Mouse C2C12 Cells Research Articles

Genome-wide mapping of the binding sites of myocyte enhancer factor 2A in chicken primary myoblasts

Myocyte enhancer factor 2A (MEF2A) is a transcription factor that plays a critical role in cell proliferation, differentiation and apoptosis. In contrast to the wide characterization of its regulation mechanism in mammalian skeletal muscle, its role in chickens is limited. Especially, its wide target genes remain to be identified. Therefore, we utilized Cleavage Under Targets and Tagmentation (CUT&Tag) technology to reveal the genome-wide binding profile of MEF2A in chicken primary myoblasts thus gaining insights into its potential role in muscle development. Our results revealed that MEF2A binding sites were primarily distributed in intergenic and intronic regions. Within the promoter region, although only 8.87% of MEF2A binding sites were found, these binding sites were concentrated around the transcription start site (TSS). Following peak annotation, a total of 1903 genes were identified as potential targets of MEF2A. Gene Ontology (GO) enrichment analysis further revealed that MEF2A target genes may be involved in the regulation of embryonic development in multiple organ systems, including muscle development, gland development, and visual system development. Moreover, a comparison of the MEF2A target genes identified in chicken primary myoblasts with those in mouse C2C12 cells revealed 388 target genes are conserved across species, 1515 target genes are chicken specific. Among these conserved genes, ankyrin repeat and SOCS box containing 5 (ASB5), transmembrane protein 182 (TMEM182), myomesin 2 (MYOM2), leucyl and cystinyl aminopeptidase (LNPEP), actinin alpha 2 (ACTN2), sorbin and SH3 domain containing 1 (SORBS1), ankyrin 3 (ANK3), sarcoglycan delta (SGCD), and ORAI calcium release-activated calcium modulator 1 (ORAI1) exhibited consistent expression patterns with MEF2A during embryonic muscle development. Finally, TMEM182, as an important negative regulator of muscle development, has been validated to be regulated by MEF2A by dual-luciferase and quantitative real-time PCR (qPCR) assays. In summary, our study for the first time provides a wide landscape of MEF2A target genes in chicken primary myoblasts, which supports the active role of MEF2A in chicken muscle development.

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.

Effect of lactate supplementation to skeletal muscle cells on fiber type transition and cellular bioenergetics

It is well-known that lactate (LAC) is the primary cellular end-product of glycolysis. However, during intense exercise and transient hypoxic conditions, the intracellular LAC levels are significantly elevated. Recently, our in vitro biochemical and biophysical studies have revealed that LAC avidly binds to oxy-myoglobin (oxy-Mb) in acidic conditions and rapidly releases oxygen (O2). Based on these results, we hypothesize that LAC plays a significant role in tissue O2 management combating hypoxic conditions in high demanding exercising tissues, such as Mb rich skeletal muscles type I oxidative fibers. Here, we have investigated three types of mouse C2C12 cell line models, i.e. Wild-type (WT) cells, Mb knock-out (MbKO) cells, and Empty vector (EV) cells. 5-day old differentiated C2C12 cells were supplemented with varying concentrations (3 mM, 5 mM and 8 mM) of sodium lactate added to the DMEM growth medium. Thereafter, cells were incubated in temperature regulated CO2 incubator for varying time periods (3 h, 6 h, and 24 h) to evaluate the early and prolonged effect on tissue fiber type transition and cellular bioenergetics. Metabolic assays such as hexokinase activity, LAC dehydrogenase (LDH) activity, and glucose uptake were performed using the intracellular cell lysate whereas extracellular LAC levels were measured using LAC assay kit. Seahorse experiments were conducted to measure the cellular glycolytic and mitochondrial ATP production rates. Our findings revealed that, there was no significant difference in myotube width in C2C12 cells either with or without LAC supplementation across all the test conditions. Intriguingly, at lower LAC supplementation (1.8 mM) conditions, the extracellular LAC levels were found to be significantly increased in WT cells compared to MbKO cells. However, at mimicking hyperlactatemia (5 mM LAC) and lactic acidosis (8 mM LAC) conditions, distinct extracellular LAC levels were recorded with WT and MbKO cells. Our results clearly suggests that Mb containing cells effciently utilize LAC for cellular bioenergetics and other biochemical processes. Similarly, in response to increasing LAC supplementation levels, LDH activity was also found to higher in WT cells compared to MbKO cells. Furthermore, glucose uptake in MbKO cells was significantly decreased. Finally, significantly lower rates of glycolytic ATP production was observed when compared to mitochondrial ATP production in WT cells with similar overall ATP production rates in all treatment conditions and incubation periods. However, marginal decrease in both glycolytic and mitochondrial ATP production rates were observed in MbKO cells. Overall, this study clearly demonstrated that external supplementation of LAC to C2C12 cells had no significant effect on fiber type transition but reveals that Mb plays a significant role in LAC utilization. USDA-ARS project 6026-51000-012-06S, Sturgis Foundation Pilot Grant G1-54750-01 and ACRI-ABI Investigator Initiated Grant Award GR037175-4450S ABI. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Leu promotes C2C12 cell differentiation by regulating the GSK3β/β-catenin signaling pathway through facilitating the interaction between SESN2 and RPN2.

Leucine (Leu) is an essential amino acid that facilitates skeletal muscle satellite cell differentiation, yet its mechanism remains underexplored. Sestrin2 (SESN2) serves as a Leu sensor, binding directly to Leu, while ribophorin II (RPN2) acts as a signaling factor in multiple pathways. This study aimed to elucidate Leu's impact on mouse C2C12 cell differentiation and skeletal muscle injury repair by modulating RPN2 expression through SESN2, offering a theoretical foundation for clinical skeletal muscle injury prevention and treatment. Leu addition promoted C2C12 cell differentiation compared to the control, enhancing early differentiation via myogenic determinant (MYOD) up-regulation. Sequencing revealed SESN2 binding to and interacting with RPN2. RPN2 overexpression up-regulated MYOD, myogenin and myosin heavy chain 2, concurrently decreased p-GSK3β and increased nuclear β-catenin. Conversely, RPN2 knockdown yielded opposite results. Combining RPN2 knockdown with Leu rescued increased p-GSK3β and decreased nuclear β-catenin compared to Leu absence. Hematoxylin and eosin staining results showed that Leu addition accelerated mouse muscle damage repair, up-regulating Pax7, MYOD and RPN2 in the cytoplasm, and nuclear β-catenin, confirming that the role of Leu in muscle injury repair was consistent with the results for C2C12 cells. Leu, bound with SESN2, up-regulated RPN2 expression, activated the GSK3β/β-catenin pathway, enhanced C2C12 differentiation and expedited skeletal muscle damage repair. © 2024 Society of Chemical Industry.

METTL21C mediates autophagy and formation of slow-twitch muscle fibers in mice after exercise.

Homeostasis is essential for muscle repair and regeneration after skeletal muscle exercise. This study investigated the role of methyltransferase-like 21C (METTL21C) in skeletal muscle of mice after exercise and the potential mechanism. First, muscle samples were collected at 2, 4 and 6 weeks after exercise, and liver glycogen, muscle glycogen, blood lactic acid and triglyceride were assessed. Moreover, the expression levels of autophagy markers and METTL21C in skeletal muscle were analyzed. The results showed that the expression levels of METTL21C and MYH7 in the gastrocnemius muscle of mice in the exercise group were significantly higher after exercise than those in the control group, which suggested that long-term exercise promoted the formation of slow-twitch muscle fibers in mouse skeletal muscle. Likewise, the autophagy capacity was enhanced with the prolongation of exercise in muscles. The findings were confirmed in mouse C2C12 cells. We discovered that knockdown of Mettl21c reduced the expression of MYH7 and the autophagy level in mouse myoblasts. These findings indicate that METTL21C promotes skeletal muscle homeostasis after exercise by enhancing autophagy, and also contributes to myogenic differentiation and the formation of slow muscle fibers.

Serine synthesis pathway enzyme PHGDH is critical for muscle cell biomass, anabolic metabolism, and mTORC1 signaling.

Cells use glycolytic intermediates for anabolism, e.g., via the serine synthesis and pentose phosphate pathways. However, we still understand poorly how these metabolic pathways contribute to skeletal muscle cell biomass generation. The first aim of this study was therefore to identify enzymes that limit protein synthesis, myotube size, and proliferation in skeletal muscle cells. We inhibited key enzymes of glycolysis, the pentose phosphate pathway, and the serine synthesis pathway to evaluate their importance in C2C12 myotube protein synthesis. Based on the results of this first screen, we then focused on the serine synthesis pathway enzyme phosphoglycerate dehydrogenase (PHGDH). We used two different PHGDH inhibitors and mouse C2C12 and human primary muscle cells to study the importance and function of PHGDH. Both myoblasts and myotubes incorporated glucose-derived carbon into proteins, RNA, and lipids, and we showed that PHGDH is essential in these processes. PHGDH inhibition decreased protein synthesis, myotube size, and myoblast proliferation without cytotoxic effects. The decreased protein synthesis in response to PHGDH inhibition appears to occur mainly mechanistic target of rapamycin complex 1 (mTORC1)-dependently, as was evident from experiments with insulin-like growth factor 1 and rapamycin. Further metabolomics analyses revealed that PHGDH inhibition accelerated glycolysis and altered amino acid, nucleotide, and lipid metabolism. Finally, we found that supplementing an antioxidant and redox modulator, N-acetylcysteine, partially rescued the decreased protein synthesis and mTORC1 signaling during PHGDH inhibition. The data suggest that PHGDH activity is critical for skeletal muscle cell biomass generation from glucose and that it regulates protein synthesis and mTORC1 signaling.NEW & NOTEWORTHY The use of glycolytic intermediates for anabolism was demonstrated in both myoblasts and myotubes, which incorporate glucose-derived carbon into proteins, RNA, and lipids. We identify phosphoglycerate dehydrogenase (PHGDH) as a critical enzyme in those processes and also for muscle cell hypertrophy, proliferation, protein synthesis, and mTORC1 signaling. Our results thus suggest that PHGDH in skeletal muscle is more than just a serine-synthesizing enzyme.

Expression and electrophysiological characteristics of VGSC during mouse myoblasts differentiation

Voltage-gated sodium channels (VGSC) are essential for triggering and relaying action potentials (AP), which perform critical functions in a variety of physiological processes, such as controlling muscle contractions and facilitating the release of neurotransmitters. In this study, we used a mouse C2C12 cell differentiation model to study the molecular expression and channel dynamics of VGSC and to investigate the exact role of VGSC in the development of muscle regeneration. Immunofluorescence, Real-time quantitative polymerase chain reaction, Western blot, and whole-cell patch clamp were employed for this purpose in mouse myoblasts. The findings revealed an increase in intracellular sodium concentration, NaV1.4 gene expression, and protein expression with the progress of differentiation (days 0, 1, 3, 5 and 7). Furthermore, VGSC dynamics exhibit the following characteristics: ① The increase of sodium current (INa); ② The decrease in the activation threshold and the voltage trigger maximum of INa; ③ A positive shift in the steady-state inactivation curve; ④ The recovery of INa during repolarization is delayed, the activity-dependent decay rate of INa was accelerated, and the proportionate amount of the fraction of activated channels was reduced. Based on these results, it is postulated that the activation threshold of AP could be decreased, and the refractory period could be extended with the extension of differentiation duration, which may contribute to muscle contraction. Taken together, VGSC provides a theoretical and empirical basis for exploring potential targets for neuromuscular diseases and other therapeutic muscle regeneration dysfunctions.

Lactate promotes myogenesis via activating H3K9 lactylation-dependent up-regulation of Neu2 expression.

Lactate, a glycolytic metabolite mainly produced in muscles, has been suggested to regulate myoblast differentiation, although the underlying mechanism remains elusive. Recently, lactate-mediated histone lactylation is identified as a novel epigenetic modification that promotes gene transcription. We used mouse C2C12 cell line and 2-month-old male mice as in vitro and in vivo models, respectively. These models were treated with lactate to explore the biological function and latent mechanism of lactate-derived histone lactylation on myogenic differentiation by quantitative real-time PCR, western blotting, immunofluorescence staining, chromatin immunoprecipitation, cleavage under targets and tagmentation assay and RNA sequencing. Using immunofluorescence staining and western blotting, we proposed that lactylation might occur in the histones. Inhibition of lactate production or intake both impaired myoblast differentiation, accompanied by diminished lactylation in the histones. Using lactylation site-specific antibodies, we demonstrated that lactate preferentially increased H3K9 lactylation (H3K9la) during myoblast differentiation (CT VS 5, 10, 15, 20, 25mM lactate treatment, P=0.0012, P=0.0007, and the rest of all P<0.0001). Notably, inhibiting H3K9la using P300 antagonist could block lactate-induced myogenesis. Through combined omics analysis using cleavage under targets and tagmentation assay and RNA sequencing, we further identified Neu2 as a potential target gene of H3K9la. IGV software analysis (P=0.0013) and chromatin immunoprecipitation-qPCR assay (H3K9la %Input, LA group=9.0076, control group=2.7184, IgG=0.3209) confirmed that H3K9la is enriched in the promoter region of Neu2. Moreover, siRNAs or inhibitors against Neu2 both abrogated myoblast differentiation despite lactate treatment, suggesting that Neu2 is required for lactate-mediated myoblast differentiation. Our findings provide novel understanding of histone lysine lactylation, suggesting its role in myogenesis, and as potential therapeutic targets for muscle diseases.

The Role of SOCS3 in Regulating Meat Quality in Jinhua Pigs

Meat quality is an important economic trait that influences the development of the pig industry. Skeletal muscle development and glycolytic potential (GP) are two crucial aspects that significantly impact meat quality. It has been reported that abnormal skeletal muscle development and high glycogen content results in low meat quality. However, the genetic mechanisms underlying these factors are still unclear. Compared with intensive pig breeds, Chinese indigenous pig breeds, such as the Jinhua pig, express superior meat quality characteristics. The differences in the meat quality traits between Jinhua and intensive pig breeds make them suitable for uncovering the genetic mechanisms that regulate meat quality traits. In this study, the Jinhua pig breed and five intensive pig breeds, including Duroc, Landrace, Yorkshire, Berkshire, and Pietrain pig breeds, were selected as experimental materials. First, the FST and XP-EHH methods were used to screen the selective signatures on the genome in the Jinhua population. Then, combined with RNA-Seq data, the study further confirmed that SOCS3 could be a key candidate gene that influences meat quality by mediating myoblast proliferation and glycometabolism because of the down-regulated expression of SOCS3 in Jinhua pigs compared with Landrace pigs. Finally, through SOCS3 knockout (KO) and overexpression (OE) experiments in mouse C2C12 cells, the results showed that SOCS3 regulated the cell proliferation of myoblasts. Moreover, SOCS3 is involved in regulating glucose uptake by the IRS1/PI3K/AKT signaling pathway. Overall, these findings provide a basis for the genetic improvement of meat quality traits in the pig industry.

Effect of electrochemotherapy on myogenesis of mouse C2C12 cells in vitro

Electrochemotherapy (ECT) is a local ablative therapy for the treatment of different skin and subcutaneous tumors and certain tumors in internal organs. Skeletal muscle represents a major tumor- surrounding tissue, exposed to side effects of ECT. At the cellular level, side-effects of ECT on skeletal muscle and underlying mechanisms have not been examined yet. Thus, we aimed to determine the effect of ECT in the mouse muscle cell line C2C12 during in vitro myogenesis. We evaluated the electroporation efficiency and viability of C2C12 myotubes at increasing voltages (200–1300 V/cm) using propidium iodide (PI). Permeabilization of PI into the cells was voltage-dependent accounting up to 97 % efficiency at the highest voltage. High cell viability and myotube integrity were maintained until 4 days after electroporation. ECT with the cytostatic drugs bleomycin and cisplatin decreased the viability of C2C12 myoblasts and myotubes in a dose-dependent manner. However, myoblasts were more sensitive to ECT than myotubes. Increased secretion of IL-6, observed 3 days after ECT, confirming its effects on early myogenesis. Only minor effects of ECT were observed in treated myotubes. These results contribute to the safety profile of ECT in tumor treatment.

Propolis Ethanolic Extract Attenuates D-gal-induced C2C12 Cell Injury by Modulating Nrf2/HO-1 and p38/p53 Signaling Pathways.

Previous study has shown that propolis ethanolic extract (PEE) has a protective effect on aging skeletal muscle atrophy. However, the exact molecular mechanism remains unclear. This study aimed to investigate the effect of PEE on D-galactose (D-gal)-induced damage in mouse C2C12 cells. The results revealed that PEE increased the viability of senescent C2C12 cells, decreased the number of senescence-associated β-galactosidase (SA-β-Gal)-positive cells and promoted the differentiation of C2C12 cells. PEE resisted oxidative stress caused by D-gal by activating the Nrf2/HO-1 signaling pathway and maintained the differentiation ability of C2C12 cells. PEE inhibited apoptosis by suppressing p38 phosphorylation and reducing p53 expression. In summary, our findings reveal the molecular mechanism by which PEE protects D-gal-induced C2C12 cells, providing a theoretical basis for the development of PEE for the alleviation of muscle atrophy.

5′-CMP and 5′-UMP alleviate dexamethasone-induced muscular atrophy in C2C12 myotubes

Atrogin-1 and muscle RING finger 1 (MuRF1) are ubiquitin ligases specifically expressed during skeletal muscle atrophy and mediate muscle protein degradation. In contrast, PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which is a master regulator of mitochondrial biosynthesis, protects skeletal muscle from atrophy. Pyrimidine nucleoside 5′-monophosphates, such as cytidine 5′-monophosphate (5′-CMP) and uridine 5′-monophosphate (5′-UMP), induce PGC-1α expression and promote myotube formation in mouse C2C12 cells. In this study, we determined the effect of 5′-CMP and 5′-UMP on muscular atrophy in C2C12 myotube cells. 5′-UMP decreased Atrogin-1 and MuRF1 mRNA levels that were upregulated by dexamethasone treatment. 5′-CMP and 5′-UMP ameliorated dexamethasone-mediated atrophy in C2C12 myotubes. Furthermore, the combination of 5′-CMP and 5′-UMP further alleviated dexamethasone-mediated atrophy. In addition, cytidine and uridine, the precursors of 5′-CMP and 5′-UMP, markedly ameliorated dexamethasone-mediated atrophy. Considering nucleotide metabolism and absorption, the active metabolites underlying the observed effects of 5′-CMP and 5′-UMP appear to be cytidine and uridine. Our results indicate that 5′-CMP alleviates muscle atrophy by activating PGC-1α and differentiation, and 5′-UMP alleviates muscle atrophy by suppressing the activation of the myolytic system, whereas the combined use of both enhances the muscle atrophy inhibitory effect. 5′-CMP and 5′-UMP may be an effective and safe treatment for muscular atrophy.

Effects of Rosemary Extract on C2C12 Myoblast Differentiation and 5-Aminoimidazole-4-carboxamide Ribonucleoside (AICAR)-Induced Muscle Cell Atrophy

Sarcopenia is an aging-related disease that involves the gradual loss of muscle mass and function. However, no suitable therapeutic drugs are currently available. Accordingly, the purpose of the present study was to evaluate the effectiveness of rosemary extract (RE) in inducing myotube differentiation and inhibiting 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced muscle atrophy in mouse C2C12 cells. Morphological changes associated with the onset of RE-induced differentiation were evaluated by measuring myotube diameter, and the expression of proteins related to muscle differentiation and atrophy was measured using western blot analysis. Treatment with RE increased myotube thickness and the expression of the myogenic differentiation 1 (MyoD) and myogenin proteins. The effect of RE treatment on 5′-adenosine monophosphate-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), MyoD, myogenin, muscle atrophy factors forkhead box O3a (FoxO3a), MAFbx/atrogin-1, and muscle RING finger-1 (MuRF-1) protein expression in AICAR-induced muscle-atrophied C2C12 cells was evaluated using western blot analysis. Treatment with RE reduced FoxO3a, MAFbx/atrogin-1, and MuRF-1 expression and significantly increased MyoD and myogenin expression. These findings suggest that RE has the potential to be used as an active ingredient in sarcopenia treatments.

TBBPA induces inflammation, apoptosis, and necrosis of skeletal muscle in mice through the ROS/Nrf2/TNF-α signaling pathway

Tetrabromobisphenol A (TBBPA) is present in large quantities in the environment due to its widespread use. And TBBPA is capable of accumulating in animals, entering the ecological chain and causing widespread damage to organisms. TBBPA is capable of causing the onset of oxidative stress, which induces tissue damage and cell death, which in turn affects the physiological function of tissues. Skeletal muscle is a critical tissue for maintaining growth, movement, and health in the body. However, the mechanism of TBBPA-induced skeletal muscle injury remains unclear. In this study, we constructed mouse skeletal muscle models (10, 20, and 40 mg/kg TBBPA) and mouse myoblasts (C2C12) cell models (2,4, and 8 μg/L TBBPA) at different concentrations. The results of this experiment showed that under TBBPA treatment, the levels of reactive oxygen species (ROS) and Malondialdehyde (MDA) in mouse skeletal and C2C12 cells were increased significantly, but the activities of some antioxidant enzymes decreased. TBBPA can inhibit Nuclear factor E2-related factor 2 (Nrf2) entry into the nucleus, thus affecting the expression of the Nrf2 downstream factors. With the increase of TBBPA concentration, the expression levels of inflammatory factors were significantly increased, while the anti-apoptotic factors were significantly decreased. The expression of pro-apoptotic factors increased in a dose-dependent manner. Programmed necrosis-related factors were also significantly elevated. Our results suggest that TBBPA induces oxidative stress and inflammation, apoptosis, and necrosis in the skeletal muscle of mice by regulating Nrf2/ROS/TNF-α signaling pathway.

The Promotion of Migration and Myogenic Differentiation in Skeletal Muscle Cells by Quercetin and Underlying Mechanisms.

Aging and muscle disorders frequently cause a decrease in myoblast migration and differentiation, leading to losses in skeletal muscle function and regeneration. Several studies have reported that natural flavonoids can stimulate muscle development. Quercetin, one such flavonoid found in many vegetables and fruits, has been used to promote muscle development. In this study, we investigated the effect of quercetin on migration and differentiation, two processes critical to muscle regeneration. We found that quercetin induced the migration and differentiation of mouse C2C12 cells. These results indicated quercetin could induce myogenic differentiation at the early stage through activated p-IGF-1R. The molecular mechanisms of quercetin include the promotion of myogenic differentiation via activated transcription factors STAT3 and the AKT signaling pathway. In addition, we demonstrated that AKT activation is required for quercetin induction of myogenic differentiation to occur. In addition, quercetin was found to promote myoblast migration by regulating the ITGB1 signaling pathway and activating phosphorylation of FAK and paxillin. In conclusion, quercetin can potentially be used to induce migration and differentiation and thus improve muscle regeneration.

FATP1 Exerts Variable Effects on Adipogenic Differentiation and Proliferation in Cells Derived From Muscle and Adipose Tissue.

In livestock, intramuscular adipose tissue is highly valued whereas adipose tissue in other depots is considered as waste. Thus, genetic factors that favor fat deposition in intramuscular compartments over that in other adipose depots are highly desirable in meat-producing animals. Fatty acid transport 1 (FATP1) has been demonstrated to promote cellular fatty acid uptake and metabolism; however, whether it also influences cellular lipid accumulation remains unclear. In the present study, we investigated the effects of FATP1 on the differentiation and proliferation of adipocytes in five types of cells derived from muscle and adipose tissue and estimated the effects of FATP1 on intramuscular fat (IMF) deposition. We showed that FATP1 is mainly expressed in heart and muscle tissue in buffaloes as well as cells undergoing adipogenic differentiation. Importantly, we found that FATP1 promoted the adipogenic differentiation of muscle-derived cells (buffalo myocytes and intramuscular preadipocytes and mouse C2C12 cells) but did not affect, or even inhibited, that of adipose-derived cells (buffalo subcutaneous preadipocytes and mouse 3T3-L1 cells, respectively). Correspondingly, our results further indicated that FATP1 promotes IMF deposition in mice in vivo. Meanwhile, FATP1 was found to enhance the proliferative activity of all the assessed cells, except murine 3T3-L1 cells. These results provide new insights into the potential effects of FATP1 on IMF deposition, especially regarding its positive effects on meat quality in buffaloes and other livestock.

Cellular Aquaculture: Prospects and Challenges.

Aquaculture plays an important role as one of the fastest-growing food-producing sectors in global food and nutritional security. Demand for animal protein in the form of fish has been increasing tremendously. Aquaculture faces many challenges to produce quality fish for the burgeoning world population. Cellular aquaculture can provide an alternative, climate-resilient food production system to produce quality fish. Potential applications of fish muscle cell lines in cellular aquaculture have raised the importance of developing and characterizing these cell lines. In vitro models, such as the mouse C2C12 cell line, have been extremely useful for expanding knowledge about molecular mechanisms of muscle growth and differentiation in mammals. Such studies are in an infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines, except a few fish muscle cell lines that have not yet been used for cellular aquaculture. The Prospect of cell-based aquaculture relies on the development of appropriate muscle cells, optimization of cell conditions, and mass production of cells in bioreactors. Hence, it is required to develop and characterize fish muscle cell lines along with their cryopreservation in cell line repositories and production of ideal mass cells in suitably designed bioreactors to overcome current cellular aquaculture challenges.

Specific amino acids regulate Sestrin2 mRNA and protein levels in an ATF4-dependent manner in C2C12 myocytes

BackgroundSestrin2 is a conserved protein in several species, and its expression is upregulated in cells under various environmental stresses. Sestrin2 content is involved in the function of mechanistic target of rapamycin complex 1 (mTORC1) in mouse embryonic fibroblasts and C2C12 cells. MethodsC2C12 cells were treated with amino acid-free DMEM (AF-DMEM) for 5 h. The effects of the addition of specific amino acids to AF-DMEM on Sestrin2 mRNA and protein expression were examined using RT-qPCR and western blotting, respectively. The mechanism by which amino acids regulate Sestrin2 mRNA expression was examined using blocking and siRNA experiments. ResultsAF-DMEM increased the mRNA and protein levels of both Sestrin2 and activating transcription factor 4 (ATF4). The addition of a specific amino acid changed Sestrin2 mRNA and protein levels. The response pattern of Sestrin2 to specific amino acids was similar to that of ATF4. ATF4 siRNA reduced Sestrin2 mRNA levels. AF-DMEM increased eukaryotic initiation factor 2α (eIF2α) phosphorylation as early as 10 min after the treatment; however, ATF4 and Sestrin2 were increased 300 min after the treatment. Nuclear factor erythroid 2-related factor 2 and pancreatic and duodenal homeobox 1 siRNA did not affect ATF4 and Sestrin2 mRNA expression. ConclusionsSpecific Amino acids regulate Sestrin2 levels in an ATF4-dependent manner in C2C12 cells. General significanceThe results of the present study indicate that amino acids regulate levels of Sestrin2, which might cause phenotypic alterations, including mTORC1 activity, in C2C12 cells.

2D structured graphene nanosheets decorated by monodispersed superparamagnetic Fe3O4 nanoparticles for differentiation of mouse cells

In the present study, the reduced graphene oxide (RGO) decorated by monodispersed superparamagnetic Fe3O4 (RGO-IO) nanocomposite was prepared and confirmed with various spectroscopic and microscopic techniques. The elemental spectral mapping depicts the composition of carbon, oxygen and iron in confirmed the successful formulation and homogenous distribution. The X-ray photoelectron spectroscopy (XPS) analysis of nanocomposite confirms the existence of elements such as C, O and Fe. The Raman spectroscopy analysis showed the characteristic peak for RGO i.e. D-band and G-band and A1 g phonon and Eg phonon peaks for IO is indicating the formation of RGO-IO. Moreover, the HR-TEM microscopic analysis evidently revealed the 2D structured RGO nanosheets are well decorated by a large quantity of IO nanoparticles. The in vitro cytotoxic activity of nanocomposite showed less toxicity and increased the cellular growth of mouse C2C12 and 3T3-L1 cells at higher concentration. The treatment of nanocomposite increases the ROS generation due to the excellent electrochemical performance and induces the formation of myotubes and mature adipocytes. The nanocomposite upregulates the expression of molecular marker proteins responsible for the myogenesis and adipogenesis was confirmed. Together, the synthesized RGO-IO nanocomposites provide surplus support for the use of nanomaterials in tissue engineering and regenerative medicine.

In Vitro Effect of CCL11 on Myogenic Differentiation and Its Relevance With Sarcopenia Parameters in Older Adults

Background: The C-C motif chemokine ligand 11 (CCL11) has been receiving attention as a potential pro-aging factor and thus may be also involved in muscle metabolism and sarcopenia, a key component of aging phenotypes. To clarify this possibility, we investigated the effects of CCL11 on in vitro muscle biology and clinical relevance with sarcopenia parameters in older adults. Methods: Primary mouse myoblasts and C2C12 cells were used for experimental research. Blood samples were collected from 79 participants who underwent a functional assessment, and CCL11 level was measured using a quantikine ELISA kit. Sarcopenia was diagnosed using the Asian-specific cut-off points. Results: CCL11 treatment stimulated the differentiation of both primary myoblasts and C2C12 cells into mature myotubes, and consistently increased the expression of myogenic differentiation markers, such as myosin heavy chain and myogenin. Among C-C chemokine receptors (CCRs), CCR5, not CCR2 and CCR3, was predominantly expressed in muscle cells, and CCR5 inhibitor blocked CCL11-stimulated myogenesis. In a clinical study, after adjustment for sex, age, and body mass index, serum CCL11 level was not significantly different according to the status of sarcopenia, low muscle mass, low muscle strength, and low physical performance, and was not associated with any of skeletal muscle index, grip strength, gait speed, time to complete 5 chair stands, and short physical performance battery score. Conclusions: Contrary to expectations, CCL11 showed the beneficial effects on muscle metabolism at least in vitro system. However, the role of CCL11 on muscle health in humans was not evident, suggesting that circulating CCL11 level may not be a useful biomarker for sarcopenia risk assessment in older adults.