RESUMO
Previously, we reported that piperine, one of the major pungent components in black pepper, attenuates adipogenesis by repressing PPARγ activity in 3T3-L1 preadipocytes. However, the epigenetic mechanisms underlying this activity remain unexplored. Here, gene set enrichment analysis using microarray data indicated that there was significant downregulation of adipogenesis-associated and PPARγ target genes and upregulation of genes bound with H3K27me3 in response to piperine. As shown by Gene Ontology analysis, the upregulated genes are related to lipid oxidation and polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assays revealed that PPARγ (and its coactivators), H3K4me3, and H3K9ac were less enriched at the PPAR response element of three adipogenic genes, whereas increased accumulation of H3K9me2, H3K27me3, and Ezh2 was found, which likely led to the reduced gene expression. Further analysis using three lipolytic genes revealed the opposite enrichment pattern of H3K4me3 and H3K27me3 at the Ezh2 binding site. Treatment with GSK343, an Ezh2 inhibitor, elevated lipolytic gene expression by decreasing the enrichment of H3K27me3 during adipogenesis, which confirms that Ezh2 plays a repressive role in lipolysis. Overall, these results suggest that piperine regulates the expression of adipogenic and lipolytic genes by dynamic regulation of histone modifications, leading to the repression of adipocyte differentiation.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/fisiologia , Alcaloides/uso terapêutico , Benzodioxóis/uso terapêutico , Código das Histonas/fisiologia , Piperidinas/uso terapêutico , Alcamidas Poli-Insaturadas/uso terapêutico , Alcaloides/farmacologia , Benzodioxóis/farmacologia , Diferenciação Celular , Humanos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologiaRESUMO
Oleuropein, a bitter glucoside found in green olive leaves, and its metabolite hydroxytyrosol display powerful antioxidant activity both in vivo and in vitro. In this study, we hypothesized that the antioxidant activity of oleuropein could attenuate hepatic steatosis. To test this hypothesis, we established steatotic hepatocytes using HepG2 and FL83B cells treated with free fatty acids (FFAs) (oleate:palmitate, 2:1). To confirm hepatic steatosis, the intracellular lipid levels were quantitatively measured by Nile Red staining, and the sizes and distributions of lipid droplets were visualized by transmission electron microscopy. The expression of PAT family proteins as well as of adipose differentiation-related protein and tail interacting protein (TIP47) was evaluated by reverse transcriptase polymerase chain reaction and immunoblotting. To examine the cellular and molecular events associated with oleuropein, annexin V/propidium iodide staining and immunoblotting were performed. Oleuropein decreased the number and size of lipid droplets in FFA-treated cells and reduced intracellular triglyceride accumulation. However, it did not affect the expression of lipid droplets-associated PAT family proteins, including adipose differentiation-related protein and TIP47. In addition, oleuropein reduced FFA-induced extracellular signal-regulated kinase activation but had no effect on c-Jun N-terminal kinase or AKT activation. Given its protective effects against FFA-induced hepatocellular steatosis, oleuropein may be a lipid-lowering agent.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fígado Gorduroso/metabolismo , Hepatócitos/efeitos dos fármacos , Olea/química , Extratos Vegetais/farmacologia , Piranos/farmacologia , Aciltransferases/metabolismo , Adipogenia/efeitos dos fármacos , Fígado Gorduroso/prevenção & controle , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Hipolipemiantes/farmacologia , Hipolipemiantes/uso terapêutico , Glucosídeos Iridoides , Iridoides , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Membrana/metabolismo , Perilipina-2 , Perilipina-3 , Fitoterapia , Extratos Vegetais/uso terapêutico , Folhas de Planta , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piranos/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coloração e Rotulagem , Triglicerídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Rhizoma Polygonati falcatum (RPF) has been used as a traditional herbal medicine in Asia, because of its anti-hyperglycemic, anti-triglycemic, and anti-tumor activity. In this study, we determined the anti-adipogenic potential of RPF extract and its component kaempferol in 3T3-L1 adipocytes, and the underlying molecular mechanism(s) using microarray analysis. Adipocyte differentiation of 3T3-L1 cells was significantly impaired by RPF extract and kaempferol as monitored by Oil Red O staining and quantitative measurement of lipid accumulation. Additionally, the mRNA expression of adipogenesis genes decreased on treatment with kaempferol. The role of kaempferol at the genome-wide level was further assessed by a microarray approach. Our analysis indicated that kaempferol decreased the expression of adipogenic transcription factors (Pparγ, Cebpß, Srebp1, Rxrß, Lxrß, Rorα) and genes involved in triglyceride biosynthesis (Gpd1, Agpat2, Dgat2), while increasing lipolysis-related genes, such as Tnfα, Lsr, and Cel. Finally, co-transfection assays using luciferase reporter gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis using peroxisome proliferator-activated receptor-γ (PPARγ) target genes indicated that kaempferol significantly repressed rosiglitazone-induced PPARγ transcriptional activity. Overall, our data suggests that kaempferol, a major component of RPF, may be beneficial in obesity, by reducing adipogenesis and balancing lipid homeostasis partly through the down-regulation of PPARγ.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Quempferóis/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Polygonatum/química , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Animais , Fármacos Antiobesidade/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Homeostase , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Quempferóis/uso terapêutico , Metabolismo dos Lipídeos/genética , Lipólise/efeitos dos fármacos , Lipólise/genética , Camundongos , Análise em Microsséries , Obesidade/genética , Obesidade/metabolismo , Obesidade/prevenção & controle , PPAR gama/metabolismo , Fitoterapia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rizoma , Rosiglitazona , Tiazolidinedionas/farmacologia , Fatores de Transcrição/metabolismo , Triglicerídeos/biossíntese , Triglicerídeos/genéticaRESUMO
This study investigated the antiadipogenic activity of black pepper extract and its constituent piperine in 3T3-L1 preadipocytes as well as the underlying molecular mechanisms. Both black pepper extract and piperine, without affecting cytotoxicity, strongly inhibited the adipocyte differentiation of 3T3-L1 cells. The mRNA expression of the master adipogenic transcription factors, PPARγ, SREBP-1c, and C/EBPß, was markedly decreased. Intriguingly, mRNA levels of PPARγ target genes were also down-regulated. Moreover, a luciferase reporter assay indicated that pipierine significantly represses the rosiglitazone-induced PPARγ transcriptional activity. Finally, GST-pull down assays demonstrated that piperine disrupts the rosiglitazone-dependent interaction between PPARγ and coactivator CBP. Genome-wide analysis using microarray further supports the role of piperine in regulating genes associated with lipid metabolism. Overall, these results suggest that piperine, a major component of black pepper, attenuates fat cell differentiation by down-regulating PPARγ activity as well as suppressing PPARγ expression, thus leading to potential treatment for obesity-related diseases.
Assuntos
Adipogenia/efeitos dos fármacos , Alcaloides/farmacologia , Benzodioxóis/farmacologia , Regulação para Baixo/efeitos dos fármacos , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Piper nigrum/química , Piperidinas/farmacologia , Extratos Vegetais/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Camundongos , PPAR gama/genéticaRESUMO
We purified phytoestrogens from Pueraria root (Pueraria mirifica from Thailand and Pueraria lobata from Korea), which is used as a rejuvenating folk medicine in Thailand and China. Dried, powdered plant material was extracted with 100% ethanol and further separated by concentration, filtration, and thin layer silica gel chromatography. Using the fractions obtained during separation, we first investigated their cytotoxicity in several cancer cell lines from various tissues. The ethanol-extracted components (PE1, PE4) had significant antiproliferative effects on breast cancer cell lines, including MCF-7, ZR-75-1, MDA-MB-231, SK-BR-3, and Hs578T. Second, we compared these results with the cytotoxic effects of known flavonoids, sterols, and coumarins from Pueraria root. The known compounds were not as effective, and occurred in a different polarity region on HPLC. Third, further separation resulted in the isolation of eight different components (Sub PE-A to -H). One of these, PE-D, affected the growth of some breast cancer cell lines (MCF-7, MDA- MB-231) in a dose- and time-dependent manner, as well as the growth of ovarian (2774) and cervical cancer cells (HeLa). Finally, a transfection assay showed that this component had an estrogenic effect similar to 17beta - estradiol, which activates both estrogen receptor alpha (ERalpha) and ERbeta. The NMR analysis determined that spinasterol (stigmasta-7, 22-dien-3beta-ol) is an active cytotoxic component of Pueraria root.
Assuntos
Antineoplásicos/farmacologia , Raízes de Plantas/química , Pueraria/química , Estigmasterol/análogos & derivados , Antineoplásicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Feminino , Humanos , Preparações de Plantas/uso terapêutico , Estigmasterol/isolamento & purificação , Estigmasterol/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
Thrombospondin-1 (TSP-1) level is tightly regulated at the transcriptional level. To determine the detailed molecular mechanisms of TSP-1 expression, nine serial 5'-deletion constructs of the human genomic tsp-1 promoter (nucleotides -2,220 to +756) were prepared, inserted into luciferase reporter plasmids, and transiently transfected into the Hep3B human hepatocarcinoma cell. Among the nine 5'-deletion constructs, pTSP-Luc-4 (-767 approximately +756) had consistently decreased luciferase activity with or without PMA stimulation, whereas a further truncated construct [pTSP-Luc-4' (-407 approximately +756)] had increased levels of expression. By searching the nucleotides from -767 to -407, a consensus binding sequence (5'-CCATTTT-3') for the repressor Yin Yang-1 (YY-1) at nucleotide -440 was identified. The suppression induced by this site was weakened in the presence of the region upstream of nucleotide -767 (pTSP-Luc-1 and -2). Nuclear protein directly bound to an oligonucleotide containing the repressive YY-1 sequence but the binding capacity of the sequence was decreased by the increased c-Jun levels. Moreover, proteins immunoprecipitated with anti-YY-1 revealed an interaction between c-Jun and YY-1 factor. These data suggest that the repressive YY-1 site of the tsp-1 promoter could not be functional via activating positive cis-elements on the upstream from this site and weakened via c-Jun/YY-1 interactions.