RESUMEN
n-3 PUFA such as EPA and DHA as well as oestrogen have been reported to decrease blood levels of cholesterol, but their underlying mechanism is unclear. The purpose of this study was to determine the effects of the combination of n-3 PUFA supplementation and oestrogen injection on hepatic cholesterol metabolism. Rats were fed a modified AIN-93G diet with 0, 1 or 2 % n-3 PUFA (EPA+DHA) relative to the total energy intake for 12 weeks. Rats were surgically ovariectomised at week 8, and, after 1-week recovery, rats were injected with 17ß-oestradiol-3-benzoate (E2) or maize oil for the last 3 weeks. Supplementation with n-3 PUFA and E2 injection significantly increased the ratio of the hepatic expression of phosphorylated AMP activated protein kinase (p-AMPK):AMP activated protein kinase (AMPK) and decreased sterol regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase and proprotein convertase subtilisin/kexin type 9. Supplementation with n-3 PUFA increased hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), sterol 12α-hydroxylase (CYP8B1) and sterol 27-hydroxylase (CYP27A1); however, E2 injection decreased CYP7A1 and CYP8B1 but not CYP27A1. Additionally, E2 injection increased hepatic expression of oestrogen receptor-α and ß. In conclusion, n-3 PUFA supplementation and E2 injection had synergic hypocholesterolaemic effects by down-regulating hepatic cholesterol synthesis (n-3 PUFA and oestrogen) and up-regulating bile acid synthesis (n-3 PUFA) in ovariectomised rats.
Asunto(s)
Envejecimiento , Anticolesterolemiantes/uso terapéutico , Suplementos Dietéticos , Estrógenos/uso terapéutico , Ácidos Grasos Omega-3/uso terapéutico , Hipercolesterolemia/prevención & control , Hígado/efectos de los fármacos , Animales , Anticolesterolemiantes/administración & dosificación , Colestanotriol 26-Monooxigenasa/química , Colestanotriol 26-Monooxigenasa/metabolismo , Colesterol 7-alfa-Hidroxilasa/química , Colesterol 7-alfa-Hidroxilasa/metabolismo , Terapia Combinada , Dieta con Restricción de Grasas , Estradiol/administración & dosificación , Estradiol/análogos & derivados , Estradiol/uso terapéutico , Estrógenos/administración & dosificación , Ácidos Grasos Omega-3/administración & dosificación , Femenino , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hipercolesterolemia/enzimología , Hipercolesterolemia/metabolismo , Hígado/enzimología , Hígado/metabolismo , Ovariectomía , Proproteína Convertasa 9 , Distribución Aleatoria , Ratas Wistar , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Esteroide 12-alfa-Hidroxilasa/química , Esteroide 12-alfa-Hidroxilasa/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
In mammals, cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) are rate-limiting enzymes in bile acid synthesis. In addition, a small heterodimer partner (SHP) is also known to inhibit bile acid synthesis via the suppression of CYP7A1 and CYP8B1 expression. However, little information is currently available regarding primary structure of the genes involved in bile acid synthesis in fish. We therefore cloned cyp7a1, cyp8b1 and shp genes from rainbow trout and obtained cDNAs encoding two isoforms each of Cyp7a1 (-1 and -2), Cyp8b1 (-1 and -2) and Shp (-1 and -2). Both cyp7a1-1 and -2 encoded proteins of 512 amino acids. Trout cyp7a1-1 was expressed not only primarily in the kidney, pyloric caecum and mid-gut, but also weakly in the liver, eye, gill and ovary. cyp7a1-2 was highly expressed in the liver, pyloric caecum and mid-gut. cyp8b1-1 and -2, which encoded proteins of 512 and 509 amino acids, respectively, were principally expressed in the liver. Both shp-1 and -2, which encoded proteins of 288 and 290 amino acids, respectively, were strongly expressed in the liver, but shp-2 was also highly expressed in the gallbladder and digestive tract. The temporal changes in the expression of cyp7a1-1/-2, cyp8b1-1/-2 and shp-1/-2 in the liver were assessed after consumption of a single meal. Expression of cyp7a1-1/-2 and cyp8b1-1/-2 increased within 3h post feeding (hpf) when the stomach was still approximately 84% full and the gallbladder was almost completely empty. Although the expression of shp-1 did not change after feeding, the expression pattern of shp-2 was inversely related to the expression patterns of cyp7a1-1/-2 and cyp8b1-1/-2. Specifically, shp-2 expression decreased until 3 hpf before returning to initial levels at 24 hpf. These findings suggest that Cyp7a1s/8b1s and Shp-2 function antagonistically in bile acid synthesis in rainbow trout.
Asunto(s)
Colesterol 7-alfa-Hidroxilasa/metabolismo , Proteínas de Peces/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Esteroide 12-alfa-Hidroxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Ácidos y Sales Biliares/biosíntesis , Colesterol 7-alfa-Hidroxilasa/química , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Vesícula Biliar/enzimología , Expresión Génica , Intestinos/enzimología , Hígado/enzimología , Datos de Secuencia Molecular , Oncorhynchus mykiss , Especificidad de Órganos , Filogenia , Periodo Posprandial , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/química , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Esteroide 12-alfa-Hidroxilasa/química , Estómago/enzimologíaRESUMEN
Hormonal status can influence diverse metabolic pathways. Small heterodimer partner (SHP) is an orphan nuclear receptor that can modulate the activity of several transcription factors. Estrogens are here shown to directly induce expression of the SHP in the mouse and rat liver and in human HepG2 cells. SHP is rapidly induced within 2 h following treatment of mice with ethynylestradiol (EE) or the estrogen receptor alpha (ERalpha)-selective compound propyl pyrazole triol (PPT). SHP induction by these estrogens is completely absent in ERalphaKO mice. Mutation of the human SHP promoter defined HNF-3, HNF-4, GATA, and AP-1 sites as important for basal activity, whereas EE induction required two distinct elements located between -309 and -267. One of these elements contains an estrogen response element half-site that bound purified ERalpha, and ERalpha with a mutated DNA binding domain was unable to stimulate SHP promoter activity. This ERalpha binding site overlaps the known farnesoid X receptor (FXR) binding site in the SHP promoter, and the combination of EE plus FXR agonists did not produce an additive induction of SHP expression in mice. Surprisingly, induction of SHP by EE did not inhibit expression of the known SHP target genes cholesterol 7alpha-hydroxylase (CYP7A1) or sterol 12alpha-hydroxylase (CYP8B1). However, the direct regulation of SHP expression may provide a basis for some of the numerous biological effects of estrogens.
Asunto(s)
Estradiol/análogos & derivados , Regulación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Colesterol 7-alfa-Hidroxilasa/química , Proteínas de Unión al ADN/química , Dimerización , Relación Dosis-Respuesta a Droga , Estradiol/farmacología , Receptor alfa de Estrógeno , Eliminación de Gen , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Fenoles , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Pirazoles/farmacología , ARN/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esteroide 12-alfa-Hidroxilasa/química , Esteroide 12-alfa-Hidroxilasa/metabolismo , Factores de Tiempo , Factores de Transcripción/química , TransfecciónRESUMEN
Sterol 12alpha-hydroxylase is an important enzyme in bile acid biosynthesis, responsible for the balance between formation of cholic acid and chenodeoxycholic acid. The enzyme has been purified to apparent homogeneity from rabbit liver (Ishida, H., Noshiro, M., Okuda, K., and Coon, M. J. (1992) J. Biol. Chem. 267, 21319-21323), and we here describe the cloning and sequencing of a cDNA coding for this enzyme. After tryptic digestion of purified protein in a polyacrylamide gel, eight different peptides were isolated and sequenced. Using oligonucleotides deduced from the amino acid sequences, clones were isolated from a rabbit liver cDNA library. In addition to several overlapping clones, one full-length clone was obtained that coded for a polypeptide of 500 amino acids, corresponding to a molecular mass of 57 kDa. All of the eight peptides and the reported NH2-terminal amino acid sequence were matched against the sequence. The peptide sequence showed a 39% similarity with human prostacyclin synthase (CYP8) and 31% similarity with the rate-limiting enzyme in over-all synthesis of bile acids, the cholesterol 7alpha-hydroxylase (CYP7) of the rabbit. The similarity with most other sterol cytochrome P-450 hydroxylases was less. Thus, this species of cytochrome P-450 should belong to a group of its own, here denoted CYP12. Transfection of COS cells with the coding part of the cDNA resulted in a significant expression of sterol 12alpha-hydroxylase activity toward 7alpha-hydroxy-4-cholesten-3-one. Northern blotting showed that the enzyme was exclusively expressed in the liver. The major mRNA fraction in rabbit liver had a size of approximately 2.9 kilobases, and those found in rat and human liver were about 2.5 and 4.5 kilobases, respectively. Fasting of rats and mice led to a severalfold increase in both enzyme activity and mRNA levels. In contrast, starvation of rabbits had little or no stimulatory effect on enzyme activity and mRNA levels.