RESUMEN
Transintestinal cholesterol efflux (TICE) provides an attractive target to increase body cholesterol excretion. At present, the cholesterol donor responsible for direct delivery of plasma cholesterol to the intestine is unknown. In this study, we investigated the role of HDL in TICE. ATP-binding cassette protein A1 deficient (Abca1(-/-)) mice that lack HDL and wild-type (WT) mice were intravenously injected with chylomicron-like emulsion particles that contained radiolabeled cholesterol that is liberated in the liver and partly reenters the circulation. Both groups secreted radiolabeled cholesterol from plasma into intestinal lumen and TICE was unaltered between the two mouse models. To further investigate the role of HDL, we injected HDL with radiolabeled cholesterol in WT mice and Abca1(-/-)×Sr-b1(-/-) mice that lack HDL and are also unable to clear HDL via the liver. The intestines of both mice were unable to take up and secrete radiolabeled cholesterol from HDL via TICE. Although a generally accepted major player in the hepatobiliary route-based cholesterol excretion, HDL plays no significant role in TICE in mice.
Asunto(s)
Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Lipoproteínas HDL/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismoRESUMEN
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
BACKGROUND: Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency. METHODS: In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors. RESULTS: Intravenous administration of 1 x 10(13) gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I(-/-) mice resulted in hApoA-I expression levels of 634 +/- 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice. CONCLUSIONS: The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.
Asunto(s)
Apolipoproteína A-I/sangre , Apolipoproteína A-I/deficiencia , HDL-Colesterol/sangre , Dependovirus/genética , Terapia Genética , Animales , Apolipoproteína A-I/genética , Peso Corporal , Citomegalovirus/genética , Dependovirus/clasificación , Elementos de Facilitación Genéticos/genética , Vectores Genéticos/genética , Humanos , Inyecciones Intravenosas , Hígado/metabolismo , Ratones , Mutagénesis Insercional , Especificidad de Órganos , Fenotipo , Plásmidos/genética , Regiones Promotoras Genéticas/genética , Serotipificación , Aumento de PesoRESUMEN
Peroxisome proliferator-activated receptor delta (PPARdelta) is involved in regulation of energy homeostasis. Activation of PPARdelta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary cholesterol secretion, nor by reduced cholesterol absorption. To test the hypothesis that PPARdelta activation leads to stimulation of transintestinal cholesterol efflux (TICE), we quantified it by intestine perfusions in FVB mice treated with PPARdelta agonist GW610742. To exclude the effects on cholesterol absorption, mice were also treated with cholesterol absorption inhibitor ezetimibe or ezetimibe/GW610742. GW601742 treatment had little effect on plasma lipid levels but stimulated both fecal neutral sterol excretion ( approximately 200%) and TICE ( approximately 100%). GW610742 decreased intestinal Npc1l1 expression but had no effect on Abcg5/Abcg8. Interestingly, expression of Rab9 and LIMPII, encoding proteins involved in intracellular cholesterol trafficking, was increased upon PPARdelta activation. Although treatment with ezetimibe alone had no effect on TICE, it reduced the effect of GW610742 on TICE. These data show that activation of PPARdelta stimulates fecal cholesterol excretion in mice, primarily by the two-fold increase in TICE, indicating that this pathway provides an interesting target for the development of drugs aiming at the prevention of atherosclerosis.
Asunto(s)
Transporte Biológico/efectos de los fármacos , Colesterol/metabolismo , Mucosa Intestinal/metabolismo , PPAR delta/metabolismo , Animales , Ingestión de Alimentos/efectos de los fármacos , Intestinos/efectos de los fármacos , Lipoproteínas/metabolismo , Hígado/metabolismo , Masculino , Ratones , PPAR delta/genética , ARN Mensajero , Tiazoles/farmacologíaRESUMEN
Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux (TICE) contributes significantly to cholesterol removal in mice. Our aim was to investigate whether the activity of this novel pathway can be influenced by dietary factors. In addition, we studied the role of cholesterol acceptors at the luminal side of the enterocyte. Mice were fed a Western-type diet (0.25% wt/wt cholesterol; 16% wt/wt fat), a high-fat diet (no cholesterol; 24% wt/wt fat), or high-cholesterol diet (2% wt/wt), and TICE was measured by isolated intestinal perfusion. Bile salt-phospholipid mixtures served as cholesterol acceptor. Western-type and high-fat diet increased TICE by 50 and 100%, respectively. In contrast, the high-cholesterol diet did not influence TICE. Intestinal scavenger receptor class B type 1 (Sr-B1) mRNA and protein levels correlated with the rate of TICE. Unexpectedly, although confirming a role for Sr-B1, TICE was significantly increased in Sr-B1-deficient mice. Apart from the long-term effect of diets on TICE, acute effects by luminal cholesterol acceptors were also investigated. The phospholipid content of perfusate was the most important regulator of TICE; bile salt concentration or hydrophobicity of bile salts had little effect. In conclusion, TICE can be manipulated by dietary intervention. Specific dietary modifications might provide means to stimulate TICE and, thereby, to enhance total cholesterol turnover.
Asunto(s)
Colesterol/metabolismo , Mucosa Intestinal/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Acilcoenzima A/genética , Acilcoenzima A/metabolismo , Animales , Bilis/metabolismo , Ácidos y Sales Biliares/administración & dosificación , Ácidos y Sales Biliares/farmacología , Colesterol en la Dieta/administración & dosificación , Dieta , Grasas de la Dieta/farmacología , Suplementos Dietéticos , Intestinos/efectos de los fármacos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Fosfolípidos/administración & dosificación , Fosfolípidos/farmacología , ARN Mensajero/metabolismo , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/metabolismo , Transducción de SeñalRESUMEN
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: Hepatobiliary secretion is generally believed to be an integral step in the pathway of cholesterol excretion from the body. Here we have investigated the validity of this paradigm in mice. METHODS: Cholesterol balance was assessed by measuring intake, excretion, and biliary output in different mouse models. Direct secretion of cholesterol from the luminal side of enterocytes was studied by perfusion of isolated segments of the small intestine in mice. RESULTS: Cholesterol input and output measurements in different mouse models revealed that fecal neutral sterol excretion was higher than the sum of dietary cholesterol intake and biliary cholesterol secretion indicating the existence of an alternative pathway. Here we show that substantial amounts of cholesterol can be secreted directly by enterocytes. Transintestinal cholesterol secretion is a specific process observed throughout the small intestine (proximal > medial > distal). Secretion depended on the presence of a cholesterol acceptor and was strongly stimulated by bile salts and phospholipids. The capacity of the pathway was sufficient to account for the missing cholesterol in the balance studies. The contribution of this pathway to cholesterol excretion in mice is approximately twice that of the biliary pathway. CONCLUSIONS: In mice, the intestine plays a significant role in removal of cholesterol from the body.
Asunto(s)
Colesterol/análisis , Colesterol/metabolismo , Heces/química , Intestino Delgado/metabolismo , Esteroles/metabolismo , Animales , Transporte Biológico/fisiología , Colesterol en la Dieta/metabolismo , Enterocitos/citología , Enterocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Modelos Biológicos , PerfusiónRESUMEN
Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.
Asunto(s)
Síndrome de Crigler-Najjar/terapia , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/farmacología , Glucuronosiltransferasa/deficiencia , Animales , Bilis/fisiología , Bilirrubina/sangre , Síndrome de Crigler-Najjar/genética , Dependovirus/clasificación , Dependovirus/inmunología , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/inmunología , Hígado/efectos de los fármacos , Hígado/patología , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Gunn , Serotipificación , Distribución TisularRESUMEN
Lipoprotein lipase (LPL) deficiency causes hypertriglyceridemia and recurrent, potentially life-threatening pancreatitis. There currently is no adequate treatment for this disease. Previously, we showed that intramuscular administration of an adeno-associated virus serotype 1 (AAV1) vector encoding the human LPL(S447X) variant cDNA (AAV1-LPL(S447X)) normalized the dyslipidemia of LPL-/- mice for more than 1 year. In preparation for a clinical trial, we evaluated the safety and biodistribution of AAV1-LPL(S447X) in wild-type mice and fully characterized six LPL-deficient patients. Toxicological analysis in mice showed that intramuscular administration was well tolerated. Acute inflammatory response markers were transiently increased, and anti- AAV1 antibodies were generated. Histological analyses indicated a dose-dependent reversible spleen hyperplasia, and myositis at the injection sites. Biodistribution data showed short-term vector leakage from injection sites into the circulation, followed by liver-mediated clearance. Persistence of vector DNA was limited to the injected muscle and draining lymph nodes, and spread to reproductive organs was limited. Characterization of LPL-deficient patients showed that all patients presented with hypertriglyceridemia and recurrent pancreatitis. LPL catalytic activity was absent, but LPL protein levels were 20-100% of normal. Myoblasts derived from skeletal muscle biopsies of these patients were efficiently transduced by AAV1-LPL(S447X) and secreted active LPL. These data support the initiation of a clinical trial in LPL-deficient patients, for which regulatory approval has been granted.
Asunto(s)
Terapia Genética , Hiperlipoproteinemia Tipo I/terapia , Lipoproteína Lipasa/genética , Animales , Dependovirus/genética , Estudios de Factibilidad , Femenino , Terapia Genética/efectos adversos , Vectores Genéticos , Inyecciones Intramusculares , Lipoproteína Lipasa/administración & dosificación , Lipoproteína Lipasa/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Distribución TisularRESUMEN
Human lipoprotein lipase (LPL) deficiency causes profound hypertriglyceridemia and life-threatening pancreatitis. We recently developed an adult murine model for LPL deficiency: LPL -/- mice display grossly elevated plasma triglyceride (TG) levels (>200-fold) and very low high-density lipoprotein cholesterol (HDL-C < 10% of normal). We used this animal model to test the efficacy of adeno-associated virus-mediated expression of hLPL(S447X) (AAV1-LPL(S447X)) in muscle for the treatment of LPL deficiency. Intramuscular administration of AAV1-LPL(S447X) resulted in dose-dependent expression of hLPL protein and LPL activity (up to 33% of normal murine levels) in postheparin plasma. Remarkably, visible hyperlipidemia was resolved within 1 week; plasma TG was reduced to near-normal levels (from 99.0 to 1.8 mmol/L), and plasma HDL-C was increased 6-fold (from 0.2 to 1.1 mmol/L). At 8 months after administration of AAV1-LPL(S447X), an intravenous lipid challenge showed efficient, near-normal clearance of plasma TG. Histologic analyses of injected muscle further indicated that abnormal muscle morphology observed in LPL -/- mice was reversed after treatment. Expression of therapeutic levels of LPL(S447X), and the subsequent beneficial effect on plasma lipid levels, has lasted for more than 1 year. We therefore conclude that AAV1-mediated transfer of LPL(S447X) into murine skeletal muscle results in long-term near-correction of dyslipidemia associated with LPL deficiency.