RESUMEN
MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules about 22 nucleotides in length and are encoded by endogenous genes, and are involved in the regulation of post-transcriptional gene expression in animals and plants. Many studies have shown that microRNAs regulate the development of skeletal muscle, mainly manifested in the activation of muscle satellite cells and biological processes such as proliferation, differentiation, and formation of muscle tubes. In this study, miRNA sequencing screening of longissimus dorsi (LD, mainly fast-twitch fibers) and soleus muscle (Sol, dominated by slow-twitch fibers) identified the miR-196b-5p as a differentially expressed and highly conserved sequence in different skeletal muscles. Studies of miR-196b-5p in skeletal muscle have not been reported. In this study, miR-196b-5p mimics and inhibitor were used in miR-196b-5p overexpression and interference experiments in C2C12 cells. The effect of miR-196b-5p on myoblast proliferation and differentiation was analyzed by western blotting, real-time quantitative RT-PCR, flow cytometry, immunofluorescence staining, and the target gene of miR-196b-5p was identified by bioinformatics prediction and analyzed by dual luciferase reporter assays. The results showed that overexpression of miR-196b-5p could significantly increase the mRNA and protein expression of Cyclin B, Cyclin D and Cyclin E (P<0.05); Cell cycle analysis showed that overexpression of miR-196b-5p significantly increased the proportion of cells in the S phase (P<0.05), indicating that miR-196b-5p could accelerate cell cycle progress. Results of EdU staining showed that overexpression of miR-196b-5p significantly promoted cell proliferation. Conversely, inhibition of miR-196b-5p expression could significantly reduce the proliferation capacity of myoblasts. Further, overexpression of miR-196b-5p could significantly increase the expression levels of myogenic marker genes MyoD, MoyG and MyHC (P<0.05), thereby promoting myoblast fusion and accelerating C2C12 cell differentiation. Bioinformatics predictions and dual luciferase experiments demonstrated that miR-196b-5p could target and inhibit the expression of the Sirt1 gene. Altering the Sirt1 expression could not rescue the effects of miR-196b-5p on the cell cycle, but could weaken the promoting effects of miR-196b-5p on myoblast differentiation, suggesting that miR-196b-5p promoted myoblast differentiation by targeting Sirt1.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Mioblastos , Animales , Ratones , Línea Celular , MicroARNs/genética , Mioblastos/citología , Mioblastos/metabolismoRESUMEN
Adipogenesis, a differentiation process that transitions preadipocytes to adipocytes, is key to understanding the biology of fat accumulation and obesity. During this process, there many crucial transcription factors, such as PPARγ and the C/EBP family. Here we show a transcription factor in preadipocytes --- Sox5, that has a function in porcine adipogenesis. In our porcine subcutaneous-derived preadipocyte differentiation model, we found Sox5 expression displayed a significant upregulation after initial induction and decreased afterwards, which resembles the PPARγ expression pattern. siRNA knockdown of Sox5 in porcine preadipocytes significantly promoted cell growth and accelerated cell cycle progression. After inducing differentiation, knockdown of Sox5 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired lipid accumulation. Mechanistically, the deletion of Sox5 down-regulated the BMP R-Smads signal pathway, a crucial signal pathway for controlling preadipocyte fate commitment and adipogenesis. After using BMP4 recombinant protein to activate the BMP R-Smads signal, Sox5 function was partially rescued. In conclusion, our findings uncovered a function of Sox5 in porcine adipogenesis and reveal an interaction between Sox5 and BMP signaling.
Asunto(s)
Adipogénesis , Proteínas Morfogenéticas Óseas/metabolismo , Factores de Transcripción SOXD/genética , Transducción de Señal , Proteínas Smad/metabolismo , Porcinos/fisiología , Animales , Proteínas Morfogenéticas Óseas/genética , Células Cultivadas , Regulación hacia Abajo , Interferencia de ARN , Factores de Transcripción SOXD/metabolismo , Proteínas Smad/genética , Porcinos/genética , Regulación hacia ArribaRESUMEN
Differentiation from preadipocytes into mature adipocytes is a complex biological process in which miRNAs play an important role. Previous studies showed that miR-214-3p facilitates adipocyte differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. The detailed function and molecular mechanism of miR-214-3p in adipocyte development is unclear. In this study, the 3T3-L1 cell line was used to analyze the function of miR-214-3p in vitro. Using 5-Ethynyl-2'-deoxyuridine (EdU) staining and the CCK-8 assay, we observed that transfection with the miR-214-3p agomir visibly promoted proliferation of 3T3-L1 preadipocytes by up-regulating the expression of cell cycle-related genes. Interestingly, overexpression of miR-214-3p promoted 3T3-L1 preadipocyte differentiation and up-regulated the expression of key genes for lipogenesis: PPARγ, FABP4, and Adiponectin. Conversely, inhibition of miR-214-3p repressed 3T3-L1 preadipocyte proliferation and differentiation, and down-regulated the expression of cell cycle-related genes and adipogenic markers. Furthermore, we proved that miR-214-3p regulates 3T3-L1 preadipocyte differentiation by directly targeting the 3'-untranslated regions (3'UTR) of Ctnnb1, which is an important transcriptional regulatory factor of the Wnt/ß-Catenin pathway. Taken together, the data indicate that miR-214-3p may positively regulate preadipocyte proliferation and enhance differentiation through the Wnt/ß-Catenin signaling pathway.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , Diferenciación Celular/genética , MicroARNs/genética , Vía de Señalización Wnt , beta Catenina/genética , Regiones no Traducidas 3' , Células 3T3-L1 , Adipogénesis/genética , Animales , Secuencia de Bases , Proliferación Celular , Ratones , Interferencia de ARNRESUMEN
It is well-documented that CL316,243 (a ß3 agonist) or rosiglitazone (a PPARγ agonist) can induce white adipocyte populations to brown-like adipocytes, thus increasing energy consumption and combating obesity. However, whether there is a combined effect remains unknown. In the present study, stromal vascular cells of inguinal white adipose tissue (iWAT-SVCs for short) from mice were cultured and induced into browning by CL316,243, rosiglitazone, or both. Results showed that a combination of CL316,243 and rosiglitazone significantly upregulated the expression of the core thermogenic gene Ucp1 as well as genes related with mitochondrial function (Cidea, Cox5b, Cox7a1, Cox8b, and Cycs), compared with the treatment of CL316,243 or rosiglitazone alone. Moreover, co-treatment with rosiglitazone could reverse the downregulation of Adiponectin resulting from CL316,243 stimuli alone. Taken together, a combination of rosiglitazone and CL316,243 can produce an additive effect of promoting thermogenic gene expression and an improvement of insulin sensitivity in mouse iWAT-SVCs.
Asunto(s)
Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Adipogénesis , Hipoglucemiantes/farmacología , Mitocondrias/metabolismo , Adipocitos Marrones/citología , Adipocitos Marrones/efectos de los fármacos , Adipocitos Blancos/citología , Adipocitos Blancos/efectos de los fármacos , Adiponectina/genética , Adiponectina/metabolismo , Animales , Células Cultivadas , Dioxoles/farmacología , Ratones , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Rosiglitazona , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Tiazolidinedionas/farmacologíaRESUMEN
Intramuscular fat (IMF) content affects the tenderness, juiciness, and flavor of pork. An increasing number of studies are focusing on the functions of microRNAs (miRs) during porcine intramuscular preadipocyte development. Previous studies have proved that miR-425-5p was enriched in porcine skeletal muscles and played important roles in multiple physiological processes; however, its functions during intramuscular adipogenesis remain unclear. To explore the role of miR-425-5p in porcine intramuscular adipogenesis, miR-425-5p agomir and inhibitor were used to perform miR-425-5p overexpression and knockdown in intramuscular preadipocytes, respectively. Our results showed that the agomir of miR-425-5p dramatically inhibited intramuscular adipogenic differentiation and downregulated the expression levels of adipogenic marker genes PPARγ, FABP4, and FASN, whereas its inhibitor promoted adipogenesis. Interestingly, the agomir repressed proliferation of porcine intramuscular preadipocytes by downregulation of cyclin B and cyclin E. Furthermore, we demonstrated that miR-425-5p inhibited adipogenesis via targeting and repressing the translation of KLF13. Taken together, our findings identified that miR-425-5p is a novel inhibitor of porcine intramuscular adipogenesis possibly through targeting KLF13 and subsequently downregulating PPARγ.
Asunto(s)
Adipogénesis/fisiología , Adipocitos/metabolismo , Adipogénesis/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , MicroARNs/genética , PPAR gamma/genética , PPAR gamma/metabolismo , PorcinosRESUMEN
MicroRNAs (miRNAs) are crucial regulatory molecules for adipogenesis. They contribute to the controlling of proliferation and differentiation of preadipocytes. Previous studies revealed an important role of miR-429 in cell invasion, migration, and apoptosis. Our previous work has shown that the expression of miR-429 in subcutaneous fat can be observed in newly born (3-day-old) Rongchang piglets rather than their adult counterparts (180-day-old). This expression pattern suggests that miR-429 might be functionally related to postnatal adipogenesis. However, we currently lack a mechanistic understanding of miR-429 within the context of preadipocyte differentiation. In this study, we investigated the function of miR-429 in porcine subcutaneous and intramuscular preadipocyte proliferation and differentiation. In our porcine preadipocyte differentiation model, miR-429 expression decreased remarkably upon adipogenic induction. Overexpression of miR-429 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired the triglyceride accumulation, while the expression of lipolytic gene ATGL was not affected. In addition, we observed that miR-429 significantly promoted the proliferation of porcine preadipocytes. We also found that miR-429 could directly bind to the 3'-UTRs of KLF9 and p27, which have been well documented to promote preadipocyte differentiation and repress cell cycle progression. Taken together, our data support a novel role of miR-429 in regulating porcine preadipocyte differentiation and proliferation, and KLF9 and p27 are potent targets of miR-429 during these processes.
Asunto(s)
Adipocitos/citología , Adipocitos/metabolismo , MicroARNs/genética , Adipogénesis/genética , Adipogénesis/fisiología , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , PPAR gamma/genética , PPAR gamma/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , PorcinosRESUMEN
C1q/tumor necrosis factor-related protein 6 (CTRP6), an adipose-tissue secretory factor, plays an important role in inflammatory reaction and carcinogenesis. However, the biological function of CTRP6 in adipogenesis remains unclear. In this study, we examined the effects of CTRP6 knockdown on lipogenesis of 3T3-L1 adipocytes. The results showed that after 3T3-L1 adipocytes transfected with anti-CTRP6 small interfering RNA (siRNA), not only levels of secreted CTRP6 protein in the culture medium but also the expression level of the CTRP6 protein in the 3T3-L1 adipocytes was significantly reduced (P < 0.01). In addition, the number of lipid droplets in the adipocytes was reduced, as well as the OD values reflecting the fat content being significantly decreased (P < 0.01). Meanwhile the levels of adipogenic markers, including peroxisome proliferator activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), CCAAT/enhancer-binding protein ß (C/EBPß) and adipocyte fatty acid-binding protein 4 (aP2), were decreased after treatment with anti-CTRP6 siRNA, whereas the expression of adipose triglyceride lipase (ATGL) and triacylglycerol hydrolase (TGH) were increased. Furthermore, after transfection, activity of phosphorylated Erk1/2 (p-Erk1/2) was inhibited in the early stage of differentiation, but in terminal differentiation of adipocytes, its activity was activated. Taken together, the results indicate that knockdown of CTRP6 can inhibit adipogenesis of 3T3-L1 adipocytes through lipogenic marker genes and Erk1/2 signaling pathway.
Asunto(s)
Adipogénesis/genética , Adipoquinas/genética , Lipólisis/genética , Sistema de Señalización de MAP Quinasas/genética , Factores de Necrosis Tumoral/genética , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/fisiología , Adipogénesis/efectos de los fármacos , Adipoquinas/antagonistas & inhibidores , Adipoquinas/metabolismo , Animales , Biomarcadores/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Técnicas de Silenciamiento del Gen , Lipólisis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/farmacología , Inhibidores del Factor de Necrosis TumoralRESUMEN
3,3'-Diindolylmethane (DIM), a major acid-condensation product or metabolite of indole-3-carbinol which is found in cruciferous vegetables, has been shown to have anticancer, anti-inflammatory, and multiple immune stimulating effects. However, its function in bone metabolism is poorly understood. This study evaluated the effect of DIM on bone mass in mice under physiological and pathological conditions. Eight-week-old female mice received injections of a vehicle or 0.1mg/g of DIM, twice a week for four weeks. We found that DIM treatment significantly increased bone mass as assessed by dual-energy X-ray absorptiometry (DEXA) and micro-computed tomography (µCT). Further, Bone histomorphometric analyses showed that this treatment significantly reduced bone resorption parameters, but did not increase bone formation parameters. Furthermore, we use ovariectomized (OVX)-induced osteoporotic mouse model, and explore function of DIM in skeletal pathological processes. Bone phenotype analyses revealed that the administration of DIM in this study effectively prevented OVX-induced bone loss resulting from increased bone resorption. Our results demonstrated that DIM increased bone mass by suppressing osteoclastic bone resorption in bone metabolism under both physiological and pathological conditions. Accordingly, DIM may be of value in the treatment and the possible prevention of bone diseases characterized by bone loss, such as postmenopausal osteoporosis.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Resorción Ósea/prevención & control , Indoles/farmacología , Indoles/uso terapéutico , Osteoporosis Posmenopáusica/tratamiento farmacológico , Animales , Densidad Ósea/fisiología , Huesos/anatomía & histología , Huesos/efectos de los fármacos , Huesos/fisiología , Femenino , Humanos , Ratones , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteogénesis/fisiología , Osteoporosis Posmenopáusica/fisiopatología , OvariectomíaRESUMEN
Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.
Asunto(s)
Suplementos Dietéticos , Dipéptidos/farmacología , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Complejos Multiproteicos/metabolismo , Músculo Esquelético/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , alfa-Glucosidasas/deficiencia , Animales , Línea Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Humanos , Insulina/farmacología , Cifosis/enzimología , Cifosis/patología , Cifosis/fisiopatología , Cifosis/prevención & control , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular/enzimología , Atrofia Muscular/patología , Atrofia Muscular/fisiopatología , Atrofia Muscular/prevención & control , Mioblastos/efectos de los fármacos , Mioblastos/enzimología , Interferencia de ARN , Transfección , alfa-Glucosidasas/genéticaRESUMEN
The process of preadipocytes differentiation plays a vital role in adipose tissue expansion and many factors are involved in this event. Cathepsin B (CTSB), secreted from lysosome, has been reported in regulating a variety of physiological processes. In this study, we demonstrated CTSB promotes lipid accumulation and adipogenic genes expression in porcine primary preadipocytes by degrading fibronectin (Fn), a key component of extracellular matrix. Lithium chloride (LiCl) is an activator of Wnt/ß-catenin signaling through stabilizing ß-catenin. We found that CTSB can relieve the anti-adipogenic effects of LiCl, indicating that CTSB could impact Wnt/ß-catenin signaling pathway. Interestingly, Fn is an important target gene of Wnt/ß-catenin. So we considered that CTSB promote preadipocytes differentiation by suppressing these two pathways.
Asunto(s)
Adipocitos/citología , Catepsina B/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Cloruro de Litio/farmacología , Adipogénesis , Animales , Catepsina B/genética , Diferenciación Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Porcinos , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor which plays an important role in adipocyte differentiation. Our previous Solexa sequencing results indicated a high expression of miR-125a in adult pig backfat. In this study, we predicated and experimentally validated ERRα as a target of miR-125a. To explore the role of miR-125a in porcine preadipocytes differentiation, miRNA agomir and antagomir were used to perform miR-125a overexpression or knockdown, respectively. Our results showed that overexpression of miR-125a could dramatically reduce the mRNA expression of adipogenic markers PPARγ, LPL, and aP2, as well as its target gene ERRα. Western blotting showed the protein level of aP2 and ERRα was also significantly down-regulated. The overexpression of miR-125a also led to a notable reduction in lipid accumulation which was detected by Oil Red O staining. In contrast, we observed promoted differentiation of porcine preadipocytes upon miR-125a inhibition. In conclusion, we verified miR-125a inhibits porcine preadipocytes differentiation through targeting ERRα for the first time, which may provide new insights in pork quality improvement and obesity control.
Asunto(s)
Adipocitos/fisiología , MicroARNs/genética , MicroARNs/metabolismo , Receptores de Estrógenos/genética , Sus scrofa/fisiología , Adipocitos/citología , Animales , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Modelos Biológicos , Receptores de Estrógenos/metabolismo , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Adipose tissue is an important energy reservoir, and its over-development results in obesity in humans or body fat over-deposition in livestock. Loss of preadipocytes through apoptosis has been proposed as an alternative way to reduce adipose tissue mass. At present, the effect and regulatory mechanism of Sirt1 and camptothecin on porcine preadipocyte apoptosis are still largely unknown. Here, we evaluated whether Sirt1 had any role in the basal cellular and camptothecin-induced conditions in porcine preadipocytes. Flow cytometric analysis shows that viable cells decrease as well as early apoptotic and late apoptotic cells increase after knockdown of Sirt1 in porcine preadipocytes. Camptothecin induces porcine preadipocyte apoptosis in a dose-dependent manner, assessed with the Hoechst staining and western blot analysis. Interestingly, silencing of Sirt1 significantly enhances sensitivity of porcine preadipocytes to camptothecin, which may be due to upregulation of p53, acetylated p53, Bax, cleaved caspase-3 and downregulation of Bcl-2. On the contrary, under the Sirt1 overexpression condition viable cells' number significantly increases when challenging with camptothecin, and the protein levels of cleaved caspase-3, p53, acetylated p53 and Bax are downregulated. We also find that hyperacetylated p53 is the major effect of Sirt1 knockdown by overexpression of a mutated p53, whereas Sirt1 overexpression prevents camptothecin-induced apoptosis through p53 deacetylation in preadipocytes. Furthermore, repressing preadipocyte apoptosis of Sirt1 is mediated by direct interaction with cleaved caspase-3 using immunoprecipitation and inhibition of caspase-3 transcriptional activity using luciferase reporter assays.
Asunto(s)
Adipocitos/efectos de los fármacos , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Camptotecina/farmacología , Caspasa 3/metabolismo , Sirtuina 1/metabolismo , Acetilación , Adipocitos/citología , Adipocitos/metabolismo , Animales , Western Blotting , Sinergismo Farmacológico , Citometría de Flujo , Lentivirus/genética , Luciferasas/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Sirtuina 1/antagonistas & inhibidores , Sirtuina 1/genética , Porcinos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Low-density lipoproteins (LDL) is known to protect boar sperm during freezing-thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing-thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen-thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen-thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen-thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.
Asunto(s)
Acrosoma/efectos de los fármacos , Yema de Huevo/química , Lipoproteínas LDL/farmacología , Motilidad Espermática/efectos de los fármacos , Espermatozoides/citología , Espermatozoides/fisiología , Animales , Células Cultivadas , Criopreservación , Congelación , Masculino , PorcinosRESUMEN
MicroRNAs (miRNAs), a class of small non-coding RNAs, have emerged as novel and potent regulators of adipogenesis. However, few miRNAs have been fully investigated in porcine adipogenesis, given the fact that pig is not only an apropos model of human obesity research, but also a staple meat source of human diet. In this study, we showed that miRNA-199a-5p is highly expressed in porcine subcutaneous fat deposits compared to several other tissue types and organs measured alongside. Overexpression of miR-199a-5p in porcine preadipocytes significantly promoted cell proliferation while attenuating the lipid deposition in porcine adipocytes. By target gene prediction and experimental validation, we demonstrated that caveolin-1 (Cav-1) may be a bona fide target of miR-199a-5p in porcine adipocytes, accounting for some of miR-199a-5p's functions. Taken together, our data established a role of miR-199a-5p in porcine preadipocyte proliferation and differentiation, which is at least partially played by downregulating Cav-1.
Asunto(s)
Adipocitos/citología , MicroARNs/metabolismo , Regiones no Traducidas 3' , Adipocitos/metabolismo , Adipogénesis , Animales , Secuencia de Bases , Caveolina 1/química , Caveolina 1/genética , Caveolina 1/metabolismo , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Humanos , MicroARNs/química , ARN Mensajero/metabolismo , Alineación de Secuencia , PorcinosRESUMEN
Lymphoid enhancer binding factor 1 (LEF-1) is a member of the T-cell specific factor (TCF) family, which plays a key role in the development of breast endothelial cells. Moreover, LEF-1 gene has been identified as a candidate gene for teat number trait. In the present study, we detected two novel mutations (NC_010450.3:g. 99514A>G, 119846C>T) by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism in exon 4 and intron 9 of LEF-1 in Guanzhong Black, Hanjiang Black, Bamei and Large White pigs. Furthermore, we analyzed the association between the genetic variations with teat number trait in these breeds. The 99514A>G mutation showed an extremely significant statistical relevance between different genotypes and teat number trait in Guanzhong (p<0.001) and Large White (p = 0.002), and significant relevance in Hanjiang (p = 0.017); the 119846C>T mutation suggested significant association in Guanzhong Black pigs (p = 0.042) and Large White pigs (p = 0.003). The individuals with "AG" or "GG" genotype displayed more teat numbers than those with "AA"; the individuals with "TC" or "CC" genotype showed more teat numbers than those with "TT". Our findings suggested that the 99514A>G and 119846C>T mutations of LEF-1 affected porcine teat number trait and could be used in breeding strategies to accelerate porcine teat number trait improvement of indigenous pigs breeds through molecular marker assisted selection.
RESUMEN
PU.1 is an Ets family transcription factor involved in the myelo-lymphoid differentiation. We have previously demonstrated that PU.1 is also expressed in the adipocyte lineage. However, the expression levels of PU.1 mRNA and protein in preadipocytes do not match the levels in mature adipocytes. PU.1 mRNA level is higher in preadipocytes, whereas its protein is expressed in the adipocytes but not in the preadipocytes. The underlying mechanism remains elusive. Here, we find that miR-155 knockdown or overexpression has no effect on the levels of PU.1 mRNA and protein in preadipocytes or adipocytes. MiR-155 regulates adipogenesis not through PU.1, but via C/EBPß which is another target of miR-155. We also checked the expression levels of PU.1 mRNA and antisense long non-coding RNA (AS lncRNA). Interestingly, compared with the level of PU.1 mRNA, the level of PU.1 AS lncRNA is much higher in preadipocytes, whereas it is opposite in the adipocytes. We further discover that PU.1 AS lncRNA binds to its mRNA forming an mRNA/AS lncRNA compound. The knockdown of PU.1 AS by siRNA inhibits adipogenesis and promotes PU.1 protein expression in both preadipocytes and adipocytes. Furthermore, the repression of PU.1 AS decreases the expression and secretion of adiponectin. We also find that the effect of retroviral-mediated PU.1 AS knockdown on adipogenesis is consistent with that of PU.1 AS knockdown by siRNA. Taken together, our results suggest that PU.1 AS lncRNA promotes adipogenesis through preventing PU.1 mRNA translation via binding to PU.1 mRNA to form mRNA/AS lncRNA duplex in preadipocytes.
Asunto(s)
Adipogénesis/fisiología , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/genética , Transactivadores/metabolismo , Células 3T3-L1 , Adipogénesis/genética , Adiponectina/genética , Adiponectina/metabolismo , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Proteínas Proto-Oncogénicas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transactivadores/genéticaRESUMEN
Retinol binding protein 4 (RBP4), a novel cytokine, is mainly secreted by hepatocytes and adipocytes. RBP4 reportedly induces insulin resistance and RBP4 secretion is increased in the adipocytes of animals or humans with type 2 diabetes, obesity, and metabolic syndrome, but its role in preadipocyte differentiation remains unclear. In this study, we investigated the effect of RBP4 on the differentiation of porcine preadipocytes into adipocytes. The results suggest that RBP4 significantly suppresses the differentiation of porcine preadipocytes into adipocytes, including those treated with the hormone cocktail methylisobutylxanthine-dexamethasone-insulin. RBP4 also weakened the activity of normal threonine 308, the phosphorylation of serine/threonine kinase AKT, and downstream insulin signaling, including the mammalian target of rapamycin (mTOR) and ß-catenin. Moreover, the activation of insulin signaling mediated by knockdown RBP4 in porcine preadipocytes was recovered in the suppression of LY294002. RBP4 also had a suppressive effect on the differentiation of porcine preadipocytes by decreasing the activation of insulin signaling pathways.
Asunto(s)
Adipocitos/citología , Adipogénesis , Insulina/metabolismo , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Tejido Adiposo/metabolismo , Animales , Diferenciación Celular , Cromonas/farmacología , Dexametasona/administración & dosificación , Silenciador del Gen , Insulina/administración & dosificación , Resistencia a la Insulina , Lentivirus/genética , Morfolinas/farmacología , Transducción de Señal , Porcinos , Serina-Treonina Quinasas TOR/metabolismo , Xantina/administración & dosificaciónRESUMEN
The effects of Sirt1 gene and resveratrol on porcine preadipocyte apoptosis have not been characterized. Here, we investigated the apoptotic effects of Sirt1 and resveratrol on porcine preadipocytes, finding that resveratrol-induced preadipocyte apoptosis and up-regulated protein levels of Sirt1. Intriguingly, Sirt1 knockdown by RNAi also resulted in preadipocyte apoptosis. Combining resveratrol treatment and Sirt1 knockdown has an additive effect to promote porcine preadipocyte death. We found that resveratrol treatment alone dose-dependently increased caspase-3 cleavage, as well as the levels of Bax, p53 and acetylated-p53. Interestingly, the ratio of acetylated-p53 over p53 was declined owing to deacetylation by increased Sirt1 expression. Down-regulation of Sirt1 also elevated the cleavage of caspase-3 with a decrease of p53 acetylation. These data indicate that although resveratrol treatment up-regulates Sirt1 expression, it augments porcine preadipocyte apoptosis in a Sirt1-independent manner. The regulation of apoptosis by resveratrol and Sirt1 may provide a novel insight to control preadipocyte number through cellular apoptosis.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Sirtuina 1/genética , Estilbenos/farmacología , Adipocitos , Animales , Caspasa 3/metabolismo , Células Cultivadas , Silenciador del Gen , Vectores Genéticos , Lentivirus/genética , ARN Interferente Pequeño/metabolismo , Resveratrol , Sirtuina 1/metabolismo , PorcinosRESUMEN
MicroRNAs constitute a class of ~22-nucleotide non-coding RNAs. They modulate gene expression by associating with the 3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs). Although multiple miRNAs are known to be regulated during myoblast differentiation, their individual roles in muscle development are still not fully understood. In this study, we showed that miR-199a-3p was highly expressed in skeletal muscle and was induced during C2C12 myoblasts differentiation. We also identified and confirmed several genes of the IGF-1/AKT/mTOR signal pathway, including IGF-1, mTOR, and RPS6KA6, as important cellular targets of miR-199a-3p in myoblasts. Overexpression of miR-199a-3p partially blocked C2C12 myoblast differentiation and the activation of AKT/mTOR signal pathway, while interference of miR-199a-3p by antisense oligonucleotides promoted C2C12 differentiation and myotube hypertrophy. Thus, our studies have established miR-199a-3p as a potential regulator of myogenesis through the suppression of IGF-1/AKT/mTOR signal pathway.