Advanced glycation end products promote ROS production via PKC/p47 phox axis in skeletal muscle cells.

Advanced glycation end products (AGEs) are risk factors for various diseases, including sarcopenia. One of the deleterious effects of AGEs is the induction of abnormal reactive oxygen species (ROS) production in skeletal muscle. However, the underlying mechanism remains poorly understood. Therefore, the aim of this study was to elucidate how AGEs induce ROS production in skeletal muscle cells. This study demonstrated that AGEs treatment promoted ROS production in myoblasts and myotubes while PKC inhibitor abolished ROS production by AGEs stimulation. Phosphorylation of p47 phox by kinases such as PKCα is required to form the Nox2 complex, which induces ROS production. In this study, AGEs treatment promoted the phosphorylation of PKCα and p47 phox in myoblasts and myotubes. Our findings suggest that AGEs promote ROS production through the phosphorylation of PKCα and p47 phox in skeletal muscle cells.

The Adrenal Pheochromocytoma Cell Line PC12 Efficiently Promotes the Regeneration Capability of Adipose Tissue-Derived Mesenchymal Stem Cells in Myogenesis: A Particular Approach to Improving Skeletal Muscle Cell Regeneration.

Background: Researchers are looking for a way to improve the myogenic differentiation of stem cells. Adipose-derived stem cells (ADSCs), known for their multipotency and regenerative capabilities, have been extensively studied for their therapeutic potential. Meanwhile, PC12 cells, derived from rat pheochromocytoma, have been found pivotal in neuroscience research, particularly as a neuronal model system. The current study investigated the effect of the PC12 adrenal pheochromocytoma cell line on the myogenic differentiation of ADSCs. Methods: This experimental study was conducted during 2019-2022 (Ahvaz, Iran). Differentiation of ADSCs was induced by using 3 µg/mL 5-azacytidine for 24 hours. Then, the culture media was changed with Dulbecco's Modified Eagle-High Glucose (DMEM-HG) containing 5% horse serum (HS) and kept for 7 days. Different percentages of differentiated ADSCs and PC12 (100:0, 70:30, 50:50, 30:70) were cocultured for 7 days in DMEM-HG containing 5% HS. PC12 was labeled with cell tracker C7000. The real-time polymerase chain reaction and Western blotting techniques were utilized to assess gene and protein expression. All experiments were repeated three times. Data were analyzed using GraphPad Prism 8.0.2 software with a one-way analysis of variance. P<0.05 was considered statistically significant. Results: PC12 visualization confirmed the accuracy of the co-culture process. The differentiated cells showed an aligned, multinucleated shape. The differentiated ADSCs revealed significantly elevated levels of Myh1, Myh2, and Chrn-α1 gene expression compared with undifferentiated ADSCs (P<0.0001). The ADSCs cocultured with PC12 cells showed significantly higher Myh1, Myh2, and Chrn-α1 gene expression than differentiated ADSCs (P<0.001). ADSCs cocultured with 50% PC12 revealed significantly higher MYH and nAchR protein expression than the differentiated group (P<0.01 and P<0.001). Conclusion: Coculturing PC12 cells and ADSCs improves the efficiency of myogenic differentiation. However, the effectiveness of myogenic differentiation depends on the proportions of administered PC12 cells.

Circulating Amino Acid Concentration after the Consumption of Pea or Whey Proteins in Young and Older Adults Affects Protein Synthesis in C2C12 Myotubes.

As older adults tend to reduce their intake of animal-source proteins, plant-source proteins may offer valuable resources for better protein intake. The aim of this study was to assess whether the pea proteins can be used to achieve blood amino acid levels that stimulate muscle protein synthesis. We measured variations in plasma amino acid concentrations in young and older adults given pea (NUTRALYS® S85 Plus) or whey proteins either alone or in a standardized meal. The effect of amino acid concentrations on protein synthesis in C2C12 myotubes was determined. In terms of results, plasma amino acid concentrations reflected the difference between the amino acid contents of whey and pea proteins. Blood leucine showed a greater increase of 91 to 130% with whey protein compared to pea protein, while the opposite was observed for arginine (A greater increase of 147 to 210% with pea compared to whey). Culture media prepared with plasmas from the human study induced age-dependent but not protein-type-dependent changes in myotube protein synthesis. In conclusion, pea and whey proteins have the same qualities in terms of their properties to maintain muscle protein synthesis. Pea proteins can be recommended for older people who do not consume enough animal-source proteins.

Aronia-derived anthocyanins and metabolites ameliorate TNFα-induced disruption of myogenic differentiation in satellite cells.

This study investigates the effects of Aronia berries, their primary anthocyanins and other second metabolites-mimicking dietary anthocyanin consumption-on enhancing muscular myogenesis under chronic inflammation. Murine muscle satellite cells (MuSCs) were cultured ex vivo, allowing for expansion and differentiation into myotubes. Myogenic differentiation was disrupted by TNFα at both early and terminal stages, with treatment using Aronia berries applied at physiologically relevant concentrations alongside TNFα. The results demonstrated that Aronia berries treatments, particularly phenolic metabolites, significantly stimulated the proliferative capacity of MuSCs. Furthermore, Aronia berries treatment enhanced early-stage myogenesis, marked by increased MymX and MyoG expression and nascent myotube formation, with metabolites showing the most pronounced effects. Aronia berry powder and individual anthocyanins exerted milder regulatory effects. Similar trends were observed during terminal differentiation, where Aronia berries treatment promoted myotube growth and inhibited TNFα-induced inflammatory atrophic ubiquitin-conjugating activity. Additionally, the secondary metabolites of Aronia berries significantly prevented muscle-specific ubiquitination in the dexamethasone-induced atrophy model. Overall, the treatment with Aronia berries enhanced myogenesis in a cellular model of chronic muscular inflammation, with Aronia-derived metabolites showing the strongest response, likely through TLR4/NF-κB modulation. In this case, enhanced regeneration capacity and anti-atrophy potential were associated with TLR4/NF-κB modulation.

Inhibition of MAT2A Impairs Skeletal Muscle Repair Function.

The regenerative capacity of muscle, which primarily relies on anabolic processes, diminishes with age, thereby reducing the effectiveness of therapeutic interventions aimed at treating age-related muscle atrophy. In this study, we observed a decline in the expression of methionine adenosine transferase 2A (MAT2A), which synthesizes S-adenosylmethionine (SAM), in the muscle tissues of both aged humans and mice. Considering MAT2A's critical role in anabolism, we hypothesized that its reduced expression contributes to the impaired regenerative capacity of aging skeletal muscle. Mimicking this age-related reduction in the MAT2A level, either by reducing gene expression or inhibiting enzymatic activity, led to inhibiting their differentiation into myotubes. In vivo, inhibiting MAT2A activity aggravated BaCl2-induced skeletal muscle damage and decreased the number of satellite cells, whereas supplementation with SAM improved these effects. RNA-sequencing analysis further revealed that the Fas cell surface death receptor (Fas) gene was upregulated in Mat2a-knockdown C2C12 cells. Suppressing MAT2A expression or activity elevated Fas protein levels and increased the proportion of apoptotic cells. Additionally, inhibition of MAT2A expression or activity increased p53 expression. In conclusion, our findings demonstrated that impaired MAT2A expression or activity compromised the regeneration and repair capabilities of skeletal muscle, partially through p53-Fas-mediated apoptosis.

Valsartan Rescues Suppressed Mitochondrial Metabolism during Insulin Resistance in C2C12 Myotubes.

Elevated circulating branched-chain amino acids (BCAA) have been linked with the severity of insulin resistance across numerous populations, implicating heightened BCAA metabolism as a potential therapy for insulin resistance. Recently, the angiotensin II type 1 receptor (AT1R) inhibitor Valsartan (VAL) was identified as a potent inhibitor of branched-chain alpha-keto acid dehydrogenase kinase (BCKDK), a negative regulator of BCAA metabolism. This work investigated the effect of VAL on myotube metabolism and insulin sensitivity under both insulin sensitive and insulin resistant conditions. C2C12 myotubes were treated with or without VAL at 8 µM for 24 h, both with and without hyperinsulinemic-induced insulin resistance. Oxygen consumption and extracellular acidification were used to measure mitochondrial and glycolytic metabolism, respectively. Gene expression was assessed via qRT-PCR, and insulin sensitivity was assessed via Western blot. Insulin resistance significantly reduced both basal and peak mitochondrial function which were rescued to control levels by concurrent VAL. Changes in mitochondrial function occurred without substantial changes in mitochondrial content or related gene expression. Insulin sensitivity and glycolytic metabolism were unaffected by VAL, as was lipogenic signaling and lipid content. Additionally, both VAL and insulin resistance depressed Bckdha expression. Interestingly, an interaction effect was observed for extracellular isoleucine, valine, and total BCAA (but not leucine), suggesting VAL may alter BCAA utilization in an insulin sensitivity-dependent manner. Insulin resistance appears to suppress mitochondrial function in a myotube model which can be rescued by VAL. Further research will be required to explore the implications of these findings in more complex models.

N-acetylaspartate promotes glycolytic-to-oxidative fiber-type switch and resistance to atrophic stimuli in myotubes.

N-acetylaspartate (NAA) is a neuronal metabolite that can be extruded in extracellular fluids and whose blood concentration increases in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Aspartoacylase (ASPA) is the enzyme responsible for NAA breakdown. It is abundantly expressed in skeletal muscle and most other human tissues, but the role of NAA catabolism in the periphery is largely neglected. Here we demonstrate that NAA treatment of differentiated C2C12 muscle cells increases lipid turnover, mitochondrial biogenesis and oxidative metabolism at the expense of glycolysis. These effects were ascribed to NAA catabolism, as CRISPR/Cas9 ASPA KO cells are insensitive to NAA administration. Moreover, the metabolic switch induced by NAA was associated with an augmented resistance to atrophic stimuli. Consistently with in vitro results, SOD1-G93A ALS mice show an increase in ASPA levels in those muscles undergoing the glycolytic to oxidative switch during the disease course. The impact of NAA on the metabolism and resistance capability of myotubes supports a role for this metabolite in the phenotypical adaptations of skeletal muscle in neuromuscular disorders.

Bacterioruberin extract from Haloarchaea Haloferax marinum: Component identification, antioxidant activity and anti-atrophy effect in LPS-treated C2C12 myotubes.

Carotenoids are natural pigments utilized as colourants and antioxidants across food, pharmaceutical and cosmetic industries. They exist in carbon chain lengths of C30, C40, C45 and C50, with C40 variants being the most common. Bacterioruberin (BR) and its derivatives are part of the less common C50 carotenoid group, synthesized primarily by halophilic archaea. This study analysed the compositional characteristics of BR extract (BRE) isolated from 'Haloferax marinum' MBLA0078, a halophilic archaeon isolated from seawater near Yeoungheungdo Island in the Republic of Korea, and investigated its antioxidant activity and protective effect on lipopolysaccharide (LPS)-induced C2C12 myotube atrophy. The main components of BRE included all-trans-BR, monoanhydrobacterioruberin, 2-isopentenyl-3,4-dehydrorhodopin and all-trans-bisanhydrobacterioruberin. BRE exhibited higher antioxidant activity and DNA nicking protection activity than other well-known C40 carotenoids, such as ß-carotene, lycopene and astaxanthin. In C2C12 myotubes, LPS treatment led to a reduction in myotube diameter and number, as well as the hypertranscription of the muscle-specific ubiquitin ligase MAFbx and MuRF1. BRE mitigated these changes by activating the Akt/mTOR pathway. Furthermore, BRE abolished the elevated cellular reactive oxygen species levels and the inflammation response induced by LPS. This study demonstrated that 'Hfx. marinum' is an excellent source of natural microbial C50 carotenoids with strong antioxidant capacity and may offer potential protective effects against muscle atrophy.

Comparative transcriptomics of broad-spectrum and synthetic cannabidiol treated C2C12 skeletal myotubes.

Cannabidiol (CBD) is widely used in sports for recovery, pain management, and sleep improvement, yet its effects on muscle are not well understood. This study aimed to determine the transcriptional response of murine skeletal muscle myotubes to broad-spectrum CBD and synthetic CBD (sCBD). Differentiated C2C12 myotubes were treated with 10 µM CBD, sCBD, or vehicle control (DMSO) for 24 h before RNA extraction. Poly-A tail-enriched mRNA libraries were constructed and sequenced using 2 × 50 bp paired-end sequencing. CBD and sCBD treatment induced 4489 and 1979 differentially expressed genes (DEGs; p < 0.001, FDR step-up <0.05), respectively, with common upregulation of 857 genes and common downregulation of 648 genes. Common upregulated DEGs were associated with "response to unfolded protein," "cell redox homeostasis," "endoplasmic reticulum stress," "oxidative stress," and "cellular response to hypoxia." Common downregulated DEGs were linked to "sarcomere organization," "skeletal muscle tissue development," "regulation of muscle contraction," and "muscle contraction." CBD treatment induced unique DEGs compared to sCBD. The data indicate CBD may induce mild cellular stress, activating pathways associated with altered redox balance, unfolded protein response, and endoplasmic reticulum stress. We hypothesize that CBD interacts with muscle and may elicit a "mitohormetic" effect that warrants further investigation.

Filbertone Reduces Senescence in C2C12 Myotubes Treated with Doxorubicin or H2O2 through MuRF1 and Myogenin.

It has been demonstrated that filbertone, the principal flavor compound of hazelnuts, exhibits preventive effects against hypothalamic inflammation, obesity, neurodegenerative diseases, and muscle lipid accumulation. However, its influence on muscle aging has yet to be elucidated. The objective of this study was to investigate the effects of filbertone on muscle aging in C2C12 myotubes subjected to senescence induction by either doxorubicin or hydrogen peroxide. To ascertain the mechanisms by which filbertone exerts its effects, we conducted a series of experiments, including Western blot analysis, reverse transcription quantitative polymerase chain reaction (qRT-PCR), and senescence-associated ß-galactosidase (SA-ß-gal) staining. Filbertone was markedly observed to decrease not only the protein levels of p53 (p < 0.01) in senescence-induced skeletal muscle cells, but also the gene expression levels of p21 (p < 0.05), a direct target of p53. The expression of muscle-related genes, including myogenin and muscle RING-finger protein-1 (MuRF1), was found to be significantly enhanced in senescent muscle cells following treatment with filbertone (p < 0.05). In addition, the number of senescent skeletal muscle cells exhibiting ß-galactosidase activity was found to be markedly reduced in the presence of filbertone (p < 0.01). Collectively, these findings suggest that filbertone plays a pivotal role in the regulation of muscle aging.

Daidzein Inhibits Muscle Atrophy by Suppressing Inflammatory Cytokine- and Muscle Atrophy-Related Gene Expression.

BACKGROUND: Sarcopenic obesity, which is associated with a poorer prognosis than that of sarcopenia alone, may be positively affected by soy isoflavones, known inhibitors of muscle atrophy. Herein, we hypothesize that these compounds may prevent sarcopenic obesity by upregulating the gut metabolites with anti-inflammatory effects. METHODS: To explore the effects of soy isoflavones on sarcopenic obesity and its mechanisms, we employed both in vivo and in vitro experiments. Mice were fed a high-fat, high-sucrose diet with or without soy isoflavone supplementation. Additionally, the mouse C2C12 myotube cells were treated with palmitic acid and daidzein in vitro. RESULTS: The isoflavone considerably reduced muscle atrophy and the expression of the muscle atrophy genes in the treated group compared to the control group (Fbxo32, p = 0.0012; Trim63, p < 0.0001; Foxo1, p < 0.0001; Tnfa, p = 0.1343). Elevated levels of daidzein were found in the muscles and feces of the experimental group compared to the control group (feces, p = 0.0122; muscle, p = 0.0020). The real-time PCR results demonstrated that the daidzein decreased the expression of the palmitate-induced inflammation and muscle atrophy genes in the C2C12 myotube cells (Tnfa, p = 0.0201; Il6, p = 0.0008; Fbxo32, p < 0.0001; Hdac4, p = 0.0002; Trim63, p = 0.0114; Foxo1, p < 0.0001). Additionally, it reduced the palmitate-induced protein expression related to the muscle atrophy in the C2C12 myotube cells (Foxo1, p = 0.0078; MuRF1, p = 0.0119). CONCLUSIONS: The daidzein suppressed inflammatory cytokine- and muscle atrophy-related gene expression in the C2C12 myotubes, thereby inhibiting muscle atrophy.

Glucocorticoid Deflazacort Normalizes the Ultrastructure of Skeletal Muscles and the State of the Colon Microbiota in Dystrophin-Deficient Mice.

We studied the effect of enteral administration of the glucocorticoid deflazacort (DFC, 1.2 mg/kg per day, 28 days) on the state of skeletal muscles and tissue ultrastructure, as well as the composition of the colon microbiota in dystrophin-deficient mdx mice. DFC has been shown to reduce the intensity of degeneration/regeneration cycles in muscle fibers of mdx mice. This effect of DFC was accompanied by normalization of the size of sarcomeres of skeletal muscles of mdx mice, improvement of the ultrastructure of the subsarcolemmal population of mitochondria, and an increase in the number of organelles, as well as normalization of the number of contact interactions between the sarcoplasmic reticulum and mitochondria. In addition, DFC had a corrective effect on the colon microbiota of mdx mice, which manifested in an increase in the number of the Bifidobacterium genus microorganisms and a decrease in the level of E. coli with reduced enzymatic activity.

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.

KIC (ketoisocaproic acid) and leucine have divergent effects on tissue insulin signaling but not on whole-body insulin sensitivity in rats.

Plasma levels of branched-chain amino acids and their metabolites, the branched-chain ketoacids are increased in insulin resistance. Our previous studies showed that leucine and its metabolite KIC suppress insulin-stimulated glucose uptake in L6 myotubes along with the activation of the S6K1-IRS-1 pathway. Because other tissue and fiber types can be differentially regulated by KIC, we analyzed the effect of KIC gavage on whole-body insulin sensitivity and insulin signaling in vivo. We hypothesized that KIC gavage would reduce whole-body insulin sensitivity and increase S6K1-IRS-1 phosphorylation in various tissues and muscle fibers. Five-week-old male Sprague-Dawley rats were starved for 24 hours and then gavaged with 0.75ml/100g of water, leucine (22.3g/L) or KIC (30g/L) twice, ten minutes apart. They were then euthanized at different time points post-gavage (0.5-3h), and muscle, liver, and heart tissues were dissected. Other sets of gavaged animals underwent an insulin tolerance test. Phosphorylation (ph) of S6K1 (Thr389), S6 (Ser235/6) and IRS-1 (Ser612) was increased at 30 minutes post leucine gavage in skeletal muscles irrespective of fiber type. Ph-S6 (Ser235/6) was also increased in liver and heart 30 minutes after leucine gavage. KIC gavage increased ph-S6 (Ser235/6) in the liver. Neither Leucine nor KIC influenced whole-body insulin tolerance, nor ph-Akt (Ser473) in skeletal muscle and heart. BCKD-E1 α abundance was highest in the heart and liver, while ph-BCKD-E1 α (Ser293) was higher in the gastrocnemius and EDL compared to the soleus. Our data suggests that only leucine activates the S6K1-IRS-1 signaling axis in skeletal muscle, liver and heart, while KIC only does so in the liver. The effect of leucine and KIC on the S6K1-IRS-1 signaling pathway is uncoupled from whole-body insulin sensitivity. These results suggest that KIC and leucine may not induce insulin resistance, and the contributions of other tissues may regulate whole-body insulin sensitivity in response to leucine/KIC gavage.

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.

JAK inhibition with tofacitinib rapidly increases contractile force in human skeletal muscle.

Reduction in muscle contractile force associated with many clinical conditions incurs serious morbidity and increased mortality. Here, we report the first evidence that JAK inhibition impacts contractile force in normal human muscle. Muscle biopsies were taken from patients who were randomized to receive tofacitinib (n = 16) or placebo (n = 17) for 48 h. Single-fiber contractile force and molecular studies were carried out. The contractile force of individual diaphragm myofibers pooled from the tofacitinib group (n = 248 fibers) was significantly higher than those from the placebo group (n = 238 fibers), with a 15.7% greater mean maximum specific force (P = 0.0016). Tofacitinib treatment similarly increased fiber force in the serratus anterior muscle. The increased force was associated with reduced muscle protein oxidation and FoxO-ubiquitination-proteasome signaling, and increased levels of smooth muscle MYLK. Inhibition of MYLK attenuated the tofacitinib-dependent increase in fiber force. These data demonstrate that tofacitinib increases the contractile force of skeletal muscle and offers several underlying mechanisms. Inhibition of the JAK-STAT pathway is thus a potential new therapy for the muscle dysfunction that occurs in many clinical conditions.

Anti-muscle atrophy effect of fermented Tenebrio molitor larvae extract by modulating the PI3K-Akt-mTOR/FoxO3α pathway in mice treated with dexamethasone.

This study investigated the anti-sarcopenic effect of fermented Tenebrio molitor larvae (mealworms) extract (FME) in both dexamethasone (DEX)-treated C2C12 cells and mice. FME (100 µg/mL) increased the diameter of myotubes and inhibited the gene and protein expression of atrogin-1 compared to DEX- or non-fermented mealworms extract (ME)-treated C2C12 cells. Male C57BL/6N mice were divided into five groups: Normal Control (NC), DEX (10 mg/kg, intraperitoneal), and three groups of DEX+FME (100, 200, or 500 mg FME/kg/day, oral) for two weeks. FME at doses of 200 and 500 mg/kg effectively improved grip strength when compared to the DEX group. Histological analysis of the quadriceps muscle showed a larger muscle fiber size in the DEX+FME groups compared to DEX group. FME (200 and 500 mg/kg) significantly increased cross-sectional area of the muscle fiber compared to DEX group. FME (500 mg/kg) significantly decreased the ubiquitin, atrogin-1 and MuRF-1 protein levels, and increased levels of MHC and MyoG in DEX-treated mice. The puromycin labeling assay revealed that FME increased protein synthesis in DEX-induced muscle atrophy. The FME treatment demonstrated significant upregulation in phosphorylation levels, including mTOR, FoxO3α, Akt, and PI3K compared to DEX group. In conclusion, FME inhibited the increase in proteins associated with muscle atrophy, including, atrogin-1 and MuRF-1, by regulating the PI3K-Akt-FoxO3α pathway. FME improved the PI3K-Akt-mTOR signaling pathway, which was reduced by DEX. This study suggests that FME has the potential for use in sarcopenia therapy, possibly serving as a natural agent that counteracts the negative effects of DEX on muscle tissue.

Vigeo Promotes Myotube Differentiation and Protects Dexamethasone-Induced Skeletal Muscle Atrophy via Regulating the Protein Degradation, AKT/mTOR, and AMPK/Sirt-1/PGC1α Signaling Pathway In Vitro and In Vivo.

Sarcopenia, a condition caused by an imbalance between muscle growth and loss, can severely affect the quality of life of elderly patients with metabolic, inflammatory, and cancer diseases. Vigeo, a nuruk-fermented extract of three plants (Eleutherococcus senticosus Maxim (ESM), Achyranthes japonica (Miq.) Nakai (AJN), and Atractylodes japonica Koidzumi (AJK)) has been reported to have anti-osteoporotic effects. However, evidence of the effects of Vigeo on muscle atrophy is not available. Here, in the in vivo model of dexamethasone (Dex)-induced muscle atrophy, Vigeo treatment significantly reversed Dex-induced decreases in calf muscle volume, gastrocnemius (GA) muscle weight, and histological cross-section area. In addition, in mRNA and protein analyses isolated from GA muscle, we observed that Vigeo significantly protected against Dex-induced mouse muscle atrophy by inhibiting protein degradation regulated by atrogin and MuRF-1. Moreover, we demonstrated that Vigeo significantly promoted C2C12 cell line differentiation, as evidenced by the increased width and length of myotubes, and the increased number of fused myotubes with three or more nuclei. Vigeo alleviated the formation of myotubes compared to the control group. Vigeo also significantly increased the mRNA and protein expression of myosin heavy chain (MyHC), MyoD, and myogenin compared to that in the control. Vigeo treatment significantly reduced the mRNA and protein expression of muscle degradation markers atrogin-1 and muscle RING Finger 1 (MuRF-1) in the C2C12 cell line in vitro. Vigeo also activated the AMP-activated protein kinase (AMPK)/silent information regulator 1 (Sirt-1)/peroxisome proliferator-activated receptor-γ co-activator-1α (PGC1α) mitochondrial biogenesis pathway and the Akt/mTOR protein synthesis signaling pathway in Dex-induced myotube atrophy. These findings suggest that Vigeo may have protective effects against Dex-induced muscle atrophy. Therefore, we propose Vigeo as a supplement or potential therapeutic agent to prevent or treat sarcopenia accompanied by muscle atrophy and degeneration.

Ameliorative Effects of Fermented Red Ginseng Extract on Muscle Atrophy in Dexamethasone-Induced C2C12 Cell And Hind Limb-Immobilized C57BL/6J Mice.

Fermented red ginseng (FRG) enhances the bioactivity and bioavailability of ginsenosides, which possess various immunomodulatory, antiaging, anti-obesity, and antidiabetic properties. However, the effects of FRG extract on muscle atrophy and the underlying molecular mechanisms remain unclear. This study aimed to elucidate the effects of FRG extract on muscle atrophy using both in vitro and in vivo models. In vitro experiments used dexamethasone (DEX)-induced C2C12 myotubes to assess cell viability, myotube diameter, and fusion index. In vivo experiments were conducted on hind limb immobilization (HI)-induced mice to evaluate grip strength, muscle mass, and fiber cross-sectional area (CSA) of the gastrocnemius (GAS), quadriceps (QUA), and soleus (SOL) muscles. Molecular mechanisms were investigated through the analysis of key signaling pathways associated with muscle protein synthesis, energy metabolism, and protein degradation. FRG extract treatment enhanced viability of DEX-induced C2C12 myotubes and restored myotube diameter and fusion index. In HI-induced mice, FRG extract improved grip strength, increased muscle mass and CSA of GAS, QUA, and SOL muscles. Mechanistic studies revealed that FRG extract activated the insulin-like growth factor 1/protein kinase B (Akt)/mammalian target of rapamycin signaling pathway, promoted muscle energy metabolism via the sirtuin 1/peroxisome proliferator-activated receptor gamma-coactivator-1α pathway, and inhibited muscle protein degradation by suppressing the forkhead box O3a, muscle ring-finger 1, and F-box protein (Fbx32) signaling pathways. FRG extract shows promise for ameliorating muscle atrophy by modulating key molecular pathways associated with muscle protein synthesis, energy metabolism, and protein degradation, offering insights for future drug development.

Cadmium exposure induces skeletal muscle insulin resistance through the reactive oxygen species-mediated PINK1/Parkin pathway.

Epidemiological studies have suggested a positive association between environmental cadmium (Cd) exposure and type 2 diabetes mellitus (T2DM). Skeletal muscle insulin resistance (IR) plays a critical role in the pathogenesis of T2DM. This study aimed to investigate the effects of chronic low-level Cd exposure on skeletal muscle IR and its potential mechanism. Rats were exposed to drinking water containing 2 or 10 mg/L Cd for 24 weeks. Differentiated L6 myotubes were treated with Cd for 72 h. Immunofluorescence, flow cytometry assay, RNA-sequencing, and Seahorse analysis were conducted to determine the effects of Cd and its underlying mechanism on relevant parameters, including insulin sensitivity, glucose uptake, oxidative stress, mitophagy, and mitochondrial function in skeletal muscle and L6 myotubes. N-acetyl-cysteine (NAC), a scavenger of reactive oxygen species (ROS), and mitophagy inhibitor Cyclosporin A (CsA) were used to confirm the role of oxidative stress in mitophagy and mitochondrial dysfunction caused by Cd. We found that rats exposed to 10 mg/L Cd exhibited hyperglycemia and skeletal muscle IR. Cd markedly increased IRS-1 phosphorylation at Ser612, while decreased levels of phosphorylated PI3K, Akt, AS160, inhibited GLUT4 translocation and glucose uptake. Mechanistically, Cd increased the intracellular ROS, hydrogen peroxide, and malondialdehyde levels and decreased antioxidase activity in L6 myotubes. Furthermore, Cd upregulated the mRNA and protein levels of LC3II/I, PINK1, and Parkin. In addition, Cd induced the formation of mitophagosomes, reduced the mitochondrial membrane potential, decreased the adenosine triphosphate content, and impaired the mitochondrial respiratory capacity. Strikingly, NAC ameliorated oxidative stress, excessive mitophagy, and the associated reduction in myotube insulin sensitivity, while inhibition of mitophagy by CsA alleviated skeletal muscle IR. In conclusion, this study reveals a previously unrecognized mechanism that chronic low-level Cd exposure may induce mitophagy by activating the PINK1/Parkin signal pathway by increasing ROS, thus causing skeletal muscle IR and elevated blood glucose.