RESUMEN
Sargassum horneri (S. horneri) is a well-known brown seaweed widely distributed worldwide. Several biological activities of S. horneri have been reported. However, its effects on lipid metabolism and the underlying mechanisms remain elusive. In the present study, we examined the inhibitory effect of the active compound "(-)-loliolide ((6S,7aR)-6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydro-1-benzofuran-2(4H)-one (HTT))" from S. horneri extract on lipid accumulation in differentiated adipocytes. MTT assays demonstrated that (-)-loliolide is not toxic to 3T3-L1 adipocytes in a range of concentrations. (-)-loliolide significantly reduced intracellular lipid accumulation in the differentiated phase of 3T3-L1 adipocytes as shown by Oil Red O staining. Western blot analysis revealed that (-)-loliolide increased the expression of lipolytic protein phospho-hormone-sensitive lipase (p-HSL) and thermogenic protein peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1). Additionally, (-)-loliolide decreased expression of adipogenic and lipogenic proteins, including sterol regulatory element-binding protein-1 (SREBP-1), peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding protein-α (C/EBP-α), and fatty acid-binding protein 4 (FABP4) in 3T3-L1 adipocytes. These results indicate that (-)-loliolide from S. horneri could suppress lipid accumulation via regulation of antiadipogenic and prolipolytic mechanisms in 3T3-L1 cells. Considering the multifunctional effect of (-)-loliolide, it can be useful as a lipid-lowering agent in the management of patients who suffer from obesity.
Asunto(s)
Benzofuranos/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Sargassum/química , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Lipólisis/efectos de los fármacos , Ratones , Obesidad/tratamiento farmacológico , PPAR gamma/antagonistas & inhibidores , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Termogénesis/efectos de los fármacosRESUMEN
Deoxynivalenol (DON)-a type B trichothecene mycotoxin, mainly produced by the secondary metabolism of Fusarium-has toxic effects on animals and humans. Although DON's toxicity in many organs including the adrenal glands, thymus, stomach, spleen, and colon has been addressed, its effects on adipocytes have not been investigated. In this study, 3T3-L1 cells were chosen as the cell model and treated with less toxic doses of DON (100 ng/mL) for 7 days. An inhibition of adipogenesis and decrease in triglycerides (TGs) were observed. DON exposure significantly downregulated the expression of PPARγ2 and C/EBPα, along with that of other adipogenic marker genes in 3T3-L1 cells and BALB/c mice. The anti-adipogenesis effect of DON and the downregulation of the expression of adipogenic marker genes were effectively reversed by PPARγ2 overexpression. The repression of PPARγ2's expression is the pivotal event during DON exposure regarding adipogenesis. DON exposure specifically decreased the di-/trimethylation levels of Histone 3 at lysine 4 in 3T3-L1 cells, therefore weakening the enrichment of H3K4me2 and H3K4me3 at the Pparγ2 promoter and suppressing its expression. Conclusively, DON exposure inhibited PPARγ2 expression via decreasing H3K4 methylation, downregulated the expression of PPARγ2-regulated adipogenic marker genes, and consequently suppressed the intermediate and late stages of adipogenesis. Our results broaden the current understanding of DON's toxic effects and provide a reference for addressing the toxicological mechanism of DON's interference with lipid homeostasis.
Asunto(s)
Adipogénesis , Diferenciación Celular , Regulación de la Expresión Génica/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , Tricotecenos/farmacología , Células 3T3-L1 , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , PPAR gamma/genética , PPAR gamma/metabolismoRESUMEN
Atherosclerosis (AS) is a risky cardiovascular disease with limited treatment options. Various pan or type-selective histone deacetylase (HDAC) inhibitors are reportedly atheroprotective against atherosclerosis (AS); however, the key effectors and the main cellular processes that mediate the protective effects remain poorly defined. Here, we report that PPARγ (Peroxisome proliferator-activated receptor gamma), a transcription factor actively involved in lipid metabolism with strong tissue protective and anti-inflammation properties, is a critical mediator of the anti-AS effects by HDAC inhibition. We showed that a well-known pan-HDAC inhibitor TSA (Trichostatin A) reduced foam cell formation of macrophages that is accompanied by a marked elevation of PPARγ and its downstream cholesterol efflux transporter ABCA1 (ATP-binding membrane cassette transport protein A1) and ABCG1. In an AS model of ApoE-/- mice fed on high-fat diet, TSA treatment alleviated AS lesions, similarly increased PPARγ and the downstream cholesterol transporters and mitigated the induction of inflammatory cytokine TNFα and IL-1ß. Exploring the potential cause of PPARγ elevation revealed that TSA induced the acetylation of C/EBPα (CCAAT enhancer binding protein alpha), the upstream regulator of PPARγ, through which it increased PPARγ transactivation. More importantly, we generated a strain of PPARγ/ApoE double knockout mice and demonstrated that lack of PPARγ abrogated the protective effects of TSA on foam cell formation of peritoneal macrophages and the AS pathogenesis. Taken together, these results unravel that C/EBPα and PPARγ are the HDAC-sensitive components of an epigenetic signaling pathway mediating foam cell formation and AS development, and suggest that targeting C/EBPα/PPARγ axis by HDAC inhibitors possesses therapeutic potentials in retarding the progression of AS and the related cardiovascular diseases.
Asunto(s)
Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/prevención & control , Células Espumosas/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , PPAR gamma/efectos de los fármacos , Transportador 1 de Casete de Unión a ATP/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/antagonistas & inhibidores , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Dieta Alta en Grasa , Epigénesis Genética/efectos de los fármacos , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Noqueados , Células RAW 264.7RESUMEN
The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is downregulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring.
Asunto(s)
Alquinos/farmacología , Antineoplásicos/farmacología , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Quinazolinonas/farmacología , Alquinos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Leucemia Mieloide Aguda/metabolismo , Estructura Molecular , Quinazolinonas/síntesis química , Quinazolinonas/química , Relación Estructura-ActividadRESUMEN
Adipose tissue stores energy and plays an important role in energy homeostasis. CCAAT/enhancer-binding protein ß (C/EBPß) is an important early transcription factor for 3T3-L1 preadipocyte differentiation, facilitating mitotic clonal expansion (MCE) and transactivating C/EBPα and peroxisome proliferator-activated receptor-γ (PPARγ) to promote adipogenesis. C/EBPß is induced early, but the expression of antimitotic C/EBPα and PPARγ is not induced until â¼48 h. The delayed expression of C/EBPα and PPARγ is thought to ensure MCE progression, but the molecular mechanism for this delay remains elusive. Here, we show that the zinc-finger transcription factor Krüppel-like factor 10 (KLF10) is induced after adipogenic induction and that its expression positively correlates with that of C/EBPß but inversely correlates with expression of C/EBPα and PPARγ. C/EBPß bound to the KLF10 promoter and transactivated its expression during MCE. KLF10 overexpression in 3T3-L1 preadipocyte repressed adipogenesis and decreased C/EBPα and PPARγ expression, whereas siRNA-mediated down-regulation of KLF10 enhanced adipogenesis and increased C/EBPα and PPARγ expression. Luciferase assays revealed an inhibitory effect of KLF10 on C/EBPα promoter activity. Using promoter deletion and mutation analysis, we identified a KLF10-binding site within the proximal promoter region of C/EBPα. Furthermore, KLF10 interacted with and recruited histone deacetylase 1 (HDAC1) to the C/EBPα promoter, decreasing acetylated histone H4 on the C/EBPα promoter and inactivating C/EBPα transcription. Because C/EBPα can transactivate PPARγ, our results suggest a mechanism by which expression of C/EBPα and PPARγ is delayed via KLF10 expression and shed light on the negative feedback loop for C/EBPß-regulated adipogenesis in 3T3-L1 preadipocyte.
Asunto(s)
Adipogénesis , Proteína alfa Potenciadora de Unión a CCAAT/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de Tipo Kruppel/genética , Activación Transcripcional , Células 3T3-L1 , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Diferenciación Celular , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Retroalimentación Fisiológica , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , PPAR gamma/metabolismo , Factores de TiempoRESUMEN
Caffeine is a methylxanthine derived from plant foods such as coffee beans and tea leaves, and has multiple biological activities against physiological response and several diseases. Although there are some reports about the direct effect of caffeine against anti-lipid accumulation in vitro, the effect of caffeine on lipid accumulation in adipocytes through stimulating intestinal epithelial cells is unknown. Since direct treatment with caffeine to 3T3-L1 cells did not affect lipid accumulation, we determined whether caffeine-stimulated intestinal epithelial Caco-2 cells influence the lipid accumulation in 3T3-L1 adipocytes. Caco-2 cells were cultured on a transwell insert with or without caffeine for 24 h. Subsequently, the basolateral component of the Caco-2 cell culture on the transwell was collected and termed caffeine-conditioning medium (CCM). When 3T3-L1 adipocytes were incubated with CCM, CCM decreased lipid accumulation and suppressed gene expression of proliferator activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α in 3T3-L1 adipocytes. Furthermore, CCM decreased the expression of C/EBPß and C/EBPδ at the protein level, but not at the mRNA level. We observed that a proteasome inhibitor, MG132, inhibited CCM-caused down-expression of C/EBPß and C/EBPδ proteins, and that CCM promoted the ubiquitination level of C/EBPß and C/EBPδ proteins. Protein microarray analysis showed caffeine suppresses the secretion of inflammatory cytokines, interleukin-8 and plasminogen activator inhibitor-1 from Caco-2 cells. These results suggest that caffeine indirectly suppresses lipid accumulation in 3T3-L1 adipocytes through decreasing secretion of inflammatory cytokines from Caco-2 cells.
Asunto(s)
Adipocitos/metabolismo , Adipogénesis , Cafeína/metabolismo , Regulación hacia Abajo , Enterocitos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Metabolismo de los Lípidos , Células 3T3-L1 , Adipocitos/citología , Adipocitos/inmunología , Adipogénesis/efectos de los fármacos , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Células CACO-2 , Polaridad Celular , Estimulantes del Sistema Nervioso Central/metabolismo , Medios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Enterocitos/efectos de los fármacos , Enterocitos/inmunología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidores de Proteasoma/farmacologíaRESUMEN
The aim of this study was to investigate whether fermented Platycodon grandiflorum (FPG) inhibits lipid accumulation in 3T3-L1 adipocytes and mice with high-fat diet (HFD)-induced obesity. We evaluated the effect of FPG on antiadipogenic activity via regulation of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), as well as protein expression of their target genes, fatty acid binding protein 4 (FABP4). We further examined the antiobesity effects of FPG on HFD-induced obesity in mice. The FPG was orally administered to mice with a HFD at 50, 100, or 200 mg/kg/day for 8 weeks. Our results show that FPG significantly inhibited fat accumulation during 3T3-L1 adipogenesis through downregulating adipogenic transcript factors. Moreover, FPG markedly reduced the final body weight with a decrease in epididymal adipose tissue mass and adipocyte size compared with the untreated HFD-induced group. The effects of FPG on HFD-induced obesity were primarily responsible for inhibiting adipogenesis in adipose tissue and regulating lipid metabolism, such as through lipogenesis and fatty acid oxidation. Additionally, FPG ameliorated serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels. Hence, FPG may be an alternative treatment for controlling obesity through downregulating lipid accumulation.
Asunto(s)
Adipocitos/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Dieta Alta en Grasa/efectos adversos , Producto de la Acumulación de Lípidos/efectos de los fármacos , Obesidad/tratamiento farmacológico , Extractos Vegetales/farmacología , Platycodon/química , Células 3T3-L1 , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión a Ácidos Grasos/efectos de los fármacos , Proteínas de Unión a Ácidos Grasos/genética , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Modelos Animales , Obesidad/metabolismo , PPAR gamma/antagonistas & inhibidores , PPAR gamma/metabolismo , Platycodon/metabolismo , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/efectos de los fármacosRESUMEN
OBJECTIVE: Celastrol, a triterpene from the root bark of the Chinese medicinal plant Tripterygium wilfordii, has been shown to exhibit anti-oxidant, anti-inflammatory, anti-cancer and insecticidal activities. Also, it has been demonstrated that celastrol has obesity-controlling effects in diet-induced obesity mice. However, direct evidence that celastrol contributes to the development of adipocyte differentiation and lipolysis has not been fully elucidated. Moreover, no previous studies have evaluated whether celastrol may regulate adipogenic transcriptional markers in adipocytes. MATERIALS/METHODS: In order to address the questions above, we extended previous observations and investigated in vitro celastrol signaling study whether celastrol may regulate differentiation, lipolysis and key adipogenic transcriptional pathways in 3T3-L1 adipocytes. RESULTS: Treatment of celastrol not only inhibited adipocyte differentiation (lipid accumulation, glyceraldehyde-3-phosphate dehydrogenase activity and triglyceride content) but also increased lipolysis (glycerol release and free fatty acid release) in 3T3-L1 adipocytes. In addition, all celastrol-regulated functional activities were controlled by PPARγ(2) and C/EBPα signaling pathways in duration of celastrol's treatment in 3T3-L1 adipocytes. CONCLUSION: Our initial data from in vitro celastrol signaling studies suggest novel insights into the role of PPARγ(2) and C/EBPα as probable mediators of the action of celastrol in regulating adipocyte differentiation and lipolysis in 3T3-L1 adipocytes.
Asunto(s)
Adipocitos Blancos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Fármacos Antiobesidad/farmacología , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Lipólisis/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , Triterpenos/farmacología , Células 3T3-L1 , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/farmacología , Fármacos Antiobesidad/efectos adversos , Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/farmacología , Antioxidantes/efectos adversos , Antioxidantes/farmacología , Biomarcadores/química , Biomarcadores/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Supervivencia Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Insecticidas/toxicidad , Cinética , Ratones , PPAR gamma/genética , PPAR gamma/metabolismo , Triterpenos Pentacíclicos , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Triterpenos/efectos adversosRESUMEN
C/EBPα (p42 and p30 isoforms) is commonly dysregulated in cancer via the action of oncogenes, and specifically in acute myeloid leukaemia (AML) by mutation. Elevated TRIB2 leads to the degradation of C/EBPα p42, leaving p30 intact in AML. Whether this relationship is a cooperative event in AML transformation is not known and the molecular mechanism involved remains elusive. Using mouse genetics, our data reveal that in the complete absence of C/EBPα, TRIB2 was unable to induce AML. Only in the presence of C/EBPα p42 and p30, were TRIB2 and p30 able to cooperate to decrease the latency of disease. We demonstrate that the molecular mechanism involved in the degradation of C/EBPα p42 requires site-specific direct interaction between TRIB2 and C/EBPα p42 for the K48-specific ubiquitin-dependent proteasomal degradation of C/EBPα p42. This interaction and ubiquitination is dependent on a critical C terminal lysine residue on C/EBPα. We show effective targeting of this pathway pharmacologically using proteasome inhibitors in TRIB2-positive AML cells. Together, our data show that excess p30 cooperated with TRIB2 only in the presence of p42 to accelerate AML, and the direct interaction and degradation of C/EBPα p42 is required for TRIB2-mediated AML.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia Mieloide Aguda/genética , Isoformas de Proteínas/genética , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Ratones , Mutación , Inhibidores de Proteasoma/administración & dosificación , Isoformas de Proteínas/biosíntesisRESUMEN
Obesity is a medical condition of excess body fat negatively influencing morbidity and mortality via non-communicable disease risks. Adipogenesis, the process in which preadipocytes differentiate into adipocytes, plays a pivotal role in obesity. Our previous study proved that tannic acid (TA) showed anti-adipogenesis effect in 3T3-L1 preadipocytes. However, the precise mechanism involved in the inhibition in adipocytes differentiation by TA is unclear, and thus this is the subject of the present investigation. In this study, we determined the effect of TA on different stages of 3T3-L1 preadipocytes differentiation, and found that when treating in the early stage of differentiation, TA reduced lipid accumulation significantly. However, TA did not reduce lipid accumulation when treating in mid- and late-stages of adipocyte differentiation. To further study which gene TA had an impact on in the early stage of differentiation, we identified a number of genes associated with lipid metabolism. The results showed that compared to the control group, the mRNA levels of FAS, C/EBPα, and PPARγ were significantly decreased (p < 0.05), whereas the mRNA levels of adipsin, ap2 were increased (p < 0.05). However, TA had no effect on mRNA levels of ACC1 and ACC2. Western blot results showed that TA down-regulated the expression of PPARγ, which is a major factor in preadipocyte differentiation. In addition, TA did not affect the PI3 K/AKT pathway. These results indicate that the anti-adipogenesis effect of TA involves down-regulation of PPARγ in the early stage of 3T3-L1 preadipocyte differentiation. Some potential limitations of this study should be considered. All the results in this study were based on cell experiments. However, the human bioavailability of TA is not clear. In the present study, the concentration of TA was 5 µM; therefore, there were concerns about whether oral intake of TA could reach the effective concentrations. This important point needs to be clarified in vivo.
Asunto(s)
Adipocitos Blancos/metabolismo , Adipogénesis , Fármacos Antiobesidad/metabolismo , Suplementos Dietéticos , Regulación hacia Abajo , PPAR gamma/antagonistas & inhibidores , Taninos/metabolismo , Células 3T3-L1 , Adipocitos Blancos/citología , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proliferación Celular , Factor D del Complemento/química , Factor D del Complemento/genética , Factor D del Complemento/metabolismo , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Proteínas de Unión a Ácidos Grasos/agonistas , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Cinética , Metabolismo de los Lípidos , Ratones , PPAR gamma/genética , PPAR gamma/metabolismo , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
Myeloid progenitors lose their potential to generate neutrophils when they adopt the mononuclear phagocyte lineage. The mechanism underlying this lineage restriction remains unknown. We here report that the protein expression of IRF8, an essential transcription factor for the development of dendritic cells (DCs) and monocytes, sharply increases at the monocyte-DC progenitor (MDP) stage and remains high in common monocyte progenitors (cMoPs). Irf8(-/-) MDPs and cMoPs accumulate but fail to efficiently generate their downstream populations, instead giving rise to neutrophils in vivo. IRF8 physically interacts with the transcription factor C/EBPα and prevents its binding to chromatin in MDPs and cMoPs, blocking the ability of C/EBPα to stimulate transcription and neutrophil differentiation. A partial inhibition of C/EBP activity in Irf8(-/-) haematopoietic progenitors alleviates the neutrophil overproduction in vivo. Thus, IRF8 not only bestows monocyte and DC differentiation potential upon mononuclear phagocyte progenitors but also restrains these progenitors from differentiating into neutrophils.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Regulación de la Expresión Génica , Factores Reguladores del Interferón/metabolismo , Leucocitos Mononucleares/citología , Neutrófilos/citología , Fagocitos/citología , Animales , Células de la Médula Ósea/citología , Diferenciación Celular , Cromatina/metabolismo , Femenino , Citometría de Flujo , Genes Reporteros , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre/citología , TranscriptomaRESUMEN
VPA (valproic acid), a short-chain fatty acid that is a HDAC (histone deacetylase) inhibitor, is known to suppress adipogenesis. In the present study, we identified the molecular mechanism of VPA-mediated suppression of adipogenesis in adipocytes. VPA suppressed the accumulation of intracellular triacylglycerol. The expression levels of PPARγ (peroxisome-proliferator-activated receptor γ) and C/EBPα (CCAAT/enhancer-binding protein α), which are key regulators of adipogenesis, as well as the expression of SCD (stearoyl-CoA desaturase), were decreased by the treatment with VPA. Moreover, glycerol release was decreased in the VPA-treated cells, even though the transcription levels of ATGL (adipose triacylglycerol lipase), HSL (hormone-sensitive lipase) and MGL (monoacylglycerol lipase), all of which are involved in lipolysis, were elevated by the treatment with VPA. It is noteworthy that the expression level of FAS (fatty acid synthase) was significantly suppressed when the cells were cultured in medium containing VPA. Furthermore, VPA-mediated suppression of the accumulation of the intracellular triacylglycerols was prevented by the treatment with palmitic acid, a major product of FAS. The results of promoter-luciferase and chromatin immunoprecipitation assays demonstrated that USF1(upstream stimulating factor 1) bound to the E-box of the promoter region of the FAS gene. In addition, the expression of USF1 was decreased by the treatment with VPA. siRNA-mediated knockdown of the expression of the USF1 gene repressed adipogenesis along with the decreased expression of the FAS gene. The overexpression of USF1 enhanced both adipogenesis and the expression of FAS in VPA-treated cells. These results indicate that VPA suppressed adipogenesis through the down-regulation of USF1-activated fatty acid synthesis in adipocytes.
Asunto(s)
Adipocitos Blancos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Factores Estimuladores hacia 5'/antagonistas & inhibidores , Ácido Valproico/farmacología , Células 3T3-L1 , Adipocitos Blancos/citología , Adipocitos Blancos/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Acido Graso Sintasa Tipo I/química , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Grasos no Esterificados/metabolismo , Genes Reporteros/efectos de los fármacos , Ratones , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Ácido Palmítico/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo , Triglicéridos/biosíntesis , Factores Estimuladores hacia 5'/genética , Factores Estimuladores hacia 5'/metabolismo , Ácido Valproico/antagonistas & inhibidoresRESUMEN
We investigated the effects of 6-gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) on the inhibition of rosiglitazone (RGZ)-induced adipogenesis in 3T3-L1 cells. The morphological changes were photographed based on staining lipid accumulation by Oil-Red O in RGZ (1 µmol/l)-treated 3T3-L1 cells without or with various concentrations of 6-gingerol on differentiation day 8. Quantitation of triglycerides content was performed in cells on day 8 after differentiation induction. Differentiated cells were lysed to detect mRNA and protein levels of adipocyte-specific transcription factors by real-time reverse transcription-polymerase chain reaction and Western blot analysis, respectively. 6-gingerol (50 µmol/l) effectively suppressed oil droplet accumulation and reduced the sizes of the droplets in RGZ-induced adipocyte differentiation in 3T3-L1 cells. The triglyceride accumulation induced by RGZ in differentiated 3T3-L1 cells was also reduced by 6-gingerol (50 µmol/l). Treatment of differentiated 3T3-L1 cells with 6-gingerol (50 µmol/l) antagonized RGZ-induced gene expression of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein α. Additionally, the increased levels of mRNA and protein in adipocyte-specific fatty acid binding protein 4 and fatty acid synthase induced by RGZ in 3T3-L1 cells were decreased upon treatment with 6-gingerol. Our data suggests that 6-gingerol may be beneficial in obesity, by reducing adipogenesis partly through the down-regulating PPARγ activity.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Catecoles/farmacología , Alcoholes Grasos/farmacología , Tiazolidinedionas/efectos adversos , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Ratones , Obesidad , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rosiglitazona , Triglicéridos/metabolismoRESUMEN
Obesity is a result of adipocyte hypertrophy followed by hyperplasia. It is a risk factor for several metabolic disorders such as dyslipidemia, type-2 diabetes, hypertension, and cardiovascular diseases. Coagulanolides, particularly coagulin-L isolated from W. coagulan has earlier been reported for anti-hyperglycemic activity. In this study, we investigated the effect of coagulin-L on in vitro models of adipocyte differentiation including 3T3-L1 pre-adipocyte, mouse stromal mesenchymal C3H10T1/2 cells and bone marrow derived human mesenchymal stem cells (hMSCs). Our results showed that, coagulin-L reduces the expressions of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), the major transcription factors orchestrating adipocyte differentiation. Detailed analysis further proved that early exposure of coagulin-L is sufficient to cause significant inhibition during adipogenesis. Coagulin-L inhibited mitotic clonal expansion (MCE) by delayed entry in G1 to S phase transition and S-phase arrest. This MCE blockade was caused apparently by decreased phosphorylation of C/EBPß, modulation in expression of cell cycle regulatory proteins, and upregulation of Wnt/ß-catenin pathway, the early stage regulatory proteins of adipogenic induction. Taken together all evidences, a known anti-hyperglycemic agent coagulin-L has shown potential to inhibit adipogenesis significantly, which can be therapeutically exploited for treatment of obesity and metabolic syndrome.
Asunto(s)
Adipogénesis/efectos de los fármacos , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Withania , Witanólidos/farmacología , Células 3T3-L1 , Animales , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Ratones , Mitosis/efectos de los fármacos , Obesidad/tratamiento farmacológico , PPAR gamma/antagonistas & inhibidores , Fitoterapia , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Células Madre/efectos de los fármacos , Witanólidos/uso terapéutico , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus etiologically associated with adult T-cell leukemia (ATL). The HTLV-1 bZIP factor (HBZ), which is encoded by minus strand of provirus, is expressed in all ATL cases and supports the proliferation of ATL cells. However, the precise mechanism of growth promoting activity of HBZ is poorly understood. RESULTS: In this study, we showed that HBZ suppressed C/EBPα signaling activation induced by either Tax or C/EBPα. As mechanisms of HBZ-mediated C/EBPα inhibition, we found that HBZ physically interacted with C/EBPα and diminished its DNA binding capacity. Luciferase and immunoprecipitation assays revealed that HBZ repressed C/EBPα activation in a Smad3-dependent manner. In addition, C/EBPα was overexpressed in HTLV-1 infected cell lines and fresh ATL cases. HBZ was able to induce C/EBPα transcription by enhancing its promoter activity. Finally, HBZ selectively modulated the expression of C/EBPα target genes, leading to the impairment of C/EBPα-mediated cell growth suppression. CONCLUSION: HBZ, by suppressing C/EBPα signaling, supports the proliferation of HTLV-1 infected cells, which is thought to be critical for oncogenesis.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proliferación Celular , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/fisiología , Linfocitos T/virología , Proteínas Virales/metabolismo , Adulto , Anciano , Línea Celular , ADN/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Unión Proteica , Proteínas de los RetroviridaeRESUMEN
This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBPß, as well as the C/EBPα and PPARγ genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3ß, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPß and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBPß, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Arándanos Azules (Planta)/química , Frutas/química , Obesidad/tratamiento farmacológico , Preparaciones de Plantas/farmacología , Células 3T3-L1 , Adipocitos/metabolismo , Adipocitos/patología , Adipogénesis/genética , Animales , Peso Corporal/efectos de los fármacos , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular , Dieta Alta en Grasa , Ingestión de Alimentos/efectos de los fármacos , Regulación de la Expresión Génica , Masculino , Ratones , Obesidad/metabolismo , Obesidad/fisiopatología , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , PPAR gamma/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
We previously identified a quantitative trait locus for adiposity, non-insulin-dependent diabetes 5 (Nidd5), on mouse chromosome 2. In the current study, we identified the actual genetic alteration at Nidd5 as a nonsense mutation of the Acvr1c gene encoding activin receptor-like kinase 7 (ALK7), one of the type I transforming growth factor-ß receptors, which results in a COOH-terminal deletion of the kinase domain. We further showed that the ALK7 dysfunction causes increased lipolysis in adipocytes and leads to decreased fat accumulation. Conversely, ALK7 activation inhibits lipolysis by suppressing the expression of adipose lipases. ALK7 and activated Smads repress those lipases by downregulating peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBP) α. Although PPARγ and C/EBPα act as adipogenic transcription factors during adipocyte differentiation, they are lipolytic in sum in differentiated adipocytes and are downregulated by ALK7 in obesity to accumulate fat. Under the obese state, ALK7 deficiency improves glucose tolerance and insulin sensitivity by preferentially increasing fat combustion in mice. These findings have uncovered a net lipolytic function of PPARγ and C/EBPα in differentiated adipocytes and point to the ALK7-signaling pathway that is activated in obesity as a potential target of medical intervention.
Asunto(s)
Receptores de Activinas Tipo I/fisiología , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Lipólisis , Obesidad/metabolismo , PPAR gamma/antagonistas & inhibidores , Células 3T3-L1 , Receptores de Activinas Tipo I/genética , Animales , Proteína alfa Potenciadora de Unión a CCAAT/fisiología , Codón sin Sentido , Regulación hacia Abajo , Lipasa/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , PPAR gamma/fisiología , Proteínas Smad/fisiología , Triglicéridos/metabolismoRESUMEN
BACKGROUND: Kefir, a traditional fermented milk composed of microbial symbionts, is reported to have various health benefits such as anti-tumour, anti-inflammatory, anti-neoplastic and pro-digestive effects. In this study, to elucidate the effects of kefir on adipocyte differentiation and lipid accumulation, three fractions were prepared from kefir culture broth. The inhibitory effects of kefir liquid culture broth fraction (Fr-1), soluble fraction (Fr-2) and insoluble fraction (Fr-3), prepared by sonication of kefir solid culture broth, on adipocyte differentiation in 3T3-L1 preadipocytes were examined. RESULTS: Fr-3 (0.1 mg mL(-1)) significantly decreased lipid accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity by 60 and 68% respectively without affecting cell viability. In addition, Fr-3 treatment down-regulated the mRNA expression of adipogenic transcription factors including C/EBPα (32%), PPARγ (46%) and SREBP-1c (34%) during adipocyte differentiation compared with untreated control cells. The mRNA expression of adipocyte-specific genes (aP2, FAS and ACC) was also clearly decreased. CONCLUSION: The results suggest that the insoluble fraction of kefir (Fr-3) mediates anti-adipogenic effects through the inhibition of adipocyte differentiation, partly via suppression of the C/EBPα-, SREBP-1c- and PPARγ-dependent pathways.
Asunto(s)
Adipocitos/citología , Adipogénesis/genética , Diferenciación Celular/fisiología , Productos Lácteos Cultivados/fisiología , Factores de Transcripción/genética , Células 3T3-L1 , Animales , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína alfa Potenciadora de Unión a CCAAT/genética , Productos Lácteos Cultivados/química , Regulación hacia Abajo , Glicerolfosfato Deshidrogenasa/antagonistas & inhibidores , Ratones , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , ARN Mensajero/genética , Solubilidad , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
BACKGROUND: Centipede grass (CG) originates from China and South America and is reported to contain several C-glycosyl flavones and phenolic constituents, including maysin and luteolin derivatives. This study aimed to investigate, for the first time, the antiobesity activity of CG and its potential molecular mechanism in 3T3-L1 cells. METHODS: To study the effect of CG on adipogenesis, differentiating 3T3-L1 cells were treated every day with CG at various concentrations (0-100 µg/ml) for six days. Oil-red O staining and triglyceride content assay were performed to determine the lipid accumulation in 3T3-L1 cells. The expression of mRNAs or proteins associated with adipogenesis was measured using RT-PCR and Western blotting analysis. We examined the effect of CG on level of phosphorylated Akt in 3T3-L1 cells treated with CG at various concentration s during adipocyte differentiation. RESULTS: Differentiation was investigated with an Oil-red O staining assay using CG-treated 3T3-L1 adipocytes. We found that CG suppressed lipid droplet formation and adipocyte differentiation in 3T3-L1 cells in a dose-dependent manner. Treatment of the 3T3-L1 adipocytes with CG resulted in an attenuation of the expression of adipogenesis-related factors and lipid metabolic genes. The expression of C/EBPα and PPARγ, the central transcriptional regulators of adipogenesis, was decreased by the treatment with CG. The expression of genes involved in lipid metabolism, aP2 were significantly inhibited following the CG treatment. Moreover, the CG treatment down-regulated the phosphorylation levels of Akt and GSK3ß. CONCLUSIONS: Taken collectively, these data indicated that CG exerts antiadipogenic activity by inhibiting the expression of C/EBPß, C/EBPα, and PPARγ and the Akt signaling pathway in 3T3-L1 adipocytes.
Asunto(s)
Adipogénesis/efectos de los fármacos , Proteínas Potenciadoras de Unión a CCAAT/antagonistas & inhibidores , Expresión Génica/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , Extractos Vegetales/farmacología , Poaceae , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Animales , Fármacos Antiobesidad/farmacología , Proteína alfa Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Flavonoides/farmacología , Glucósidos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Metabolismo de los Lípidos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Luteolina/farmacología , Ratones , Fosforilación , Polifenoles/farmacología , Transducción de SeñalRESUMEN
Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs. Conversion begins by glucocorticoid and cAMP signals raising C/EBPß levels above a critical threshold, triggering three consecutive positive feedback loops: from PPARγ to C/EBPα, then to C/EBPß, and last to the insulin receptor. Experiments and modeling show that these feedbacks create a robust, irreversible transition to a terminally differentiated state by rejecting short- and low-amplitude stimuli. After the differentiation switch is triggered, insulin controls fat accumulation in a graded fashion. Altogether, our study introduces a regulatory motif that locks cells in a differentiated state by engaging a sequence of positive feedback loops.