RESUMEN
Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.
Asunto(s)
Hepatitis B , Simportadores , Hepatitis B/tratamiento farmacológico , Virus de la Hepatitis B/fisiología , Hepatocitos/metabolismo , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/uso terapéutico , Simportadores/genética , Simportadores/metabolismo , Simportadores/uso terapéuticoRESUMEN
Toxicological approaches in screening drugs that cause drug-induced liver injury (DILI) are urgently needed to reduce the risk of developing DILI and avoid immense costs resulting from late-stage drug withdrawal from clinical trials. Cholestatic DILI is characterized by bile acid (BA) accumulation in hepatocytes, typically caused by drug-induced inhibition of important bile transporters, such as bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4). Therefore, NTCP expression is essential for construction of an in vitro hepatocellular toxicity evaluation system. Here, we investigated whether sandwich-cultured HepG2-hNTCP-C4 (SCHepG2-hNTCP-C4) cells were applicable for evaluation of cholestatic DILI. In SCHepG2-hNTCP-C4 cells, NTCP and MRP2/4 expression levels were comparable to those in human primary hepatocytes; however, BSEP expression was low. In addition, the substrates tauro-nor-THCA-24 DBD and CDF confirmed the functionality of NTCP and MRP2, respectively. When 22 known hepatotoxins were exposed to BAs to evaluate cholestatic DILI, cytotoxicity in SCHepG2-hNTCP-C4 cells was more frequent than that in SCHepG2 cells. Thus, SCHepG2-hNTCP-C4 cells may be useful preclinical screening tools to predict the risk of cholestatic DILI induced by drug candidates. However, further studies are needed to determine why the cholestatic cytotoxicity of some compounds would be still insufficient in SCHepG2-hNTCP-C4 cells.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Colestasis/inducido químicamente , Evaluación Preclínica de Medicamentos/métodos , Ácidos y Sales Biliares/toxicidad , Técnicas de Cultivo de Célula , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Colestasis/genética , Colesterol 7-alfa-Hidroxilasa/genética , Células Hep G2 , Humanos , Proteínas de Transporte de Membrana/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Receptores Citoplasmáticos y Nucleares/genética , Simportadores/genéticaRESUMEN
OBJECTIVE: To investigate the beneficial role of prebiotics on endothelial dysfunction, an early key marker of cardiovascular diseases, in an original mouse model linking steatosis and endothelial dysfunction. DESIGN: We examined the contribution of the gut microbiota to vascular dysfunction observed in apolipoprotein E knockout (Apoe-/-) mice fed an n-3 polyunsaturated fatty acid (PUFA)-depleted diet for 12â weeks with or without inulin-type fructans (ITFs) supplementation for the last 15â days. Mesenteric and carotid arteries were isolated to evaluate endothelium-dependent relaxation ex vivo. Caecal microbiota composition (Illumina Sequencing of the 16S rRNA gene) and key pathways/mediators involved in the control of vascular function, including bile acid (BA) profiling, gut and liver key gene expression, nitric oxide and gut hormones production were also assessed. RESULTS: ITF supplementation totally reverses endothelial dysfunction in mesenteric and carotid arteries of n-3 PUFA-depleted Apoe-/- mice via activation of the nitric oxide (NO) synthase/NO pathway. Gut microbiota changes induced by prebiotic treatment consist in increased NO-producing bacteria, replenishment of abundance in Akkermansia and decreased abundance in bacterial taxa involved in secondary BA synthesis. Changes in gut and liver gene expression also occur upon ITFs suggesting increased glucagon-like peptide 1 production and BA turnover as drivers of endothelium function preservation. CONCLUSIONS: We demonstrate for the first time that ITF improve endothelial dysfunction, implicating a short-term adaptation of both gut microbiota and key gut peptides. If confirmed in humans, prebiotics could be proposed as a novel approach in the prevention of metabolic disorders-related cardiovascular diseases.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Fructanos/farmacología , Microbioma Gastrointestinal/efectos de los fármacos , Prebióticos , Aminopeptidasas/genética , Animales , Péptidos Catiónicos Antimicrobianos/genética , Bacterias/efectos de los fármacos , Ácidos y Sales Biliares/biosíntesis , Ácidos y Sales Biliares/sangre , Arterias Carótidas/fisiología , Ciego/microbiología , Suplementos Dietéticos , Modelos Animales de Enfermedad , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Omega-3/deficiencia , Expresión Génica/efectos de los fármacos , Péptido 1 Similar al Glucagón/biosíntesis , Masculino , Arterias Mesentéricas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Neurotensina/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Proglucagón/genética , Simportadores/genética , VasodilataciónRESUMEN
Angiopoietin-like 4 (ANGPTL4) raises plasma triglyceride levels by inhibiting lipoprotein lipase. A set of compounds that are able to reduce plasma triglyceride levels are bile acids (BA). Because BA have been shown to decrease ANGPTL4 secretion by intestinal cells, we hypothesized that BA lower plasma triglycerides (partly) via ANGPTL4. To test that hypothesis, wild-type and Angptl4-/- mice were fed chow supplemented with taurocholic acid (TCA) for seven days. TCA supplementation effectively lowered plasma triglycerides in wild-type and Angptl4-/- mice, indicating that ANGPTL4 is not required for plasma triglyceride-lowering by BA. Intriguingly, however, plasma and hepatic BA concentrations were significantly lower in TCA-supplemented Angptl4-/- mice than in TCA-supplemented wild-type mice. These changes in the Angptl4-/- mice were accompanied by lower BA levels in ileal scrapings and decreased expression of FXR-target genes in the ileum, including the BA transporter Slc10a2. By contrast, faecal excretion of specifically primary BA was higher in the Angptl4-/- mice, suggesting that loss of ANGPTL4 impairs intestinal BA absorption. Since the gut microbiota converts primary BA into secondary BA, elevated excretion of primary BA in Angptl4-/- mice may reflect differences in gut microbial composition and/or functionality. Indeed, colonic microbial composition was markedly different between Angptl4-/- and wild-type mice. Suppression of the gut bacteria using antibiotics abolished differences in plasma, hepatic, and faecal BA levels between TCA-supplemented Angptl4-/- and wild-type mice. In conclusion, 1) ANGPTL4 is not involved in the triglyceride-lowering effect of BA; 2) ANGPTL4 promotes BA absorption during TCA supplementation via a mechanism dependent on the gut microbiota.
Asunto(s)
Proteína 4 Similar a la Angiopoyetina/metabolismo , Ácidos y Sales Biliares/metabolismo , Suplementos Dietéticos , Microbioma Gastrointestinal/fisiología , Absorción Intestinal/efectos de los fármacos , Ácido Taurocólico , Proteína 4 Similar a la Angiopoyetina/genética , Animales , Ácidos y Sales Biliares/genética , Absorción Intestinal/genética , Ratones , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/farmacocinética , Ácido Taurocólico/farmacología , Triglicéridos/sangreRESUMEN
Dioscorea bulbifera L. (DB) is a traditional Chinese herb used in thyroid disease and cancer. However, the clinical use of DB remains a challenge due to its hepatotoxicity, which is caused, in part, by the presence of Diosbulbin B (DIOB), a toxin commonly found in DB extracts. As abnormal expression of hepatobiliary transporters plays an important role in drug-induced liver injury, we assessed the hepatotoxicity induced by DB and DIOB, and explored their impacts on hepatobiliary transporter expression levels. Following liquid chromatography-tandem mass analysis of the DIOB content of DB extract, male ICR mice were randomly orally administered DB or DIOB for 14days. Liver injury was assessed by histopathological and biochemical analysis of liver fuction. The levels of transporter protein and mRNA were determined by western blotting and real-time PCR. Liver function and histopathological analysis indicated that both DB and DIOB could induce liver injury in mice, and that DIOB might be the primary toxic compound in DB. Moreover, down-regulation of Mrp2 blocked the excretion of bilirubin, glutathione disulfide, and bile acids, leading to the accumulation of toxic substrates in the liver and a redox imbalance. We identified down-regulated expression of Mrp2 as potential factors linked to increased serum bilirubin levels and decreased levels of glutathione in the liver and increased liver injury severity. In summary, our study indicates that down-regulation of Mrp2 represents the primary mechanism of DB- and DIOB-induced hepatotoxicity, and provides insight into novel therapies that could be used to prevent DB- and DIOB-mediated liver injury.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Dioscorea/química , Medicamentos Herbarios Chinos/toxicidad , Compuestos Heterocíclicos de 4 o más Anillos/toxicidad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Simportadores/metabolismo , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Medicamentos Herbarios Chinos/aislamiento & purificación , Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/aislamiento & purificación , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos ICR , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Proteínas de Transporte de Catión Orgánico/genética , Simportadores/genéticaRESUMEN
Sodium-taurocholate co-transporting polypeptide (Ntcp/NTCP) is the major uptake transporter of bile salts in mouse and human livers. In certain diseases, including endotoxemia, cholestasis, diabetes, and hepatocarcinoma, Ntcp/NTCP expression is markedly reduced, which interferes with enterohepatic circulation of bile salts, impairing the absorption of lipophilic compounds. Therefore, normal Ntcp/NTCP expression in the liver is physiologically important. Berberine is an herbal medicine used historically to improve liver function and has recently been shown to repress STAT signaling. However, berberine effects on Ntcp/NTCP expression are unknown, prompting use to investigate this possible connection. Our results showed that berberine dose-dependently increased Ntcp expression in male mouse liver and decreased taurocholic acid levels in serum but increased them in the liver. In mouse and human hepatoma cells, berberine induced Ntcp/NTCP mRNA and protein expression and increased cellular uptake of [3H] taurocholate. Mechanistically, berberine decreased nuclear protein levels of phospho-JAK2 and phospho-STAT5, thus disrupting the JAK2-STAT5 signaling. Moreover, berberine stimulated luciferase reporter expression from the mouse Ntcp promoter when one putative STAT5 response element (RE) (-1137 bp) was deleted and from the human NTCP promoter when three putative STAT5REs (-2898, -2164, and -691 bp) were deleted. Chromatin immunoprecipitation demonstrated that berberine decreased binding of phospho-STAT5 protein to the-2164 and -691 bp STAT5REs in the human NTCP promoter. In summary, berberine-disrupted STAT5 signaling promoted mouse and human Ntcp/NTCP expression, resulting in enhanced bile acid uptake. Therefore, berberine may be a therapeutic candidate compound for maintaining bile acid homeostasis.
Asunto(s)
Berberina/farmacología , Hígado/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Simportadores/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Línea Celular , Vesícula Biliar/efectos de los fármacos , Vesícula Biliar/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Transportadores de Anión Orgánico Sodio-Dependiente/análisis , Transportadores de Anión Orgánico Sodio-Dependiente/genética , ARN Mensajero/genética , Simportadores/análisis , Simportadores/genética , Ácido Taurocólico/metabolismoRESUMEN
CONTEXT: Drug-induced liver injury (DILI) is associated with altering expression of hepatobiliary membrane transporters. Monoammonium glycyrrhizin (MAG) is commonly used for hepatic protection and may have a correlation with the inhibition effect of multidrug resistance-associated protein 2 (Mrp2). OBJECTIVE: This study evaluates the dynamic protective effect of MAG on rifampicin (RIF)- and isoniazid (INH)-induced hepatotoxicity in rats. MATERIALS AND METHODS: Male Wistar rats were randomly divided into four groups of 15 rats. Liver injury was induced by co-treatment with RIF (60 mg/kg) and INH (60 mg/kg) by gavage administration; MAG was orally pretreated at the doses of 45 or 90 mg/kg 3 h before RIF and INH. Rats in each group were sacrificed at 7, 14, and 21 d time points after drug administration. RESULTS: Liver function, histopathological analysis, and oxidative stress factors were significantly altered in each group. The expression of Mrp2 was significantly increased 230, 760, and 990% at 7, 14, and 21 time points, respectively, in RIF- and INH-treated rats. Compared with the RIF and INH groups, Mrp2 was reduced and Ntcp was significantly elevated by 180, 140, and 160% in the MAG high-dose group at the three time points, respectively. The immunoreaction intensity of Oatp1a4 was increased 170, 190, and 370% in the MAG low-dose group and 160, 290, and 420% in the MAG high-dose group at the three time points, respectively, compared with the RIF and INH groups. DISCUSSION AND CONCLUSION: These results indicated that MAG has a protective effects against RIF- and INH-induced hepatotoxicity. The underlying mechanism may have correlation with its effect on regulating the expression of hepatobiliary membrane transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Antituberculosos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Regulación de la Expresión Génica/efectos de los fármacos , Ácido Glicirrínico/uso terapéutico , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico/genética , Simportadores/genética , Administración Oral , Animales , Antituberculosos/administración & dosificación , Antituberculosos/farmacocinética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Ácido Glicirrínico/administración & dosificación , Isoniazida/administración & dosificación , Isoniazida/farmacocinética , Isoniazida/toxicidad , Masculino , Ratas Wistar , Rifampin/administración & dosificación , Rifampin/farmacocinética , Rifampin/toxicidadRESUMEN
The human sodium taurocholate cotransporting polypeptide (NTCP) is a hepatic bile acid transporter. Inhibition of NTCP uptake may potentially also prevent hepatitis B virus (HBV) infection. The first objective was to develop a quantitative pharmacophore for NTCP inhibition. Recent studies showed that hepatotoxic drugs could inhibit bile acid uptake into hepatocytes, without inhibiting canalicular efflux, and cause bile acid elevation in plasma. Hence, a second objective was to examine whether NTCP inhibition is associated with drug induced liver injury (DILI). Twenty-seven drugs from our previous study were used as the training set to develop a quantitative pharmacophore. From secondary screening from a drug database, six retrieved drugs and three drugs not retrieved by the model were tested for NTCP inhibition. Tertiary screening involved drugs known to cause DILI and not cause DILI. Overall, ninety-four drugs were assessed for hepatotoxicity and were assessed relative to NTCP inhibition. The quantitative pharmacophore possessed one hydrogen bond acceptor, one hydrogen bond donor, a hydrophobic feature, and excluded volumes. From 94 drugs, NTCP inhibitors and non-inhibitors were approximately equally distributed across the drugs of most DILI concern, less DILI concern, and no DILI concern, indicating no relationship between NTCP inhibition and DILI risk. Hence, an approach to treat HBV via NTCP inhibition is not expected to be associated with DILI.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/antagonistas & inhibidores , Simportadores/metabolismo , Biología Computacional , Bases de Datos Factuales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Conformación Proteica , Relación Estructura-Actividad , Simportadores/genéticaRESUMEN
BACKGROUND/AIMS: During a single pass through the kidneys, more than 80% of glutathione (GSH) is excreted, indicating not only glomerular filtration, but also tubular secretion. The first step in tubular secretion is the uptake of a substance across the basolateral membrane of proximal tubule cells by sodium-dependent and -independent transporters. Due to the dicarboxylate-like structure, we postulated that GSH uptake across the basolateral membrane is mediated by the sodium-dependent dicarboxylate transporter 3 (NaDC3). METHODS: Tracer uptake and electrophysiologic measurements using a two-electrode voltage clamp device were performed in Xenopus laevis oocytes expressing the human (h)NaDC3. RESULTS: Uptake of succinate, the reference substrate of hNaDC3, was inhibited by GSH in a dose-dependent manner with an IC50 of 1.88 mM. GSH evoked potential-dependent inward currents, which were abolished under sodium-free conditions. At -60 mV, GSH currents showed saturation kinetics with a KM of 1.65 mM. CONCLUSION: hNaDC3 present at the basolateral membrane of proximal tubule cells mediates sodium-dependent GSH uptake. The kinetic data show that NaDC3 is a low-affinity GSH transporter.
Asunto(s)
Glutatión/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Ácido Succínico/metabolismo , Simportadores/metabolismo , Animales , Humanos , Túbulos Renales Proximales/metabolismo , Hígado/metabolismo , Oocitos/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , ARN Complementario/genética , Simportadores/genética , Xenopus laevis/genéticaRESUMEN
To investigate the hypocholesterolemic mechanism of barley in vivo, six-week-old C57BL/6J mice were fed a high-fat diet (HFD) or high-fat diet containing barley (HFD-B) for seven weeks. Total and LDL cholesterol concentrations were significantly reduced in the HFD-B group while fecal cholesterol and bile acid was increased. Real-time PCR and immunoblot analysis revealed the induction of FXR expression, which in turn suppressed the expression of ASBT and NPC1L1 in the HFD-B group compared with the controls. In the liver, the expression of HMG-CoA reductase was significantly reduced while LDL receptor expression was unaltered in the HFD-B group compared with the controls. Our data suggest that the hypocholesterolemic effects of barley are primarily the result of reduced dietary cholesterol uptake and bile acid resorption. Reduced expression of intestinal ASBT and NPC1L1 may play a key role in the regulation of dietary cholesterol and bile acid metabolism in mice consuming a diet containing barley.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Regulación hacia Abajo , Hordeum/química , Hipercolesterolemia/dietoterapia , Mucosa Intestinal/metabolismo , Proteínas de Transporte de Membrana/genética , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Preparaciones de Plantas/administración & dosificación , Simportadores/genética , Animales , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismoRESUMEN
The sodium-dependent vitamin C transporter (SVCT) 2 is crucial for ascorbate uptake in metabolically active and specialized tissues. This study focused on the gene regulation of SVCT2 exon 1b, which is ubiquitously expressed in human and mouse tissues. Although the human SVCT2 exon 1b promoter does not contain a classical TATA box, we found that it does contain a functional initiator that binds Yin Yang-1 (YY1) and interacts with upstream Sp1/Sp3 elements in the proximal promoter region. These elements in turn play a critical role in regulating YY1-mediated transcription of exon 1b. Formation of YY1/Sp complexes on the promoter is required for its optional function. YY1 with Sp1 or Sp3 synergistically enhanced exon 1b promoter activity as well as the endogenous SVCT2 protein expression. Further, in addition to Sp1/Sp3, both EGR-1 and EGR-2 were detected in the protein complexes that bound the three GC boxes bearing overlapping binding sites for EGR/WT1 and Sp1/3. The EGR family factors WT1 and MAZ were found to differentially regulate exon 1b promoter activity. These results show that differential occupancy of transcription factors on the GC-rich consensus sequences in the SVCT2 exon 1b promoter contributes to the regulation of cell and tissue expression of SVCT2.
Asunto(s)
Regulación de la Expresión Génica , Transportadores de Anión Orgánico Sodio-Dependiente , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Simportadores , Transcripción Genética , Factor de Transcripción YY1/metabolismo , Animales , Ácido Ascórbico/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Inmunoprecipitación de Cromatina , Drosophila melanogaster , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción de la Respuesta de Crecimiento Precoz/metabolismo , Exones , Secuencia Rica en GC , Humanos , Datos de Secuencia Molecular , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportadores de Sodio Acoplados a la Vitamina C , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/genética , Simportadores/genética , Simportadores/metabolismo , Transfección , Células Tumorales Cultivadas , Factor de Transcripción YY1/genética , Dedos de ZincRESUMEN
Ascorbic acid, the active form of vitamin C, is a vital antioxidant in the human liver, yet the molecular mechanisms involved in the regulation of ascorbic acid transporters [human sodium-dependent vitamin C transporters (hSVCT) 1 and 2] in liver cells are poorly understood. Therefore, we characterized the minimal promoter regions of hSVCT1 and 2 in cultured human liver epithelial cells (HepG2) and examined the effects of ascorbic acid deprivation and supplementation on activity and regulation of the transport systems. Identified minimal promoters required for basal activity were found to include multiple cis regulatory elements, whereas mutational analysis demonstrated that HNF-1 sites in the hSVCT1 promoter and KLF/Sp1 sites in the hSVCT2 promoter were essential for activities. When cultured in ascorbic acid deficient or supplemented media, HepG2 cells demonstrated significant (P<.01) and specific reciprocal changes in [(14)C]-Ascorbic acid uptake, and in hSVCT1 mRNA and protein levels as well as hSVCT1 promoter activity. However, no significant changes in hSVCT2 expression or promoter activity were observed during ascorbic acid deficient or supplemented conditions. We mapped the ascorbic acid responsive region in the hSVCT1 promoter and determined that HNF-1 sites are important for the adaptive regulation response. The results of these studies further characterize the hSVCT1 and 2 promoters establish that ascorbic acid uptake by human liver epithelial cells is adaptively regulated and show that transcriptional mechanisms via HNF-1 in the hSVCT1 promoter may, in part, be involved in this regulation.
Asunto(s)
Transportadores de Anión Orgánico Sodio-Dependiente/genética , Simportadores/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacología , Deficiencia de Ácido Ascórbico/fisiopatología , Regulación de la Expresión Génica , Células Hep G2 , Factor Nuclear 1 del Hepatocito/fisiología , Humanos , Regiones Promotoras Genéticas/fisiología , Transportadores de Sodio Acoplados a la Vitamina CRESUMEN
OBJECTIVE: To assess the role of combined deficiencies of vitamins C and E on the earliest stages of atherosclerosis (an inflammatory condition associated with oxidative stress), 4 combinations of vitamin supplementation (low C/low E, low C/high E, high C/low E, and high C/high E) were studied in atherosclerosis-prone apolipoprotein E-deficient mice also unable to synthesize their own vitamin C (gulonolactone oxidase(-/-)); and to evaluate the effect of a more severe depletion of vitamin C alone in a second experiment using gulonolactone oxidase(-/-) mice carrying the hemizygous deletion of SVCT2 (the vitamin C transporter). METHODS AND RESULTS: After 8 weeks of a high-fat diet (16% lard and 0.2% cholesterol), atherosclerosis developed in the aortic sinus areas of mice in all diet groups. Each vitamin-deficient diet significantly decreased liver and brain contents of the corresponding vitamin. Combined deficiency of both vitamins increased lipid peroxidation, doubled plaque size, and increased plaque macrophage content by 2- to 3-fold in male mice, although only plaque macrophage content was increased in female mice. A more severe deficiency of vitamin C in gulonolactone oxidase(-/-) mice with defective cellular uptake of vitamin C increased both oxidative stress and atherosclerosis in apolipoprotein E(-/-) mice compared with littermates receiving a diet replete in vitamin C, again most clearly in males. CONCLUSIONS: Combined deficiencies of vitamins E and C are required to worsen early atherosclerosis in an apolipoprotein E-deficient mouse model. However, a more severe cellular deficiency of vitamin C alone promotes atherosclerosis when vitamin E is replete.
Asunto(s)
Enfermedades de la Aorta/etiología , Apolipoproteínas E/deficiencia , Deficiencia de Ácido Ascórbico/complicaciones , Aterosclerosis/etiología , Deficiencia de Vitamina E/complicaciones , Animales , Enfermedades de la Aorta/tratamiento farmacológico , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacología , Deficiencia de Ácido Ascórbico/tratamiento farmacológico , Deficiencia de Ácido Ascórbico/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Encéfalo/metabolismo , Suplementos Dietéticos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , L-Gulonolactona Oxidasa/deficiencia , L-Gulonolactona Oxidasa/genética , Peroxidación de Lípido , Hígado/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/deficiencia , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Estrés Oxidativo , Índice de Severidad de la Enfermedad , Factores Sexuales , Transportadores de Sodio Acoplados a la Vitamina C , Simportadores/deficiencia , Simportadores/genética , Factores de Tiempo , Vitamina E/metabolismo , Vitamina E/farmacología , Deficiencia de Vitamina E/tratamiento farmacológico , Deficiencia de Vitamina E/metabolismo , Vitaminas/metabolismo , Vitaminas/farmacologíaRESUMEN
Levels of the necessary nutrient vitamin C (ascorbate) are tightly regulated by intestinal absorption, tissue accumulation, and renal reabsorption and excretion. Ascorbate levels are controlled in part by regulation of transport through at least 2 sodium-dependent transporters: Slc23a1 and Slc23a2 (also known as Svct1 and Svct2, respectively). Previous work indicates that Slc23a2 is essential for viability in mice, but the roles of Slc23a1 for viability and in adult physiology have not been determined. To investigate the contributions of Slc23a1 to plasma and tissue ascorbate concentrations in vivo, we generated Slc23a1-/- mice. Compared with wild-type mice, Slc23a1-/- mice increased ascorbate fractional excretion up to 18-fold. Hepatic portal ascorbate accumulation was nearly abolished, whereas intestinal absorption was marginally affected. Both heterozygous and knockout pups born to Slc23a1-/- dams exhibited approximately 45% perinatal mortality, and this was associated with lower plasma ascorbate concentrations in dams and pups. Perinatal mortality of Slc23a1-/- pups born to Slc23a1-/- dams was prevented by ascorbate supplementation during pregnancy. Taken together, these data indicate that ascorbate provided by the dam influenced perinatal survival. Although Slc23a1-/- mice lost as much as 70% of their ascorbate body stores in urine daily, we observed an unanticipated compensatory increase in ascorbate synthesis. These findings indicate a key role for Slc23a1 in renal ascorbate absorption and perinatal survival and reveal regulation of vitamin C biosynthesis in mice.
Asunto(s)
Ácido Ascórbico/metabolismo , Riñón/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/fisiología , Mortalidad Perinatal , Simportadores/fisiología , Absorción , Animales , Animales Recién Nacidos , Ácido Ascórbico/administración & dosificación , Femenino , Intestino Delgado/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Polimorfismo de Nucleótido Simple , Embarazo , Transportadores de Sodio Acoplados a la Vitamina C , Simportadores/genéticaRESUMEN
Green tea catechins exhibit hypocholesterolemic effects probably via their inhibitory effects on intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) is responsible for reabsorption of bile acids. The present studies were, therefore, designed to investigate the modulation of ASBT function and membrane expression by green tea catechins in human embryonic kidney HEK-293 cells stably transfected with ASBT-V5 fusion protein and intestinal Caco-2 monolayers. Our data showed that ASBT activity was significantly decreased by (-)-epigallocatechin-3-gallate (EGCG) but not other green tea catechins. Inhibition of PKC, phosphatidylinositol 3-kinase, and MAPK-dependent pathways failed to block the reduction in ASBT activity by EGCG. Kinetics studies showed a significant decrease in the V(max) of the transporter, whereas total ASBT content on the plasma membrane was unaltered by EGCG. Concomitant with the decrease in ASBT function, EGCG significantly reduced ASBT pool in the detergent-insoluble fraction, while increasing its presence in the detergent-soluble fraction of plasma membrane. Furthermore, EGCG decreased the association of ASBT with floating lipid raft fractions of cellular membrane on Optiprep density gradient. In conclusion, our data demonstrate a novel role of lipid rafts in the modulation of ASBT function by the dietary component EGCG, which may underlie the hypocholesterolemic effects of green tea.
Asunto(s)
Catequina/análogos & derivados , Íleon/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Simportadores/antagonistas & inhibidores , Té/química , Transporte Biológico/efectos de los fármacos , Biotinilación , Células CACO-2 , Catequina/farmacología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Íleon/citología , Íleon/efectos de los fármacos , Cinética , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Octoxinol/farmacología , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/metabolismo , TransfecciónRESUMEN
The human apical sodium-dependent bile acid transporter (ASBT; SLC10A2) is the primary mechanism for intestinal bile acid reabsorption. In the colon, secondary bile acids increase the risk of cancer. Therefore, drugs that inhibit ASBT have the potential to increase the risk of colon cancer. The objectives of this study were to identify FDA-approved drugs that inhibit ASBT and to derive computational models for ASBT inhibition. Inhibition was evaluated using ASBT-MDCK monolayers and taurocholate as the model substrate. Computational modeling employed a HipHop qualitative approach, a Hypogen quantitative approach, and a modified Laplacian Bayesian modeling method using 2D descriptors. Initially, 30 compounds were screened for ASBT inhibition. A qualitative pharmacophore was developed using the most potent 11 compounds and applied to search a drug database, yielding 58 hits. Additional compounds were tested, and their K(i) values were measured. A 3D-QSAR and a Bayesian model were developed using 38 molecules. The quantitative pharmacophore consisted of one hydrogen bond acceptor, three hydrophobic features, and five excluded volumes. Each model was further validated with two external test sets of 30 and 19 molecules. Validation analysis showed both models exhibited good predictability in determining whether a drug is a potent or nonpotent ASBT inhibitor. The Bayesian model correctly ranked the most active compounds. In summary, using a combined in vitro and computational approach, we found that many FDA-approved drugs from diverse classes, such as the dihydropyridine calcium channel blockers and HMG CoA-reductase inhibitors, are ASBT inhibitors.
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Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Simportadores/antagonistas & inhibidores , Animales , Inteligencia Artificial , Teorema de Bayes , Ácidos y Sales Biliares/metabolismo , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/toxicidad , Línea Celular , Neoplasias del Colon/etiología , Diuréticos/química , Diuréticos/toxicidad , Perros , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/toxicidad , Técnicas In Vitro , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Modelos Biológicos , Modelos Moleculares , Modelos Estadísticos , Transportadores de Anión Orgánico Sodio-Dependiente/química , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Relación Estructura-Actividad Cuantitativa , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Factores de Riesgo , Simportadores/química , Simportadores/genética , TransfecciónRESUMEN
BACKGROUND/AIMS: Vitamin C transporter proteins SVCT1 and SVCT2 are required for the absorption and transport of vitamin C in humans. This study aims to determine whether common SVCT genotypes modify the association between dietary vitamin C and serum ascorbic acid. METHODS: Non-smoking men and women (n=1,046) aged 20-29 were participants of the Toronto Nutrigenomics and Health Study. Overnight fasting blood samples were collected to determine serum ascorbic acid concentrations by HPLC and to genotype for two SVCT1 (rs4257763 and rs6596473) and two SVCT2 (rs6139591 and rs2681116) polymorphisms. RESULTS: No diet-gene interactions were observed for the vitamin C transporter polymorphisms, however, the average (mean+/-SE) serum ascorbic acid concentrations differed between rs4257763 genotypes (GG: 24.4+/-1.3, GA: 26.8+/-1.1, AA: 29.7+/-1.4 micromol/l; p=0.002). For this polymorphism, the correlation between dietary vitamin C and serum ascorbic acid was only significant in subjects with a G allele. The SVCT2 polymorphisms also appeared to modify the strength of the diet-serum correlation. CONCLUSIONS: Our findings demonstrate that genetic variation in SVCT1 can influence serum ascorbic acid concentrations and that SVCT1 and SVCT2 genotypes modify the strength of the correlation between dietary vitamin C and serum ascorbic acid.
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Ácido Ascórbico/sangre , Ácido Ascórbico/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Polimorfismo Genético , Simportadores/genética , Adulto , Presión Sanguínea , HDL-Colesterol/metabolismo , Estudios Transversales , Suplementos Dietéticos , Femenino , Genotipo , Humanos , Masculino , Estaciones del Año , Transportadores de Sodio Acoplados a la Vitamina C , Adulto Joven , alfa-Tocoferol/sangreRESUMEN
Vectorial transport of bile acids across hepatocytes is a major driving force for bile flow, and bile acid retention in the liver causes hepatotoxicity. The basolateral and apical transporters for bile acids are thought to be targets of drugs that induce cholestasis. Previously, we constructed polarized LLC-PK1 cells that express both a major bile acid uptake transporter human Na+/taurocholate cotransporting polypeptide (SLC10A1) (NTCP) and the bile acid efflux transporter human bile salt export pump (ABCB 11) (BSEP) and showed that monolayers of such cells can be used to characterize vectorial transcellular transport of bile acids. In the present study, we investigated whether cholestasis-inducing drugs could inhibit bile acid transport in such cells. Because fluorescent substrates allow the development of a high-throughput screening method, we examined the transport by NTCP and BSEP of fluorescent bile acids as well as taurocholate. The aminofluorescein-tagged bile acids, chenodeoxycholylglycylamidofluorescein and cholylglycylamidofluorescein, were substrates of both NTCP and BSEP, and their basal-to-apical transport rates across coexpressing cell monolayers were 4.3 to 4.5 times those of the vector control, although smaller than for taurocholate. The well known cholestatic drugs, rifampicin, rifamycin SV, glibenclamide, and cyclosporin A, reduced the basal-to-apical transport and the apical efflux clearance of taurocholate across NTCP- and BSEP-coexpressing cell monolayers. Further analysis indicated that the drugs inhibited both NTCP and BSEP. Our study suggests that such coexpressing cells can provide a useful system for the identification of inhibitors of these two transport systems, including potential drug candidates.
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Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Ciclosporina/toxicidad , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Rifampin/toxicidad , Rifamicinas/toxicidad , Simportadores/antagonistas & inhibidores , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Colestasis/etiología , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Fluoresceínas/metabolismo , Cinética , Células LLC-PK1 , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Oxadiazoles/metabolismo , Porcinos , Simportadores/genética , Simportadores/metabolismo , Ácido Taurocólico/análogos & derivados , Ácido Taurocólico/metabolismo , TransfecciónRESUMEN
Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium-vitamin C cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high-affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.
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Ácido Ascórbico/metabolismo , Epéndimo/metabolismo , Hipotálamo/metabolismo , Neuroglía/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Animales , Ácido Ascórbico/farmacocinética , Transporte Biológico Activo/fisiología , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Líquido Cefalorraquídeo/metabolismo , Citoprotección/fisiología , Epéndimo/ultraestructura , Hipotálamo/ultraestructura , Hibridación in Situ , Cinética , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Neuroglía/ultraestructura , Neuronas/citología , Neuronas/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Isoformas de Proteínas/fisiología , ARN Mensajero/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C , Simportadores/genética , Tercer Ventrículo/metabolismo , Tercer Ventrículo/ultraestructuraRESUMEN
AIM: We have previously demonstrated that cholangiocytes, the epithelial cells lining intrahepatic bile ducts, encode two functional bile acid transporters via alternative splicing of a single gene to facilitate bile acid vectorial transport. Cholangiocytes possess ASBT, an apical sodium-dependent bile acid transporter to take up bile acids, and t-ASBT, a basolateral alternatively spliced and truncated form of ASBT to efflux bile acids. Though hepatocyte and ileal bile acid transporters are in part regulated by the flux of bile acids, the effect of alterations in bile acid flux on the expression of t-ASBT in terminal ileocytes remains unclear. Thus, we tested the hypothesis that expression of ASBT and t-ASBT in cholangiocytes and ileocytes was regulated by bile acid flux. METHODS: Expression of ASBT and t-ASBT message and protein in cholangiocytes and ileocytes isolated from pair-fed rats given control (C) and 1% taurocholate (TCA) or 5% cholestyramine (CY) enriched diets, were assessed by both quantitative RNase protection assays and quantitative immunoblotting. The data obtained from each of the control groups were pooled to reflect the changes observed following TCA and CY treatments with respect to the control diets. Cholangiocyte taurocholate uptake was determined using a novel microperfusion technique on intrahepatic bile duct units (IBDUs) derived from C, TCA and CY fed rats. RESULTS: In cholangiocytes, both ASBT and t-ASBT message RNA and protein were significantly decreased in response to TCA feeding compared to C diet. In contrast, message and protein of both bile acid transporters significantly increased following CY feeding compared to C diet. In the ileum, TCA feeding significantly up-regulated both ASBT and t-ASBT message and protein compared to C diet, while CY feeding significantly down-regulated message and protein of both bile acid transporters compared to C diet. As anticipated from alterations in cholangiocyte ASBT expression, the uptake of taurocholate in microperfused IBDUs derived from rats on TCA diet decreased 2.7-fold, whereas it increased 1.7-fold in those on CY diet compared to C diet fed groups. CONCLUSION: These data demonstrate that expression of ASBT and t-ASBT in cholangiocytes is regulated by a negative feedback loop while the expression of these transporters in terminal ileum is modified via positive feedback. Thus, while transcriptional regulatory mechanisms in response to alterations in bile acid pool size are operative in both cholangiocytes and ileocytes, each cell type responds differently to bile acid supplementation and depletion.