RESUMEN
BACKGROUND AND AIMS: Faecal bile acid elimination greatly contributes to cholesterol homeostasis. Synthesised from cholesterol in the liver, bile acids are actively reclaimed in the ileum by the apical sodium dependent bile acid transporter (ASBT). Although the expression level of ASBT affects body cholesterol balance, the impact of cholesterol on ASBT gene expression remains unclear. In this study, the effect of cholesterol on ASBT expression and ileal bile acid uptake was explored in vivo and in vitro. METHODS: ASBT gene expression was assessed by real time quantitative polymerase chain reaction and northern or western blotting, or both, in mice subjected to a 2% cholesterol diet for two weeks, in mouse ileal explants, or in human enterocyte-like Caco-2 cells cultured in sterol enriched or depleted media. Bile acid uptake was determined by measuring [3H]-taurocholic acid influx into in situ isolated ileal loops from mice or into differentiated Caco-2 cells. Molecular analysis of mouse and human ASBT promoters was undertaken with reporter assays, site directed mutagenesis, and electrophoretic mobility shift assays. RESULTS: In mice, cholesterol enriched diet triggered a downregulation of ASBT expression (mRNA and protein), a fall in ileal bile acid uptake, and a rise in the faecal excretion of bile acids. This effect was direct as it was reproduced ex vivo using mouse ileal explants and in vitro in differentiated Caco-2 cells. CONCLUSIONS: This regulation, which involves an original partnership between SREBP-2 and HNF-1alpha transcription factors, affects ileal bile acid recycling and thus might participate in the maintenance of body cholesterol homeostasis.
Asunto(s)
Colesterol en la Dieta/farmacología , Regulación hacia Abajo/efectos de los fármacos , Transportadores de Anión Orgánico Sodio-Dependiente/biosíntesis , Simportadores/biosíntesis , Animales , Secuencia de Bases , Ácidos y Sales Biliares/metabolismo , Células CACO-2 , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Humanos , Íleon/metabolismo , Absorción Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Técnicas de Cultivo de Órganos , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/fisiología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/fisiología , Simportadores/genética , Simportadores/fisiología , TransfecciónRESUMEN
AIMS/HYPOTHESIS: Dietary supplementation with conjugated linoleic acids (CLA) has a fat-reducing effect in various species, but induces severe hyperinsulinaemia and hepatic steatosis in the mouse. This study aimed to determine the causes of the deleterious effects of CLA on insulin homeostasis. METHODS: The chronology of adipose and liver weight, hepatic triglyceride accumulation and selected blood parameters, including lipids, insulin, leptin and adiponectin, was determined in C57BL/6J female mice fed a 1% isomeric mixture of CLA for various periods of time ranging from 2 to 28 days. Insulin secretion was measured in 1-h static incubations of pancreatic islets, and pancreas morphometric parameters were determined in mice fed CLA for 28 days. RESULTS: Plasma levels of leptin and adiponectin sharply decreased after 2 days of CLA feeding, although adipose tissue mass only decreased after day 6. Hyperinsulinaemia developed at day 6 and consistently worsened up to day 28, in parallel with increases in hepatic lipid content. Islets from CLA-fed mice displayed three- to four-fold increased rates of glucose-stimulated insulin secretion, both in the absence and presence of isobutyl methylxanthine or carbachol. The increased insulin-releasing capacity of islets from CLA-fed mice was explained by an increase in beta cell mass and number. CONCLUSIONS/INTERPRETATION: The data suggest that CLA supplementation induces a profound reduction of leptinaemia and adiponectinaemia, followed by hyperinsulinaemia due to the increased secretory capacity of pancreatic islets, leading, in turn, to liver steatosis. These observations cast doubt on the safety of dietary supplements containing CLA.
Asunto(s)
Hiperinsulinismo/inducido químicamente , Péptidos y Proteínas de Señalización Intercelular/sangre , Islotes Pancreáticos/patología , Leptina/sangre , Ácidos Linoleicos Conjugados/efectos adversos , Adiponectina , Tejido Adiposo/anatomía & histología , Animales , Peso Corporal , Modelos Animales de Enfermedad , Femenino , Hiperplasia , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Hígado/anatomía & histología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Triglicéridos/metabolismoRESUMEN
Liver fatty-acid-binding protein (L-FABP) is a cytoplasmic polypeptide that binds with strong affinity especially to long-chain fatty acids (LCFAs). It is highly expressed in both the liver and small intestine, where it is thought to have an essential role in the control of the cellular fatty acid (FA) flux. Because expression of the gene encoding L-FABP is increased by both fibrate hypolipidaemic drugs and LCFAs, it seems to be under the control of transcription factors, termed peroxisome-proliferator-activated receptors (PPARs), activated by fibrate or FAs. However, the precise molecular mechanism by which these regulations take place remain to be fully substantiated. Using transfection assays, we found that the different PPAR subtypes (alpha, gamma and delta) are able to mediate the up-regulation by FAs of the gene encoding L-FABP in vitro. Through analysis of LCFA- and fibrate-mediated effects on L-FABP mRNA levels in wild-type and PPARalpha-null mice, we have found that PPARalpha in the intestine does not constitute a dominant regulator of L-FABP gene expression, in contrast with what is known in the liver. Only the PPARdelta/alpha agonist GW2433 is able to up-regulate the gene encoding L-FABP in the intestine of PPARalpha-null mice. These findings demonstrate that PPARdelta can act as a fibrate/FA-activated receptor in tissues in which it is highly expressed and that L-FABP is a PPARdelta target gene in the small intestine. We propose that PPARdelta contributes to metabolic adaptation of the small intestine to changes in the lipid content of the diet.
Asunto(s)
Proteínas Portadoras/genética , Regulación de la Expresión Génica/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Ácido Linoleico/farmacología , Hígado/efectos de los fármacos , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Bezafibrato/farmacología , Butiratos/farmacología , Cartilla de ADN , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Genes Reporteros , Hipolipemiantes , Intestino Delgado/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Compuestos de Fenilurea/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Transcripción Genética/fisiologíaRESUMEN
The effects of dietary oil intake and fatty acid infusions on the expression of intestinal and liver fatty acid-binding proteins (I-FABP and L-FABP, respectively) were investigated in the small intestine of mice. A daily force-feeding for 7 days with 0.2 ml sunflower oil specifically increased L-FABP mRNA and protein levels in duodenum and proximal jejunum. This upregulation was mediated in time- and dose-dependent manners by a minute quantity of linoleic acid, the main fatty acid found in sunflower oil. The L-FABP induction was only found with long-chain fatty acids, with the nonmetabolizable, substituted fatty acid alpha-bromopalmitate being far more active. A hormonally mediated effect is unlikely because long-chain fatty acids induced L-FABP mRNA in the Caco-2 cell line cultured in serum-free medium. Therefore, long-chain fatty acids are strong inducers of L-FABP gene expression in the small intestine. In contrast to data found in the rat, I-FABP gene expression appears to be unaffected by a lipid-enriched diet in the mouse.