Extract of Phyllanthus emblica L. fruit stimulates basal glucose uptake and ameliorates palmitate-induced insulin resistance through AMPK activation in C2C12 myotubes.

BACKGROUND: The fruit of Phyllanthus emblica L., a traditional medicine in China and India, is used to treat diabetes mellitus. Its water extract (WEPE) has demonstrated hypoglycemic effects in diabetic rats, but its mechanisms on glucose utilization and insulin resistance in skeletal muscle remain unclear. Therefore, this study aims to investigate the effects and underlying mechanisms of WEPE on glucose utilization and insulin resistance using C2C12 myotubes. METHODS: Effects of WEPE on glucose uptake, GLUT4 translocation, and AMPK and AKT phosphorylation were investigated in C2C12 myotubes and palmitate-treated myotubes. An AMPK inhibitor and siRNA were used to explore the mechanisms of WEPE. Glucose uptake was determined using a 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake assay, and protein expression and GLUT4 translocation were assessed via western blotting. RESULTS: In normal myotubes, WEPE significantly stimulated glucose uptake and GLUT4 translocation to the plasma membrane at concentrations of 125 and 250 µg/mL. This was accompanied by an increase in the phosphorylation of AMPK and its downstream targets. However, both compound C and AMPK siRNA blocked the WEPE-induced GLUT4 translocation and glucose uptake. Moreover, pretreatment with STO-609, a calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor, inhibited WEPE-induced AMPK phosphorylation and attenuated the WEPE-stimulated glucose uptake and GLUT4 translocation. In myotubes treated with palmitate, WEPE prevented palmitate-induced insulin resistance by enhancing insulin-mediated glucose uptake and AKT phosphorylation. It also restored the insulin-mediated translocation of GLUT4 from cytoplasm to membrane. However, these effects of WEPE on glucose uptake and GLUT4 translocation were blocked by pretreatment with compound C. CONCLUSIONS: WEPE significantly stimulated basal glucose uptake though CaMKKß/AMPK pathway and markedly ameliorated palmitate-induced insulin resistance by activating the AMPK pathway in C2C12 myotubes.

Comparative Metabolomic Analysis of Moringa oleifera Leaves of Different Geographical Origins and Their Antioxidant Effects on C2C12 Myotubes.

Moringa oleifera is widely grown throughout the tropics and increasingly used for its therapeutic and nutraceutical properties. These properties are attributed to potent antioxidant and metabolism regulators, including glucosinolates/isothiocyanates as well as flavonoids, polyphenols, and phenolic acids. Research to date largely consists of geographically limited studies that only examine material available locally. These practices make it unclear as to whether moringa samples from one area are superior to another, which would require identifying superior variants and distributing them globally. Alternatively, the finding that globally cultivated moringa material is essentially functionally equivalent means that users can easily sample material available locally. We brought together accessions of Moringa oleifera from four continents and nine countries and grew them together in a common garden. We performed a metabolomic analysis of leaf extracts (MOLE) using an LC-MSMS ZenoTOF 7600 mass spectrometry system. The antioxidant capacity of leaf samples evaluated using the Total Antioxidant Capacity assay did not show any significant difference between extracts. MOLE samples were then tested for their antioxidant activity on C2C12 myotubes challenged with an oxidative insult. Hydrogen peroxide (H2O2) was added to the myotubes after pretreatment with different extracts. H2O2 exposure caused an increase in cell death that was diminished in all samples pretreated with moringa extracts. Our results show that Moringa oleifera leaf extract is effective in reducing the damaging effect of H2O2 in C2C12 myotubes irrespective of geographical origin. These results are encouraging because they suggest that the use of moringa for its therapeutic benefits can proceed without the need for the lengthy and complex global exchange of materials between regions.

Monascus pigment prevent the oxidative cytotoxicity in myotube derived hydrogen peroxide.

The amounts of Reactive oxygen species (ROS) become higher by strenuous exercises which consume larger amounts of oxygen in active muscles. Since these ROS directly injured muscles, the high ROS concentration involves muscle fatigue. Thus, an immediate ROS scavenging system in the muscle is desired. Since Monascus pigment (MP) involves physiologically active substances which scavenge ROS, it may be a clue to save the muscle injury. However, there are no reports examining MP effects on oxidative stress in skeletal muscle. In this study, we investigated the effect and mechanism of MP on skeletal muscle cells damaged by oxidative stress. The ability to directly eliminate ROS was evaluated by mixing MP solutions with •OH and O2 •-, a type of ROS. The effect of peroxidation in C2C12 cells was evaluated by cell viability assay and Western blotting. MP scavenges •OH and O2 •-. MP treatment increases the survival rate under oxidative stress. At that time, the expression of catalase was increased: the enzyme change H2O2 into H2O to rescue the cells under oxidative stress. We conclude that monascus pigment suppressed myotube damage under oxidative stress by both non-enzymatic ROS scavenging and up-regulation of catalase expression.

Taurine Alleviates Ferroptosis-Induced Metabolic Impairments in C2C12 Myoblasts by Stabilizing the Labile Iron Pool and Improving Redox Homeostasis.

Ferroptosis adversely affects the viability, differentiation, and metabolic integrity of C2C12 myoblasts, contributing to the decline in skeletal muscle health. The intricate mechanisms behind this process are not fully understood. In this study, we induced ferroptosis in myoblasts using targeted inducers and found a marked decrease in specific redox metabolites, particularly taurine. Taurine supplementation effectively reversed the deleterious effects of ferroptosis, significantly increased cellular glutathione levels, reduced MDA and ROS levels, and rejuvenated impaired myogenic differentiation. Furthermore, taurine downregulated HO-1 expression and decreased intracellular Fe2+ levels, thereby stabilizing the labile iron pool. Using NMR metabolomic analysis, we observed that taurine profoundly promoted glycerophospholipid metabolism, which is critical for cell membrane repair, and enhanced mitochondrial bioenergetics, thereby increasing the energy reserves essential for muscle satellite cell regeneration. These results suggest that taurine is a potent ferroptosis inhibitor that attenuates key drivers of this process, strengthens oxidative defenses, and improves redox homeostasis. This combined effect protects cells from ferroptosis-induced damage. This study highlights the potential of taurine as a valuable ferroptosis inhibitor that protects skeletal muscle from ferroptosis-induced damage and provides a basis for therapeutic strategies to rejuvenate and facilitate the regeneration of aging skeletal muscle.

Actin-organizing protein palladin modulates C2C12 cell fate determination.

Background: Cell confluency and serum deprivation promote the transition of C2C12 myoblasts into myocytes and subsequence fusion into myotubes. However, despite all myoblasts undergoing the same serum deprivation trigger, their responses vary: whether they become founder myocytes, remain proliferative, or evolve into fusion-competent myocytes remains unclear. We have previously shown that depletion of the scaffolding protein palladin in myoblasts inhibits cell migration and promotes premature muscle differentiation, pointing to its potential significance in muscle development and the necessity for a more in-depth examination of its function in cellular heterogeneity. Methods and results: Here, we showed that the subcellular localization of palladin might contribute to founder-fate cell decision in the early differentiation process. Depleting palladin in C2C12 myoblasts depleted integrin-ß3 plasma membrane localization of and focal adhesion formation at the early stage of myogenesis, decreased kindlin-2 and metavinculin expression during the myotube maturation process, leading to the inability of myocytes to fuse into preexisting mature myotubes. This aligns with previous findings where early differentiation into nascent myotubes occurred but compromised maturation. In contrast, wildtype C2C12 overexpressing the 140-kDa palladin isoform developed a polarized morphology with star-like structures toward other myoblasts. However, this behaviour was not observed in palladin-depleted cells, where the 140-kDa palladin overexpression could not recover cell migration capacity, suggesting other palladin isoforms are also needed to establish cell polarity. Conclusion: Our study identifies a counter-intuitive role for palladin in regulating myoblast-to-myocyte cell fate decisions and impacting their ability to form mature multinucleated myotubes by influencing cell signalling pathways and cytoskeletal organization, necessary for skeletal muscle regeneration and repair studies.

Korean Mint (Agastache rugosa) Extract and Its Bioactive Compound Tilianin Alleviate Muscle Atrophy via the PI3K/Akt/FoxO3 Pathway in C2C12 Myotubes.

Skeletal muscle atrophy, which is characterized by diminished muscle mass, strength, and function, is caused by malnutrition, physical inactivity, aging, and diseases. Korean mint (Agastache rugosa Kuntze) possesses various biological functions, including anti-inflammatory, antioxidant, anticancer, and antiosteoporosis activities. Moreover, it contains tilianin, which is a glycosylated flavone that exerts antioxidant, anti-inflammatory, antidiabetic, and neuroprotective activities. However, no studies have analyzed the inhibitory activity of A. rugosa extract (ARE) and tilianin on muscle atrophy. Thus, the present study investigated the potential of ARE and tilianin on muscle atrophy and their underlying mechanisms of action in C2C12 myotubes treated with tumor necrosis factor-α (TNF-α). The results showed that ARE and tilianin promoted the phosphatidylinositol 3-kinase/protein kinase B pathway, thereby activating mammalian target of rapamycin (a protein anabolism-related factor) and its downstream factors. Moreover, ARE and tilianin inhibited the mRNA expression of muscle RING-finger protein-1 and atrogin-1 (protein catabolism-related factors) by blocking Forkhead box class O3 translocation. ARE and tilianin also mitigated inflammatory responses by downregulating nuclear factor-kappa B expression levels, thereby diminishing the expression levels of inflammatory cytokines, including TNF-α and interleukin-6. Additionally, ARE and tilianin enhanced the expression levels of antioxidant enzymes, including catalase, superoxide dismutase, and glutathione peroxidase. Overall, these results suggest that ARE and tilianin are potential functional ingredients for preventing or improving muscle atrophy.

Lactiplantibacillus plantarum LM1001 Improves Digestibility of Branched-Chain Amino Acids in Whey Proteins and Promotes Myogenesis in C2C12 Myotubes.

Lactiplantibacillus plantarum is a valuable potential probiotic species with various proven health-beneficial effects. L. plantarum LM1001 strain was selected among ten strains of L. plantarum based on proteolytic activity on whey proteins. L. plantarum LM1001 produced higher concentrations of total free amino acids and branched-chain amino acids (Ile, Leu, and Val) than other L. plantarum strains. Treatment of C2C12 myotubes with whey protein culture supernatant (1%, 2% and 3%, v/v) using L. plantarum LM1001 significantly increased the expression of myogenic regulatory factors, such as Myf-5, MyoD, and myogenin, reflecting the promotion of myotubes formation (p<0.05). L. plantarum LM1001 displayed ß-galactosidase activity but did not produce harmful ß-glucuronidase. Thus, the intake of whey protein together with L. plantarum LM1001 has the potential to aid protein digestion and utilization.

Ankrd1 inhibits the FAK/Rho-GTPase/F-actin pathway by downregulating ITGA6 transcriptional to regulate myoblast functions.

Skeletal muscle constitutes the largest percentage of tissue in the animal body and plays a pivotal role in the development of normal life activities in the organism. However, the regulation mechanism of skeletal muscle growth and development remains largely unclear. This study investigated the effects of Ankrd1 on the proliferation and differentiation of C2C12 myoblasts. Here, we identified Ankrd1 as a potential regulator of muscle cell development, and found that Ankrd1 knockdown resulted in the proliferation ability decrease but the differentiation level increase of C2C12 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyzes as well as RNA-seq results showed that Ankrd1 knockdown activated focal adhesion kinase (FAK)/F-actin signal pathway with most genes significantly enriched in this pathway upregulated. The integrin subunit Itga6 promoter activity is increased when Ankrd1 knockdown, as demonstrated by a dual-luciferase reporter assay. This study revealed the molecular mechanism by which Ankrd1 knockdown enhanced FAK phosphorylation activity through the alteration of integrin subunit levels, thus activating FAK/Rho-GTPase/F-actin signal pathway, eventually promoting myoblast differentiation. Our data suggested that Ankrd1 might serve as a potential regulator of muscle cell development. Our findings provide new insights into skeletal muscle growth and development and valuable references for further study of human muscle-related diseases.

Muscle-Protective Effect of Carnosine against Dexamethasone-Induced Muscle Atrophy in C2C12 Myotube.

This study investigated the protective effect of carnosine and its components (L-histidine and ß-alanine [HA]) against dexamethasone (Dex)-induced muscle atrophy in C2C12 myotubes. Myotubes were treated with Dex (10 µM) to induce muscle atrophy manifested by decreased myotube diameter, low myosin heavy chain content, and increased expression of muscle atrophy-associated ubiquitin ligases (Atrogin-1, MuRF-1, and Cbl-b). Carnosine (20 mM) treatment significantly improved the myotube diameter and MyHC protein expression level in Dex-treated C2C12 myotubes. It also downregulated the expression of Atrogin-1, MuRF-1, and Cbl-b and suppressed the expression of forkhead box O3 (FoxO3a) mediated by Dex. Furthermore, reactive oxygen species production was increased by Dex but was ameliorated by carnosine treatment. However, HA (20 mM), the component of carnosine, treatment was found ineffective in preventing Dex-induced protein damage. Therefore, based on above results it can be suggested that carnosine could be a potential therapeutic agent to prevent Dex-induced muscle atrophy compared to its components HA.

Dasatinib promotes muscle differentiation and disrupts normal muscle regeneration.

Dasatinib is one of the second-generation tyrosine kinase inhibitors used to treat chronic myeloid leukemia and has a broad target spectrum, including KIT, PDGFR, and SRC family kinases. Due to its broad drug spectrum, dasatinib has been reported at the basic research level to improve athletic performance by eliminating senescent cell removal and to have an effect on muscle diseases such as Duchenne muscular dystrophy, but its effect on myoblasts has not been investigated. In this study, we evaluated the effects of dasatinib on skeletal muscle both under normal conditions and in the regenerating state. Dasatinib suppressed the proliferation and promoted the fusion of C2C12 myoblasts. During muscle regeneration, dasatinib increased the gene expressions of myogenic-related genes (Myod, Myog, and Mymx), and caused abnormally thin muscle fibers on the CTX-induced muscle injury mouse model. From these results, dasatinib changes the closely regulated gene expression pattern of myogenic regulatory factors during muscle differentiation and disrupts normal muscle regeneration. Our data suggest that when using dasatinib, its effects on skeletal muscle should be considered, particularly at regenerating stages.

Cost-Efficient Expression of Human Cardiac Myosin Heavy Chain in C2C12 Cells with a Non-Viral Transfection Reagent.

Production of functional myosin heavy chain (MHC) of striated muscle myosin II for studies of isolated proteins requires mature muscle (e.g., C2C12) cells for expression. This is important both for fundamental studies of molecular mechanisms and for investigations of deleterious diseases like cardiomyopathies due to mutations in the MHC gene (MYH7). Generally, an adenovirus vector is used for transfection, but recently we demonstrated transfection by a non-viral polymer reagent, JetPrime. Due to the rather high costs of JetPrime and for the sustainability of the virus-free expression method, access to more than one transfection reagent is important. Here, we therefore evaluate such a candidate substance, GenJet. Using the human cardiac ß-myosin heavy chain (ß-MHC) as a model system, we found effective transfection of C2C12 cells showing a transfection efficiency nearly as good as with the JetPrime reagent. This was achieved following a protocol developed for JetPrime because a manufacturer-recommended application protocol for GenJet to transfect cells in suspension did not perform well. We demonstrate, using in vitro motility assays and single-molecule ATP turnover assays, that the protein expressed and purified from cells transfected with the GenJet reagent is functional. The purification yields reached were slightly lower than in JetPrime-based purifications, but they were achieved at a significantly lower cost. Our results demonstrate the sustainability of the virus-free method by showing that more than one polymer-based transfection reagent can generate useful amounts of active MHC. Particularly, we suggest that GenJet, due to its current ~4-fold lower cost, is useful for applications requiring larger amounts of a given MHC variant.

Eugenol mimics exercise to promote skeletal muscle fiber remodeling and myokine IL-15 expression by activating TRPV1 channel.

Metabolic disorders are highly prevalent in modern society. Exercise mimetics are defined as pharmacological compounds that can produce the beneficial effects of fitness. Recently, there has been increased interest in the role of eugenol and transient receptor potential vanilloid 1 (TRPV1) in improving metabolic health. The aim of this study was to investigate whether eugenol acts as an exercise mimetic by activating TRPV1. Here, we showed that eugenol improved endurance capacity, caused the conversion of fast-to-slow muscle fibers, and promoted white fat browning and lipolysis in mice. Mechanistically, eugenol promoted muscle fiber-type transformation by activating TRPV1-mediated CaN signaling pathway. Subsequently, we identified IL-15 as a myokine that is regulated by the CaN/nuclear factor of activated T cells cytoplasmic 1 (NFATc1) signaling pathway. Moreover, we found that TRPV1-mediated CaN/NFATc1 signaling, activated by eugenol, controlled IL-15 levels in C2C12 myotubes. Our results suggest that eugenol may act as an exercise mimetic to improve metabolic health via activating the TRPV1-mediated CaN signaling pathway.

Kaempferol-Enhanced Migration and Differentiation of C2C12 Myoblasts via ITG1B/FAK/Paxillin and IGF1R/AKT/mTOR Signaling Pathways.

SCOPE: Kaempferol (KMP), a bioactive flavonoid compound found in fruits and vegetables, contributes to human health in many ways but little is known about its relationship with muscle mass. The effect of KMP on C2C12 myoblast differentiation and the mechanisms that might underlie that effect are studied. METHODS AND RESULTS: This study finds that KMP (1, 10 µM) increases the migration and differentiation of C2C12 myoblasts in vitro. Studying the possible mechanism underlying its effect on migration, the study finds that KMP activates Integrin Subunit Beta 1 (ITGB1) in C2C12 myoblasts, increasing p-FAK (Tyr398) and its downstream cell division cycle 42 (CDC42), a protein previously associated with cell migration. Regarding differentiation, KMP upregulates the expression of myosin heavy chain (MHC) and activates IGF1/AKT/mTOR/P70S6K. Interestingly, pretreatment with an AKT inhibitor (LY294002) and siRNA knockdown of IGF1R leads to a decrease in cell differentiation, suggesting that IGF1/AKT activation is required for KMP to induce C2C12 myoblast differentiation. CONCLUSION: Together, the findings suggest that KMP enhances the migration and differentiation of C2C12 myoblasts through the ITG1B/FAK/paxillin and IGF1R/AKT/mTOR pathways. Thus, KMP supplementation might potentially be used to prevent or delay age-related loss of muscle mass and help maintain muscle health.

mTORC1 and BMP-Smad1/5 regulation of serum-stimulated myotube hypertrophy: a role for autophagy.

Protein synthesis regulation is critical for skeletal muscle hypertrophy, yet other established cellular processes are necessary for growth-related cellular remodeling. Autophagy has a well-acknowledged role in muscle quality control, but evidence for its role in myofiber hypertrophy remains equivocal. Both mammalian target of rapamycin complex I (mTORC1) and bone morphogenetic protein (BMP)-Smad1/5 (Sma and Mad proteins from Caenorhabditis elegans and Drosophila, respectively) signaling are reported regulators of myofiber hypertrophy; however, gaps remain in our understanding of how this regulation is integrated with growth processes and autophagy regulation. Therefore, we investigated the mTORC1 and Smad1/5 regulation of protein synthesis and autophagy flux during serum-stimulated myotube growth. Chronic serum stimulation experiments were performed on day 5 differentiated C2C12 myotubes incubated in differentiation medium [2% horse serum (HS)] or growth medium [5% fetal bovine serum (FBS)] for 48 h. Rapamycin or LDN193189 was dosed for 48 h to inhibit mTORC1 and BMP-Smad1/5 signaling, respectively. Acute serum stimulation was examined in day 7 differentiated myotubes. Protein synthesis was measured by puromycin incorporation. Bafilomycin A1 and immunoblotting for LC3B were used to assess autophagy flux. Chronic serum stimulation increased myotube diameter 22%, total protein 21%, total RNA 100%, and Smad1/5 phosphorylation 404% and suppressed autophagy flux. Rapamycin, but not LDN193189, blocked serum-induced myotube hypertrophy and the increase in total RNA. Acute serum stimulation increased protein synthesis 111%, Smad1/5 phosphorylation 559%, and rpS6 phosphorylation 117% and suppressed autophagy flux. Rapamycin increased autophagy flux during acute serum stimulation. These results provide evidence for mTORC1, but not BMP-Smad1/5, signaling being required for serum-induced myotube hypertrophy and autophagy flux by measuring LC3BII/I expression. Further investigation is warranted to examine the role of autophagy flux in myotube hypertrophy.NEW & NOTEWORTHY The present study demonstrates that myotube hypertrophy caused by chronic serum stimulation requires mammalian target of rapamycin complex 1 (mTORC1) signaling but not bone morphogenetic protein (BMP)-Smad1/5 signaling. The suppression of autophagy flux was associated with serum-induced myotube hypertrophy and mTORC1 regulation of autophagy flux by measuring LC3BII/I expression. Rapamycin is widely investigated for beneficial effects in aging skeletal muscle and sarcopenia; our results provide evidence that rapamycin can regulate autophagy-related signaling during myotube growth, which could benefit skeletal muscle functional and metabolic health.

Muscle-specific PGC-1α modulates mitochondrial oxidative stress in aged sarcopenia through regulating Nrf2.

BACKGROUND: Aged sarcopenia is characterized by loss of skeletal muscle mass and strength, and mitochondrial dysregulation in skeletal myocyte is considered as a major factor. Here, we aimed to analyze the effects of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) on mitochondrial reactive oxygen species (ROS) and nuclear factor erythroid 2-related factor 2 (Nrf2) in aged skeletal muscles. METHODS: C2C12 cells were stimulated by 50 µM 7ß-hydroxycholesterol (7ß-OHC) to observe the changes of cellular ROS, mitochondrial ROS, and expression of PGC-1α and Nrf2. Different PGC-1α expression in cells was established by transfection with small interfering RNA (siRNA) or plasmids overexpressing PGC-1α (pEX-3-PGC-1α). The effects of different PGC-1α expression on cellular ROS, mitochondrial ROS and Nrf2 expression were measured in cells. Wild type (WT) mice and PGC-1α conditional knockout (CKO) mice were used to analyze the effects of PGC-1α on aged sarcopenia and expression of Nrf2 and CD38 in gastrocnemius muscles. Diethylmaleate, a Nrf2 activator, was used to analyze the connection between PGC-1α and Nrf2 in cells and in mice. RESULTS: In C2C12 cells, the expressions of PGC-1α and Nrf2 were declined by the 7ß-OHC treatment or PGC-1α silence. Moreover, PGC-1α silence increased the harmful ROS and decreased the Nrf2 protein expression in the 7ß-OHC-treated cells. PGC-1α overexpression decreased the harmful ROS and increased the Nrf2 protein expression in the 7ß-OHC-treated cells. Diethylmaleate treatment decreased the harmful ROS in the 7ß-OHC-treated or PGC-1α siRNA-transfected cells. At the same age, muscle-specific PGC-1α deficiency aggravated aged sarcopenia, decreased Nrf2 expression and increased CD38 expression in gastrocnemius muscles compared with the WT mice. Diethylmaleate treatment improved the muscle function and decreased the CD38 expression in the old two genotypes. CONCLUSIONS: Our study demonstrated that PGC-1α modulated mitochondrial oxidative stress in aged sarcopenia through regulating Nrf2.

Amber (Succinite) Extract Enhances Glucose Uptake through the Up-Regulation of ATP and Down-Regulation of ROS in Mouse C2C12 Cells.

Traditionally, amber (Succinite) has been used to alleviate all types of pain, skin allergies, and headaches. However, no studies have been conducted on its antidiabetic and antioxidant effects. In this study, differentiated skeletal muscle C2C12 cells were used to demonstrate the protective effects of amber (AMB) against H2O2-induced cell death. In addition, the effects of AMB on glucose uptake and ATP production were investigated. Our results showed that AMB at 10, 25, and 50 µg/mL suppressed the elevation of ROS production induced by H2O2 in a dose-dependent manner. Moreover, AMB enhanced glucose utilization in C2C12 cells through the improvement of ATP production and an increase in PGC-1α gene expression resulting in an amelioration of mitochondrial activity. On the other hand, AMB significantly increased the gene expression of glucose transporters GLUT4 and GLUT1. Our finding suggests that AMB can be used as a natural supplement for diabetes treatment and for the promotion of skeletal muscle function.

Dexamethasone and Insulin Modulate Alanine Aminotransferase (ALT) Activity and Alanine Oxidation in C2C12 Cells in a Dose-Dependent Manner.

BACKGROUND: The muscle cells myocytes are differentiated for the purpose of contraction function, which plays a major role in body metabolism and energy haemostasis, through different metabolic pathways, such as glucose and protein metabolic pathways. Alanine aminotransferase (ALT) plays a crucial role by reversibly catalysing transamination between alanine and a-ketoglutarate to form pyruvate and glutamate and by mediating the conversion of these four major intermediate metabolites. ALT plays important roles for energy homeostasis during fasting and prolonged exercise anaerobically, when muscle protein must first be broken down into its constituent amino acids. METHODS: Mouse skeletal myoblast cell line C2C12 was cultured in Dulbecco's modified eagle medium (DMEM) growth medium, supplied with 2% horse serum supplemented with 1 uM insulin, 2 mM glutamine and penicillin and streptomycin antibiotics for seven days. The differentiation medium is refreshed every 24 hours. Then, C2C12 cells were treated with insulin and dexamethasone to examine their effects on myocytes' ALT activity. RESULTS: In our study, we found an impact on ALT activity under different influences, including C2C12 differentiation, dexamethasone and insulin treatments, which shed light on the dynamic interplay between ALT activity, alanine metabolism, and cellular states, like differentiation and stress responses. CONCLUSION: The study provides valuable insights into the dynamic regulation of ALT activity and alanine metabolism in C2C12 cells across differentiation and drug treatments. Further research is encouraged to explore the underlying mechanisms and their implications for muscle function, differentiation and potential therapeutic interventions in metabolic disorders.

Crusting-fabricated three-dimensional soy-based scaffolds for cultured meat production: A preliminary study.

Crusting has been developed as a non-chemical and non-machine intensive scaffold fabrication method. This method is based on the self-assembling ability of soy biomolecules, allowing the fabrication of a three-dimensional network for cell growth. Preliminary characterization revealed differences in pore size, water absorption, and degradation between pure soy-based scaffold (Y2R) and with added glycerol (Y2G). The Fourier-transform infrared spectrum absorbance peaks of functional groups related to proteins, carbohydrates, and lipids hinted the integration of soy biomolecules potentially via the Maillard reaction, as supported by the visible browning of the scaffold surface. Microscopic images revealed aligned myotubes in both scaffolds, with Y2G myotubes having greater proximity after 72 h of proliferation. Both spontaneous and electro-stimulated contractions were recorded as early as 72 h in proliferation medium. Crusting-fabricated soy-based scaffolds can further be explored for its application in cultured meat production.

Saururus chinensis (Lour.) Baill. extract promotes skeletal muscle cell differentiation by positively regulating mitochondrial biogenesis and AKT/mTOR signaling in vitro.

Promotion of myoblast differentiation by activating mitochondrial biogenesis and protein synthesis signaling pathways provides a potential alternative strategy to balance energy and overcome muscle loss and muscle disorders. Saururus chinensis (Lour.) Baill. extract (SCE) has been used extensively as a traditional herbal medicine and has several physiological activities, including anti­asthmatic, anti­oxidant, anti­inflammatory, anti­atopic, anticancer and hepatoprotective properties. However, the effects and mechanisms of action of SCE on muscle differentiation have not yet been clarified. In the present study, it was investigated whether SCE affects skeletal muscle cell differentiation through the regulation of mitochondrial biogenesis and protein synthesis in murine C2C12 myoblasts. The XTT colorimetric assay was used to determine cell viability, and myosin heavy chain (MyHC) levels were determined using immunocytochemistry. SCE was applied to C2C12 myotube at different concentrations (1, 5, or 10 ng/ml) and times (1,3, or 5 days). Reverse transcription­quantitative PCR and western blotting were used to analyze the mRNA and protein expression change of factors related to differentiation, mitochondrial biogenesis and protein synthesis. Treatment of C2C12 cells with SCE at 1,5, and 10 ng/ml did not affect cell viability. SCE promoted C2C12 myotube formation and significantly increased MyHC expression in a concentration­ and time­dependent manner. SCE significantly increased the mRNA and protein expression of muscle differentiation­specific markers, such as MyHC, myogenic differentiation 1, myogenin, Myogenic Factor 5, and ß­catenin, mitochondrial biosynthesis­related factors, such as peroxisome proliferator­activated receptor­gamma coactivator­1α, nuclear respirator factor­1, AMP­activated protein kinase phosphorylation, and histone deacetylase 5 and AKT/mTOR signaling factors related to protein synthesis. SCE may prevent skeletal muscle dysfunction by enhancing myoblast differentiation through the promotion of mitochondrial biogenesis and protein synthesis.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.