RESUMEN
Cultured meat, an emerging meat production technology, has reduced environmental burden as well as provide healthier and more sustainable method of meat culture. Fat in cultured meat is essential for enhancing texture, taste, and tenderness. However, current cultured meat production method is limited to single-cell type. To meet the consumer demands for cultured meat products, it is crucial to develop new methods for producing cultured meat products that contain both muscle and fat. In this study, cell viability and differentiation were promoted by controlling the ratio and cultivation conditions of myocytes and adipocytes. The total digestibility of cultured meat exceeded 37%, higher than that of beef (34.7%). Additionally, the texture, appearance, and taste of the co-cultured meat were improved. Collectively, this research has great promise for preparing rich-nutritious and digestion cultured meat.
Asunto(s)
Adipocitos , Técnicas de Cocultivo , Animales , Bovinos , Adipocitos/citología , Adipocitos/metabolismo , Humanos , Diferenciación Celular , Células Musculares/metabolismo , Células Musculares/citología , Productos de la Carne/análisis , Supervivencia Celular , Carne/análisis , Carne in VitroRESUMEN
Research conducted previously has demonstrated that apoptosis significantly influences the chicken quality. While ROS are acknowledged as significant activators of apoptosis, the precise mechanism by which they influence muscle cell apoptosis in the post-mortem remains unclear. In this study, chicken samples were treated with rosemarinic acid and H2O2 to induce varying ROS levels, and the ROS-triggered apoptosis mechanism in chicken muscle cells in post-mortem was analyzed. The TUNEL results revealed that elevated ROS levels in chicken were associated with a greater degree of muscle cell apoptosis. Western-blot results suggested that sarcoplasmic ROS could initiate apoptosis through the mitochondrial pathway by activating the MAPK-JNK signaling pathway. Moreover, TEM and shear force results demonstrated that muscle cell apoptosis initiates myofiber fragmentation and structural damage to sarcomeres, ultimately reducing chicken tenderness. This study enhances our understanding of post-mortem muscle cell apoptosis, providing valuable insights for regulating chicken quality.
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Apoptosis , Pollos , Especies Reactivas de Oxígeno , Animales , Apoptosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Carne/análisis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Musculares/metabolismo , Células Musculares/citología , Cambios Post Mortem , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacologíaRESUMEN
Current studies on cultured meat mainly focus on the muscle tissue reconstruction in vitro, but lack the formation of intramuscular fat, which is a crucial factor in determining taste, texture, and nutritional contents. Therefore, incorporating fat into cultured meat is of superior value. In this study, we employed the myogenic/lipogenic transdifferentiation of chicken fibroblasts in 3D to produce muscle mass and deposit fat into the same cells without the co-culture or mixture of different cells or fat substances. The immortalized chicken embryonic fibroblasts were implanted into the hydrogel scaffold, and the cell proliferation and myogenic transdifferentiation were conducted in 3D to produce the whole-cut meat mimics. Compared to 2D, cells grown in 3D matrix showed elevated myogenesis and collagen production. We further induced fat deposition in the transdifferentiated muscle cells and the triglyceride content could be manipulated to match and exceed the levels of chicken meat. The gene expression analysis indicated that both lineage-specific and multifunctional signalings could contribute to the generation of muscle/fat matrix. Overall, we were able to precisely modulate muscle, fat, and extracellular matrix contents according to balanced or specialized meat preferences. These findings provide new avenues for customized cultured meat production with desired intramuscular fat contents that can be tailored to meet the diverse demands of consumers.
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Transdiferenciación Celular , Pollos , Fibroblastos , Carne , Animales , Fibroblastos/metabolismo , Fibroblastos/citología , Tejido Adiposo/citología , Células Musculares/citología , Desarrollo de Músculos , Proliferación Celular , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Carne in VitroRESUMEN
Myocyte fusion is a pivotal process in the development and regeneration of skeletal muscle. Failure during fusion can lead to a range of developmental as well as pathological consequences. This review aims to comprehensively explore the intricate processes underlying myocyte fusion, from the molecular to tissue scale. We shed light on key players, such as the muscle-specific fusogens - Myomaker and Myomixer, in addition to some lesser studied molecules contributing to myocyte fusion. Conserved across vertebrates, Myomaker and Myomixer play a crucial role in driving the merger of plasma membranes of fusing myocytes, ensuring the formation of functional muscle syncytia. Our multiscale approach also delves into broader cell and tissue dynamics that orchestrate the timing and positioning of fusion events. In addition, we explore the relevance of muscle fusogens to human health and disease. Mutations in fusogen genes have been linked to congenital myopathies, providing unique insights into the molecular basis of muscle diseases. We conclude with a discussion on potential therapeutic avenues that may emerge from manipulating the myocyte fusion process to remediate skeletal muscle disorders.
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Fusión Celular , Humanos , Animales , Músculo Esquelético/metabolismo , Músculo Esquelético/citología , Células Musculares/metabolismo , Células Musculares/citología , Proteínas Musculares/metabolismo , Proteínas Musculares/genéticaRESUMEN
Cardiac patch implantation helps maximize the paracrine function of grafted cells and serves as a reservoir of soluble proangiogenic factors required for the neovascularization of infarcted hearts. We have previously fabricated a cardiac patch, EF-HAM, composed of a human amniotic membrane (HAM) coated with aligned PLGA electrospun fibers (EF). In this study, we aimed to evaluate the biocompatibility and angiogenic effects of EF-HAM scaffolds with varying fiber thicknesses on the paracrine behavior of skeletal muscle cells (SkM). Conditioned media (CM) obtained from SkM-seeded HAM and EF-HAM scaffolds were subjected to multiplex analysis of angiogenic factors and tested on HUVECs for endothelial cell viability, migration, and tube formation analyses. All three different groups of EF-HAM scaffolds demonstrated excellent biocompatibility with SkM. CM derived from SkM-seeded EF-HAM 7 min scaffolds contained significantly elevated levels of proangiogenic factors, including angiopoietin-1, IL-8, and VEGF-C compared to plain CM, which was obtained from SkM cultured on the plain surface. CM obtained from all SkM-seeded EF-HAM scaffolds significantly increased the viability of HUVECs compared to plain CM after five days of culture. However, only EF-HAM 7 min CM induced a higher migration capacity in HUVECs and formed a longer and more elaborate capillary-like network on Matrigel compared with plain CM. Surface roughness and wettability of EF-HAM 7 min scaffolds might have influenced the proportion of skeletal myoblasts and fibroblasts growing on the scaffolds and subsequently potentiated the angiogenic paracrine function of SkM. This study demonstrated the angioinductive properties of EF-HAM composite scaffold and its potential applications in the repair and regeneration of ischemic tissues.
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Isquemia/terapia , Neovascularización Fisiológica , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Regeneración/fisiología , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Amnios , Angiopoyetina 1/metabolismo , Materiales Biocompatibles/química , Movimiento Celular , Supervivencia Celular , Medios de Cultivo Condicionados/farmacología , Fibroblastos/citología , Fibroblastos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interleucina-8/metabolismo , Isquemia/patología , Células Musculares/citología , Células Musculares/metabolismo , Células Musculares/ultraestructura , Músculo Esquelético/citología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cross-talk between peripheral tissues is essential to ensure the coordination of nutrient intake with disposition during the feeding period, thereby preventing metabolic disease. This mini-review considers the interactions between the key peripheral tissues that constitute the metabolic clock, each of which is considered in a separate mini-review in this collation of articles published in Endocrinology in 2020 and 2021, by Martchenko et al (Circadian rhythms and the gastrointestinal tract: relationship to metabolism and gut hormones); Alvarez et al (The microbiome as a circadian coordinator of metabolism); Seshadri and Doucette (Circadian regulation of the pancreatic beta cell); McCommis et al (The importance of keeping time in the liver); Oosterman et al (The circadian clock, shift work, and tissue-specific insulin resistance); and Heyde et al (Contributions of white and brown adipose tissues to the circadian regulation of energy metabolism). The use of positive- and negative-feedback signals, both hormonal and metabolic, between these tissues ensures that peripheral metabolic pathways are synchronized with the timing of food intake, thus optimizing nutrient disposition and preventing metabolic disease. Collectively, these articles highlight the critical role played by the circadian clock in maintaining metabolic homeostasis.
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Relojes Circadianos/fisiología , Ritmo Circadiano , Conducta Alimentaria , Homeostasis , Hígado/fisiología , Adipocitos/citología , Animales , Endocrinología/métodos , Ingestión de Energía , Metabolismo Energético/fisiología , Retroalimentación Fisiológica , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Intestinos/fisiología , Islotes Pancreáticos/citología , Mamíferos/fisiología , Enfermedades Metabólicas/metabolismo , Microbiota , Modelos Biológicos , Células Musculares/citología , Músculo Esquelético/fisiologíaRESUMEN
The ascidian larval tail contains muscle cells for swimming. Most of these muscle cells differentiate autonomously. The genetic program behind this autonomy has been studied extensively and the genetic cascade from maternal factors to initiation of expression of a muscle structural gene, Myl.c, has been uncovered; Myl.c expression is directed initially by transcription factor Tbx6-r.b at the 64-cell stage and then by the combined actions of Tbx6-r.b and Mrf from the gastrula to early tailbud stages. In the present study, we showed that transcription of Myl.c continued in late tailbud embryos and larvae, although a fusion protein of Tbx6-r.b and GFP was hardly detectable in late tailbud embryos. A knockdown experiment, reporter assay, and in vitro binding assay indicated that an essential cis-regulatory element of Myl.c that bound Tbx6-r.b in early embryos bound Tbx15/18/22 in late embryos to maintain expression of Myl.c. We also found that Tbx15/18/22 was controlled by Mrf, which constitutes a regulatory loop with Tbx6-r.b. Therefore, our data indicated that Tbx15/18/22 was activated initially under control of this regulatory loop as in the case of Myl.c, and then Tbx15/18/22 maintained the expression of Myl.c after Tbx6-r.b had disappeared. RNA-sequencing of Tbx15/18/22 morphant embryos revealed that many muscle structural genes were regulated similarly by Tbx15/18/22. Thus, the present study revealed the mechanisms of maintenance of transcription of muscle structural genes in late embryos in which Tbx15/18/22 takes the place of Tbx6-r.b.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Expresión Génica , Músculos/embriología , Músculos/metabolismo , Proteínas de Dominio T Box/metabolismo , Urocordados/embriología , Urocordados/genética , Animales , Sitios de Unión , Diferenciación Celular/genética , Femenino , Gástrula/metabolismo , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Células Musculares/citología , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Oviparidad/genética , Proteínas de Dominio T Box/genética , Transcripción Genética/genéticaRESUMEN
Clostridium sporogenes (C. sporogenes), as a potential probiotic, metabolizes tryptophan and produces an anti-inflammatory metabolite, indole-3-propionic acid (IPA). Herein, we studied the effects of C. sporogenes and its bioactive metabolite, IPA, on skeletal muscle development and chronic inflammation in mice. In the in vivo study, the muscle tissues and serum samples of mice with C. sporogenes supplementation were used to analyze the effects of C. sporogenes on muscle metabolism; the IPA content was determined by metabonomics and ELISA. In an in vitro study, C2C12 cells were exposed to lipopolysaccharide (LPS) alone or LPS + IPA to verify the effect of IPA on muscle cell inflammation by transcriptome, and the involved mechanism was revealed by different functional assays. We observed that C. sporogenes colonization significantly increased the body weight and muscle weight gain, as well as the myogenic regulatory factors' (MRFs) expression. In addition, C. sporogenes significantly improved host IPA content and decreased pro-inflammatory cytokine levels in the muscle tissue of mice. Subsequently, we confirmed that IPA promoted C2C12 cells' proliferation by activating MRF signaling. IPA also effectively protected against LPS-induced C2C12 cells inflammation by activating Pregnane X Receptor and restoring the inhibited miR-26a-2-3p expression. miR-26a-2-3p serves as a novel muscle inflammation regulatory factor that could directly bind to the 3'-UTR of IL-1ß, a key initiator factor in inflammation. The results suggested that C. sporogenes with its functional metabolite IPA not only helps muscle growth development, but also protects against inflammation, partly by the IPA/ miR-26a-2-3p /IL-1ß cascade.
Asunto(s)
Clostridium/metabolismo , Indoles/metabolismo , Interleucina-1beta/genética , MicroARNs/genética , Células Musculares/efectos de los fármacos , Receptor X de Pregnano/genética , Propionatos/metabolismo , Regiones no Traducidas 3' , Animales , Línea Celular , Microbioma Gastrointestinal/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Indoles/farmacología , Inflamación/prevención & control , Interleucina-1beta/metabolismo , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Células Musculares/citología , Células Musculares/metabolismo , Desarrollo de Músculos/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Receptor X de Pregnano/metabolismo , Probióticos/metabolismo , Propionatos/farmacología , Transducción de Señal , Transcriptoma , Triptófano/metabolismoRESUMEN
Non-centrosomal microtubule-organizing centers (MTOCs) are pivotal for the function of multiple cell types, but the processes initiating their formation are unknown. Here, we find that the transcription factor myogenin is required in murine myoblasts for the localization of MTOC proteins to the nuclear envelope. Moreover, myogenin is sufficient in fibroblasts for nuclear envelope MTOC (NE-MTOC) formation and centrosome attenuation. Bioinformatics combined with loss- and gain-of-function experiments identified induction of AKAP6 expression as one central mechanism for myogenin-mediated NE-MTOC formation. Promoter studies indicate that myogenin preferentially induces the transcription of muscle- and NE-MTOC-specific isoforms of Akap6 and Syne1, which encodes nesprin-1α, the NE-MTOC anchor protein in muscle cells. Overexpression of AKAP6ß and nesprin-1α was sufficient to recruit endogenous MTOC proteins to the nuclear envelope of myoblasts in the absence of myogenin. Taken together, our results illuminate how mammals transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify AKAP6 as a novel NE-MTOC component in muscle cells.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Centro Organizador de los Microtúbulos/fisiología , Células Musculares/metabolismo , Miogenina/metabolismo , Células 3T3 , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Células Musculares/citología , Membrana NuclearRESUMEN
Higher-order chromatin structure regulates gene expression, and mutations in proteins mediating genome folding underlie developmental disorders known as cohesinopathies. However, the relationship between three-dimensional genome organization and embryonic development remains unclear. Here we define a role for bromodomain-containing protein 4 (BRD4) in genome folding, and leverage it to understand the importance of genome folding in neural crest progenitor differentiation. Brd4 deletion in neural crest results in cohesinopathy-like phenotypes. BRD4 interacts with NIPBL, a cohesin agonist, and BRD4 depletion or loss of the BRD4-NIPBL interaction reduces NIPBL occupancy, suggesting that BRD4 stabilizes NIPBL on chromatin. Chromatin interaction mapping and imaging experiments demonstrate that BRD4 depletion results in compromised genome folding and loop extrusion. Finally, mutation of individual BRD4 amino acids that mediate an interaction with NIPBL impedes neural crest differentiation into smooth muscle. Remarkably, loss of WAPL, a cohesin antagonist, rescues attenuated smooth muscle differentiation resulting from BRD4 loss. Collectively, our data reveal that BRD4 choreographs genome folding and illustrates the relevance of balancing cohesin activity for progenitor differentiation.
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Diferenciación Celular , Genoma , Cresta Neural/citología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Integrasas/metabolismo , Ratones , Modelos Biológicos , Células Madre Embrionarias de Ratones/metabolismo , Células Musculares/citología , Cresta Neural/metabolismo , Unión Proteica , Dominios Proteicos , Proteolisis , Factores de Transcripción/química , Transcripción Genética , CohesinasRESUMEN
Pluripotent adult stem cells have potential applications in cell therapy and tissue engineering. Urine-derived stem cells (UDSCs) differentiate into various cell types. Here, we attempted to differentiate human UDSCs (hUDSCs) into smooth muscle cells (SMCs) using transforming growth factor-beta 1 (TGF-ß1) and/or PD98059, an extracellular signal-regulated kinase (ERK) inhibitor. Both quantitative polymerase chain reaction (qPCR) and Western blot analysis showed that the expression of messenger ribonucleic acid (mRNA) and proteins for alpha-smooth muscle actin (α-SMA), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC), which are specific markers for SMCs, increased on day 9 after differentiation and again on day 14. The differentiated cells from human UDSCs (hUDSCs) with a combination of TGF-ß1 and PD98059 showed the highest expression of SMC marker proteins. Immunocytochemical staining performed to assess the molecular expression revealed CNN and α-SMA colocalizing in the cytoplasm. The cells that differentiated from hUDSCs with a combination of TGF-ß1 and PD98059 showed the strongest expression for CNN1, α-SMA, and SM-MHC. Functional testing of the differentiated cells revealed a stronger contractile capacity for the cells differentiated with a combination of PD98059 and TGF-ß1 than those differentiated with a single factor. These results suggest the combination of PD98059 and TGF-ß1 to be a more effective differentiation method and that differentiated SMCs could be used for restoring the functions of the sphincter muscle or bladder.
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Diferenciación Celular/efectos de los fármacos , Flavonoides/farmacología , Células Musculares , Células Madre , Factor de Crecimiento Transformador beta1/farmacología , Orina/citología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células Musculares/citología , Células Musculares/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
MicroRNAs (miRNAs) play an essential role in the regulation of a number of physiological functions. miR-133a and other muscular miRs (myomiRs) play a key role in muscle cell growth and in some type of cancers. Here, we show that miR133a is upregulated in individuals that undertake physical exercise. We used a skeletal muscle differentiation model to dissect miR-133a's role and to identify new targets, identifying Tropomyosin-4 (TPM4). This protein is expressed during muscle differentiation, but importantly it is an essential component of microfilament cytoskeleton and stress fibres formation. The microfilament scaffold remodelling is an essential step in cell transformation and tumour progression. Using the muscle system, we obtained valuable information about the microfilament proteins, and the knowledge on these molecular players can be transferred to the cytoskeleton rearrangement observed in cancer cells. Further investigations showed a role of TPM4 in cancer physiology, specifically, we found that miR-133a downregulation leads to TPM4 upregulation in colon carcinoma (CRC), and this correlates with a lower patient survival. At molecular level, we demonstrated in myocyte differentiation that TPM4 is positively regulated by the TA isoform of the p63 transcription factor. In muscles, miR-133a generates a myogenic stimulus, reducing the differentiation by downregulating TPM4. In this system, miR-133a counteracts the differentiative TAp63 activity. Interestingly, in CRC cell lines and in patient biopsies, miR-133a is able to regulate TPM4 activity, while TAp63 is not active. The downregulation of the miR leads to TPM4 overexpression, this modifies the architecture of the cell cytoskeleton contributing to increase the invasiveness of the tumour and associating with a poor prognosis. These results add data to the interesting question about the link between physical activity, muscle physiology and protection against colorectal cancer. The two phenomena have in common the cytoskeleton remodelling, due to the TPM4 activity, that is involved in stress fibres formation.
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Diferenciación Celular/genética , Neoplasias del Colon/genética , MicroARNs/genética , Factores de Transcripción/genética , Tropomiosina/genética , Proteínas Supresoras de Tumor/genética , Citoesqueleto de Actina/genética , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias del Colon/patología , Citoesqueleto/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células Musculares/citología , Desarrollo de Músculos/genética , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/metabolismo , Fibras de Estrés/genéticaRESUMEN
Muscular dystrophies are a heterogeneous group of inherited diseases characterized by the progressive degeneration and weakness of skeletal muscles, leading to disability and, often, premature death. To date, no effective therapies are available to halt or reverse the pathogenic process, and meaningful treatments are urgently needed. From this perspective, it is particularly important to establish reliable in vitro models of human muscle that allow the recapitulation of disease features as well as the screening of genetic and pharmacological therapies. We herein review and discuss advances in the development of in vitro muscle models obtained from human induced pluripotent stem cells, which appear to be capable of reproducing the lack of myofiber proteins as well as other specific pathological hallmarks, such as inflammation, fibrosis, and reduced muscle regenerative potential. In addition, these platforms have been used to assess genetic correction strategies such as gene silencing, gene transfer and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), as well as to evaluate novel small molecules aimed at ameliorating muscle degeneration. Furthermore, we discuss the challenges related to in vitro drug testing and provide a critical view of potential therapeutic developments to foster the future clinical translation of preclinical muscular dystrophy studies.
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Diferenciación Celular/fisiología , Descubrimiento de Drogas/métodos , Terapia Genética/métodos , Células Madre Pluripotentes Inducidas/fisiología , Células Musculares/fisiología , Distrofias Musculares/terapia , Animales , Distrofina/genética , Distrofina/fisiología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Musculares/citología , Distrofias Musculares/genética , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/terapiaRESUMEN
Lymphatic muscle cells (LMCs), with unique characteristics resembling a combination of both cardiac and smooth muscle cells, play an essential role in the spontaneous contraction of the lymphatic vessels to pump fluid forward. However, our understanding of the more detailed molecular phenotypes of LMCs is limited. Here, we described a method to isolate the LMCs from rat mesentery and then culture the cells in vitro, which will serve a lot more molecular biology study of LMCs and significantly improve our knowledge about the unique characteristics of LMCs.
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Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Disección/métodos , Mesenterio/citología , Células Musculares/citología , Animales , Técnica del Anticuerpo Fluorescente , Células Musculares/metabolismo , RatasRESUMEN
Momordica charantia is a popular vegetable associated with effective complementary and alternative diabetes management in some parts of the world. However, the molecular mechanism is less commonly investigated. In this study, we investigated the association between a major cucurbitane triterpenoid isolated from M. charantia, 3ß,7ß,25-trihydroxycucurbita-5,23(E)-dien-19-al (THCB) and peroxisome proliferator activated receptor gamma (PPARγ) activation and its related activities using cell culture and molecular biology techniques. In this study, we report on both M. charantia fruit crude extract and THCB in driving the luciferase activity of Peroxisome Proliferator Response Element, associated with PPARγ activation. Other than that, THCB also induced adipocyte differentiation at far less intensity as compared to the full agonist rosiglitazone. In conjunction, THCB treatment on adipocytes also resulted in upregulation of PPAR gamma target genes expression; AP2, adiponectin, LPL and CD34 at a lower magnitude compared to rosiglitazone's induction. THCB also induced glucose uptake into muscle cells and the mechanism is via Glut4 translocation to the cell membrane. In conclusion, THCB acts as one of the many components in M. charantia to induce hypoglycaemic effect by acting as PPARγ ligand and inducing glucose uptake activity in the muscles by means of Glut4 translocation.
Asunto(s)
Momordica/química , PPAR gamma/metabolismo , Triterpenos/química , Células 3T3-L1 , Adipocitos/citología , Animales , Diferenciación Celular , Membrana Celular/metabolismo , Glucosa/metabolismo , Hepatocitos/citología , Hipoglucemia/tratamiento farmacológico , Insulina/química , Ligandos , Ratones , Células Musculares/citología , Dominios Proteicos , Rosiglitazona/farmacología , Triterpenos/farmacologíaRESUMEN
OBJECTIVE: IGF-I and branched-chain amino acids have been reported to promote muscle hypertrophy via the stimulation of protein synthesis. Sestrin2, the function of which is regulated by leucine, has been reported to attenuate the activity of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) that stimulates protein synthesis. The objective of this study was to examine whether IGF-I modulates Sestrin2 abundance and to clarify the involvement of Sestrin2 in the effect of IGF-I and leucine on mTROC1. DESIGN: C2C12 and L6 myocytes were stimulated by leucine (1 mM) with or without pretreatment with IGF-I (100 ng/mL). Phosphorylation of p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), both of which are targets of the mTORC1, was examined by western blotting. Effects of Sestrin2 small interfering RNA (siRNA) on the actions of leucine and IGF-I were examined. Sestrin2 mRNA and protein levels were also determined after Sestrin2 siRNA. RESULTS: Leucine increased the phosphorylation of S6K and 4E-BP1 in a dose-dependent manner. Pretreatment with IGF-I for 5 h further increased the stimulatory effect of leucine on the phosphorylation of S6K and 4E-BP1 in C2C12 cells. IGF-I increased Sestrin2 protein and messenger RNA levels. Sestrin2 siRNA increased or tended to increase basal phosphorylation of 4E-BP1 and decreased the leucine-induced phosphorylation in C2C12 and L6 cells, in particular after IGF-I treatment, suggesting the involvement of Sestrin2 in the action of leucine and IGF-I. The net increase in leucine-induced 4E-BP1 phosphorylation appeared to be attenuated by Sestrin2 siRNA. Likewise, Sestrin2 siRNA attenuated leucine-induced S6K phosphorylation in L6 cells. However, Sestrin2 siRNA did not influence leucine-induced S6K phosphorylation in C2C12 cells. CONCLUSIONS: IGF-I and leucine cooperatively increased mTORC1 activity in C2C12 cells. IGF-I increased Sestrin2. Sestrin2 siRNA experiments showed that Sestrin2 was involved in the effect of leucine and IGF-I on mTORC1 activity in C2C12 and L6 cells, and suggested that increased Sestrin2 by IGF-I pretreatment might play a role in enhancing the effect of leucine on mTORC1.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Leucina/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células Musculares/metabolismo , Peroxidasas/metabolismo , Animales , Células Cultivadas , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Células Musculares/citología , Células Musculares/efectos de los fármacos , Peroxidasas/genética , Fosforilación , Transducción de SeñalRESUMEN
This study aimed to investigate the function of estrogen receptors (ERs) in myoregeneration and intermuscular adipogenesis. Ovariectomized (OVX) ERα knockout (KO) mice and ERß KO mice were used to assess the effect of estrogen on the myoregenerative process. Tibialis anterior muscle was collected on days 7, 10, and 14 after cardiotoxin injection to assess myotube morphology and adipogenesis area. Regenerated myotubes from OVX-ERß KO mice were consistently smaller in diameter than those from OVX-ERα KO and OVX-wild-type mice, whereas the adipogenesis area of OVX-ERß KO mice was consistently greater than that of the other types. Therefore, ERß may be an influential factor in promoting myoregeneration and adipogenesis inhibition compared to ERα.
Asunto(s)
Adipogénesis , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Células Musculares/citología , Regeneración , Animales , Estradiol , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Estrógenos , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovariectomía/veterinariaRESUMEN
Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self-renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC-specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol-anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down-regulated in bone marrow-derived MSCs cultured on plastic, making it difficult to examine the self-renewal and differentiation of Meflin-positive cells at the single-cell level. Here, we traced the lineage of Meflin-positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin-positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin-positive cells or their lineage-committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Inmunoglobulinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Condrocitos/citología , Condrocitos/metabolismo , Inmunoglobulinas/genética , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Células Musculares/citología , Células Musculares/metabolismo , Osteocitos/citología , Osteocitos/metabolismoRESUMEN
The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.
Asunto(s)
Huesos/fisiología , Vesículas Extracelulares/metabolismo , Hidrodinámica , Células Musculares/citología , Resistencia al Corte , Estrés Mecánico , Animales , Fenómenos Biomecánicos , Línea Celular , RatonesRESUMEN
Muscle tissue is often removed during hamstring tendon graft preparation for anterior cruciate ligament (ACL) reconstruction. The purpose of the study was to test whether preservation of muscle remnants on a tendon graft is beneficial to the graft healing process following ACL reconstruction. Co-culturing of tendon-derived cells (TDCs) and muscle-derived cells (MDCs) was performed at various ratios, and their potential for cell viability and multilineage differentiation was compared to a single TDC cell group. Ligamentous and chondrogenic differentiation was most enhanced when a small population of MDCs was co-cultured with TDCs (6:2 co-culture group). Cell viability and osteogenic differentiation were proportionally enhanced with increasing MDC population size. MDCs co-cultured with TDCs possess both the ability to enhance cell viability and differentiate into other cell lineages.