C2C12 Myotubes Research Articles

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

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

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

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

Equisetum arvense standardized dried extract hinders age-related osteosarcopenia

Age-associated osteosarcopenia is an unresolved syndrome characterized by the concomitant loss of bone (osteopenia) and skeletal muscle (sarcopenia) tissues increasing falls, immobility, morbidity, and mortality. Unbalanced resorption of bone in the remodeling process and excessive protein breakdown, especially fast type II myosin heavy chain (MyHC-II) isoform and myofiber metabolic shift, are the leading causes of bone and muscle deterioration in the elderly, respectively. Equisetum arvense (EQ) is a plant traditionally recommended for many pathological conditions due to its anti-inflammatory properties. Thus, considering that a chronic low-grade inflammatory state predisposes to both osteoporosis and sarcopenia, we tested a standardized hydroalcoholic extract of EQ in in vitro models of muscle atrophy [C2C12 myotubes treated with proinflammatory cytokines (TNFα/IFNγ), excess glucocorticoids (dexamethasone), or the osteokine, receptor activator of nuclear factor kappa-B ligand (RANKL)] and osteoclastogenesis (RAW 264.7 cells treated with RANKL). We found that EQ counteracted myotube atrophy, blunting the activity of several pathways depending on the applied stimulus, and reduced osteoclast formation and activity. By in silico target fishing, IKKB-dependent nuclear factor kappa-B (NF-κB) inhibition emerges as a potential common mechanism underlying EQ′s anti-atrophic effects. Consumption of EQ (500 mg/kg/day) by pre-geriatric C57BL/6 mice for 3 months translated into: i) maintenance of muscle mass and performance; ii) restrained myofiber oxidative shift; iii) slowed down age-related modifications in osteoporotic bone, significantly preserving trabecular connectivity density; iv) reduced muscle- and spleen-related inflammation. EQ can preserve muscle functionality and bone remodeling during aging, potentially valuable as a natural treatment for osteosarcopenia.

Coculture with Colon-26 cancer cells decreases the protein synthesis rate and shifts energy metabolism toward glycolysis dominance in C2C12 myotubes.

Cancer cachexia is the result of complex interorgan interactions initiated by cancer cells and changes in patient behavior such as decreased physical activity and energy intake. Therefore, it is crucial to distinguish between the direct and indirect effects of cancer cells on muscle mass regulation and bioenergetics to identify novel therapeutic targets. In this study, we investigated the direct effects of Colon-26 cancer cells on the molecular regulating machinery of muscle mass and its bioenergetics using a coculture system with C2C12 myotubes. Our results demonstrated that coculture with Colon-26 cells induced myotube atrophy and reduced skeletal muscle protein synthesis and its regulating mechanistic target of rapamycin complex 1 signal transduction. However, we did not observe any activating effects on protein degradation pathways including ubiquitin-proteasome and autophagy-lysosome systems. From a bioenergetic perspective, coculture with Colon-26 cells decreased the complex I-driven, but not complex II-driven, mitochondrial ATP production capacity, while increasing glycolytic enzyme activity and glycolytic metabolites, suggesting a shift in energy metabolism toward glycolysis dominance. Gene expression profiling by RNA sequencing showed that the increased activity of glycolytic enzymes was consistent with changes in gene expression. However, the decreased ATP production capacity of mitochondria was not in line with the gene expression. The potential direct interaction between cancer cells and skeletal muscle cells revealed in this study may contribute to a better fundamental understanding of the complex pathophysiology of cancer cachexia.NEW & NOTEWORTHY We explored the potential direct interplay between colon cancer cells (Colon-26) and skeletal muscle cells (C2C12 myotubes) employing a noncontact coculture experimental model. Our findings reveal that coculturing with Colon-26 cells substantially impairs the protein synthesis rate, concurrently instigating a metabolic shift toward glycolytic dominance in C2C12 myotubes. This research unveils critical insights into the intricate cellular cross talk underpinning the complex pathophysiology of cancer cachexia.

Differential effects of cholecalciferol and calcitriol on muscle proteolysis and oxidative stress in angiotensin II-induced C2C12 myotube atrophy.

Renin-angiotensin system activation contributes to skeletal muscle atrophy in aging individuals with chronic diseases. We aimed to explore the effects of cholecalciferol (VD3) and calcitriol (1,25VD3) on signaling of muscle proteolysis and oxidative stress in myotubes challenged with angiotensin II (AII). The mouse C2C12 myotubes were assigned to vehicle, AII, AII + VD3, AII + 1,25VD3, and AII + losartan groups. The expression levels of muscle-specific E3 ubiquitin ligase proteins, autophagy-related proteins, and oxidative stress markers were investigated. We demonstrated the diverse effects of VD3 and 1,25VD3 on AII-induced myotube atrophy. The myotube diameter was preserved by treatment with 100 nM VD3 and losartan, while 1 and 10 nM 1,25VD3 increased levels of FoxO3a, MuRF1, and atrogin-1 protein expression in myotubes exposed to AII. Treatment with AII + 10 nM 1,25VD3 resulted in the upregulation of LC3B-II, LC3B-II/LC3B-I, and mature cathepsin L, which are autophagic marker proteins. The p62/SQSTM1 protein was downregulated and vitamin D receptor was upregulated after treatment with AII + 10 nM 1,25VD3. A cellular redox imbalance was observed as AII + 10 nM 1,25VD3-induced reactive oxygen species and NADPH oxidase-2 overproduction, and these changes were associated with an inadequate response of antioxidant superoxide dismutase-1 and catalase proteins. Collectively, these findings provide a translational perspective on the role of vitamin D3 in alleviating muscle atrophy related to high levels of AII.

Impact of MG132 induced-proteotoxic stress on αB-crystallin and desmin phosphorylation and O-GlcNAcylation and their partition towards cytoskeleton

Small Heat Shock Proteins are considered as the first line of defense when proteostasis fails. Among them, αB-crystallin is expressed in striated muscles in which it interacts with desmin intermediate filaments to stabilize them, maintaining cytoskeleton's integrity and muscular functionalities. Desmin is a key actor for muscle health; its targeting by αB-crystallin is thus crucial, especially in stress conditions.αB-crystallin is phosphorylated and O-GlcNAcylated. Its phosphorylation increases consecutively to various stresses, correlated with its recruitment for cytoskeleton's safeguarding. However, phosphorylation as unique signal for cytoskeleton translocation remains controversial; indeed, O-GlcNAcylation was also proposed to be involved. Thus, there are still some gaps for a deeper comprehension of how αB-crystallin functions are finely regulated by post-translational modifications. Furthermore, desmin also bears both post-translational modifications; while desmin phosphorylation is closely linked to desmin intermediates filaments turnover, it is unclear whereas its O-GlcNAcylation could impact its proper function.In the herein paper, we aim at identifying whether phosphorylation and/or O-GlcNAcylation are involved in αB-crystallin targeting towards cytoskeleton in proteotoxic stress induced by proteasome inhibition in C2C12 myotubes. We demonstrated that proteotoxicity led to αB-crystallin's phosphorylation and O-GlcNAcylation patterns changes, both presenting a dynamic interplay depending on protein subfraction. Importantly, both post-translational modifications showed a spatio-temporal variation correlated with αB-crystallin translocation towards cytoskeleton. In contrast, we did not detect any change of desmin phosphorylation and O-GlcNAcylation. All together, these data strongly support that αB-crystallin phosphorylation/O-GlcNAcylation interplay rather than changes on desmin is a key regulator for its cytoskeleton translocation, preserving it towards stress.

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Kaempferol Improves Exercise Performance by Regulating Glucose Uptake, Mitochondrial Biogenesis, and Protein Synthesis via PI3K/AKT and MAPK Signaling Pathways.

Kaempferol is a natural flavonoid with reported bioactivities found in many fruits, vegetables, and medicinal herbs. However, its effects on exercise performance and muscle metabolism remain inconclusive. The present study investigated kaempferol's effects on improving exercise performance and potential mechanisms in vivo and in vitro. The grip strength, exhaustive running time, and distance of mice were increased in the high-dose kaempferol group (p < 0.01). Also, kaempferol reduced fatigue-related biochemical markers and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) related to antioxidant capacity. Kaempferol also increased the glycogen and adenosine triphosphate (ATP) content in the liver and skeletal muscle, as well as glucose in the blood. In vitro, kaempferol promoted glucose uptake, protein synthesis, and mitochondrial function and decreased oxidative stress in both 2D and 3D C2C12 myotube cultures. Moreover, kaempferol activated the PI3K/AKT and MAPK signaling pathways in the C2C12 cells. It also upregulated the key targets of glucose uptake, mitochondrial function, and protein synthesis. These findings suggest that kaempferol improves exercise performance and alleviates physical fatigue by increasing glucose uptake, mitochondrial biogenesis, and protein synthesis and by decreasing ROS. Kaempferol's molecular mechanism may be related to the regulation of the PI3K/AKT and MAPK signaling pathways.

Sulforaphane Ameliorates High-Fat-Diet-Induced Metabolic Abnormalities in Young and Middle-Aged Obese Male Mice.

Type 2 diabetes (T2D) is still a fast-growing health problem globally. It is evident that chronic insulin resistance (IR) and progressive loss of β-cell mass and function are key features of T2D etiology. Obesity is a leading pathogenic factor for developing IR. The aim of the present study was to determine whether sulforaphane (SFN), a natural compound derived from cruciferous vegetables, can prevent (prevention approach) or treat (treatment approach) obesity and IR in mouse models. We show that dietary intake of SFN (0.5 g/kg of HFD) for 20 weeks suppressed high-fat diet (HFD)-induced fat accumulation by 6.04% and improved insulin sensitivity by 23.66% in young male mice. Similarly, dietary provision of SFN (0.25 g/kg) significantly improved blood lipid profile, glucose tolerance, and insulin sensitivity of the middle-aged male mice while it had little effects on body composition as compared with the HFD group. In the treatment study, oral administration of SFN (40 mg/kg) induced weight loss and improved insulin sensitivity and plasma lipid profile in the diet-induced-obesity (DIO) male mice. In all three studies, the metabolic effects of SFN administration were not associated with changes in food intake. In vitro, SFN increased glucose uptake in C2C12 myotubes and increased fatty acid and pyruvate oxidation in primary human skeletal muscle cells. Our results suggest that SFN may be a naturally occurring insulin-sensitizing agent that is capable of improving the metabolic processes in HFD-induced obesity and IR and thereby may be a promising compound for T2D prevention.

ADAR2 deficiency ameliorates non-alcoholic fatty liver disease and muscle atrophy through modulating serum amyloid A1.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. Sarcopenia is a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength, which is commonly associated with NAFLD. Adenosine-to-inosine editing, catalysed by adenosine deaminase acting on RNA (ADAR), is an important post-transcriptional modification of genome-encoded RNA transcripts. Three ADAR gene family members, including ADAR1, ADAR2 and ADAR3, have been identified. However, the functional role of ADAR2 in obesity-associated NAFLD and sarcopenia remains unclear. ADAR2+/+/GluR-BR/R mice (wild type [WT]) and ADAR2-/-/GluR-BR/R mice (ADAR2 knockout [KO]) were subjected to feeding with standard chow or high-fat diet (HFD) for 20weeks at the age of 5weeks. The metabolic parameters, hepatic lipid droplet, grip strength test, rotarod test, muscle weight, fibre cross-sectional area (CSA), fibre types and protein associated with protein degradation were examined. Systemic and local tissues serum amyloid A1 (SAA1) were measured. The effects of SAA1 on C2C12 myotube atrophy were investigated. ADAR2 KO mice fed with HFD exhibited lower body weight (-7.7%, P<0.05), lower liver tissue weight (-20%, P<0.05), reduced liver lipid droplets in concert with a decrease in hepatic triglyceride content (-24%, P<0.001) and liver injury (P<0.01). ADAR2 KO mice displayed protection against HFD-induced glucose intolerance, insulin resistance and dyslipidaemia. Skeletal muscle mass (P<0.01), muscle strength (P<0.05), muscle endurance (P<0.001) and fibre size (CSA; P<0.0001) were improved in ADAR2 KO mice fed with HFD compared with WT mice fed with HFD. Muscle atrophy-associated transcripts, such as forkhead box protein O1, muscle atrophy F-box/atrogin-1 and muscle RING finger 1/tripartite motif-containing 63, were decreased in ADAR2 KO mice fed with HFD compared with WT mice fed with HFD. ADAR2 deficiency attenuates HFD-induced local liver and skeletal muscle tissue inflammation. ADAR2 deficiency abolished HFD-induced systemic (P<0.01), hepatic (P<0.0001) and muscular (P<0.001) SAA1 levels. C2C12 myotubes treated with recombinant SAA1 displayed a decrease in myotube length (-37%, P<0.001), diameter (-20%, P<0.01), number (-39%, P<0.001) and fusion index (-46%, P<0.01). Myogenic markers (myosin heavy chain and myogenin) were decreased in SAA1-treated myoblast C2C12 cells. These results provide novel evidence that ADAR2 deficiency may be important in obesity-associated sarcopenia and NAFLD. Increased SAA1 might be involved as a regulatory factor in developing sarcopenia in NAFLD.

Compound Z526 alleviates chemotherapy-induced cachectic muscle loss by ameliorating oxidative stress-driven protein metabolic imbalance and apoptosis

Chemotherapy is one of the primary and indispensable intervention against cancers though it is always accompanied by severe side effects especially cachexia. Cachexia is a fatal metabolic disorder syndrome, mainly characterized by muscle loss. Oxidative stress is the key factor that trigger cachectic muscle loss by inducing imbalance in protein metabolism and apoptosis. Here, we showed an oral compound (Z526) exhibited potent alleviating effects on C2C12 myotube atrophy induced by various chemotherapeutic agents in vitro as well as mice muscle loss and impaired grip force induced by oxaliplatin in vivo. Furthermore, Z526 also could ameliorate C2C12 myotube atrophy induced by the combination of chemotherapeutic agents with conditioned medium of various tumor cells in vitro as well as mice muscle atrophy of C26 tumor-bearing mice treated with oxaliplatin. The pharmacological effects of Z526 were based on its potency in reducing oxidative stress in cachectic myocytes and muscle tissues, which inhibited the activation of NF-κB and STAT3 to decrease Atrogin-1-mediated protein degradation, activated the AKT/mTOR signaling pathway to promote protein synthesis, regulated Bcl-2/BAX ratio to reduce Caspase-3-triggered apoptosis. Our work suggested Z526 to be an optional strategy for ameliorating cachexia muscle atrophy in the multimodality treatment of cancers.

Polygonatum sibiricum polysaccharide ameliorates skeletal muscle aging via mitochondria-associated membrane-mediated calcium homeostasis regulation

BackgroundSarcopenia, an age-related disease, is characterized by a gradual loss of muscle mass, strength, and function. It has been linked to abnormal organelle function in myotubes, including the mitochondria and endoplasmic reticulum (ER). Recent studies revealed that mitochondria-associated membranes (MAM), the sites connecting mitochondria and the ER, may be implicated in skeletal muscle aging. In this arena, the potential of Polygonatum sibiricum polysaccharide (PSP) emerges as a beacon of hope. PSP, with its remarkable antioxidant and anti-senescence properties, is on the cusp of a therapeutic revolution, offering a promising strategy to mitigate the impacts of sarcopenia. PurposeThe objective of this research is to explore the effects of PSP on age-related muscle dysfunction and the underlying mechanisms involved both in vivo and in vitro. MethodsIn this investigation, we used in vitro experiments using D-galactose (D-gal)-induced aging in C2C12 myotubes and in vivo experiments on aged mice. Key indices were assessed, including reactive oxygen species (ROS) levels, mitochondrial function, the expression of aging-related markers, and the key proteins of mitochondria and MAM fraction. Differentially expressed genes (DEGs) related to mitochondria and ER were identified, and bioinformatic analyses were performed to explore underlying mechanisms. Muscle mass and function were determined to evaluate the quantity and quality of skeletal muscle in vivo. ResultsPSP treatment effectively mitigated oxidative stress and mitochondrial malfunction caused by D-gal in C2C12 myotubes, preserving mitochondrial fitness and reducing MAM formation. Besides, PSP attenuated D-gal-induced increases in Ca2+ concentrations intracellularly by modulating the calcium-related proteins, which were also confirmed by gene ontology (GO) analysis of DEGs. In aged mice, PSP increased muscle mass and improved grip strength, hanging time, and other parameters while reducing ROS levels and increasing antioxidant enzyme activities in skeletal muscle tissue. ConclusionPSP offers protection against age-associated muscle impairments. The proposed mechanism suggests that modulation of calcium homeostasis via regulation of the MAM results in a favorable functional outcome during skeletal muscle aging. The results of this study highlight the prospect of PSP as a curative intervention for sarcopenia and affiliated pathological conditions, warranting further investigation.

Abstract 382: Pancreatic cancer-derived organoids alter muscle fiber type and increased energy consumption leading to cachexia

Abstract Cachexia, marked by continuous skeletal muscle mass loss and severe metabolic disturbances, presents a substantial challenge for pancreatic ductal adenocarcinoma (PDAC) patients. Despite its prevalence and the adverse impact on quality of life and survival rates, effective clinical treatments for cachexia remain elusive. This study aims to bridge this gap by employing innovative in vitro models to investigate the underlying mechanisms of cachexia and identify potential therapeutic targets. To enhance the relevance of cell culture models, modifications were introduced to replicate in vivo conditions, where the muscle is exposed to the ongoing kinetics of constant tumor secretion of active factors. Patient-derived PDAC cells were cultured in a dense 3D stiffness extracellular matrix (ECM) to mimic the hypoxic desmoplastic tumor microenvironment. The conditioned medium from prolonged cultured patient-derived organoids (PDOs) was then used to supplement C2C12-derived myotubes. The investigation focuses on dynamic adaptations in muscle fiber type and metabolism when exposed to tumor paracrine factors. Metabolomic investigations of PDO-conditioned medium (PDO CM) on myotubes revealed distinct metabolic differences compared to non-treatment controls. Discriminatory metabolites included those associated with glycolysis, lipid, amino acid, and fatty acid metabolism. PDO CM induced a significant shift in muscle fiber type from slow-twitch to fast-twitch phenotypes, accompanied by de novel expression of embryonic and neonatal Myosin Heavy Chain (MyHC) isoforms. Fast fiber (F59) staining also confirmed the protein expression. Concurrently, PGC-1a, a master regulator of mitochondrial biogenesis and oxidative metabolism, significantly decreased, along with reduced ATP and Mito tracker staining, indicating a metabolic switch under the influence of tumor paracrine factors. Metabolome results on C2C12 myotubes treated with PDO CM also revealed significantly changed ABC transporter-related pathways, an adaptive process required to optimize substrate use under the influence of tumor secretome. However, the genes involved in the proteasome degradation process, such as MAFbx and MuRF1, did not show altered expression. The establishment of an in vitro cachexia model, focusing on changes in energy metabolism and utilizing muscle fiber type alterations as markers, will deepen our understanding of the mechanisms of cancer-associated cachexia. The insights gained may pave the way for identifying potential therapeutic targets to mitigate muscle wasting in PDAC patients and improve overall outcomes. Citation Format: Bo Han, Shuqing Zhao, zhi Yang, Jose Trevino, Steven Grossman, Heinz-Josef Lenz, Ba Xuan Hoang. Pancreatic cancer-derived organoids alter muscle fiber type and increased energy consumption leading to cachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 382.

Abstract 4457: Skeletal muscle-infiltrating CD8 T cells exacerbate muscle atrophy in a syngeneic lung cancer cachexia mouse model

Abstract Cancer-associated cachexia (CAC) is a devastating syndrome of irreversible body weight loss and muscle wasting. CAC has been reported to often lead to poor outcomes of immune checkpoint inhibitors (ICIs), emerging as a standard treatment for various types of cancer. Previously, we have demonstrated that KRAS-mutant lung tumor tissues display an immunosuppressive microenvironment with decreased CD8 T cells and increased regulatory T cells (Tregs). In this orthotopic lung cancer mouse model, treatment with an anti-PD-L1 antibody, a representative ICI, reduced tumor burden, whereas accelerated loss of body weight and skeletal muscle mass. We thus assumed that CD8 T cells could facilitate skeletal muscle wasting in lung tumor-bearing mice, a key feature of CAC. First, we established a syngeneic CAC mouse model through orthotopic transplantation of murine lung cancer cells. At 7 weeks post-injection, these mice developed CAC, displaying over 20 % body weight loss, almost total loss of epididymal fats, and profound skeletal muscle wasting. In line with our assumption, CAC mice showed an increased number of CD8 T cells but a reduced number of Tregs in skeletal muscles, compared to tumor-free control mice. We then investigated whether skeletal muscle-infiltrating CD8 T cells directly induce muscle atrophy indeed. In CAC mice, CD8 neutralization led to restoration of muscle mass and downregulation of muscle atrophy markers, but was not able to significantly affect lung tumor burden. Moreover, direct co-culture with splenic CD8 T cells exacerbated C2C12 myotube atrophy in a conditioned medium from lung cancer cells, further supporting the involvement of CD8 T cells in CAC. To elucidate its underlying mechanism, RNA sequencing analysis was conducted in both skeletal muscles and lung tumors of CAC mice. Based on the differently expressed gene analysis, Cxcl13 was upregulated in skeletal muscles of CAC mice, while downregulated in lung tumors. It is well documented that CXCL13 can mediate recruitment of CD8 T cells. In this regard, CXCL13 might be a plausible regulator in CD8 T cell-induced skeletal muscle atrophy, which requires further investigation to fully understand the molecular mechanism. Citation Format: Se-Young Park, Bomi Kim, Myunggyo Lee, Haeseung Lee, Na-Young Song. Skeletal muscle-infiltrating CD8 T cells exacerbate muscle atrophy in a syngeneic lung cancer cachexia mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4457.

Acetyl genistin modulates myotube differentiation and attenuates dexamethasone-induced muscle atrophy through the FoxO1/3 signaling pathway in C2C12 myotubes

Muscle atrophy, a debilitating condition characterized by loss of muscle mass and strength, is a major concern in various clinical settings. Acetyl genistin (AG), a bioactive compound, was evaluated for its role in muscle cell differentiation and its potential protective effects against dexamethasone (dexa)-induced muscle atrophy. Our study demonstrated that AG significantly promoted C2C12 myotube differentiation, as evidenced by enhanced myotube width and increased fusion index. Notably, AG treatment upregulated the expression of myogenic markers, including MHC, MyoD, and MyoG. Moreover, AG displayed protective properties by attenuating dexa-induced muscle atrophy, mainly by suppressing the expression of the atrophy-related genes MAFbx and MuRF1. AG's protective effects are mechanistically attributed to its regulation of the AMPK/FoxO-dependent signaling pathway. Our results highlighted the dual benefits of AG in fostering muscle differentiation and safeguarding against muscle atrophy, positioning it as a promising agent for muscle health and therapeutic applications.

The myokine CCL5 recruits subcutaneous preadipocytes and promotes intramuscular fat deposition in obese mice.

Intramuscular fat (IMF) refers to the lipid stored in skeletal muscle tissue. The number and size of intramuscular adipocytes are the primary factors that regulate IMF content. Intramuscular adipocytes can be derived from either in situ or ectopic migration. In this study, it was discovered that the regulation of IMF levels is achieved through the chemokine (C-C motif) ligand 5 (CCL5)/chemokine (C-C motif) receptor 5 (CCR5) pathway by modulating adipocyte migration. In coculture experiments, C2C12 myotubes were more effective in promoting the migration of 3T3-L1 preadipocytes than C2C12 myoblasts, along with increasing CCL5. Correspondingly, overexpressing the CCR5, one of the receptors of CCL5, in 3T3-L1 preadipocytes facilitated their migration. Conversely, the application of the CCL5/CCR5 inhibitor, MARAVIROC (MVC), reduced this migration. In vivo, transplanted experiments of subcutaneous adipose tissue (SCAT) from transgenic mice expressing green fluorescent protein (GFP) provided evidence that injecting recombinant CCL5 (rCCL5) into skeletal muscle promotes the migration of subcutaneous adipocytes to the skeletal muscle. The level of CCL5 in skeletal muscle increased with obesity. Blocking the CCL5/CCR5 axis by MVC inhibited IMF deposition, whereas elevated skeletal muscle CCL5 promoted IMF deposition in obese mice. These results establish a link between the IMF and the CCL5/CCR5 pathway, which could have a potential application for modulating IMF through adipocyte migration.NEW & NOTEWORTHY C2C12 myotubes attract 3T3-L1 preadipocyte migration regulated by the chemokine (C-C motif) ligand 5 (CCL5)/ chemokine (C-C motif) receptor 5 (CCR5) axis. High levels of skeletal muscle-specific CCL5 promote the migration of subcutaneous adipocytes to skeletal muscle and induce the intramuscular fat (IMF) content.

Orientin Promotes Antioxidant Capacity, Mitochondrial Biogenesis, and Fiber Transformation in Skeletal Muscles through the AMPK Pathway.

The sleep-breathing condition obstructive sleep apnea (OSA) is characterized by repetitive upper airway collapse, which can exacerbate oxidative stress and free radical generation, thereby detrimentally impacting both motor and sensory nerve function and inducing muscular damage. OSA development is promoted by increasing proportions of fast-twitch muscle fibers in the genioglossus. Orientin, a water-soluble dietary C-glycosyl flavonoid with antioxidant properties, increased the expression of slow myosin heavy chain (MyHC) and signaling factors associated with AMP-activated protein kinase (AMPK) activation both in vivo and in vitro. Inhibiting AMPK signaling diminished the effects of orientin on slow MyHC, fast MyHC, and Sirt1 expression. Overall, orientin enhanced type I muscle fibers in the genioglossus, enhanced antioxidant capacity, increased mitochondrial biogenesis through AMPK signaling, and ultimately improved fatigue resistance in C2C12 myotubes and mouse genioglossus. These findings suggest that orientin may contribute to upper airway stability in patients with OSA, potentially preventing airway collapse.

Abrus precatorius Leaf Extract Stimulates Insulin-mediated Muscle Glucose Uptake: In vitro Studies and Phytochemical Analysis.

Diabetes mellitus, linked with insulin resistance and hyperglycaemia, is a leading cause of mortality. Glucose uptake through glucose transporter type 4, especially in skeletal muscle, is crucial for maintaining euglycaemia and is a key pathway targeted by antidiabetic medication. Abrus precatorius is a medicinal plant with demonstrated antihyperglycaemic activity in animal models, but its mechanisms are unclear.This study evaluated the effect of a 50% ethanolic (v/v) A.precatorius leaf extract on (1) insulin-stimulated glucose uptake and (2) related gene expression in differentiated C2C12 myotubes using rosiglitazone as a positive control, and (3) generated a comprehensive phytochemical profile of A.precatorius leaf extract using liquid chromatography-high resolution mass spectrometry to elucidate its antidiabetic compounds. A.precatorius leaf extract significantly increased insulin-stimulated glucose uptake, and insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression; however, it had no effect on glucose transporter type 4 gene expression. At 250 µg/mL A.precatorius leaf extract, the increase in glucose uptake was significantly higher than 1 µM rosiglitazone. Fifty-five phytochemicals (primarily polyphenols, triterpenoids, saponins, and alkaloids) were putatively identified, including 24 that have not previously been reported from A.precatorius leaves. Abrusin, precatorin I, glycyrrhizin, hemiphloin, isohemiphloin, hispidulin 4'-O-β-D-glucopyranoside, homoplantaginin, and cirsimaritin were putatively identified as known major compounds previously reported from A.precatorius leaf extract. A.precatorius leaves contain antidiabetic phytochemicals and enhance insulin-stimulated glucose uptake in myotubes via the protein kinase B/phosphoinositide 3-kinase pathway by regulating insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression. Therefore, A.precatorius leaves may improve skeletal muscle insulin sensitivity and hyperglycaemia. Additionally, it is a valuable source of bioactive phytochemicals with potential therapeutic use for diabetes.

Bidirectional effect of uric acid on C2C12 myotubes and its partial mechanism.

To explore the effects and mechanisms of different concentrations of uric acid on skeletal muscle cells. C2C12 myoblasts were differentiated into myotubes and then exposed to a medium containing uric acid (0 μM, 200 μM, 400 μM, 600 μM, 800 μM, 1000 μM, 1200 μM, 1400 μM). The myotube diameters were observed under light microscopy; the expressions of myosin heavy chain (MyHC), autophagy-related proteins (LC3BII/LC3BI, P62), cGAS, and p-Sting/Sting proteins were analyzed using Western blotting or immunoprecipitation; and oxidative stress and mitochondrial damage were evaluated using ROS, mtDNA and JC-1 assays. Cell viability was measured via CCK8 assay, and 1000-μM uric acid was selected for follow-up experiments. Furthermore, C2C12 myotubes were divided into a blank control group (Ctrl), a high-uric-acid group (HUA), and an HUA plus cGASn inhibitor group (HUA + RU.521). Then, the myotube diameter was observed, oxidative stress and mitochondrial damage were evaluated, and MyHC and autophagy-related protein expressions were analysed. C2C12 myotubes cultured in 400-μM uric acid medium had the greatest myotube diameter and the highest MyHC protein expression. At 1000-μM uric acid, the diameter and MyHC protein expression were significantly decreased, LCB3II/LCB3I expression was notably increased, and the level of p62 protein expression was considerably decreased. RU.521 partially alleviated the HUA-induced C2C12 myotubes changes. Uric acid bidirectionally affected C2C12 myotubes: 400-μΜ uric acid promoted myotube growth, while 1000-μΜ uric acid triggered myotube atrophy with increased autophagy. Inhibiting cGAS-Sting signaling attenuated HUA-induced C2C12 myotube autophagy and atrophy. Geriatr Gerontol Int 2024; 24: 430-439.

Secretome from Magnetically Stimulated Muscle Exhibits Anticancer Potency: Novel Preconditioning Methodology Highlighting HTRA1 Action.

Briefly (10 min) exposing C2C12 myotubes to low amplitude (1.5 mT) pulsed electromagnetic fields (PEMFs) generated a conditioned media (pCM) that was capable of mitigating breast cancer cell growth, migration, and invasiveness in vitro, whereas the conditioned media harvested from unexposed myotubes, representing constitutively released secretome (cCM), was less effective. Administering pCM to breast cancer microtumors engrafted onto the chorioallantoic membrane of chicken eggs reduced tumor volume and vascularity. Blood serum collected from PEMF-exposed or exercised mice allayed breast cancer cell growth, migration, and invasiveness. A secretome preconditioning methodology is presented that accentuates the graded anticancer potencies of both the cCM and pCM harvested from myotubes, demonstrating an adaptive response to pCM administered during early myogenesis that emulated secretome-based exercise adaptations observed in vivo. HTRA1 was shown to be upregulated in pCM and was demonstrated to be necessary and sufficient for the anticancer potency of the pCM; recombinant HTRA1 added to basal media recapitulated the anticancer effects of pCM and antibody-based absorption of HTRA1 from pCM precluded its anticancer effects. Brief and non-invasive PEMF stimulation may represent a method to commandeer the secretome response of muscle, both in vitro and in vivo, for clinical exploitation in breast and other cancers.