C2C12 Myotube Cells Research Articles

Icariin promotes skeletal muscle mitochondrial biogenesis by the AMPK-PPARδ-PGC-1α signaling pathway: A potential mechanism for beneficial muscle function homeostasis

Mitochondrial biogenesis is a key factor in regulating skeletal muscle function and homeostasis. At present, a growing number of natural polyphenols, flavonoids and other compounds have been reported to have the function of regulating skeletal muscle oxidative metabolism. Here, we aimed to explore the effect of the flavonoid compound icariin purified from Epimedium koreanum Nakai in regulating mitochondrial biogenesis in mice and C2C12 myotubes. We found that icariin elevated the inverted suspension time by 1.72-fold and the distance run on the treadmill by 1.52-fold in mice. Daytime respiratory exchange ratio was significantly lower in mice after 8 weeks of icariin treatment compared to control groups. Icariin activated the mitochondrial biogenetic signaling pathway and mitochondrial marker expression, and the mitochondrial size of gastrocnemius and soleus was increased by 2.3-fold and 2.0-fold in mice, respectively. In addition, icariin elevated mitochondrial DNA copy number by 1.41-fold and 1.54-fold in gastrocnemius and C2C12 myotube cells, respectively. In vitro studies showed that inhibition of p-AMPK and PPARδ could attenuate ICA-regulated mitochondrial biogenetic pathway. Our study suggests that icariin promotes mitochondrial biogenesis and exercise endurance through AMPK-PPARδ-PGC-1α signaling pathway in C57BL/6 J mice skeletal muscle, indicating that icariin may be able to regulate muscle mitochondria function as an exercise mimetic.

Roles of programmed death-1 and muscle innate lymphoid cell-derived interleukin 13 in sepsis-induced intensive care unit-acquired weakness.

Intensive care unit-acquired weakness (ICU-AW) is a syndrome characterized by a long-term muscle weakness often observed in sepsis-surviving patients during the chronic phase. Although ICU-AW is independently associated with increased mortality, effective therapies have yet to be established. Programmed death-1 (PD-1) inhibitors have attracted attention as potential treatments for reversing immune exhaustion in sepsis; however, its impact on ICU-AW remains to be elucidated. Here, we study how PD-1 deficiency affects sepsis-induced skeletal muscle dysfunction in a preclinical sepsis model. Chronic sepsis model was developed by treating wild-type (WT) and PD-1 knockout (KO) mice with caecal slurry, followed by resuscitation with antibiotics and saline. Mice were euthanized on days 15-17. Body weights, muscle weights, and limb muscle strengths were measured. Interleukin 13 (IL-13)and PD-1 expressions were examined by flow cytometry. Messenger RNA (mRNA) expressions of slow-twitch muscles were measured by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). In an in vitro study, C2C12 myotubes were treated with lipopolysaccharide (LPS) and recombinant IL-13 followed by gene expression measurements. WT septic mice exhibited decreased muscle weight (quadriceps, P<0.01; gastrocnemius, P<0.05; and tibialis anterior, P<0.01) and long-term muscle weakness (P<0.0001), whereas PD-1 KO septic mice did not exhibit any reduction in muscle weights and strengths. Slow-twitch specific mRNAs, including myoglobin (Mb), troponin I type 1 (Tnni1), and myosin heavy chain 7 (Myh7) were decreased in WT skeletal muscle (Mb, P<0.0001; Tnni1, P<0.05; and Myh7, P<0.05) after sepsis induction, but mRNA expressions of Tnni1 and Myh7 were increased in PD-1 KO septic mice (Mb, not significant; Tnni1, P<0.0001; and Myh7, P<0.05). Treatment of C2C12 myotube cells with LPS decreased the expression of slow-twitch mRNAs, which was restored by IL-13 (Mb, P<0.0001; Tnni1, P<0.001; and Myh7, P<0.05). IL-13 production was significantly higher in ILC2s compared to T cells in skeletal muscle (P<0.05). IL-13-producing ILC2s in skeletal muscle were examined and found to increase in PD-1 KO septic mice, compared with WT septic mice (P<0.05). ILC2-derived IL-13 was increased by PD-1 KO septic mice and thought to protect the muscles from experimental ICU-AW. Long-term muscle weakness in experimental ICU-AW was ameliorated in PD-1 KO mice. ILC2-derived IL-13 production in skeletal muscles was increased in PD-1 KO mice, thereby suggesting that IL-13 alleviates muscle weakness during sepsis. This study demonstrates the effects of PD-1 blockade in preserving muscle strength during sepsis through an increase in ILC2-derived IL-13 and may be an attractive therapeutic target for sepsis-induced ICU-AW.

Profiling cells with DELs: Small molecule fingerprinting of cell surfaces

DNA-encoded small molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, it has been used to identify ligands against targets that are soluble or overexpressed on cell surfaces. Here, we report applying cell-based selection methods to profile surfaces of mouse C2C12 myoblasts and myotube cells in an unbiased, target agnostic manner. A panel of on-DNA compounds were identified and confirmed for cell binding selectivity. We optimized the cell selection protocol and employed a novel data analysis method to identify cell selective ligands against a panel of human B and T lymphocytes. We discuss the generality of using this workflow for DNA encoded small molecule library selection and data analysis against different cell types, and the feasibility of applying this method to profile cell surfaces for biomarker and target identification.

Modulation of macrophage transcript and secretion profiles by Sargassum Serratifolium extract is associated with the suppression of muscle atrophy

Recent research has emphasized the role of macrophage-secreted factors on skeletal muscle metabolism. We studied Sargassum Serratifolium ethanol extract (ESS) in countering lipopolysaccharide (LPS)-induced changes in the macrophage transcriptome and their impact on skeletal muscle. Macrophage-conditioned medium (MCM) from LPS-treated macrophages (LPS-MCM) and ESS-treated macrophages (ESS-MCM) affected C2C12 myotube cells. LPS-MCM upregulated muscle atrophy genes and reduced glucose uptake, while ESS-MCM reversed these effects. RNA sequencing revealed changes in the immune system and cytokine transport pathways in ESS-treated macrophages. Protein analysis in ESS-MCM showed reduced levels of key muscle atrophy-related proteins, TNF-α, IL-6, IL-1, and GDF-15. These proteins play crucial roles in muscle function. These findings highlight the intricate relationship between the macrophage transcriptome and their secreted factors in either impairing or enhancing skeletal muscle function. ESS treatment has the potential to reduce macrophage-derived cytokines, preserving skeletal muscle function.

Arsenic induces the C2C12 myotube cell injury by mitochondrial iron-mediated oxidative stress and inflammatory response

Arsenic induces the C2C12 myotube cell injury by mitochondrial iron-mediated oxidative stress and inflammatory response

Effect of Yeast-Derived Peptides on Skeletal Muscle Function and Exercise-Induced Fatigue in C2C12 Myotube Cells and ICR Mice.

In our previous study, the antioxidant peptides (XHY69AP, AP-D, YPLP, and AGPL) were obtained from potential probiotic yeast (Yamadazyma triangularis XHY69), which was selected by our lab from dry-cured ham. This work aimed to explore the effects of yeast-derived peptides on skeletal muscle function and muscle fatigue. Results showed that yeast-derived peptides up-regulated slow-twitch fiber expression and down-regulated fast-twitch fiber expression in C2C12 cells (p < 0.05). The peptides improved mitochondrial membrane potential, adenosine triphosphate generation, and expression of cytochrome-relative genes, thus promoting mitochondrial function. Among these peptides, YPLP up-regulated the relative gene expression of the AMP-activated protein kinase (AMPK) pathway and activated AMPK by phosphorylation. Moreover, YPLP could prolong treadmill time, increase muscle and liver glycogen contents, reduce lactic acid and urea nitrogen contents, and alleviate muscle tissue injury in ICR exercise mice. These results demonstrate that yeast-derived peptides could change the muscle fiber composition, improve muscle function, and relieve muscle fatigue.

Pancreatic Cancer-Derived Exosomal miR-Let-7b-5p Stimulates Insulin Resistance in Skeletal Muscle Cells Through RNF20/STAT3/FOXO1 Axis Regulation.

Cancers trigger systemic metabolic disorders usually associated with glucose intolerance, which is an initially apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). Our previous study reported that mice pancreatic cancer-derived exosomes could induce skeletal muscle cells (C2C12) IR, and exosomal microRNAs (miRNAs) may exert an important effect. However, the underlying mechanism remains to be further elucidated. qPCR was used to determine the expression of let-7b-5p in normal pancreatic islet cells and PC cells. Exosomes were purified from PC cell culture medium by ultracentrifugation. The role let-7b-5p on IR-mediated by PC cells-derived exosomes was asses by Oil Red O staining using miRNA inhibitor. Western blot assay was performed to examine the expression of IR-related genes and the activation of signaling pathways. A Luciferase experiment was applied to confirm how let-7b-5p regulated the expression of RNF20. IP/WB analysis further determined whether RNF20 promoted STAT3 ubiquitination. Rescue experiment using RNF20 overexpression plasmid was performed to confirm the role of RNF20 on IR-mediated using PC cell-derived exosomes in C2C12 myotube cells. miRNA-let-7b-5p was identified as the key exosomal miRNA, which could promote the IR in C2C12 myotube cells supported the lipid accumulation, the activation of STAT3/FOXO1 axis, and the decreased expression of IRS-1 and GLUT4. RNF20, an E3 ubiquitin ligase, was confirmed as the target gene of let-7b-5p and was found to improve IR by downregulating STAT3 protein expression via ubiquitination-mediated protein degradation. The ectopic expression of RNF20 could effectively attenuate the IR mediated by the pancreatic cancer-derived exosomes in C2C12 myotube cells. Our data suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells by targeting RNF20 to activate the STAT3/FOXO1 axis.

GLP-1RA Liraglutide and Semaglutide Improves Obesity-Induced Muscle Atrophy via SIRT1 Pathway.

Obesity is related to the loss of skeletal muscle mass and function (sarcopenia). The co-existence of obesity and sarcopenia is called sarcopenic obesity (SO). Glucagon like peptide-1 receptor agonists (GLP-1RA) are widely used in the treatment of diabetes and obesity. However, the protective effects of GLP-1RA on skeletal muscle in obesity and SO are not clear. This study investigated the effects of GLP-1RA liraglutide and semaglutide on obesity-induced muscle atrophy and explored the underlying mechanisms. Thirty-six male C57BL/6J mice were randomly divided into two groups and fed a regular diet and a high-fat diet for 18 weeks, respectively. After establishing an obesity model, mice were further divided into six groups: control group, liraglutide (LIRA) group, semaglutide (SEMA) group, high-fat diet (HFD) group, HFD + LIRA group, HFD + SEMA group, and subcutaneous injection for 4 weeks. The body weight, muscle mass, muscle strength, glycolipid metabolism, muscle atrophy markers, myogenic differentiation markers, GLUT4 and SIRT1 were analyzed. C2C12 myotube cells treated with palmitic acid (PA) were divided into four groups: control group, PA group, PA + LIRA group, PA + SEMA group. The changes in glucose uptake, myotube diameter, lipid droplet infiltration, markers of muscle atrophy, myogenic differentiation markers, GLUT4 and SIRT1 were analyzed, and the changes in related indicators were observed after the addition of SIRT1 inhibitor EX527. Liraglutide and semaglutide reduced HFD-induced body weight gain, excessive lipid accumulation and improved muscle atrophy. Liraglutide and semaglutide eliminated the increase of muscle atrophy markers in skeletal muscle and C2C12 myotubes. Liraglutide and semaglutide restored impaired glucose tolerance and insulin resistance. However, these beneficial effects were attenuated by inhibiting SIRT1 expression. Liraglutide and semaglutide protects skeletal muscle against obesity-induced muscle atrophy via the SIRT1 pathway.

1640-P: Meroterpenoid-Rich Ethanolic Extract of Sargassum serratifolium (MES) Alters Macrophage Transcriptome and Secretion Profile toward Improving Muscle Atrophy and Glucose Intolerance

Skeletal muscle plays an essential role in glucose homeostasis. Of many cell types in the skeletal muscle, recent studies indicate that secretion factors derived from macrophages are important for glucose metabolism in the skeletal muscle. Here, RNA Sequencing analysis showed that Lipopolysaccharide (LPS) treatment broadly altered the transcriptome of macrophages, of which signaling related to secretion factors was markedly altered toward elevating unfavorable cytokines to maintain skeletal muscle metabolism. On the other hand, the treatment of the Meroterpenoid-Rich Extract of Sargassum Serratifolium (MES), a strong anti-inflammatory and antioxidant extract found in our previous studies, reversed the LPS-induced signaling pathways. We further tested whether the treatment of conditioned media (CM) derived from LPS-macrophage (LPS-CM) or CM derived from LPS-macrophage treated with MES (MES-CM) affects glucose homeostasis in differentiated C2C12 myotube cells. LPS-CM treatment notably increased mRNA levels of muscle atrophy-associated genes including MuRF1, MAFbx, and Myostatin, and decreased glucose uptake, and MES-CM treatment reversed these characteristics, indicating that secretion factors induced by LPS or MES either impair or improve muscle glucose homeostasis, respectively. We further analyzed secretion factors inside by proteome array and data showed that LPS-CM includes GDF-15, IL-1 α/β, RAGE, etc related to muscle atrophy and glucose intolerance, which were significantly reduced in MES-CM. These data indicate that LPS- or MES-induced changes in the transcriptome in macrophages are closely related to the alteration in secretion factors toward either impairing or improving glucose metabolism in the skeletal muscle respectively. Of many, the decrease in macrophage-derived cytokine mentioned above may be important for glucose homeostasis in the skeletal muscle. Disclosure H.Ryu: None. B.Lee: None.

Improving Mitochondrial Function in Skeletal Muscle Contributes to the Amelioration of Insulin Resistance by Nicotinamide Riboside.

High-fat diet (HFD)-induced insulin resistance (IR) in skeletal muscle is often accompanied by mitochondrial dysfunction and oxidative stress. Boosting nicotinamide adenine dinucleotide (NAD) using nicotinamide riboside (NR) can effectively decrease oxidative stress and increase mitochondrial function. However, whether NR can ameliorate IR in skeletal muscle is still inconclusive. We fed male C57BL/6J mice with an HFD (60% fat) ± 400 mg/kg·bw NR for 24 weeks. C2C12 myotube cells were treated with 0.25 mM palmitic acid (PA) ± 0.5 mM NR for 24 h. Indicators for IR and mitochondrial dysfunction were analyzed. NR treatment alleviated IR in HFD-fed mice with regard to improved glucose tolerance and a remarkable decrease in the levels of fasting blood glucose, fasting insulin and HOMA-IR index. NR-treated HFD-fed mice also showed improved metabolic status regarding a significant reduction in body weight and lipid contents in serum and the liver. NR activated AMPK in the skeletal muscle of HFD-fed mice and PA-treated C2C12 myotube cells and upregulated the expression of mitochondria-related transcriptional factors and coactivators, thereby improving mitochondrial function and alleviating oxidative stress. Upon inhibiting AMPK using Compound C, NR lost its ability in enhancing mitochondrial function and protection against IR induced by PA. In summary, improving mitochondrial function through the activation of AMPK pathway in skeletal muscle may play an important role in the amelioration of IR using NR.

Identification of natural product inhibitors of PTP1B based on high-throughput virtual screening strategy: In silico, in vitro and in vivo studies

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathway, which is a potential therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). In this study, we identified several PTP1B inhibitors with high activity by using high-throughput virtual screening and in vitro enzyme inhibition activity verification strategies. Among them, baicalin was first reported as a selective mixed inhibitor of PTP1B, with IC50 value of 3.87 ± 0.45 μM, and its inhibitory activity against homologous proteins TCPTP, SHP2, and SHP1 exceeded 50 μM. Molecular docking study found that baicalin and PTP1B could bind stably, and revealed that baicalin had a dual inhibitory effect. Cell experiments showed that baicalin was almost non-toxic and could significantly enhance the phosphorylation of IRS-1 in C2C12 myotube cells. Animal experiments showed that baicalin could significantly reduce the blood sugar of STZ-induced diabetic mice models, and had a liver protective effect. In conclusion, this study can provide new ideas for the development of PTP1B selective inhibitors.

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.

Eugenol improves high-fat diet/streptomycin-induced type 2 diabetes mellitus (T2DM) mice muscle dysfunction by alleviating inflammation and increasing muscle glucose uptake.

Eugenol has been used in dietary interventions for metabolic diseases such as diabetes and obesity. However, the protective effect of eugenol on muscle function in diabetes is unclear. In this study, a high-fat diet (HFD) with a streptozocin (STZ) injection induced type II diabetes mellitus in a mouse model. Oral eugenol lowered blood glucose and insulin resistance of HFD/STZ-treated mice. Eugenol reduced HFD/STZ-induced muscle inflammation and prevented muscle weakness and atrophy. Eugenol administration significantly increased GLUT4 translocation and AMPK phosphorylation in skeletal muscle, thereby enhancing glucose uptake. By silencing the transient receptor potential vanilloid channel 1 (TRPV1) gene in C2C12 myotube cells, eugenol was found to increase intracellular Ca2+ levels through TRPV1, which then activated calmodulin-dependent protein kinase-2 (CaMKK2) and affected AMPK protein phosphorylation. In conclusion, eugenol is a potential nutraceutical for preventing high-glucose-induced muscle impairments, which could be explained by its mediating effects on glucose absorption and inflammatory responses in the muscle.

ODP201 Fyn regulates sarcopenia via autophagy

Abstract Sarcopenia is an important element that impairs daily activity. Sarcopenia is also linked to insulin resistance, and the number of diabetic patients also presenting sarcopenia is increasing. In recent years, autophagy has been identified as one of the novel mechanisms associated with sarcopenia. We previously reported that Fyn not only participates in the metabolic syndrome but also that autophagy-regulating sarcopenia was decreased in muscle specific Fyn transgenic mice through STAT3 regulation (Cell metabolism 2010, Cell Rep. 2012). More recently, we revealed Fyn-dependent STAT3 phosphorylation by IL6, which is involved in chronic inflammation and metabolic syndrome, using mouse C2C12 myotube cells expressing shRNA for Fyn. Furthermore, using WT mice with both the denervated and the hind limb suspension (HS) that promotes disuse atrophy by suspending the hind limb, not only both Fyn and IL6 gene expressions were increased but the expression and phosphorylation levels of STAT3 were also augmented, while the autophagic activity was decreased. To confirm those regulations are Fyn dependent, we took advantage of Fyn null mice (FynKO) with HS. Compare to WT mice with HS, all the muscles of FynKO were resistant for the loss of muscle mass induced by HS. The expressions of bothatrogin-1 and muRF-1,the key muscle specific E3 ubiquitin ligases as well as the markers for sarcopenia, were not changed in the muscles of FynKO with HS. Moreover, the STAT3 phosphorylation on tyrosine 705 was decreased in the muscle of FynKO compared to WT mice after hind limb suspension, while expression levels of STAT3 were not changed. IL-6mRNA expression levels were increased in the HS muscle of control mice, but were not changed in FynKO HS muscle. Finally, the autophagic activity, examined by both LC3-2 protein levels after intraperitoneal administration of colchicine andSQSTM1/p62 immunofluorescence in muscles, was retained in FynKO after HS. These results strongly suggest that Fyn is involved not only in the metabolic syndrome but also in the pathogenesis of sarcopenia, and may lead to a better understanding of the pathology of sarcopenia-associated obesity and the development of therapeutic methods. Presentation: No date and time listed

Silibinin Alleviates Muscle Atrophy Caused by Oxidative Stress Induced by Cisplatin through ERK/FoxO and JNK/FoxO Pathways.

Cisplatin (DDP), a widely used chemotherapeutic drug in cancer treatment, causes oxidative stress, resulting in cancer cachexia and skeletal muscle atrophy. This study investigated the effects and activity of silibinin (SLI) in reducing DDP-induced oxidative stress and skeletal muscle atrophy in vivo and in vitro. SLI alleviated weight loss, food intake, muscle wasting, adipose tissue depletion, and organ weight reduction induced by DDP and improved the reduction of grip force caused by DDP. SLI can attenuated the increase in reactive oxygen species (ROS) levels, the decrease in Nrf2 expression, the decrease in the fiber cross-sectional area, and changes in fiber type induced by DDP. SLI regulated the ERK/FoxO and JNK/FoxO pathways by downregulating the abnormal increase in ROS and Nrf2 expression in DDP-treated skeletal muscle and C2C12 myotube cells. Further, SLI inhibited the upregulation of MAFbx and Mstn, the downregulation of MyHC and MyoG, the increase in protein degradation, and the decrease of protein synthesis. The protective effects of SLI were reversed by cotreatment with JNK agonists and ERK inhibitors. These results suggest that SLI can reduce DDP-induced skeletal muscle atrophy by reducing oxidative stress and regulating ERK/FoxO and JNK/FoxO pathways.

DIF-1 promotes glucose uptake in mouse C2C12 myotube cells

DIF-1 promotes glucose uptake in mouse C2C12 myotube cells

Let-7e-5p Regulates IGF2BP2, and Induces Muscle Atrophy.

Background and AimsTo understand the role of microRNAs in muscle atrophy caused by androgen-depletion, we performed microarray analysis of microRNA expression in the skeletal muscles of Sham, orchiectomized (ORX), and androgen-treated ORX mice.MethodsTo clarify role and mechanisms of let-7e-5p in the muscle, the effect of let-7e-5p overexpression or knockdown on the expression of myosin heavy chain, glucose uptake, and mitochondrial function was investigated in C2C12 myotube cells. Moreover, we examined serum let-7e-5p levels among male subjects with type 2 diabetes.ResultsWe found that the expression of the miRNA, lethal (let)-7e-5p was significantly lower in ORX mice than that in Sham mice (p = 0.027); however, let-7e-5p expression in androgen-treated ORX mice was higher (p = 0.047). Suppression of let-7e-5p significantly upregulated the expression of myosin heavy chain, glucose uptake, and mitochondrial function. Real-time PCR revealed a possible regulation involving let-7e-5p and Igf2bp2 mRNA and protein in C2C12 cells. The serum let-7e-5p levels were significantly lower, which might be in compensation, in subjects with decreased muscle mass compared to subjects without decreased muscle mass. Let-7e-5p downregulates the expression of Igf2bp2 in myotube cells and inhibits the growth of the myosin heavy chain.ConclusionsBased on our study, serum level of let-7e-5p may be used as a potential diagnostic marker for muscle atrophy.

L-Arginine Alleviates LPS-Induced Oxidative Stress and Apoptosis via Activating SIRT1-AKT-Nrf2 and SIRT1-FOXO3a Signaling Pathways in C2C12 Myotube Cells.

l-arginine (l-Arg) has been reported to possess a wide range of functions, including anti-inflammatory, anti-oxidative, and anti-apoptosis. However, the role of l-Arg in LPS-induced muscle injury and its potential protective mechanism has not been well elucidated. This study aimed to investigate the effects of l-Arg on the LPS-induced oxidative stress and apoptosis in differentiated C2C12 myotube cells. Our results demonstrated that myotube cells treated with 0.2 mg/mL LPS significantly decreased cell viability. l-Arg treatment significantly suppressed LPS induced ROS accumulation and cell apoptosis. Furthermore, l-Arg improved antioxidant-related enzymes’ activities; increased antioxidant ability via Akt-Nrf2 signaling pathway; maintained the mitochondrial membrane potential (MMP); and enhanced FOXO3a expression, leading to a decrease in the mitochondrial-associated apoptotic proteins. In addition, l-Arg exposure dramatically increased the mRNA and protein expressions of SIRT1. The cytoprotective effect of l-Arg was restricted by the SIRT1 inhibitor EX527, which led to an increase in ROS level, apoptosis rate, and decreased cell MMP. The results also demonstrated that EX527 treatment significantly eliminated the effect of l-Arg on LPS-induced oxidative damage and mitochondria-mediated cell apoptosis. Our findings revealed that l-Arg could be used as a potential nutraceutical in reducing muscle injury via regulating SIRT1-Akt-Nrf2 and SIRT1-FOXO3a-mitochondria apoptosis signaling pathways.

5-Aminolevulinic acid combined with ferrous iron improves glucose tolerance in high-fat diet-fed mice via upregulation of glucose transporter 1.

Decreased mitochondrial metabolism suppresses glucose metabolism, resulting in obesity and diabetes. The present study aimed to investigate mechanisms underlying the 5-aminolevulinic acid (5-ALA) hydrochloride-mediated increase in glucose uptake in high-fat diet (HFD)-fed mice in vivo and C2C12 myotube cells in vitro. C57BL/6N male mice (20 weeks old) were fed either HFD or normal diet (ND) for 4 weeks. A total of five HFD-fed mice were orally administered with 300 mg/kg 5-ALA hydrochloride and 47.1 mg/kg sodium ferrous citrate (SFC; HFD + 5-ALA/SFC), whereas ND and other HFD-fed mice were orally administered with saline. After 4 weeks, these mice were intraperitoneally administered with 2 g/kg glucose and 3.2 mg/kg 2-deoxyglucose (2DG) for intraperitoneal glucose tolerance test (IPGTT) and glucose uptake test. Body weights, plasma glucose levels and the area under the curve of IPGTT were lower in mice treated with HFD + 5-ALA/SFC compared with in those treated with HFD alone. 2DG uptake in the gastrocnemius muscle and heart were more significantly improved in the HFD + 5-ALA/SFC mice compared with the HFD-fed mice. Furthermore, 5-ALA/SFC increased 2DG uptake in C2C12 cells to a similar level to the insulin-treated group. Moreover, it increased glucose transport (GLUT)1 translocation in the plasma membrane by 2.5-fold relative to the controls without affecting GLUT1 expression; however, it had no effect on GLUT4 translocation. Therefore, 5-ALA/SFC enhanced gastrocnemius and cardiac glucose uptake in HFD-fed mice, and upregulated GLUT1 translocation to the plasma membrane, but not GLUT4 in C2C12 myotube cells. Therefore, it could potentially be used as a novel drug for the treatment of diabetes.

Inhibition of heat shock protein (HSP) 90 reverses signal transducer and activator of transcription (STAT) 3-mediated muscle wasting in cancer cachexia mice.

Cancer cachexia is a common cause of death among cancer patients with no currently effective treatment available. In animal models, aberrant activation of STAT3 in skeletal muscle contributes to muscle wasting. However, clinically the factors regulating STAT3 activation and the molecular mechanisms involved remain incompletely understood. The expression of HSP90 and the activation of STAT3 were detected in muscle from the patients with cancer cachexia or the tumour-bearing cachectic mice. HSP90 inhibitors, including 17DMAG (alvespimycin) and PU-H71, were administered to cachexic mice and cachexia parameters, weight loss, food intake, survival rate, body composition, serum metabolites, muscle wasting pathology and catabolic activation were analysed. The co-culture of C2C12 myotube cells with C26 conditioned media was performed to investigate the pathological mechanism involved in catabolic muscle wasting. The roles of HSP90, STAT3 and FOXO1 in myotube atrophy were explored via overexpression or knockdown. An enhanced interaction between activated STAT3 and HSP90 in the skeletal muscle of cancer cachexia patients, is a crucial for the development of cachectic muscle wasting. HSP90 inhibitors 17DMAG and PU-H71 alleviated the muscle wasting in C26 and models or the myotube atrophy of C2C12 cells induced by C26 conditional medium. Prolonged STAT3 activation transactivated FOXO1 by binding directly to its promoter and triggered the muscle wasting in a FOXO1-dependent manner in muscle cells. The HSP90/STAT3/FOXO1 axis plays a critical role in cachectic muscle wasting, which might be a potential therapeutic target for the treatment of cancer cachexia.