whole-body insulin sensitivity (assessed using a hyperinsulinemic-euglycemic clamp and glucose tracer infusion), and 24-hour serial plasma cytokine concentrations were evaluated in 3 groups stratified by adiposity and intrahepatic triglyceride (IHTG) content: (1) lean with normal IHTG content (LEAN; N= 14); (2) obese with normal IHTG content (OB-NL; N= 28); and (3) obese with NAFLD (OB-NAFLD; N= 28). The effect of plasma and SAAT-derived exosomes on insulin-stimulated Akt phosphorylation in human skeletal muscle myotubes and mouse primary hepatocytes was assessed in a subset of participants. Proinflammatory macrophages, proinflammatory CD4 and CD8 T-cell populations, and gene expression of several cytokines in SAAT were greater in the OB-NAFLD than the OB-NL and LEAN groups. However, with the exception of PAI-1, which was greater in the OB-NAFLD than the LEAN and OB-NL groups, 24-hour plasma cytokine concentration areas-under-the-curve were not different between groups. The percentage of proinflammatory macrophages and plasma PAI-1 concentration areas-under-the-curve were inversely correlated with both hepatic and whole-body insulin sensitivity. Compared with exosomes from OB-NL participants, plasma and SAAT-derived exosomes from the OB-NAFLD group decreased insulin signaling in myotubes and hepatocytes. Systemic insulin resistance in people with obesity and NAFLD is associated with increased plasma PAI-1 concentrations and both plasma and SAAT-derived exosomes. ClinicalTrials.gov number: NCT02706262 (https://clinicaltrials.gov/ct2/show/NCT02706262).

A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength

Three-dimensional (3D) in vitro models of human skeletal muscle mimic aspects of native tissue structure and function, thereby providing a promising system for disease modeling, drug discovery or pre-clinical validation, and toxicity testing. Widespread adoption of this research approach is hindered by the lack of easy-to-use platforms that are simple to fabricate and that yield arrays of human skeletal muscle micro-tissues (hMMTs) in culture with reproducible physiological responses that can be assayed non-invasively. Here, we describe a design and methods to generate a reusable mold to fabricate a 96-well platform, referred to as MyoTACTIC, that enables bulk production of 3D hMMTs. All 96-wells and all well features are cast in a single step from the reusable mold. Non-invasive calcium transient and contractile force measurements are performed on hMMTs directly in MyoTACTIC, and unbiased force analysis occurs by a custom automated algorithm, allowing for longitudinal studies of function. Characterizations of MyoTACTIC and resulting hMMTs confirms the capability of the device to support formation of hMMTs that recapitulate biological responses. We show that hMMT contractile force mirrors expected responses to compounds shown by others to decrease (dexamethasone, cerivastatin) or increase (IGF-1) skeletal muscle strength. Since MyoTACTIC supports hMMT long-term culture, we evaluated direct influences of pancreatic cancer chemotherapeutics agents on contraction competent human skeletal muscle myotubes. A single application of a clinically relevant dose of Irinotecan decreased hMMT contractile force generation, while clear effects on myotube atrophy were observed histologically only at a higher dose. This suggests an off-target effect that may contribute to cancer associated muscle wasting, and highlights the value of the MyoTACTIC platform to non-invasively predict modulators of human skeletal muscle function.

GLP‑1 improves palmitate‑induced insulin resistance in human skeletal muscle via SIRT1 activity.

The present study investigated whether glucagon like peptide‑1 (GLP‑1) improves glucose uptake through glucose transporter type 4 (GLUT4), mediated by the activation of sirtuin 1 (SIRT1), in skeletal muscle cells with palmitate induced‑insulin resistance. The levels of glucose uptake, GLUT4, protein kinase A (PKA), and cyclic adenosine monophosphate (cAMP) were determined in human skeletal muscle myotubes (HSMMs) exposed to palmitate and GLP‑1. Then, to determine whether PKA/cAMP were downstream signals of GLP‑1, a PKA inhibitor was used. To determine whether SIRT‑1 contributes to GLP‑1 action in HSMMs with palmitate‑induced insulin resistance, the levels of peroxisome proliferator‑activated receptor γ coactivator 1α (PGC1α) deacetylation and SIRT‑1 activity were assessed using a SIRT1 inhibitor and small interfering RNA (siRNA). The phosphorylation levels of protein kinase B (Akt) and insulin receptor substrate 1 (IRS‑1) as insulin signaling pathways, were assessed in GLP‑1‑treated HSMMs exposed to palmitate. The influence of SIRT1 on the GLP‑1‑induced activation of insulin signaling pathway was determined using a SIRT1 inhibitor. GLP‑1 restored the palmitate‑induced reductions in the levels of glucose uptake, GLUT4 mRNA, GLUT4 promoter activity, and GLUT4 protein in HSMMs. PKA and cAMP, as GLP‑1 downstream signals, played a role in this process. GLP‑1 increased the deacetylation levels of PGC1α, and stimulated SIRT1 in HSMMs. Moreover, the SIRT1 inhibitor and siRNA of SIRT1 suppressed the effect of GLP‑1 on GLUT4 expression in HSMMs exposed to palmitate. The SIRT1 inhibitor also prevented the GLP‑1‑induced phosphorylation of IRS‑1 and Akt in palmitate‑treated HSMMs. The present findings suggest that in palmitate‑induced insulin‑resistant HSMM, GLP‑1 activates SIRT1 through the PKA/cAMP pathway, which in turn enhances glucose uptake through GLUT4 and the insulin signaling pathway.

Sex influences DNA methylation and gene expression in human skeletal muscle myoblasts and myotubes

BackgroundSex differences are known to impact muscle phenotypes, metabolism, and disease risk. Skeletal muscle stem cells (satellite cells) are important for muscle repair and to maintain functional skeletal muscle. Here we studied, for the first time, effects of sex on DNA methylation and gene expression in primary human myoblasts (activated satellite cells) before and after differentiation into myotubes.MethodWe used an array-based approach to analyse genome-wide DNA methylation and gene expression in myoblasts and myotubes from 13 women and 13 men. The results were followed up with a reporter gene assay.ResultsGenome-wide DNA methylation and gene expression differences between the sexes were detected in both myoblasts and myotubes, on the autosomes as well as the X-chromosome, despite lack of exposure to sex hormones and other factors that differ between sexes. Pathway analysis revealed higher expression of oxidative phosphorylation and other metabolic pathways in myoblasts from women compared to men. Oxidative phosphorylation was also enriched among genes with higher expression in myotubes from women. Forty genes in myoblasts and 9 in myotubes had differences in both DNA methylation and gene expression between the sexes, including LAMP2 and SIRT1 in myoblasts and KDM6A in myotubes. Furthermore, increased DNA methylation of LAMP2 promoter had negative effects on reporter gene expression. Five genes (CREB5, RPS4X, SYAP1, XIST, and ZRSR2) showed differential DNA methylation and gene expression between the sexes in both myoblasts and myotubes. Interestingly, differences in DNA methylation and expression between women and men were also found during differentiation (myoblasts versus myotubes), e.g., in genes involved in energy metabolism. Interestingly, more DNA methylation changes occur in women compared to men on autosomes.ConclusionAll together, we show that epigenetic and transcriptional differences exist in human myoblasts and myotubes as well as during differentiation between women and men. We believe that these intrinsic differences might contribute to sex dependent differences in muscular phenotypes.

Electrical pulse stimulation induces differential responses in insulin action in myotubes from severely obese individuals.

Exercise/exercise training can enhance insulin sensitivity through adaptations in skeletal muscle, the primary site of insulin-mediated glucose disposal; however, in humans the range of improvement can vary substantially. The purpose of this study was to determine if obesity influences the magnitude of the exercise response in relation to improving insulin sensitivity in human skeletal muscle. Electrical pulse stimulation (EPS; 24h) of primary human skeletal muscle myotubes improved insulin action in tissue from both lean and severely obese individuals, but responses to EPS were blunted with obesity. EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and increased AMP accumulation and AMPK Thr172 phosphorylation, but to a lesser degree in myotubes from the severely obese. These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise-responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects. Exercise/muscle contraction can enhance whole-body insulin sensitivity; however, in humans the range of improvements can vary substantially. In order, to determine if obesity influences the magnitude of the exercise response, this study compared the effects of electrical pulse stimulation (EPS)-induced contractile activity upon primary myotubes derived from lean and severely obese (BMI≥ 40kg/m2 ) women. Prior to muscle contraction, insulin action was compromised in myotubes from the severely obese as was evident from reduced insulin-stimulated glycogen synthesis, glucose oxidation, glucose uptake, insulin signal transduction (IRS1, Akt, TBC1D4), and insulin-stimulated GLUT4 translocation. EPS (24h) increased AMP, IMP, AMPK Thr172 phosphorylation, PGC1α content, and insulin action in myotubes of both the lean and severely obese subjects. However, despite normalizing indices of insulin action to levels seen in the lean control (non-EPS) condition, responses to EPS were blunted with obesity. EPS improved insulin signal transduction in myotubes from lean but not severely obese subjects and EPS increased AMP accumulation and AMPK Thr172 phosphorylation, but to a lesser degree in myotubes from the severely obese. These data reveal that myotubes of severely obese individuals enhance insulin action and stimulate exercise-responsive molecules with contraction, but in a manner and magnitude that differs from lean subjects.

The effect of cyanidin-3-O-β-glucoside and peptides extracted from yoghurt on glucose uptake and gene expression in human primary skeletal muscle myotubes from obese and obese diabetic participants

Incidence of type II diabetes mellitus (T2DM) is rapidly increasing worldwide. This study aimed to investigate whether cyanidin-3-O-β-glucoside (C3G), or peptides with angiotensin converting enzyme (ACE) inhibitory activity, alone or in combination, alter glucose regulation in human primary myotubes derived from obese and obese T2DM participants. In the obese group, both low and high concentration of peptides and the combination of these peptides with high C3G concentration significantly enhanced glucose uptake in the presence or absence of insulin, and high concentration of peptide alone and its combination with low C3G down-regulated the mRNA expression of angiotensin II receptor, type 1 (AGTR-1), and up-regulated the mRNA expression of insulin receptor substrate 1 (IRS-1), and glucose transporter 4 (GLUT4). However, only high peptide concentration increased glucose uptake in the absence of insulin in the obese T2DM group, and the expression of AGTR-1 was decreased with high peptide and its combinations of C3G.

1,25-Dihydroxyvitamin D promotes calcium influx in human skeletal muscle myotubes

1,25-Dihydroxyvitamin D promotes calcium influx in human skeletal muscle myotubes

Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle.

Mineralocorticoid and glucocorticoid receptors are closely related steroid hormone receptors that regulate gene expression through many of the same hormone response elements. However, their transcriptional activities and effects in skeletal muscles are largely unknown. We recently identified mineralocorticoid receptors (MR) in skeletal muscles after finding that combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and MR antagonist spironolactone was therapeutic in Duchenne muscular dystrophy mouse models. The glucocorticoid receptor (GR) agonist prednisolone is the current standard-of-care treatment for Duchenne muscular dystrophy because it prolongs ambulation, likely due to its anti-inflammatory effects. However, data on whether glucocorticoids have a beneficial or detrimental direct effect on skeletal muscle are controversial. Here, we begin to define the gene expression profiles in normal differentiated human skeletal muscle myotubes treated with MR and GR agonists and antagonists. The MR agonist aldosterone and GR agonist prednisolone had highly overlapping gene expression profiles, supporting the notion that prednisolone acts as both a GR and MR agonist that may have detrimental effects on skeletal muscles. Co-incubations with aldosterone plus either nonspecific or selective MR antagonists, spironolactone or eplerenone, resulted in similar numbers of gene expression changes, suggesting that both drugs can block MR activation to a similar extent. Eplerenone treatment alone decreased a number of important muscle-specific genes. This information may be used to develop biomarkers to monitor clinical efficacy of MR antagonists or GR agonists in muscular dystrophy, develop a temporally coordinated treatment with both drugs, or identify novel therapeutics with more specific downstream targets.

Regulation of Glucose Metabolism by Contractile Activity in Human Skeletal Muscle Myotubes from Severely Obese Individuals

IntroductionExercise/ or contractile activity can enhance insulin sensitivity and oxidative capacity in skeletal muscle. However, the effects of contractile activity in skeletal muscle from severely obese individuals still remain unestablished.PurposeWe investigated insulin‐stimulated glucose metabolism in primary skeletal muscle myotubes from severely obese individuals (BMI ≥ 40 kg/m2) in response to contractile activity induced by electrical pulse stimulation.MethodFully mature myotubes from lean (BMI: 23.1 ± 0.7 kg/m2) and obese (BMI: 44.9± 2.5 kg/m2) individuals were stimulated for 24‐hours by applying an electrical pulse at 11.5 V, 1 Hz and 2 ms. Following contraction, insulin was added to examine the combined effects of muscle contraction and insulin on anabolic signaling.ResultIn myotubes from the severely obese individuals, insulin‐stimulated glycogen synthesis and glucose oxidation were normalized to the levels seen in muscle from the lean subjects without electrical stimulation. However, responses to electrical stimulation were blunted in the severely obese vs. lean subjects in terms of the magnitude of the improvements in insulin‐stimulated glucose metabolism. In addition, electrical stimulation increased insulin‐stimulated lactate production in severely obese but not lean subjects. Electrical stimulation increased adenosine monophosphate (AMP) accumulation and AMP‐activated protein kinase (AMPK) signaling, but to a lesser degree in myotubes from the severely obese donors. Electrical stimulation provided an improvement in insulin signal transduction in lean but not severely obese individuals. Especially, sites on AS160 phosphorylation (T642, S588, S341) showed enhanced response to electrical stimulation in lean but no changes in severely obese individuals.ConclusionThese data reveal that in the skeletal muscle of severely obese individuals, contractile activity can improve insulin‐stimulated glucose metabolism independent of insulin signaling. In addition, transient increase in AMPK signaling by accumulation of AMP for contractile activity appears to play a critical role in enhancing glucose metabolism in severely obese individual in response to electrical stimulation.Support or Funding InformationDK56112

Glucocorticoids increase skeletal muscle NF-κB inducing kinase (NIK): links to muscle atrophy.

Glucocorticoids (GC) are a frontline therapy for numerous acute and chronic diseases because of their demonstrated efficacy at reducing systemic inflammation. An unintended side effect of GC therapy is the stimulation of skeletal muscle atrophy. Pathophysiological mechanisms responsible for GC‐induced skeletal muscle atrophy have been extensively investigated, and the ability to treat patients with GC without unintended muscle atrophy has yet to be realized. We have reported that a single, standard‐of‐care dose of Methylprednisolone increases in vivo expression of NF‐κB‐inducing kinase (NIK), an important upstream regulatory kinase controlling NF‐κB activation, along with other key muscle catabolic regulators such as Atrogin‐1 and MuRF1 that induce skeletal muscle proteolysis. Here, we provide experimental evidence that overexpressing NIK by intramuscular injection of recombinant human NIK via adenoviral vector in mouse tibialis anterior muscle induces a 30% decrease in the average fiber cross‐sectional area that is associated with increases in mRNA expression of skeletal muscle atrophy biomarkers MuRF1, Atrogin‐1, myostatin and Gadd45. A single injection of GC induced NIK mRNA and protein within 2 h, with the increased NIK localized to nuclear and sarcolemmal locations within muscle fibers. Daily GC injections induced skeletal muscle fore limb weakness as early as 3 days with similar atrophy of muscle fibers as observed with NIK overexpression. NIK overexpression in primary human skeletal muscle myotubes increased skeletal muscle atrophy biomarkers, while NIK knockdown significantly attenuated GC‐induced increases in NIK and Atrogin‐1. These results suggest that NIK may be a novel, previously unrecognized mediator of GC‐induced skeletal muscle atrophy.

722. AAV Capsid Evolution for Enhanced Antibody Delivery to Human Skeletal Muscle for Use in Next-Generation HIV Vaccines and Muscle Gene Therapies

Our goal is to develop an AAV vector that can transduce human skeletal muscle after intramuscular administration at levels sufficient to express therapeutic levels of antibodies with broad-spectrum protection against HIV. AAV has begun being used in passive vaccines for HIV and influenza. Yet for many indications, greater human skeletal muscle transduction is needed than has been achieved with existing AAV serotypes. To bioengineer novel capsids with unparalleled human skeletal muscle transduction, we utilized primary human skeletal muscle cells from surgical resections to screen libraries of replication-competent shuffled AAV capsids. Two screens were performed in pools of primary human skeletal muscle stem cells or myotubes from six patients. Six rounds of replicating selection were carried out and the five most highly selected variants from each screen were vectorized and validated against existing serotypes with muscle tropism (AAV1, 6 and 8). In primary human muscle stem cells, variants NP22, NP66 and NP94 had significantly increased transduction that ranged from a 65 to 284-fold improvement over AAV1, 27 to 118-fold over AAV6, and 10 to 45-fold over AAV8. In primary human myotubes, NP22, NP66 and NP94 again showed significantly increased transduction that ranged from a 13 to 464-fold improvement over AAV1, 25 to 871-fold over AAV8, and NP94 showed a 15-fold improvement over AAV6. To assess human skeletal muscle transduction capabilities in vivo, we injected xenografted humanized muscle NSG mice with shuffled or control capsids expressing luciferase by intramuscular injection (1E9/leg) and assessed transduction weekly by live imaging in time course studies over two months. AAV6 was the first to uncoat but NP66 produced the highest sustained transduction levels. To control for species-specific transduction, we performed the same time course experiment in strain- and gender-matched non-transplanted mice. Strikingly, AAV6 outperformed shuffled variants when no human muscle fibers were present, highlighting the importance of performing preclinical studies in human cells and xenograft systems whenever possible to prevent misleading results which are mouse-specific. To further demonstrate the superiority of our shuffled variants and more accurately predict eventual muscle transduction in humans, we transduced human skeletal muscle explants from surgical resections ex vivo. Four adult patients (two male and two female) had healthy skeletal muscle tissue removed for ex vivo transduction analyses with luciferase. In all 4 patients, NP22 and NP66 (and NP94 in 3/4 patients) had significantly increased transduction by live fiber luciferase imaging as well as luciferase assays on lysed fibers that ranged from a 4 to 116-fold improvement over all control serotypes. To support preclinical vaccine testing in non-human primates, we assessed transduction in rhesus macaque skeletal muscle explants ex vivo. Here again we showed that NP22 and NP66 showed a 30 to 57-fold improvement in transduction over control serotypes. Taken together, our results demonstrate that capsid variants NP22 and NP66 (and in some cases NP94), have highly significant increased human skeletal muscle fiber transduction when assessed in vitro, in vivo and ex vivo. These capsids represent powerful tools to express therapeutic quantities of human monoclonal antibodies for use in passive vaccines, or transgenes for various muscle disorders in gene therapy, specifically in humans.

Association between insulin resistance and impairment of FGF21 signal transduction in skeletal muscles.

Fibroblast growth factor (FGF) 21, was identified as a potent metabolic regulator of glucose and lipid metabolism. We investigated whether the levels and signalings of FGF21 changed in the skeletal muscle of type 2 diabetes mellitus (T2DM) patients, participants with impaired glucose tolerance (IGT), human skeletal muscle myotubes (HSMMs) under insulin-resistant conditions, and mice with diet-induced obesity (DIO). A percutaneous biopsy sample of the vastus lateralis muscle of T2DM patients, IGT subjects, and participants with normal glucose tolerance was obtained and the levels and signalings of FGF21 were assessed. We determined whether the expression and signalings of FGF21 in HSMMs altered according to palmitate concentrations and exposure time. Also, we confirmed whether changes of FGF21 signal transduction resulted in the alteration of FGF21 functions. DIO mice were treated intravenously with recombinant FGF21, and the levels and signalings of FGF21 were assessed in their soleus muscles. We checked whether or not FGF21 played a role in the gene transcription related to lipid oxidation. Levels of FGF21 increased, whereas levels of phosphorylated FGF receptor (p-FGFR), phosphorylated FGFR substrates 2α (p-FRS2α), and phosphorylated extracellular signal-regulated kinases (p-ERK) decreased in the skeletal muscle of both T2DM patients and IGT subjects. In vitro, palmitate increased the levels of FGF21 and significantly reduced the levels of β-klotho, p-FGFR, p-FRS2α, and p-ERK1/2 in HSMMs exposed to palmitate. Palmitate also decreased glucose uptake and glycogen contents of FGF21. Consistently, the levels of FGF21 were significantly higher and the levels of β-klotho and p-FGFR were lower in the DIO mice than in normal lean mice. The levels of FGF21 increased but its signal transduction and actions were impaired in skeletal muscles of T2DM patients, IGT subjects, in insulin-resistant HSMMs, and DIO mice.

Ginger extract prevents high-fat diet-induced obesity in mice via activation of the peroxisome proliferator-activated receptor δ pathway

The initiation of obesity entails an imbalance wherein energy intake exceeds expenditure. Obesity is increasing in prevalence and is now a worldwide health problem. Food-derived peroxisome proliferator-activated receptor δ (PPARδ) stimulators represent potential treatment options for obesity. Ginger (Zingiber officinale Roscoe) was previously shown to regulate the PPARγ signaling pathway in adipocytes. In this study, we investigated the antiobesity effects of ginger in vivo and the mechanism of action in vitro. Energy expenditure was increased, and diet-induced obesity was attenuated in C57BL/6J mice treated with dietary ginger extract (GE). GE also increased the number of Type I muscle fibers, improved running endurance capacity and upregulated PPARδ-targeted gene expression in skeletal muscle and the liver. 6-Shogaol and 6-gingerol acted as specific PPARδ ligands and stimulated PPARδ-dependent gene expression in cultured human skeletal muscle myotubes. An analysis of cellular respiration revealed that pretreating cultured skeletal muscle myotubes with GE increased palmitate-induced oxygen consumption rate, which suggested an increase in cellular fatty acid catabolism. These results demonstrated that sustained activation of the PPARδ pathway with GE attenuated diet-induced obesity and improved exercise endurance capacity by increasing skeletal muscle fat catabolism. 6-Shogaol and 6-gingerol may be responsible for the regulatory effects of dietary ginger on PPARδ signaling.

Improved Anabolic Signaling In Human Skeletal Muscle Myotubes Following Electrical Pulse Stimulation

Improved Anabolic Signaling In Human Skeletal Muscle Myotubes Following Electrical Pulse Stimulation