C2C12 Mouse Skeletal Muscle Cells Research Articles

Genome-wide transcriptome analysis reveals that Bisphenol A activates immune responses in skeletal muscle

Cumulative human exposure to the environmental toxin, bisphenol A (BPA), has raised important health concerns in recent decades. However, the direct genomic regulation of BPA in skeletal muscles and its clinical significance are poorly understood. Therefore, we conducted a genome-wide transcriptome analysis after daily oral administration of BPA at the lowest observed adverse-effect level (LOAEL, 50 mg/kg) in male mice for six weeks to explore the gene-expression regulations in skeletal muscle induced by BPA. The primary Gene Ontology terms linked to BPA-dependent, differentially expressed genes at LOAEL comprised adaptive-immune response, positive regulation of T cell activation, and immune system process. The gene-set enrichment analysis disclosed increased complement-associated genes [complement components 3 (C3) and 4B, complement factor D, complement receptor 2, and immunoglobulin lambda constant 2] in the group administered with BPA, with a false-discovery rate of <0.05. Subsequent validation analysis conducted in BPA-fed animal skeletal muscle tissue and in vitro experiments confirmed that BPA induced immune activation, as evidenced by increased levels of C3 and C4α proteins in mice, C2C12 myoblasts, and mouse skeletal muscle cells. In addition, BPA markedly upregulated the transcription of tumor necrosis factor-α (Tnfα) in C2C12 myoblasts and mouse skeletal muscle cells, which was substantially inhibited by 5z-7-oxozeanol and parthenolide, providing further evidence of BPA-induced inflammation in muscle cells. Our bioinformatics and subsequent animal and in vitro validations demonstrate that BPA can activate inflammation in skeletal muscle, which could be a risk factor underlying chronic muscle weakness and wastage.

Soy conglycinin amyloid fibril and chitosan complex scaffold for cultivated meat application

This study addresses the critical need in cultivated meat production for suitable scaffolds that support the growth and differentiation of muscle cells. We explore the synergy of soy conglycinin (7S) amyloid protein fibrils and chitosan to create three-dimensional (3D) porous scaffolds, specifically designed for this application. By varying the molecular weight and deacetylation degrees of chitosan, we employed a controlled temperature water annealing method to produce these complex scaffolds. The formation mechanism of the 7S-chitosan scaffolds was elucidated through molecular simulation and Fourier-transform infrared (FTIR) analysis. Characterization of the scaffolds revealed pore sizes within the 50–250 μm range, porosities from 71% to 78%, and compressive moduli varying between 4.8 and 15.3 kPa, dependent on the chitosan's deacetylation degree and molecular weight. Notably, these scaffolds maintained structural integrity in refrigerated Phosphate Buffered Saline (PBS) for up to three months and effectively supported the growth and differentiation of C2C12 mouse skeletal muscle cells without necessitating additional cell adhesion enhancements. Their mechanical properties, comparable to natural muscle tissue, and compatibility with high-temperature sterilization processes, underscore their potential as innovative solutions in the field of cultivated meat production.

Irisin Improves Differentiation through Preserved Mitochondrial Function in Mouse C2C12 Skeletal Muscle Cells

PURPOSEIn response to muscle contraction, irisin, a myokine released by skeletal muscle and white adipose tissue (WAT), is cleaved from the transmembrane protein fibronectin type III domain‐containing protein 5 (FNDC5) and has been shown to upregulate expression of uncoupling of protein 1 (UCP 1), inducing the browning of WAT. Prior research has demonstrated the protective role of irisin against insulin resistance, ischemic injury, and inflammation. We aimed to investigate the effects of Irisin on the differentiation of skeletal muscle myocytes.METHODSMouse C2C12 skeletal muscle cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM) until cell confluency reached 60%. Once the plates reached 60% confluency, 2% Horse Serum was added to the medium to induce differentiation. The cells were incubated at 37 degrees C. and 5% CO2 for three days. Cells were then incubated in medium containing irisin at differing concentrations: 0 ng/mL, 10 ng/mL, and 20 ng/mL for an additional three days. Significance was established at p &lt; 0.05. The number of myotubes were compared days three and six in each group. Immunofluorescent staining of actinin, myosin heavy chain, myocyte enhancing factor‐2C (MEF2C), and mitochondrial capture was performed for each group.RESULTSThe average number of myotubes per field for the no‐irisin group was 8.95 (+/‐ 7.85), compared to 13.6 myotubes (+/‐ 11.02 myotubes) in the 10 ng/mL irisin group and 30.5 myotubes (+/‐ 9.92 myotubes) in the 20 ng/mL irisin group. There was no statistical significance between the no‐irisin group and the 10 ng/mL group, however the number of myotubes in the 20 ng/mL group was statistically greater than both the no‐irisin group and the 10 ng/mL irisin group at p &lt; 0.05.CONCLUSIONTreatment of C2C12 myocytes with irisin at 20 ng/mL concentration induced increased myotube differentiation. Irisin appears to play a significant role in muscle regeneration and growth.

Monotropein Improves Dexamethasone-Induced Muscle Atrophy via the AKT/mTOR/FOXO3a Signaling Pathways.

The present study aimed to investigate the effects of monotropein (MON) on improving dexamethasone (DEX)-induced muscle atrophy in mice and C2C12 mouse skeletal muscle cells. The body weights, grip strengths, and muscle weights of mice were assessed. The histological change in the gastrocnemius tissues was also observed through H&E staining. The expression of myosin heavy chain (MyHC), muscle ring finger 1 (MuRF1), and muscle atrophy F-box (Atrogin1) and the phosphorylation of AKT, mTOR, and FOXO3a in the muscle tissues of mice and C2C12 myotubes were analyzed using Western blotting. MON improved muscle atrophy in mice and C2C12 myotubes by regulating catabolic states via the AKT/mTOR/FOXO3a signaling pathways, and enhanced muscle function by the increases of muscle mass and strength in mice. This suggests that MON could be used for the prevention and treatment of muscle atrophy in patients.

Fimasartan Ameliorates Deteriorations in Glucose Metabolism in a High Glucose State by Regulating Skeletal Muscle and Liver Cells.

PurposeSince diabetes and hypertension frequently occur together, it is thought that these conditions may have a common pathogenesis. This study was designed to evaluate the anti-diabetic function of the anti-hypertensive drug fimasartan on C2C12 mouse skeletal muscle and HepG2 human liver cells in a high glucose state.Materials and MethodsThe anti-diabetic effects and mechanism of fimasartan were identified using Western blot, glucose uptake tests, oxygen consumption rate (OCR) analysis, adenosine 5′-triphosphate (ATP) enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining for diabetic biomarkers in C2C12 cells. Protein biomarkers for glycogenolysis and glycogenesis were evaluated by Western blotting and ELISA in HepG2 cells.ResultsThe protein levels of phosphorylated 5′ adenosine monophosphate-activated protein kinase (p-AMPK), p-AKT, insulin receptor substrate-1 (IRS-1), and glucose transporter type 4 (Glut4) were elevated in C2C12 cells treated with fimasartan. These increases were reversed by peroxisome proliferator-activated receptor delta (PPARδ) antagonist. ATP, OCR, and glucose uptake were increased in cells treated with 200 µM fimasartan. Protein levels of glycogen phosphorylase, glucose synthase, phosphorylated glycogen synthase, and glycogen synthase kinase-3 (GSK-3) were decreased in HepG2 cells treated with fimasartan. However, these effects were reversed following the addition of the PPARδ antagonist GSK0660.ConclusionIn conclusion, fimasartan ameliorates deteriorations in glucose metabolism as a result of a high glucose state by regulating PPARδ in skeletal muscle and liver cells.

New Intracellular Peptide Derived from Hemoglobin Alpha Chain Induces Glucose Uptake and Reduces Blood Glycemia.

Intracellular peptides were shown to derive from proteasomal degradation of proteins from mammalian and yeast cells, being suggested to play distinctive roles both inside and outside these cells. Here, the role of intracellular peptides previously identified from skeletal muscle and adipose tissues of C57BL6/N wild type (WT) and neurolysin knockout mice were investigated. In differentiated C2C12 mouse skeletal muscle cells, some of these intracellular peptides like insulin activated the expression of several genes related to muscle contraction and gluconeogenesis. One of these peptides, LASVSTVLTSKYR (Ric4; 600 µg/kg), administrated either intraperitoneally or orally in WT mice, decreased glycemia. Neither insulin (10 nM) nor Ric4 (100 µM) induced glucose uptake in adipose tissue explants obtained from conditional knockout mice depleted of insulin receptor. Ric4 (100 µM) similarly to insulin (100 nM) induced Glut4 translocation to the plasma membrane of C2C12 differentiated cells, and increased GLUT4 mRNA levels in epididymal adipose tissue of WT mice. Ric4 (100 µM) increased both Erk and Akt phosphorylation in C2C12, as well as in epididymal adipose tissue from WT mice; Erk, but not Akt phosphorylation was activated by Ric4 in tibial skeletal muscle from WT mice. Ric4 is rapidly degraded in vitro by WT liver and kidney crude extracts, such a response that is largely reduced by structural modifications such as N-terminal acetylation, C-terminal amidation, and substitution of Leu8 for DLeu8 (Ac-LASVSTV[DLeu]TSKYR-NH2; Ric4-16). Ric4-16, among several Ric4 derivatives, efficiently induced glucose uptake in differentiated C2C12 cells. Among six Ric4-derivatives evaluated in vivo, Ac-LASVSTVLTSKYR-NH2 (Ric4-2; 600 µg/kg) and Ac-LASVSTV[DLeu]TSKYR (Ric4-15; 600 µg/kg) administrated orally efficiently reduced glycemia in a glucose tolerance test in WT mice. The potential clinical application of Ric4 and Ric4-derivatives deserves further attention.

Schisandrin C Affects Glucose-Stimulated Insulin Secretion in Pancreatic β-Cells and Glucose Uptake in Skeletal Muscle Cells.

The aim of our study was to investigate the effect of three lignans (schisandrol A, schisandrol B, and schisandrin C) on insulin secretion in rat INS-1 pancreatic β-cells and glucose uptake in mouse C2C12 skeletal muscle cells. Schisandrol A and schisandrin C enhanced insulin secretion in response to high glucose levels with no toxic effects on INS-1 cells. The effect of schisandrin C was superior to that of gliclazide (positive control), a drug commonly used to treat type 2 diabetes (T2D). In addition, western blot analysis showed that the expression of associated proteins, including peroxisome proliferator-activated receptor γ (PPARγ), pancreatic and duodenal homeobox 1 (PDX-1), phosphatidylinositol 3-kinase (PI3K), Akt, and insulin receptor substrate-2 (IRS-2), was increased in INS-1 cells after treatment with schisandrin C. In addition, insulin secretion effect of schisandrin C were enhanced by the Bay K 8644 (L-type Ca2+ channel agonist) and glibenclamide (K+ channel blocker), were abolished by the nifedipine (L-type Ca2+ channel blocker) and diazoxide (K+ channel activator). Moreover, schisandrin C enhanced glucose uptake with no toxic effects on C2C12 cells. Western blot analysis showed that the expression of associated proteins, including insulin receptor substrate-1 (IRS-1), AMP-activated protein kinase (AMPK), PI3K, Akt, glucose transporter type 4 (GLUT-4), was increased in C2C12 cells after treatment with schisandrin C. Schisandrin C may improve hyperglycemia by enhancing insulin secretion in pancreatic β-cells and improving glucose uptake into skeletal muscle cells. Our findings may provide evidence that schisandrin C may be beneficial in devising novel anti-T2D strategies.

Reconstitution of muscle cell microtubule organization in vitro.

Skeletal muscle differentiation occurs as muscle precursor cells (myoblasts) elongate and fuse to form multinucleated syncytial myotubes in which the highly-organized actomyosin sarcomeres of muscle fibers assemble. Although less well characterized, the microtubule cytoskeleton also undergoes dramatic rearrangement during myogenesis. The centrosome-nucleated microtubule array found in myoblasts is lost as the nuclear membrane acquires microtubule nucleating activity and microtubules emerge from multiple sites in the cell, eventually rearranging into a grid-like pattern in myotubes. In order to characterize perinuclear microtubule organization using a biochemically tractable system, we isolated nuclei from mouse C2C12 skeletal muscle cells during the course of differentiation and incubated them in cytoplasmic extracts prepared from eggs of the frog Xenopus laevis. Whereas centrosomes associated with myoblast nuclei gave rise to radial microtubule arrays in extracts, myotube nuclei produced a sun-like pattern with microtubules transiently nucleating from the entire nuclear envelope. Perinuclear microtubule growth was suppressed by inhibition of Aurora A kinase or by degradation of RNA, treatments that also inhibited microtubule growth from sperm centrosomes. Myotube nuclei displayed microtubule motor-based movements leading to their separation, as occurs in myotubes. This in vitro assay therefore recapitulates key features of microtubule organization and nuclear movement observed during muscle cell differentiation.

Cerium Oxide Nanoparticle Administration to Skeletal Muscle Cells under Different Gravity and Radiation Conditions.

For their remarkable biomimetic properties implying strong modulation of the intracellular and extracellular redox state, cerium oxide nanoparticles (also termed “nanoceria”) were hypothesized to exert a protective role against oxidative stress associated with the harsh environmental conditions of spaceflight, characterized by microgravity and highly energetic radiations. Nanoparticles were supplied to proliferating C2C12 mouse skeletal muscle cells under different gravity and radiation levels. Biological responses were thus investigated at a transcriptional level by RNA next-generation sequencing. Lists of differentially expressed genes (DEGs) were generated and intersected by taking into consideration relevant comparisons, which led to the observation of prevailing effects of the space environment over those induced by nanoceria. In space, upregulation of transcription was slightly preponderant over downregulation, implying involvement of intracellular compartments, with the majority of DEGs consistently over- or under-expressed whenever present. Cosmic radiations regulated a higher number of DEGs than microgravity and seemed to promote increased cellular catabolism. By taking into consideration space physical stressors alone, microgravity and cosmic radiations appeared to have opposite effects at transcriptional levels despite partial sharing of molecular pathways. Interestingly, gene ontology denoted some enrichment in terms related to vision, when only effects of radiations were assessed. The transcriptional regulation of mitochondrial uncoupling protein 2 in space-relevant samples suggests perturbation of the intracellular redox homeostasis, and leaves open opportunities for antioxidant treatment for oxidative stress reduction in harsh environments.

Potential Antidiabetic Effects of Seaweed Extracts by Upregulating Glucose Utilization and Alleviating Inflammation in C2C12 Myotubes

Seaweed is known to have various health-promoting effects. However, the mechanisms underlying seaweed’s antidiabetic effects remain unclear. We investigated the potential antidiabetic effects of seaweed water extracts and further examined their mechanism(s) using C2C12 mouse skeletal muscle cells. Briefly, we screened the physiochemical properties of seven seaweed extracts by comparing the antioxidant and α-glucosidase inhibitory effects. Among them, three seaweed extracts, Undaria pinnatifida sporophyll (UPS), Codium fragile (CF), and Gracilaria verrucosa (GV), were selected for further testing of their possible antidiabetic effects with underlying mechanisms using C2C12 myotubes. Consistent with the superior α-glucosidase inhibition of the three seaweed extracts, the extracts also enhanced glucose utilization in myotubes compared to the control. The upregulated glucose uptake by the seaweed extracts was reversed by an AMP-activated protein kinase (AMPK) inhibitor, compound C, in the UPS- and CF-treated groups. Furthermore, all three seaweed extracts significantly promoted the phosphorylation of AMPK which was completely blocked by pretreating with compound C. In addition, all three extracts reduced lipopolysaccharide-simulated TNF-α production in C2C12 cells. Our results demonstrated that all three seaweed extracts exhibited antidiabetic properties through not only the inhibition of glucose absorption but also the promotion of glucose utilization. Moreover, the regulation of inflammatory cytokine production by the extracts suggested their potential anti-inflammatory property which might play a critical role in protecting insulin sensitivity in a chronic inflammatory state. Taken together, UPS, CF, and GV are a promising source to modulate the glucose absorption and utilization in muscle cells partially via the AMPK pathway.

Comparative multi-omics analyses reveal differential expression of key genes relevant for parasitism between non-encapsulated and encapsulated Trichinella

Genome assemblies provide a powerful basis of comparative multi-omics analyses that offer insight into parasite pathogenicity, host-parasite interactions, and invasion biology. As a unique intracellular nematode, Trichinella consists of two clades, encapsulated and non-encapsulated. Genomic correlation of the distinct differences between the two clades is still unclear. Here, we report an annotated draft reference genome of non-encapsulated Trichinella, T. pseudospiralis, and perform comparative multi-omics analyses with encapsulated T. spiralis. Genome and methylome analyses indicate that, during Trichinella evolution, the two clades of Trichinella exhibit differential expansion and methylation of parasitism-related multi-copy gene families, especially for the DNase II members of the phospholipase D superfamily and Glutathione S-transferases. Further, methylome and transcriptome analyses revealed divergent key excretory/secretory (E/S) genes between the two clades. Among these key E/S genes, TP12446 is significantly more expressed across three life stages in T. pseudospiralis. Overexpression of TP12446 in the mouse C2C12 skeletal muscle cell line could induce inhibition of myotube formation and differentiation, further indicating its key role in parasitism of T. pseudospiralis. This multi-omics study provides a foundation for further elucidation of the mechanism of nurse cell formation and immunoevasion, as well as the identification of pharmacological and diagnostic targets of trichinellosis.

Zinc Modulates Several Transcription-Factor Regulated Pathways in Mouse Skeletal Muscle Cells.

Zinc is an essential metal ion involved in many biological processes. Studies have shown that zinc can activate several molecules in the insulin signalling pathway and the concomitant uptake of glucose in skeletal muscle cells. However, there is limited information on other potential pathways that zinc can activate in skeletal muscle. Accordingly, this study aimed to identify other zinc-activating pathways in skeletal muscle cells to further delineate the role of this metal ion in cellular processes. Mouse C2C12 skeletal muscle cells were treated with insulin (10 nM), zinc (20 µM), and the zinc chelator TPEN (various concentrations) over 60 min. Western blots were performed for the zinc-activation of pAkt, pErk, and pCreb. A Cignal 45-Reporter Array that targets 45 signalling pathways was utilised to test the ability of zinc to activate pathways that have not yet been described. Zinc and insulin activated pAkt over 60 min as expected. Moreover, the treatment of C2C12 skeletal muscle cells with TPEN reduced the ability of zinc to activate pAkt and pErk. Zinc also activated several associated novel transcription factor pathways including Nrf1/Nrf2, ATF6, CREB, EGR1, STAT1, AP-1, PPAR, and TCF/LEF, and pCREB protein over 120 min of zinc treatment. These studies have shown that zinc’s activity extends beyond that of insulin signalling and plays a role in modulating novel transcription factor activated pathways. Further studies to determine the exact role of zinc in the activation of transcription factor pathways will provide novel insights into this metal ion actions.

High phosphate induces skeletal muscle atrophy and suppresses myogenic differentiation by increasing oxidative stress and activating Nrf2 signaling

Skeletal muscle wasting represents both a common phenotype of aging and a feature of pathological conditions such as chronic kidney disease (CKD). Although both clinical data and genetic experiments in mice suggest that hyperphosphatemia accelerates muscle wasting, the underlying mechanism remains unclear. Here, we showed that inorganic phosphate (Pi) dose-dependently decreases myotube size, fusion index, and myogenin expression in mouse C2C12 skeletal muscle cells. These changes were accompanied by increases in reactive oxygen species (ROS) production and Nrf2 and p62 expression, and reductions in mitochondrial membrane potential (MMP) and Keap1 expression. Inhibition of Pi entry, cytosolic ROS production, or Nrf2 activation reversed the effects of high Pi on Nrf2, p62, and myogenin expression. Overexpression of Nrf2 respectively increased and decreased the promoter activity of p62-Luc and myogenin-Luc reporters. Analysis of nuclear extracts from gastrocnemius muscles from mice fed a high-Pi (2% Pi) diet showed increased Nrf2 phosphorylation in sham-operated and 5/6 nephrectomized (CKD) mice, and both increased p62 phosphorylation and decreased myogenin expression in CKD mice. These data suggest that high Pi suppresses myogenic differentiation in vitro and promotes muscle atrophy in vivo through oxidative stress-mediated protein degradation and both canonical (ROS-mediated) and non-canonical (p62-mediated) activation of Nrf2 signaling.

Fibronectin type III domain containing four promotes differentiation of C2C12 through the Wnt/β-catenin signaling pathway.

Fibronectin type III domain containing 4 (FNDC4) belongs to the fibronectin type III domain containing protein family. FNDC5, which is highly homologous to FNDC4, can promote the differentiation of cardiac cells. We aimed to investigate the role of FNDC4 in the differentiation of C2C12 mouse skeletal muscle cells. Western blotting and immunofluorescence analysis showed that FNDC4 gradually increased with the differentiation of C2C12. Muscle injury repair experiments indicated that FNDC4 may promote the repair of injured muscles. When FNDC4 was either overexpressed or knocked down, the expression of desmin and myogenin myogenic marker molecules followed that of FNDC4, suggesting that FNDC4 can influence the differentiation of C2C12. In addition, immunoprecipitation results showed that FNDC4 can interact with the Wnt/β-catenin signaling pathway receptor low-density lipoprotein receptor-related protein 6 (LRP6), and that β-catenin levels in the nucleus decreased after knocking down FNDC4. Exogenous addition of FNDC4 protein could not restore the blocking of differentiation due to inhibition of both Wnt/β-catenin signal transduction and LRP6 activity via the β-catenin inhibitor XAV-939. Overall, our findings indicate that FDNC4 can influence the differentiation of C2C12 by activating Wnt/β-catenin signal transduction.

Beef extract supplementation promotes myoblast proliferation and myotube growth in C2C12 cells.

We previously determined that the intake of beef extract for 4weeks increases skeletal muscle mass in rats. Thus, this study aimed to clarify whether beef extract has a hypertrophic effect on muscle cells and to determine the signaling pathway underlying beef extract-induced myotube hypertrophy. We assessed the effects of beef extract supplement on mouse C2C12 skeletal muscle cell proliferation and differentiation and myotube growth. In addition, the phosphorylation of Akt, ERK1/2, and mTOR following beef extract supplementation was examined by western blotting. Furthermore, the bioactive constituents of beef extract were examined using amino acid analysis and dialysis. In the proliferative stage, beef extract significantly increased myoblast proliferation. In the differentiation stage, beef extract supplementation did not promote myoblast differentiation. In mature myotubes, beef extract supplementation increased myotube diameter and promoted protein synthesis. Although Akt and ERK1/2 levels were not affected, beef extract supplementation increased mTOR phosphorylation, which indicated that the mTOR pathway mediates beef extract-induced myotube hypertrophy. The hypertrophic activity was observed in fractions of > 7000Da. Beef extract promoted C2C12 myoblast proliferation and C2C12 myotube hypertrophy. Myotube hypertrophy was potentially induced by mTOR activation and active components in beef extract were estimated to be > 7000Da.

Comparative profiling of skeletal muscle models reveals heterogeneity of transcriptome and metabolism

Rat L6, mouse C2C12, and primary human skeletal muscle cells (HSMCs) are commonly used to study biological processes in skeletal muscle, and experimental data on these models are abundant. However, consistently matched experimental data are scarce, and comparisons between the different cell types and adult tissue are problematic. We hypothesized that metabolic differences between these cellular models may be reflected at the mRNA level. Publicly available data sets were used to profile mRNA levels in myotubes and skeletal muscle tissues. L6, C2C12, and HSMC myotubes were assessed for proliferation, glucose uptake, glycogen synthesis, mitochondrial activity, and substrate oxidation, as well as the response to in vitro contraction. Transcriptomic profiling revealed that mRNA of genes coding for actin and myosin was enriched in C2C12, whereas L6 myotubes had the highest levels of genes encoding glucose transporters and the five complexes of the mitochondrial electron transport chain. Consistently, insulin-stimulated glucose uptake and oxidative capacity were greatest in L6 myotubes. Insulin-induced glycogen synthesis was highest in HSMCs, but C2C12 myotubes had higher baseline glucose oxidation. All models responded to electrical pulse stimulation-induced glucose uptake and gene expression but in a slightly different manner. Our analysis reveals a great degree of heterogeneity in the transcriptomic and metabolic profiles of L6, C2C12, or primary human myotubes. Based on these distinct signatures, we provide recommendations for the appropriate use of these models depending on scientific hypotheses and biological relevance.

The Effects of Non‐Small Cell Lung Carcinoma Conditioned Media on Skeletal Muscle Cell Proliferation and Metabolism

Lung cancer is the second most common cancer and one of the largest causes of cancer‐related mortality worldwide. 80–85% of all new lung cancer diagnoses are Non‐Small Cell Lung Carcinoma (NSCLC), suggesting a great need to continue elucidating the metabolic mechanisms surrounding this prevalent disease. Due to the oncogenic environment surrounding NSCLC growth, factors released into the environment could have a direct impact on other tissues. In particular, skeletal muscle, a leading insulin‐sensitive and metabolically active tissue, is subject to wasting in many cancer patients. The purpose of this study was to determine how NSCLC conditioned media affects C2C12 skeletal muscle cells proliferation and metabolism. C2C12 mouse skeletal muscle cells were grown for 24 hours and serum‐starved overnight with serum‐free media to induce quiescence. Cells were treated with equal volumes of either fresh growth media or conditioned media from proliferating NSCLC cells. C2C12 myoblast proliferation was assessed every 24 hours for 3 days via an MTT assay using 3‐(4,5‐Dimethyltiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide. Proliferating C2C12 myoblasts treated with fresh growth media or conditioned media from NSCLC cells showed significant proliferation (p=0.0016) over 72 hours. Treatment with NSCLC conditioned media did not induce any significant increases in myoblast proliferation when compared to control C2C12 cells. In conclusion, these data suggest that C2C12 myoblasts did not respond adversely to NSCLC conditioned media. Experiments are ongoing to investigate alterations in metabolic and myogenic signaling pathways, as well as extended time courses.Support or Funding InformationThis work was supported by the Targeted Research Internal Seed Program, JSM.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

4-hydroxy-3-methoxy cinnamic acid accelerate myoblasts differentiation on C2C12 mouse skeletal muscle cells via AKT and ERK 1/2 activation

BackgroundThe dietary intake of plant-based supplements has a vital role in human health and development. However, the actions of secondary plant metabolites on cell growth, differentiation and their signaling mechanisms are still unclear. PurposeIn this study, we aim to investigate the C2C12 myoblast cells proliferation and differentiation by 4-hydroxy-3-methoxy cinnamic acid (=HMCA, ferulic acid) in a dose-dependent manner and to reveal its underlying mechanism of action. Methods: The effect of HMCA on C2C12 cell proliferation and differentiation were evaluated by expression of BMP's marker genes (-2, -4, -6, -7) and related myogenic proteins were analyzed by quantitative PCR and western blot techniques, respectively. ResultsThe in vitro findings confirmed that the HMCA upregulates BMPs (including BMP-2, -4, -6, and-7), gene expression in C2C12 skeletal muscle cells. Exposure to the lower dose of HMCA caused a significantly greater induction of myogenic differentiation than the higher dose during three- and six-day treatments. Further, the C2C12 myogenic differentiation signaling proteins MyoD, myogenin, JAK-1, -2, -3, STAT -2, -3, AMPK-α, ERK(1/2), and AKT were more preferentially activated by HMCA exposure cells than by untreated models. Thus, the experiment with inhibitors revealed that the HMCA induced muscle cell proliferation and differentiation through AKT and ERK (1/2) signaling cascades. Also, HMCA enhanced the C2C12 muscle cell differentiation protein markers such as myogenin, AKT and ERK (1/2) significantly (p ≤ 0.05) at day three in chemical inhibitors of LY 294002 and PD98056 treated samples. ConclusionThe HMCA has a significant effect on muscle cell differentiation through ERK(1/2) and AKT signaling activation. Also, the HMCA promotes C2C12 muscle cell proliferation and differentiation via activation of osteogenic genes and myogeneic protein markers. Therefore, this study suggests that the natural phenolic compound HMCA has a potent function in muscle cell proliferation, differentiation, and development.

12,23-Dione dammarane triterpenes from Gynostemma longipes and their muscle cell proliferation activities via activation of the AMPK pathway

The aging population is growing rapidly around the world and there is also an increase in sarcopenia, which is characterized by decreased muscle mass, strength and function in the elderly population. AMP-activated protein kinase (AMPK) is an essential sensor and regulator of glucose, lipid and energy metabolism throughout the body. Previous studies have shown that AMPK pathway activation by regular exercise and appropriate dietary control have beneficial effects on skeletal muscle. In the process of searching for new AMPK activators from medicinal plants, we isolated and characterized eight new 12,23-dione dammarane triterpenoids (1–3 and 5–9), as well as one known gypentonoside A from Gynostemma longipes. When all isolates were tested for their AMPK activation activities, seven compounds (1 and 3–8) were significantly activated AMPK phosphorylation in mouse C2C12 skeletal muscle cell lines. Since G. longipes contained a significant amount of active compound 1 (over 2.08% per dried raw plant), it suggested the potential of this plant to be developed as a functional food or botanical drug that enhances muscle proliferation by activating AMPK signaling pathways.

WNK1 regulates skeletal muscle cell hypertrophy by modulating the nuclear localization and transcriptional activity of FOXO4

With-no-lysine (K) (WNK) kinases, which are mutated in the inherited form of hypertension pseudohypoaldosteronism type II, are essential regulators of membrane ion transporters. Here, we report that WNK1 positively regulates skeletal muscle cell hypertrophy via mediating the function of the pro-longevity transcription factor forkhead box protein O4 (FOXO4) independent of the conventional WNK signaling pathway linking SPS/STE20-related proline-alanine–rich kinase (SPAK)/oxidative stress response kinase 1 (OSR1) to downstream effector ion transporters. Small interfering RNA (siRNA)-mediated silencing of WNK1, but not SPAK/OSR1 kinases, induced myotube atrophy and remarkable increases in the mRNA expression of the muscle atrophy ubiquitin ligases MAFbx and MuRF1 in C2C12 mouse skeletal muscle cells. WNK1 silencing also increased FOXO4 nuclear localization, and co-transfection of Foxo4-targeted siRNA completely reversed the myotube atrophy and upregulation of atrogene transcription induced by WNK1 silencing. We further illustrated that WNK1 protein abundance in skeletal muscle was increased by chronic voluntary wheel running exercise (hypertrophic stimulus) and markedly decreased by adenine-induced chronic kidney disease (atrophic stimulus) in mice. These findings suggest that WNK1 is involved in the physiological regulation of mammalian skeletal muscle hypertrophy and atrophy via interactions with FOXO4. The WNK1-FOXO4 axis may be a potential therapeutic target in human diseases causing sarcopenia.