Lactobacillus gasseri BNR17 Ameliorates Dexamethasone-Induced Muscle Loss in BALB/c Mice and C2C12 Myotubes.

This study aimed to investigate the effects and mechanism of Lactobacillus gasseri BNR17, a probiotic strain isolated from human breast milk, on dexamethasone-induced muscle loss in mice and cultured myotubes. BALB/c mice were intraperitoneally injected with dexamethasone, and orally administered L. gasseri BNR17 for 21 days. L. gasseri BNR17 treatment ameliorated dexamethasone-induced decline in muscle function, as evidenced by an increase in forelimb grip strength, treadmill running time, and rotarod retention time in both female and male mice. In addition, L. gasseri BNR17 treatment significantly increased the mass of the gastrocnemius and quadriceps muscles. Dual-energy X-ray absorptiometry showed a significant increase in lean body mass and a decrease in fat mass in both whole body and hind limb after treatment with L. gasseri BNR17. It was found that L. gasseri BNR17 treatment downregulated serum myostatin level and the protein degradation pathway composed of muscle-specific ubiquitin E3 ligases, MuRF1 and MAFbx, and their transcription factor FoxO3. In contrast, L. gasseri BNR17 treatment upregulated serum insulin-like growth factor-1 level and Akt-mTOR-p70S6K signaling pathway involved in protein synthesis in muscle. As a result, L. gasseri BNR17 treatment significantly increased the levels of major muscular proteins such as myosin heavy chain and myoblast determination protein 1. Consistent with in vivo results, L. gasseri BNR17 culture supernatant significantly ameliorated dexamethasone-induced C2C12 myotube atrophy in vitro. In conclusion, L. gasseri BNR17 ameliorates muscle loss by downregulating the protein degradation pathway and upregulating the protein synthesis pathway.

Royal Jelly Enhances the Ability of Myoblast C2C12 Cells to Differentiate into Multilineage Cells.

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes.

The Lipophilic Extract from Ginkgo biloba L. Leaves Promotes Glucose Uptake and Alleviates Palmitate-Induced Insulin Resistance in C2C12 Myotubes.

Ginkgo biloba L. (ginkgo) is a widely used medicinal plant around the world. Its leaves, which have been used as a traditional Chinese medicine, are rich in various bioactive components. However, most of the research and applications of ginkgo leaves have focused on terpene trilactones and flavonol glycosides, thereby overlooking the other active components. In this study, a lipophilic extract (GL) was isolated from ginkgo leaves. This extract is abundant in lipids and lipid-like molecules. Then, its effect and potential mechanism on glucose uptake and insulin resistance in C2C12 myotubes were investigated. The results showed that GL significantly enhanced the translocation of GLUT4 to the plasma membrane, which subsequently promoted glucose uptake. Meanwhile, it increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream targets. Both knockdown of AMPK with siRNA and inhibition with AMPK inhibitor compound C reversed these effects. Additionally, GL ameliorated palmitate-induced insulin resistance by enhancing insulin-stimulated glucose uptake, increasing the phosphorylation of protein kinase B (PKB/AKT), and restoring the translocation of GLUT4 from the cytoplasm to the membrane. However, pretreatment with compound C abolished these beneficial effects of GL. In conclusion, GL enhances basal glucose uptake in C2C12 myotubes and improves insulin sensitivity in palmitate-induced insulin resistant myotubes through the AMPK pathway.

Cardioprotective effects of arjunolic acid in LPS-stimulated H9C2 and C2C12 myotubes via the My88-dependent TLR4 signaling pathway.

CONTEXT: Arjunolic acid (AA) is a triterpenoid saponin found in Terminalia arjuna (Roxb.) Wight & Arn. (Combretaceae). It exerts cardiovascular protective effects as a phytomedicine. However, it is unclear how AA exerts the effects at the molecular level. OBJECTIVE: This study investigates the cardioprotective effects of arjunolic acid (AA) via MyD88-dependant TLR4 downstream signaling marker expression. MATERIALS AND METHODS: The MTT viability assay was used to assess the cytotoxicity of AA. LPS induced in vitro cardiovascular disease model was developed in H9C2 and C2C12 myotubes. The treatment groups were designed such as control (untreated), LPS control, positive control (LPS + pyrrolidine dithiocarbamate (PDTC)-25 µM), and treatment groups were co-treated with LPS and three concentrations of AA (50, 75, and 100 µM) for 24 h. The changes in the expression of TLR4 downstream signaling markers were evaluated through High Content Screening (HCS) and Western Blot (WB) analysis. RESULTS: After 24 h of co-treatment, the expression of TLR4, MyD88, MAPK, JNK, and NF-κB markers were upregulated significantly (2-6 times) in the LPS-treated groups compared to the untreated control in both HCS and WB experiments. Evidently, the HCS analysis revealed that MyD88, NF-κB, p38, and JNK were significantly downregulated in the H9C2 myotube in the AA treated groups. In HCS, the expression of NF-κB was downregulated in C2C12. Additionally, TLR4 expression was downregulated in both H9C2 and C2C12 myotubes in the WB experiment. DISCUSSION AND CONCLUSIONS: TLR4 marker expression in H9C2 and C2C12 myotubes was subsequently decreased by AA treatment, suggesting possible cardioprotective effects of AA.

7ß-Hydroxycholesterol and 7-ketocholesterol: New oxidative stress biomarkers of sarcopenia inducing cytotoxic effects on myoblasts and myotubes.

Aging is a complex biological process which can be associated with skeletal muscle degradation leading to sarcopenia. The aim of this study consisted i) to determine the oxidative and inflammatory status of sarcopenic patients and ii) to clarify the impact of oxidative stress on myoblasts and myotubes. To this end, various biomarkers of inflammation (C-reactive protein (CRP), TNF-α, IL-6, IL-8, leukotriene B4 (LTB4)) and oxidative stress (malondialdehyde, conjugated dienes, carbonylated proteins and antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase) as well as oxidized derivatives of cholesterol formed by cholesterol autoxidation (7-ketocholesterol, 7ß-hydroxycholesterol), were analyzed. Apelin, a myokine which contributes to muscle strength, was also quantified. To this end, a case-control study was conducted to evaluate the RedOx and inflammatory status in 45 elderly subjects (23 non-sarcopenic; 22 sarcopenic) from 65 years old and higher. SARCopenia-Formular (SARC-F) and Timed Up and Go (TUG) tests were used to distinguish between sarcopenic and non-sarcopenic subjects. By using red blood cells, plasma and/or serum, we observed in sarcopenic patients an increased activity of major antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) associated with lipid peroxidation and protein carbonylation (increased level of malondialdehyde, conjugated dienes and carbonylated proteins). Higher levels of 7-ketocholesterol and 7ß-hydroxycholesterol were also observed in the plasma of sarcopenic patients. Significant differences were only observed with 7ß-hydroxycholesterol. In sarcopenic patients comparatively to non-sarcopenic subjects, significant increase of CRP, LTB4 and apelin were observed whereas similar levels of TNF-α, IL-6 and IL-8 were found. The increased plasma level of 7-ketocholesterol and 7ß-hydroxycholesterol in sarcopenic patients led us to study the cytotoxic effect of these oxysterols on undifferentiated (myoblasts) and differentiated (myotubes) murine C2C12 cells. With the fluorescein diacetate and sulforhodamine 101 assays, an induction of cell death was observed both on undifferentiated and differentiated cells: the cytotoxic effects were less pronounced with 7-ketocholesterol. In addition, IL-6 secretion was never detected whatever the culture conditions, TNF-α secretion was significantly increased on undifferentiated and differentiated C2C12 cells treated with 7-ketocholesterol- and 7ß-hydroxycholesterol, and IL-8 secretion was increased on differentiated cells. 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cell death was strongly attenuated by α-tocopherol and Pistacia lentiscus L. seed oil both on myoblasts and/or myotubes. TNF-α and/or IL-8 secretions were reduced by α-tocopherol and Pistacia lentiscus L. seed oil. Our data support the hypothesis that the enhancement of oxidative stress observed in sarcopenic patients could contribute, especially via 7ß-hydroxycholesterol, to skeletal muscle atrophy and inflammation via cytotoxic effects on myoblasts and myotubes. These data bring new elements to understand the pathophysiology of sarcopenia and open new perspectives for the treatment of this frequent age-related disease.

Vitamin D supplementation is effective for olanzapine-induced dyslipidemia.

Olanzapine is an atypical antipsychotic drug that is clinically applied in patients with schizophrenia. It increases the risk of dyslipidemia, a disturbance of lipid metabolic homeostasis, usually characterized by increased low-density lipoprotein (LDL) cholesterol and triglycerides, and accompanied by decreased high-density lipoprotein (HDL) in the serum. In this study, analyzing the FDA Adverse Event Reporting System, JMDC insurance claims, and electronic medical records from Nihon University School of Medicine revealed that a co-treated drug, vitamin D, can reduce the incidence of olanzapine-induced dyslipidemia. In the following experimental validations of this hypothesis, short-term oral olanzapine administration in mice caused a simultaneous increase and decrease in the levels of LDL and HDL cholesterol, respectively, while the triglyceride level remained unaffected. Cholecalciferol supplementation attenuated these deteriorations in blood lipid profiles. RNA-seq analysis was conducted on three cell types that are closely related to maintaining cholesterol metabolic balance (hepatocytes, adipocytes, and C2C12) to verify the direct effects of olanzapine and the functional metabolites of cholecalciferol (calcifediol and calcitriol). Consequently, the expression of cholesterol-biosynthesis-related genes was reduced in calcifediol- and calcitriol-treated C2C12 cells, which was likely to be mediated by activating the vitamin D receptor that subsequently inhibited the cholesterol biosynthesis process via insulin-induced gene 2 regulation. This clinical big-data-based drug repurposing approach is effective in finding a novel treatment with high clinical predictability and a well-defined molecular mechanism.

Chemical constituents of the Ajuga multiflora bunge and their protective effects on dexamethasone-induced muscle atrophy in C2C12 myotubes.

Ajuga multiflora Bunge is a perennial ornamental herb and has been used for the treatment of fever in Korean folk medicine. In the course of searching for protective agents associated with the potential of A. multiflora against dexamethsone (DEX)-induced muscle atrophy, a new phytoecdysteroid, 29-hydroxyprecyasterone (1), together with four known compounds (2-5), were isolated from A. multiflora. The structures of the compounds were determined by spectroscopic analyses, including 1D-, 2D-NMR and HR-MS interpretation. To elucidate the effects of obtained compounds on DEX-induced muscle atrophy, the myotubes diameter, myosin heavy chain (MyHC) positive area, and fusion index were evaluated by immunofluorescence staining. Overall, each compound treatment effectively prevented the atrophic myotubes through an increase of MyHC-positive myotubes and the number of nuclei. Particularly, the measurement of myotube diameter showed that compounds 1 and 5 treatment significantly alleviated the myotube thickness.

In vitro chemotherapy-associated muscle toxicity is attenuated with nutritional support, while treatment efficacy is retained.

PURPOSE: Muscle-wasting and treatment-related toxicities negatively impact prognosis of colorectal cancer (CRC) patients. Specific nutritional composition might support skeletal muscle and enhance treatment support. In this in vitro study we assess the effect of nutrients EPA, DHA, L-leucine and vitamin D3, as single nutrients or in combination on chemotherapy-treated C2C12-myotubes, and specific CRC-tumor cells. MATERIALS AND METHODS: Using C2C12-myotubes, the effects of chemotherapy (oxaliplatin, 5-fluorouracil, oxaliplatin+5-fluorouracil and irinotecan) on protein synthesis, cell-viability, caspase-3/7-activity and LDH-activity were assessed. Addition of EPA, DHA, L-leucine and vitamin D3 and their combination (SNCi) were studied in presence of above chemotherapies. Tumor cell-viability was assessed in oxaliplatin-treated C26 and MC38 CRC cells, and in murine and patient-derived CRC-organoids. RESULTS: While chemotherapy treatment of C2C12-myotubes decreased protein synthesis, cell-viability and increased caspase-3/7 and LDH-activity, SNCi showed improved protein synthesis and cell viability and lowered LDH activity. The nutrient combination SNCi showed a better overall performance compared to the single nutrients. Treatment response of tumor models was not significantly affected by addition of nutrients. CONCLUSIONS: This in vitro study shows protective effect with specific nutrition composition of C2C12-myotubes against chemotherapy toxicity, which is superior to the single nutrients, while treatment response of tumor cells remained.

Suppressive Effects of Turmeric Extract on Muscle Atrophy in Dexamethasone-Treated Mice and Myotubes.

Sarcopenia is the decline in skeletal muscle mass, strength, and functions, which decreases the quality of life in elderly people. This study investigated the suppressive effect of turmeric (Curcuma longa) extract (TE) on muscle atrophy in dexamethasone (DEX)-treated mice and C2C12 myotubes. DEX treatment significantly decreased the muscle weight and significantly increased Fbxo32 and Murf1 expression in mice, and these changes were suppressed by the supplementation of an AIN-93 based diet with 2% TE. A similar pattern was observed in FBXO32 and MuRF1 protein expression. In C2C12 myotubes, DEX treatment significantly increased FBXO32 and MuRF1 gene and protein expression, and these increases were significantly suppressed by TE supplementation at a concentration of 200 µg/mL. Furthermore, one of the five TE fractions, which were separated by high-performance liquid chromatography had a similar effect with TE supplementation. The present study proposes the suppressive effect of turmeric on sarcopenia.

Near-infrared light-triggered polypyrrole promotes C2C12 cell differentiation and inhibits TNF-α induced myotube atrophy.

Treatment of skeletal muscle atrophy and strengthening the muscles remain a challenge in modern medicine. Studies have shown that photobiomodulation can inhibit skeletal muscle atrophy and aid in functional recovery. Near-infrared radiation (NIR) therapy has emerged as a complementary therapy for the treatment of skeletal muscle atrophy, but its underlying mechanism remains unclear. Polypyrrole (PPy) is an organic polymer with strong near-infrared absorption, which can generate heat from absorbed NIR. In this study, MHC immunofluorescence staining was performed on C2C12 myoblasts to investigate the differentiation of C2C12 cells after NIR-triggered PPy exposure. As TNF-α-induced C2C12 myotubes were used as a model of muscular atrophy. Giemsa staining was used to determine the myotube diameter. Western blot analysis was performed to examine the proteins involved in the differentiation and atrophy of muscle cells, as well as in the Akt/P70S6K signaling pathway. PPy triggered by NIR promoted the differentiation of C2C12 cells, inhibited C2C12 myotube atrophy caused by TNF-α, and downregulated the expression levels of Atrogin-1 and MuRF 1 protein. In addition, we determined that Akt/P70S6K signaling pathway activity plays a crucial role in the therapeutic effect of NIR-triggered polypyrrole, which was further confirmed by the administration of the Akt inhibitor GDC0068. The optimal conditions for these effects were a PPy concentration of 0.125 mg/ml and NIR exposure for 80 s. We show that the photothermal effect of PPy triggered by near-infrared light can increase the beneficial effects of NIR, promote the differentiation of C2C12 cells, and improve C2C12 myotube atrophy, laying a foundation for its future clinical use.

Development of a traditional Chinese medicine-based agent for the treatment of cancer cachexia.

BACKGROUND: Despite recent advances in understanding the pathophysiology of cancer cachexia, prevention/treatment of this debilitating disease remains an unmet medical need. METHODS: We developed an integrated, multi-tiered strategy involving both in vitro and in vivo muscle atrophy platforms to identify traditional Chinese medicine (TCM)-based anti-cachectic agents. In the initial screening, we used inflammatory cytokine-induced atrophy of C2C12 myotubes as a phenotypic screening platform to assess the protective effects of TCMs. The selected TCMs were then evaluated for their abilities to protect Caenorhabditis elegans from age-related reduction of mobility and contractility, followed by the C-26 colon adenocarcinoma mouse model of cachexia to confirm the anti-muscle atrophy effects (body/skeletal muscle weights, fibre size distribution, grip strengths, and serum IL-6). Transcriptome analysis, quantitative real-time polymerase chain reaction, and immunoblotting were performed to gain understanding of the potential mechanism(s) by which effective TCM protected against C26 tumour-induced muscle atrophy. RESULTS: Of 29 widely used TCMs, Dioscorea radix (DR) and Mu Dan Pi (MDP) showed a complete protection (all P values, 0.0002) vis-à-vis C26 conditioned medium control in the myotube atrophy platform. MDP exhibited a unique ability to ameliorate age-associated decreases in worm mobility, accompanied by improved total body contractions, relative to control (P < 0.0001 and <0.01, respectively), which, however, was not noted with DR. This differential in vivo protective effect between MDP and DR was also confirmed in the C-26 mouse model. MDP at 1000 mg/kg (MDP-H) was effective in protecting body weight loss (P < 0.05) in C-26 tumour-bearing mice without changing food or water intake, accompanied by the restoration of the fibre size distribution of hindleg skeletal muscles (P < 0.0001) and the forelimb grip strength (P < 0.05). MDP-treated C-26-tumour-bearing mice were alert, showed normal posture and better body conditions, and exhibited lower serum IL-6 levels (P = 0.06) relative to vehicle control. This decreased serum IL-6 was associated with the in vitro suppressive effect of MDP (25 and 50 µg/mL) on IL-6 secretion into culture medium by C26 cells. RNA-seq analysis, followed by quantitative real-time polymerase chain reaction and/or immunoblotting, shows that MDP's anti-cachectic effect was attributable to its ability to reverse the C-26 tumour-induced re-programming of muscle homoeostasis-associated gene expression, including that of two cachexia drivers (MuRF1 and Atrogin-1), in skeletal muscles. CONCLUSIONS: All these findings suggest the translational potential of MDP to foster new strategies for the prevention and/or treatment of cachexia. The protective effect of MDP on other types of muscle atrophy such as sarcopenia might warrant investigations.

Laurel Attenuates Dexamethasone-Induced Skeletal Muscle Atrophy In Vitro and in a Rat Model.

Prevention of muscle atrophy contributes to improved quality of life and life expectancy. In this study, we investigated the effects of laurel, selected from 34 spices and herbs, on dexamethasone (DEX)-induced skeletal muscle atrophy and deciphered the underlying mechanisms. Co-treatment of C2C12 myotubes with laurel for 12 h inhibited the DEX-induced expression of intracellular ubiquitin ligases-muscle atrophy F-box (atrogin-1/MAFbx) and muscle RING finger 1 (MuRF1)-and reduction in myotube diameter. Male Wistar rats were supplemented with 2% laurel for 17 days, with DEX-induced skeletal muscle atrophy occurring in the last 3 days. Laurel supplementation inhibited the mRNA expression of MuRF1, regulated DNA damage and development 1 (Redd1), and forkhead box class O 1 (Foxo1) in the muscles of rats. Mechanistically, we evaluated the effects of laurel on the cellular proteolysis machinery-namely, the ubiquitin/proteasome system and autophagy-and the mTOR signaling pathway, which regulates protein synthesis. These data indicated that the amelioration of DEX-induced skeletal muscle atrophy induced by laurel, is mainly mediated by the transcriptional inhibition of downstream factors of the ubiquitin-proteasome system. Thus, laurel may be a potential food ingredient that prevents muscle atrophy.

Comparison of the Effects of Inorganic or Amino Acid-Chelated Zinc on Mouse Myoblast Growth in vitro and Growth Performance and Carcass Traits in Growing-Finishing Pigs.

This study aimed to investigate the effects of the supplementation of different sources of zinc on mouse myoblast growth in vitro and the growth performance and carcass traits in growing-finishing pigs. In the in vitro trial, 25 or 75 mM zinc sulfate (ZnSO4), methionine-chelated zinc (ZnMet), and glycine-chelated zinc (ZnGly) were co-cultured with the myoblast during proliferation and differentiation. The results showed that the amino acid-chelated zinc supplementation, especially ZnMet, enhances cell proliferation and differentiation in mouse myoblast, and regulates the distribution in S and G2/M phases (P < 0.05). Meanwhile, the protein expression levels of the mammalian target of rapamycin pathways were up-regulated after treatment with 25 µM ZnMet (P < 0.05), which is consistent with the results of the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway in the transcriptome analysis. In the in vivo trial, 27 Duroc × (Landrace × Large White) pigs with an initial average weight of 31.62 ± 0.36 kg were divided into three groups with nine replicates per treatment. The dietary treatment groups were as follows: (1) ZnSO4 group, basal diet +75 mg/kg ZnSO4; (2) ZnMet group, basal diet +75 mg/kg ZnMet; and (3) ZnGly group, basal diet +75 mg/kg ZnGly. The whole trial lasted for 75 days. Increased final body weight, average daily gain, and decreased F/G were noted in the ZnMet group (P < 0.05). Moreover, the ZnMet group had higher carcass weight and loin eye area (P = 0.05). The ZnMet and ZnGly group both had lower serum total protein (P < 0.05), while the ZnMet group had higher serum alkaline phosphatase (P < 0.05). Also, the addition of ZnMet showed higher concentrations of zinc and iron in muscle, kidney, and serum (P < 0.05), improving the deposition and availability of micronutrients. In conclusion, amino acid-chelated zinc, particularly ZnMet, had the best effect, which could improve growth in vitro and increase growth performance while boosting bioavailability in growing-finishing pigs, ultimately, enhancing muscle mass, providing a theoretical basis and guidance for the future use of amino acid-chelated zinc to effectively replenish energy in animal nutrition and production.

Bredemolic acid restores glucose utilization and attenuates oxidative stress in palmitic acid-induced insulin-resistant C2C12 cells.

Objective. Due to insulin resistance and oxidative stress that are associated with type 2 diabetes mellitus (T2DM), T2DM has become a prevalent metabolic disorder that presents various side effects. However, alternative antidiabetic treatment has commonly been used in treating diabetes mellitus in diabetic patients. In our previous studies, bredemolic acid has been reported as an antidiabetic agent that improves glucose uptake, ameliorates insulin resistance, and oxidative stress in the liver, heart, kidney, and skeletal muscle of prediabetic rats. However, these effects have not been validated in vitro. Therefore, this study was aimed to investigate the effects of bredemolic acid on insulin-mediated glucose utilization, lipid peroxidation, and the total antioxidant capacity (TOAC) in palmitic acid-induced insulin-resistant C2C12 skeletal muscle cells in vitro. Methods. Insulin resistance was induced in the skeletal muscle cells after 4 h of exposure to palmitic acid (0.5 mmol/l). Different cell groups were incubated in culture media DMEM supplemented with fetal calf serum (10%), penicillin/streptomycin (1%), and L-glutamine (1%) and then treated with either insulin (4 µg/ml) or bredemolic acid (12.5 mmol/l) or with both. Thereafter, the cells were seeded in 24- or 96-well plates for determination of the cell viability, glucose utilization, glycogen formation, and antioxidant capacity. Results. The results showed that bredemolic acid significantly improved TOAC and promoted glucose utilization via attenuation of lipid peroxidation and increased glycogen formation in the insulin-resistant cells, respectively. Conclusion. This study showed that bredemolic acid restored the insulin resistance through improved glucose utilization, glycogen formation, and TOAC in the skeletal muscle cells.

Protective effects of milk thistle (Sylibum marianum) seed oil and α-tocopherol against 7ß-hydroxycholesterol-induced peroxisomal alterations in murine C2C12 myoblasts: Nutritional insights associated with the concept of pexotherapy.

Peroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7ß-hydroxycholesterol (7ß-OHC; 50 µM, 24 h), known to be increased in patients with age-related diseases such as sarcopenia, to trigger oxidative stress, mitochondrial and peroxisomal dysfunction was studied in murine C2C12 myoblasts. The capacity of milk thistle seed oil (MTSO, 100 µg/mL) as well as α-tocopherol (400 µM; reference cytoprotective agent) to counteract the toxic effects of 7ß-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7ß-OHC, in the presence or absence of MTSO or α-tocopherol, were studied with complementary methods: measurement of cell density and viability, quantification of reactive oxygen species (ROS) production and transmembrane mitochondrial potential (ΔΨm), evaluation of peroxisomal mass as well as topographic, morphologic and functional peroxisomal changes. Our results indicate that 7ß-OHC induces a loss of cell viability and a decrease of cell adhesion associated with ROS overproduction, alterations of mitochondrial ultrastructure, a drop of ΔΨm, and several peroxisomal modifications. In the presence of 7ß-OHC, comparatively to untreated cells, important quantitative and qualitative peroxisomal modifications were also identified: a) a reduced number of peroxisomes with abnormal sizes and shapes, mainly localized in cytoplasmic vacuoles, were observed; b) the peroxisomal mass was decreased as indicated by lower protein and mRNA levels of the peroxisomal ABCD3 transporter; c) lower mRNA level of Pex5 involved in peroxisomal biogenesis as well as higher mRNA levels of Pex13 and Pex14, involved in peroxisomal biogenesis and/or pexophagy, was found; d) lower levels of ACOX1 and MFP2 enzymes, implicated in peroxisomal ß-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal ß-oxidation, were found. These different cytotoxic effects were strongly attenuated by MTSO, in the same range of order as with α-tocopherol. These findings underline the interest of MTSO and α-tocopherol in the prevention of peroxisomal damages (pexotherapy).

Effects of American wild ginseng and Korean cultivated wild ginseng pharmacopuncture extracts on the regulation of C2C12 myoblasts differentiation through AMPK and PI3K/Akt/mTOR signaling pathway.

Targeting impaired myogenesis and mitochondrial biogenesis offers a potential alternative strategy for balancing energy to fight muscle disorders such as sarcopenia. In traditional Korean medicine, it is believed that the herb wild ginseng can help restore energy to the elderly. The present study investigated whether American wild ginseng pharmacopuncture (AWGP) and Korean cultivated wild ginseng pharmacopuncture (KCWGP) regulate energy metabolism in skeletal muscle cells. C2C12 mouse myoblasts were differentiated into myotubes using horse serum for 5 days. An MTT colorimetric assay verified cell viability. AWGP, KCWGP (0.5, 1, or 2 mg/ml), or metformin (2.5 mM) for reference were used to treat the C2C12 myotubes. The expressions of differentiation and mitochondrial biogenetic factors were measured by western blotting in C2C12 myotubes. Treatment of C2C12 cells stimulated with AWGP and KCWGP at a concentration of 10 mg/ml did not affect cell viability. AWGP and KCWGP treatments resulted in significant increases in the myogenesis proteins, myosin heavy chain, myostatin, myoblast determination protein 1 and myogenin, as well as increases to the biogenic regulatory factors, peroxisome proliferator­activated receptor­Î³ coactivator­1­α, nuclear respiratory factor 1, mitochondrial transcription factor A and Sirtuin 1, in the myotubes through AMPK and PI3K/AKT/mTOR signaling pathway activation. These results suggest that AWGP and KCWGP may be beneficial to muscle function by improving muscle differentiation and energy metabolism.

Dendropanax trifidus Sap-Mediated Suppression of Obese Mouse Body Weight and the Metabolic Changes Related with Estrogen Receptor Alpha and AMPK-ACC Pathways in Muscle Cells.

Dendropanax trifidus (DT) is a medicinal herb native to East Asia, which has been used extensively for its therapeutic properties in traditional medicine. In this study, we examined the effects of DT sap on the regulation of body weight and muscle metabolism in mice. Obese model db/db mice were administered daily with DT sap or vehicle control over a 6-week period. The effects of DT sap on muscle metabolism were studied in C2C12 muscle cells, where glycolytic and mitochondrial respiration rates were monitored. As AMP-activated protein kinase (AMPK) is a master regulator of metabolism and plays an important function as an energy sensor in muscle tissue, signaling pathways related with AMPK were also examined. We found that DT sap inhibited body weight increase in db/db, db/+, and +/+ mice over a 6-week period, while DT sap-treated muscle cells showed increased muscle metabolism and also increased phosphorylation of AMPK and Acetyl-CoA Carboxylase (ACC). Finally, we found that DT sap, which is enriched in estrogen in our previous study, significantly activates estrogen alpha receptor in a concentration-dependent manner, which can drive the activation of AMPK signaling and may be related to the muscle metabolism and weight changes observed here.

Trillin inhibits myoblast differentiation via increasing autophagy.

BACKGROUND: Trillin, an active ingredient in traditional Chinese medicine Trillium tschonoskii, is a potential small molecule compound candidate that affecting myoblast differentiation, which predicting by AI technology in our previous study. Autophagy modulating myoblast differentiation has also been studied. In addition, Trillin was shown to regulate mTOR signaling pathway, a highly conserved kinase important for autophagy regulation. PURPOSE: In this research, we aim to clarify the effect and underlying mechanism of Trillin on myoblast differentiation. STUDY DESIGN AND METHODS: Using mice C2C12 cell line to establish a myoblast differentiation model in vitro, treated with different concentration and time of Trillin, to explore the effect and latent mechanism of Trillin on myoblast differentiation by qRT-PCR, Western Blot and other molecular biological technique. RESULTS: Results showed that C2C12 differentiation was significantly inhibited by Trillin in a dose-dependent manner. The expression of MyHC, MyOG and MyoD was decreased extremely significant after 10 µM Trillin treatment. Meanwhile, autophagy level was significantly elevated with the supplement of Trillin. And C2C12 differentiation was recovered after ATG7 knockdown. Mechanically, we found that the activity of AKT/mTOR declined during the inhibition of differentiation by Trillin. CONCLUSION: Our findings suggested that Trillin attenuated C2C12 differentiation via increasing autophagy through AKT/mTOR signaling pathway. Taken together, we introduce a novel physiological function of Trillin in inhibiting skeletal muscle differentiation.

Preventing c2c12 muscular cells damage combining magnesium and potassium with vitamin D3 and curcumin.

BACKGROUND AND AIM: Physical activity is defined as any bodily movement produced by skeletal muscles which causes energy consumption; moderate and constant physical activity is known to be beneficial and to slow the muscle loss process associated with aging. The aim of the present study was to test, in an in vitro exercise model, the biological effects of a new formulation composed of magnesium and potassium combined with vitamin D and curcumin created to support muscle activity and to prevent hypercontraction damage. EXPERIMENTAL PROCEDURE: C2C12 cells were treated with vitamin D, buffered magnesium bisglycinate, curcumin, and potassium citrate. Cell viability, morpho-functional changes, calcium and magnesium movements, and the main kinases involved in glucose uptake were analyzed. The glycogen level and lactate were also evaluated. RESULTS AND CONCLUSION: Important results about a positive effect on mitochondrial activity, ATP production, oxygen consumption and in the physiological differentiation of C2C12 cells were obtained. Further experiments were performed under conditions that mimic the biological aspects of strenuous exercise. The combination of magnesium, vitamin D3, curcumin, and potassium citrate revealed beneficial effects on skeletal muscle cells under physiological conditions as well as while mimicking intense activity. In particular, in an in vitro model, they were able to control the hypercontraction, restoring ion fluxes, reducing inflammation signaling and supporting the main mechanism involved on aerobic activity. Our results have indicated for the first time that this new combination could be considered as a new nutraceutical formulation to improve physical performance and muscle recovery.

L-carnitine ameliorates the muscle wasting of cancer cachexia through the AKT/FOXO3a/MaFbx axis.

BACKGROUND: Recent studies suggest potential benefits of applying L-carnitine in the treatment of cancer cachexia, but the precise mechanisms underlying these benefits remain unknown. This study was conducted to determine the mechanism by which L-carnitine reduces cancer cachexia. METHODS: C2C12 cells were differentiated into myotubes by growing them in DMEM for 24 h (hrs) and then changing the media to DMEM supplemented with 2% horse serum. Differentiated myotubes were treated for 2 h with TNF-α to establish a muscle atrophy cell model. After treated with L-carnitine, protein expression of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K was determined by Western blotting. Then siRNA-Akt was used to determine that L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx. In vivo, the cancer cachexia model was established by subcutaneously transplanting CT26 cells into the left flanks of the BALB/c nude mice. After treated with L-carnitine, serum levels of IL-1, IL-6 and TNF-α, and the skeletal muscle content of MuRF1, MaFbx, FOXO3, p-FOXO3a, Akt, p-Akt, p70S6K and p-p70S6K were measured. RESULTS: L-carnitine increased the gastrocnemius muscle (GM) weight in the CT26-bearing cachexia mouse model and the cross-sectional fiber area of the GM and myotube diameters of C2C12 cells treated with TNF-α. Additionally, L-carnitine reduced the protein expression of MuRF1, MaFbx and FOXO3a, and increased the p-FOXO3a level in vivo and in vitro. Inhibition of Akt, upstream of FOXO3a, reversed the effects of L-carnitine on the FOXO3a/MaFbx pathway and myotube diameters, without affecting FOXO3a/MuRF-1. In addition to regulating the ubiquitination of muscle proteins, L-carnitine also increased the levels of p-p70S6K and p70S6K, which are involved in protein synthesis. Akt inhibition did not reverse the effects of L-carnitine on p70S6K and p-p70S6K. Hence, L-carnitine ameliorated cancer cachexia via the Akt/FOXO3/MaFbx and p70S6K pathways. Moreover, L-carnitine reduced the serum levels of IL-1 and IL-6, factors known to induce cancer cachexia. However, there were minimal effects on TNF-α, another inducer of cachexia, in the in vivo model. CONCLUSION: These results revealed a novel mechanism by which L-carnitine protects muscle cells and reduces inflammation related to cancer cachexia.