Cytoprotective propensity of green tea polyphenols against citrinin-induced skeletal-myotube damage in C2C12 cells

The mycotoxin citrinin, is produced by several species of Penicillium, Aspergillus and Monascus, and is capable of inducing cytotoxicity, oxidative stress and apoptosis. The aim of the present study was to investigate the effect of citrinin in mouse skeletal muscle cells (C2C12) and to overcome the cellular adverse effects by supplementing green tea extract (GTE) rich in polyphenols. C2C12 myoblasts were differentiated to myotubes and were exposed to citrinin in a dose dependent manner (0–100 µM) for 24 h and IC₅₀ value was found to be 100 µM that resulted in decreased cell viability, increased LDH leakage and compromised membrane integrity. Mitochondrial membrane potential loss, increased accumulation of intracellular ROS and sub G1 phase of cell cycle was observed. To ameliorate the cytotoxic effects of CTN, C2C12 cells were pretreated with GTE (20, 40, 80 µg/ml) for 2 h followed by citrinin (100 µM) treatment for 24 h. GTE pretreatment combated citrinin-induced cytotoxicity and oxidative stress. GTE at 40 and 80 µg/ml significantly promoted cell survival and upregulated antioxidant enzyme activities (CAT, SOD, GPx) and endogenous antioxidant GSH, while the gene and protein expression levels were significantly restored through its effective antioxidant mechanism. Present study results suggested the antioxidant properties of GTE as a herbal source in ameliorating the citrinin-induced oxidative stress.     

Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle

The uncoupling protein-3 (UCP3) is a mitochondrial protein expressed mainly in skeletal muscle. Among several hypotheses for its physiological function, UCP3 has been proposed to prevent excessive production of reactive oxygen species. In the present study, we evaluated the effect of an oxidative stress induced by hyperoxia on UCP3 expression in mouse skeletal muscle and C2C12 myotubes. We found that the hyperoxia-mediated oxidative stress was associated with a 5-fold and 3-fold increase of UCP3 mRNA and protein levels, respectively, in mouse muscle. Hyperoxia also enhanced reactive oxygen species production and UCP3 mRNA expression in C2C12 myotubes. Our findings support the view that both in vivo and in vitro UCP3 may modulate reactive oxygen species production in response to an oxidative stress.     

Prevention of hydrogen peroxide-induced oxidative stress in HDF cells by peptides derived from seaweed pipefish, Syngnathus schlegeli

Two new peptides derived from seaweed pipefish Syngnathus schlegeli, SPP-1(QLGNLGV) and SPP-2 (SVMPVVA) were assessed for their ability to prevent hydrogen peroxide induced oxidative stress in human dermal fibroblasts (HDFs). Both peptides showed a significant hydroxyl radical scavenging activity when tested by ESR technique. And also the peptides effectively suppressed the hydrogen peroxide induced ROS production and DNA damage in HDF cells. Furthermore the two peptides increase the protein expression levels of intracellular antioxidant enzymes SOD1, GSH and catalase in hydrogen peroxide stressed HDF cells. At the cellular signaling level, SPPs block the NF-κB activation which may lead to the reduction of oxidative stress mediated damage of HDF cells. These finding indicate the potential antioxidant effects of SPPs as response to H₂O₂ stimulation.     

Characterization of an Acute Muscle Contraction Model Using Cultured C2C12 Myotubes

A cultured C2C12 myotube contraction system was examined for application as a model for acute contraction-induced phenotypes of skeletal muscle. C2C12 myotubes seeded into 4-well rectangular plates were placed in a contraction system equipped with a carbon electrode at each end. The myotubes were stimulated with electric pulses of 50 V at 1 Hz for 3 ms at 997-ms intervals. Approximately 80% of the myotubes were observed to contract microscopically, and the contractions lasted for at least 3 h with electrical stimulation. Calcium ion (Ca²⁺) transient evoked by the electric pulses was detected fluorescently with Fluo-8. Phosphorylation of protein kinase B/Akt (Akt), 5′ AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38), and c-Jun NH2-terminal kinase (JNK)1/2, which are intracellular signaling proteins typically activated in exercised/contracted skeletal muscle, was observed in the electrically stimulated C2C12 myotubes. The contractions induced by the electric pulses increased glucose uptake and depleted glycogen in the C2C12 myotubes. C2C12 myotubes that differentiated after exogenous gene transfection by a lipofection or an electroporation method retained their normal contractile ability by electrical stimulation. These findings show that our C2C12 cell contraction system reproduces the muscle phenotypes that arise in vivo (exercise), in situ (hindlimb muscles in an anesthetized animal), and in vitro (dissected muscle tissues in incubation buffer) by acute muscle contraction, demonstrating that the system is applicable for the analysis of intracellular events evoked by acute muscle contraction.     

Antioxidant activity of polyphenolic extracts of Ichnocarpus frutescens

In the present study antioxidant activities by (1,1-diphenyl-2-picrylhydrazyl radical (DPPH), hydrogen peroxide, hydroxyl radical inhibition, hemolysis by hydrogen peroxide assay, reducing power and total antioxidant activities of polyphenolic extract of Ichnocarpus frutescens leaves were investigated. The flavonoids and total polyphenolic contents of the extract were also determined using standard methods. Phytochemical analyses revealed the presence of flavonoids, polyphenols, anthocyanins and simple phenolic acids. The results of antioxidant activities of polyphenol extract obtained by different in vitro methods were varied depending on the method used. Nevertheless, polyphenol extract showed significant inhibitory activities in all in vitro reactive oxygen species scavenging, might be attributed due to the high level of polyphenolic compound. Also, these various antioxidant activities were compared to α-tocopherol and l-ascorbic acid as reference antioxidant compounds. These findings provide evidence that the polyphenolic extract of I. frutescens is a natural source of antioxidant against oxidative damage.     

Induction of Oxidative Stress and Antioxidant Activity by Hydrogen Peroxide Treatment in Tolerant and Susceptible Wheat Genotypes

We induced an oxidative stress by means of exogenous hydrogen peroxide in two wheat genotypes, C 306 (tolerant to water stress) and Hira (susceptible to water stress), and investigated oxidative injury and changes in antioxidant enzymes activity. H₂O₂ treatment caused chlorophyll degradation, lipid peroxidation, decreased membrane stability and activity of nitrate reductase. Hydrogen peroxide increased the activity of antioxidant enzymes, glutathione reductase and catalase. These effects increased with increasing H₂O₂ concentrations. However, no change was observed in the activity of superoxide dismutase and proline accumulation.     

Differential thiol oxidation of the signaling proteins Akt,PTEN or PP2A determines whether Akt phosphorylation is enhanced or inhibited by oxidative stress in C2C12 myotubes derived from skeletal muscle

Oxidative stress, caused by excess reactive oxygen species (ROS), has been hypothesized to cause or exacerbate skeletal muscle wasting in a number of diseases and chronic conditions. ROS, such as hydrogen peroxide, have the potential to affect signal transduction pathways such as the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt pathway that regulates protein synthesis. Previous studies have found contradictory outcomes for the effect of ROS on the PI3K/Akt signaling pathway, where oxidative stress can either enhance or inhibit Akt phosphorylation. The apparent contradictions could reflect differences in experimental cell types or types of ROS treatments. We replicate both effects in myotubes of cultured skeletal muscle C2C12 cells, and show that increased oxidative stress can either inhibit or enhance Akt phosphorylation. This differential response could be explained: thiol oxidation of Akt, but not the phosphatases PTEN or PP2A, caused a decline in Akt phosphorylation; whereas the thiol oxidation of Akt, PTEN and PP2A increased Akt phosphorylation. These observations indicate that a more complete understanding of the effects of oxidative stress on a signal transduction pathway comes not only from identifying the proteins susceptible to thiol oxidation, but also their relative sensitivity to ROS.     

Curcumin ameliorates CKD-induced mitochondrial dysfunction and oxidative stress through inhibiting GSK-3β activity

Curcumin has been reported to attenuate muscle atrophy. However, the underling mechanism remains unclear. The aim of this study was to investigate whether curcumin could improve chronic kidney disease (CKD)-induced muscle atrophy and mitochondrial dysfunction by inhibiting glycogen synthase kinase-3β (GSK-3β) activity. The sham and CKD mice were fed either a control diet or an identical diet containing 0.04% curcumin for 12 weeks. The C2C12 myotubes were treated with H₂O₂ in the presence or absence of curcumin. In addition, wild-type and muscle-specific GSK-3β knockout (KO) CKD model mice were made by 5/6 nephrectomy, and the sham was regarded as control. Curcumin could exert beneficial effects, including weight maintenance and improved muscle function, increased mitochondrial biogenesis, alleviated mitochondrial dysfunction by increasing adenosine triphosphate levels, activities of mitochondrial electron transport chain complexes and basal mitochondrial respiration and suppressing mitochondrial membrane potential. In addition, curcumin modulated redox homeostasis by increasing antioxidant activity and suppressed mitochondrial oxidative stress. Moreover, the protective effects of curcumin had been found to be mediated via inhibiting GSK-3β activity in vitro and in vivo. Importantly, GSK-3β KO contributed to improved mitochondrial function, attenuated mitochondrial oxidative damage and augmented mitochondrial biogenesis in muscle of CKD. Overall, this study suggested that curcumin alleviated CKD-induced mitochondrial oxidative damage and mitochondrial dysfunction via inhibiting GSK-3β activity in skeletal muscle.     

Genetic modification of the manganese superoxide dismutase/glutathione peroxidase 1 pathway influences intracellular ROS generation in quiescent, but not contracting, skeletal muscle cells

Increased amounts of reactive oxygen species (ROS) are generated by skeletal muscle during contractile activity, but their intracellular source is unclear. The oxidation of 2',7'-dichlorodihydrofluorescein (DCFH) was examined as an intracellular probe for reactive oxygen species in skeletal muscle myotubes derived from muscles of wild-type mice and mice that were heterozygous knockout for manganese superoxide dismutase (Sod2(+/-)), homozygous knockout for glutathione peroxidase 1 (GPx1(-/-)), or MnSOD transgenic overexpressors (Sod2-Tg). Myoblasts were stimulated to fuse and loaded with DCFH 5-7 days later. Intracellular DCF epifluorescence was measured and myotubes were electrically stimulated to contract for 15 min. Quiescent myotubes with decreased MnSOD or GPx1 showed a significant increase in the rate of DCFH oxidation whereas those with increased MnSOD did not differ from wild type. Following contractions, myotubes from all groups showed an equivalent increase in DCF fluorescence. Thus the oxidation of DCFH in quiescent skeletal muscle myotubes is influenced by the content of enzymes that regulate mitochondrial superoxide and hydrogen peroxide content. In contrast, the increase in DCFH oxidation following contractions was unaffected by reduced or enhanced MnSOD or absent GPx1, indicating that reactive oxygen species produced by contractions were predominantly generated by nonmitochondrial sources.     

Nox2 Mediates Skeletal Muscle Insulin Resistance Induced by a High Fat Diet

Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wild-type mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H₂O₂ decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H₂O₂ in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H₂O₂. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.     

Chemerin‐induced mitochondrial dysfunction in skeletal muscle

Chemerin is a novel adipocyte‐derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin‐mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real‐time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3‐I to LC3‐II and higher expression levels of Beclin1 and autophagy‐related protein‐5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine‐like receptor 1 or the use of mitochondria‐targeting antioxidant Mito‐TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin‐stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin‐induced mitochondrial autophagy likely through an AKT‐FoxO3α‐dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.     

Modulation of redox status and calcium handling by extremely low frequency electromagnetic fields in C2C12 muscle cells: A real-time,single-cell approach

The biological effects of electric and magnetic fields, which are ubiquitous in modern society, remain poorly understood. Here, we applied a single-cell approach to study the effects of short-term exposure to extremely low frequency electromagnetic fields (ELF-EMFs) on muscle cell differentiation and function using C2C12 cells as an in vitro model of the skeletal muscle phenotype. Our focus was on markers of oxidative stress and calcium (Ca²⁺) handling, two interrelated cellular processes previously shown to be affected by such radiation in other cell models. Collectively, our data reveal that ELF-EMFs (1) induced reactive oxygen species production in myoblasts and myotubes with a concomitant decrease in mitochondrial membrane potential; (2) activated the cellular detoxification system, increasing catalase and glutathione peroxidase activities; and (3) altered intracellular Ca²⁺homeostasis, increasing the spontaneous activity of myotubes and enhancing cellular reactivity to a depolarizing agent (KCl) or an agonist (caffeine) of intracellular store Ca²⁺channels. In conclusion, our data support a possible link between exposure to ELF-EMFs and modification of the cellular redox state, which could, in turn, increase the level of intracellular Ca²⁺and thus modulate the metabolic activity of C2C12 cells.     

L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism

Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF) or growth factors and nutrients (HEPES buffered saline; HBS). Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control) or L-alanine (negative control) and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37%) and myotube diameter (HBS: +18%, SF: +29%). L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxidative stress (H₂O₂) induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.     

Selective androgen receptor modulator,S42 has anabolic and anti-catabolic effects on cultured myotubes

We previously identified a novel selective androgen receptor modulator, S42, that does not stimulate prostate growth but has a beneficial effect on lipid metabolism. S42 also increased muscle weight of the levator ani in orchiectomized Sprague–Dawley rats. These findings prompted us to investigate whether S42 has a direct effect on cultured C2C12 myotubes. S42 significantly lowered expression levels of the skeletal muscle ubiquitin ligase (muscle atrophy-related gene), atrogin1 and Muscle RING-Finger Protein 1(MuRF1) in C2C12 myotubes, as determined by real time PCR. Phosphorylation of p70 S6 kinase (p70S6K), an essential factor for promoting protein synthesis in skeletal muscle, was significantly increased by S42 to almost the same extent as by insulin, but this was significantly prevented by treatment with rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). However, phosphorylation of Akt, upstream regulator of mTORC1, was not changed by S42. S42 did not increase insulin-like growth factor 1 (Igf1) mRNA levels in C2C12 myotubes. These results suggest that S42 may have an anabolic effect through activation of mTORC1–p70S6K signaling, independent of IGF-1-Akt signaling and may exert an anti-catabolic effect through inhibition of the degradation pathway in cultured C2C12 myotubes.     

Preventive effect of dietary quercetin on disuse muscle atrophy by targeting mitochondria in denervated mice

Quercetin is a major dietary flavonoid in fruits and vegetables. We aimed to clarify the preventive effect of dietary quercetin on disuse muscle atrophy and the underlying mechanisms. We established a mouse denervation model by cutting the sciatic nerve in the right leg (SNX surgery) to lack of mobilization in hind-limb. Preintake of a quercetin-mixed diet for 14 days before SNX surgery prevented loss of muscle mass and atrophy of muscle fibers in the gastrocnemius muscle (GM). Phosphorylation of Akt, a key phosphorylation pathway of suppression of protein degradation, was activated in the quercetin-mixed diet group with and without SNX surgery. Intake of a quercetin-mixed diet suppressed the generation of hydrogen peroxide originating from mitochondria and elevated mitochondrial peroxisome proliferator-activated receptor-γ coactivator 1α mRNA expression as well as NADH dehydrogenase 4 expression in the GM with SNX surgery. Quercetin and its conjugated metabolites reduced hydrogen peroxide production in the mitochondrial fraction obtained from atrophied muscle. In C2C12 myotubes, quercetin reached the mitochondrial fraction. These findings suggest that dietary quercetin can prevent disuse muscle atrophy by targeting mitochondria in skeletal muscle tissue through protecting mitochondria from decreased biogenesis and reducing mitochondrial hydrogen peroxide release, which can be related to decreased hydrogen peroxide production and/or improvements on antioxidant capacity of mitochondria.     

PEP-1–SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages

Sirtuin 2 (SIRT2), a member of the sirtuin family of proteins, plays an important role in cell survival. However, the biological function of SIRT2 protein is unclear with respect to inflammation and oxidative stress. In this study, we examined the protective effects of SIRT2 on inflammation and oxidative stress-induced cell damage using a cell permeative PEP-1–SIRT2 protein. Purified PEP-1–SIRT2 was transduced into RAW 264.7 cells in a time- and dose-dependent manner and protected against lipopolysaccharide- and hydrogen peroxide (H₂O₂)-induced cell death and cytotoxicity. Also, transduced PEP-1–SIRT2 significantly inhibited the expression of cytokines as well as the activation of NF-κB and mitogen-activated protein kinases (MAPKs). In addition, PEP-1–SIRT2 decreased cellular levels of reactive oxygen species (ROS) and of cleaved caspase-3, whereas it elevated the expression of antioxidant enzymes such as MnSOD, catalase, and glutathione peroxidase. Furthermore, topical application of PEP-1–SIRT2 to 12-O-tetradecanoylphorbol 13-acetate-treated mouse ears markedly inhibited expression levels of COX-2 and proinflammatory cytokines as well as the activation of NF-κB and MAPKs. These results demonstrate that PEP-1–SIRT2 inhibits inflammation and oxidative stress by reducing the levels of expression of cytokines and ROS, suggesting that PEP-1–SIRT2 may be a potential therapeutic agent for various disorders related to ROS, including skin inflammation.     

Activity of antioxidant enzymes and physiological responses in ark shell,Scapharca broughtonii,exposed to thermal and osmotic stress: Effects on hemolymph and biochemical parameters

Changes in water temperature and salinity are responsible for a variety of physiological stress responses in aquatic organisms. Stress induced by these factors was recently associated with enhanced reactive oxygen species (ROS) generation, which caused oxidative damage. In the present study, we investigated the time-related effects of changes in water temperature and salinity on mRNA expression and the activities of antioxidant enzymes (SOD and CAT) and lipid peroxidation (LPO) in the gills and digestive glands of the ark shell, Scapharca broughtonii. To investigate physiological responses, hydrogen peroxide (H₂O₂), lysozyme activity, aspartate aminotransferase (AspAT), and alanine aminotransferase (AlaAT) were measured in the hemolymph. Water temperature and salinity changes significantly increased antioxidant enzyme mRNA expression and activity in the digestive glands and gills in a time-dependent manner. H₂O₂ concentrations increased significantly in the high-temperature and hyposalinity treatments. LPO, AspAT and AlaAT levels also increased significantly in a time-dependent manner, while lysozyme activity decreased. These results suggest that antioxidant enzymes play important roles in reducing oxidative stress in ark shells exposed to changes in water temperature and salinity.