RESUMEN
Pioglitazone is an insulin-sensitizing thiazolidinedione (TZD) whose use is associated with bone loss. We examined the effects of pioglitazone on components of the Wnt signaling pathway (Wnt1, ß-catenin) and markers of bone mineralization [osteoprotegerin (OPG), bone sialoprotein (BSP), fibroblast growth factor (FGF)23] as well as mineral content in human osteoblast hFOB 1.19 cells. hFOB 1.19 cells were cultured in K12/DMD medium with or without pioglitazone. PPARγ Wnt1, OPG, BSP, or FGF23 mRNA expression was measured using qRT-PCR; ß-catenin, OPG, BSP, or FGF23 using ELISA; and calcium or phosphate content using colorimetry. Treatment with pioglitazone resulted in increased expression of PPARγ mRNA in hFOB 1.19 osteoblasts. Pioglitazone decreased Wnt1 mRNA levels and suppressed components of Wnt signaling pathway as evidenced by a decrease in ß-catenin gene expression and secretion as well as ß-catenin specific activity. The expression and the activity of OPG, BSP, and FGF23 were also reduced by pioglitazone together with total (but not specific) calcium and phosphate content. Pioglitazone affects Wnt1 signaling pathway and mineral matrix regulation components in human osteoblasts.
Asunto(s)
Biomarcadores/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Tiazolidinedionas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Calcificación Fisiológica/genética , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Feto/citología , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Sialoproteína de Unión a Integrina/metabolismo , Minerales/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoprotegerina/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Fosfatos/metabolismo , Pioglitazona , ARN Mensajero/genética , ARN Mensajero/metabolismo , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
A highly sensitive HPLC method for the determination of decarboxylated S-adenosylmethionine (dc-SAM) by fluorometric detection was developed. The reaction of dc-SAM and its analogs with chloroacetaldehyde leads to the corresponding 1,N6-etheno derivatives. These highly fluorescent derivatives were fully characterized through their proton nuclear magnetic resonance spectra and/or mass spectra. This derivatization procedure has been applied to the analysis of dc-SAM in rat and human urine. After a simple cation exchange column prepurification, the urine extracts were derivatized with chloroacetaldehyde and analyzed by reversed-phase HPLC with fluorometric detection. The method allowed the determination of subpicomole amounts of dc-SAM and was shown to be highly reproducible with the use of decarboxylated S-adenosylethionine as internal standard. The application of the method to the analysis of urine of rats treated with MDL 72175, a potent ornithine decarboxylase inhibitor, showed that the dc-SAM levels increased in a dose-related fashion.
Asunto(s)
S-Adenosilmetionina/análogos & derivados , Alquinos , Animales , Fenómenos Químicos , Química , Cromatografía Líquida de Alta Presión/métodos , Descarboxilación , Diaminas/farmacología , Humanos , Masculino , Inhibidores de la Ornitina Descarboxilasa , Ratas , S-Adenosilmetionina/orina , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Treatment with ornithine decarboxylase inhibitors leads to a marked increase of decarboxylated S-adenosylmethionine (dc-SAM) in various tissues, accompanied by the concomitant formation of a metabolite of dc-SAM. This metabolite has been isolated from rat prostate samples by a combination of chromatographic procedures. The use of IH-NMR and of fast atom bombardment mass spectometry and the synthesis of an authentic sample allowed the unambiguous characterization of this unknown compound as the N-acetyl derivative of dc-SAM. A reverse-phase high performance liquid chromatography procedure was developed for the separation of dc-SAM and its N-acetyl derivative into their diastereomers resulting from the chiral sulfonium group.
