RESUMEN
BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis can be caused by mutations in ABCB4 or ATP8B1; each encodes a protein that translocates phospholipids, but in opposite directions. ABCB4 flops phosphatidylcholine from the inner to the outer leaflet, where it is extracted by bile salts. ATP8B1, in complex with the accessory protein CDC50A, flips phosphatidylserine in the reverse direction. Abcb4(-/-) mice lack biliary secretion of phosphatidylcholine, whereas Atp8b1-deficient mice have increased excretion of phosphatidylserine into bile. Each system is thought to have a role protecting the canalicular membrane from bile salts. METHODS: To investigate the relationship between the mechanisms of ABCB4 and ATP8B1, we expressed the transporters separately and together in cultured cells and studied viability and phospholipid transport. We also created mice with disruptions in ABCB4 and ATP8B1 (double knockouts) and studied bile formation and hepatic damage in mice fed bile salts. RESULTS: Overexpression of ABCB4 was toxic to HEK293T cells; the toxicity was counteracted by coexpression of the ATP8B1-CDC50A complex. In Atp8b1-deficient mice, bile salts induced extraction of phosphatidylserine and ectoenzymes from the canalicular membrane; this process was not observed in the double-knockout mice. CONCLUSIONS: ATP8B1 is required for hepatocyte function, particularly in the presence of ABCB4. This is most likely because the phosphatidylserine flippase complex of ATP8B1-CDC50A counteracts the destabilization of the membrane that occurs when ABCB4 flops phosphatidylcholine. Lipid asymmetry is therefore important for the integrity of the canalicular membrane; ABCB4 and ATP8B1 cooperate to protect hepatocytes from bile salts.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Adenosina Trifosfatasas/fisiología , Canalículos Biliares/citología , Membrana Celular/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Animales , Ácidos y Sales Biliares/farmacología , Canalículos Biliares/fisiología , Células Cultivadas , Células HEK293 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Fosfatidilcolinas/metabolismo , Proteínas de Transferencia de Fosfolípidos , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
BACKGROUNDS & AIMS: ATP8B1 is a phosphatidylserine flippase in the canalicular membrane; patients with mutations in ATP8B1 develop severe chronic (PFIC1) or periodic (BRIC1) cholestatic liver disease. We have observed that Atp8b1 deficiency leads to enhanced biliary cholesterol excretion. It has been established that biliary cholesterol excretion depends on transport by the heterodimer Abcg5/Abcg8. We hypothesized that the increased cholesterol output was due to enhanced extraction from the altered canalicular membrane rather than to higher Abcg5/Abcg8 activity. We therefore studied the relation between Abcg5/Abcg8 expression and biliary cholesterol excretion in mice lacking Atp8b1, Abcg8, or both (GF mice). METHODS: Bile formation was studied in LXR agonist-fed wild-type mice as well as mice lacking Atp8b1 or Abcg8, or in GF mice upon infusion of taurocholate. Bile samples were analyzed for cholesterol, bile salt, phospholipids, and ectoenzyme content. RESULTS: LXR agonist increased Abcg5/8 expression, and this was accompanied by increased biliary cholesterol output in both wild-type and Atp8b1(G308V/G308V) mice. However, Atp8b1(G308V/G308V) mice maintained higher cholesterol output. Although in Abcg8(-/-) mice biliary cholesterol output was severely reduced, GF mice displayed high biliary cholesterol output, which was comparable with wild-type mice. Bile of both Atp8b1(G308V/G308V) and GF mice displayed elevated levels of phosphatidylserine and sphingomyelin, indicating membrane stress. CONCLUSIONS: Our data demonstrate that the increased biliary cholesterol excretion in Atp8b1-deficient mice is independent of Abcg5/8 activity. This implicates that Atp8b1 deficiency leads to a decrease in the detergent resistance and subsequent nonspecific extraction of cholesterol from the canalicular membrane by bile salts.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Bilis/metabolismo , Colesterol/metabolismo , Lipoproteínas/metabolismo , Hígado/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Animales , Bilis/enzimología , Ácidos y Sales Biliares/sangre , Canalículos Biliares/metabolismo , Membrana Celular/metabolismo , Proteínas de Unión al ADN/agonistas , Proteínas de Unión al ADN/metabolismo , Hidrocarburos Fluorados , Lipoproteínas/deficiencia , Lipoproteínas/genética , Hígado/efectos de los fármacos , Hígado/enzimología , Receptores X del Hígado , Masculino , Fluidez de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Nucleares Huérfanos , Proteínas de Transferencia de Fosfolípidos , Fosfolípidos/metabolismo , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/metabolismo , Sulfonamidas/farmacología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
BACKGROUND/AIMS: Mutations in the ATP8B1 gene can cause Progressive Familial Intrahepatic Cholestasis type 1. We have previously reported that Atp8b1(G308V/G308V) mice, a model for PFIC1, have slightly, but significantly, higher baseline serum bile salt (BS) concentrations compared to wt mice. Upon BS feeding, serum BS concentrations strongly increased in Atp8b1-deficient mice. Despite these findings, we observed only mildly impaired canalicular BS transport. In the present report we tested the hypothesis that Atp8b1(G308V/G308V) mice hyperabsorb BS in the intestine during BS feeding. METHODS: Intestinal BS absorption was measured in intestinal perfusion and in intestinal explants. In addition, we measured BS concentrations in portal blood. Ileal expression of the Fxr-targets Asbt, Ilbp and Shp was assessed. RESULTS: In wt and Atp8b1(G308V/G308V) mice, intestinal taurocholate absorption is primarily mediated by the ileal bile salt transporter Asbt. Neither of the experimental systems revealed enhanced absorption of BS in Atp8b1(G308V/G308V) mice compared to wt mice. In line with these observations, we found no difference in the ileal protein expression of Asbt. Induction of Shp expression during BS feeding also demonstrated that Fxr signalling is intact in Atp8b1(G308V/G308V) mice. CONCLUSIONS: The accumulation of BS in plasma of Atp8b1(G308V/G308V) mice during BS feeding is not caused by increased intestinal BS absorption.
Asunto(s)
Adenosina Trifosfatasas/genética , Ácidos y Sales Biliares/metabolismo , Absorción Intestinal/fisiología , Animales , Ácidos y Sales Biliares/administración & dosificación , Ácidos y Sales Biliares/sangre , Colatos/administración & dosificación , Colestasis Intrahepática/genética , Proteínas de Unión al ADN/metabolismo , Dieta , Modelos Animales de Enfermedad , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Expresión Génica , Absorción Intestinal/genética , Ratones , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Perfusión , Proteínas de Transferencia de Fosfolípidos , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Simportadores/metabolismo , Ácido Taurocólico/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Ligand-activated Cre recombinases are widely used for studying gene function in vitro and in conditional mouse models. To compare ligand-dependent Cre recombinases, different Cre estrogen receptor fusions were introduced into the ROSA26 locus of embryonic stem (ES) cells and assayed for genotoxicity and recombination efficiency. Of the tested recombinases, the CreERT2 variant showed no toxicity and was highly responsive to ligand induction. To constitutively express CreERT2 in mice and also to clarify whether the CreERT2 system displays background activity, we generated a knock-in mouse line harboring the CreERT2 coding region under the control of the ROSA26 locus. Analysis of this ROSA26-CreERT2 deleter mouse with different reporter strains revealed ubiquitous recombination in the embryo and partial recombination in peripheral and hematopoietic tissues but no effective CreERT2 expression in the brain. Furthermore, using flow cytometry, we found low-level background recombination in noninduced bitransgenic ROSA26-CreERT2/EGFP reporter mice. To determine whether background activity poses a general problem for conducting conditional in vivo experiments with the ROSA26-CreERT2 deleter, we used a sensitive conditional skin cancer model. In this assay, cancer induction was completely restricted to induced bitransgenic CreERT2/K-Ras(V12) mice, whereas noninduced control animals did not show any sign of cancer, indicating the usefulness of the ROSA-CreERT2 system for regulating conditional gene expression in vivo. The ROSA26-CreERT2 deleter strain will be a convenient experimental tool for studying gene function under circumstances requiring partial induction of recombination in peripheral tissues and will be useful for uncovering previously unknown or unsuspected phenotypes.
Asunto(s)
Integrasas/metabolismo , Mosaicismo , Animales , Encéfalo/metabolismo , ADN/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Eliminación de Gen , Genes Reporteros , Genómica/métodos , Ligandos , Ratones , Ratones Transgénicos , Modelos Genéticos , Recombinación GenéticaRESUMEN
Coprophagy (i.e., consumption of feces) is a behavior seen in rodents and other animal species. This behavior can substantially influence the enterohepatic cycling of compounds, including bile salts. Since many studies involve the feeding of rodents with bile salt supplemented diets, it is of importance to know the influence of coprophagy on bile salt composition in such studies. We compared the peripheral and portal bile salt composition of mice in conventional and metabolic cages when fed a control diet or a diet containing 0.5% cholate. We also performed these experiments with Atp8b1-deficient mice as it has been suggested that in the absence of this transporter bile salt absorption in the intestine would be increased. In mice on a control diet there is little difference in bile salt composition between conventional housing and metabolic housing. Metabolic housing led to a near complete disappearance of the low levels of dihydroxy bile salts (i.e., deoxycholate + chenodeoxycholate) in peripheral serum. In mice fed a control diet, the portal blood concentration of unconjugated dihydroxy bile salts was extremely low (<2%), but these rose to about 10% when mice were fed a cholate-supplemented diet. In metabolic cages the portal blood content of these unconjugated dihydroxy bile salts was reduced to undetectable levels. Whether housed in conventional cages or in metabolic cages, wild-type and Atp8b1-deficient mice had similar concentrations in portal blood, suggesting that intestinal bile salt absorption is not altered in Atp8b1-deficient mice.
Asunto(s)
Adenosina Trifosfatasas/genética , Ácidos y Sales Biliares/sangre , Colatos/administración & dosificación , Coprofagia/sangre , Administración Oral , Animales , Ácidos y Sales Biliares/química , Peso Corporal/efectos de los fármacos , Conducta Alimentaria , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Proteínas de Transferencia de Fosfolípidos , Vena Porta/química , Transaminasas/sangreRESUMEN
Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasis.
Asunto(s)
Adenosina Trifosfatasas/deficiencia , Ácidos y Sales Biliares/metabolismo , Canalículos Biliares/metabolismo , Membrana Celular/metabolismo , Colestasis Intrahepática/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Canalículos Biliares/ultraestructura , Transporte Biológico , Western Blotting , Membrana Celular/ultraestructura , Colestasis Intrahepática/patología , Cromatografía en Capa Delgada , Modelos Animales de Enfermedad , Interacciones Hidrofóbicas e Hidrofílicas , Inmunohistoquímica , Técnicas In Vitro , Masculino , Ratones , Microscopía Electrónica , Proteínas de Transferencia de FosfolípidosRESUMEN
Mutations in ATP8B1, a broadly expressed P-type ATPase, result, through unknown mechanisms, in disorders of bile secretion. These disorders vary in severity from mild and episodic to progressive with liver failure. We generated Atp8b1G308V/G308V mutant mice, which carry a mutation orthologous to that present in homozygous form in patients from the Amish index kindred for severe ATP8B1 disease. In contrast to human patients, Atp8b1(G308V/G308V) mice had unimpaired bile secretion and no liver damage, but showed mild abnormalities including depressed weight at weaning and elevated serum bile salt levels. We challenged the hepatobiliary metabolism of Atp8b1G308V/G308V mice by administering exogenous bile salts. Upon bile salt feeding, Atp8b1G308V/G308V mice, but not wild-types, demonstrated serum bile salt accumulation, hepatic injury and expansion of the systemic bile salt pool. Unexpectedly, this failure of bile salt homeostasis occurred in the absence of any defect in hepatic bile secretion. Upon infusion of a hydrophobic bile salt, wild-type mice developed cholestasis while Atp8b1G308V/G308V mice maintained high biliary output and more extensively rehydroxylated the infused bile salt. Increased bile salt hydroxylation, which reduces bile salt toxicity, may explain the milder phenotype in Atp8b1G308V/G308V mice compared with humans with the equivalent mutation. These results demonstrate the key role of Atp8b1 in bile salt homeostasis and highlight the importance of bile salt hydroxylation in the prevention of cholestasis. The mouse phenotype reveals that loss of Atp8b1 disrupts bile salt homeostasis without impairment of canalicular bile secretion; in humans this process is likely to be obscured by early onset of severe liver disease.