RESUMEN
BACKGROUND: MicroRNAs (miRNAs) are epigenetically involved in regulating gene expression. They may be of importance in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to determine the role of miRNAs by their specific blocking in the CD4+CB45RBhi T-cell transfer model of chronic experimental colitis. METHODS: Colitis caused by transfer of WT CD4+CD45RBhi T cells in severe combined immunodeficiency (SCID) mice shares many features with human IBD. Colonic miRNA expression levels were measured at three time points in colitic mice, where a time-dependent upregulation of multiple miRNAs was seen. To inhibit these miRNAs, specific locked-nucleic-acid-modified (LNA) oligonucleotides were administered in further experiments at the moment the mice demonstrated the first signs of colitis. As controls, PBS and a scrambled sequence of anti-miRNA were used. Genome-wide expression analyses were also performed in order to detect candidate target genes of miR-142-5p, of which inhibition resulted in most effective amelioration of colitis. RESULTS: Anti-miR-142-5p reduced colitis and related wasting disease when administered in the T-cell transfer model, reflected in reduced weight loss and a lower disease activity index (DAI). In further validation experiments we also observed a higher survival rate and less colonic histological inflammation in the antagomir-treated mice. Moreover, by genome-wide expression analyses, we found downstream activation of the anti-inflammatory IL10RA pathway, including three genes also found in the top-20 candidate target genes of miR-142-5p. CONCLUSION: In conclusion, CD4+CD45RBhi-transfer colitis induces miR-142-5p. Blocking miR-142-5p reduced colitis and prevented wasting disease, possibly by activation of the IL10RA pathway.
Asunto(s)
Colitis/genética , Modelos Animales de Enfermedad , MicroARNs/antagonistas & inhibidores , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
BACKGROUND: There is no standardized validated experimental model used to predict human drug response, and the choice of model is not based on systematic evidence. Therefore, we decided to systematically investigate which models are currently used by selecting studies from literature that use prescribed inflammatory bowel disease medication as a positive control. METHODS: A search of PubMed was performed using terms describing experimental colitis models and the drugs used in corresponding clinical practice followed by the application of an animal filter. Each article was read and scored using a predesigned form describing the characteristics of the study (17 items), a quality assessment (10 items) completed by a meta-analysis. RESULTS: One hundred ninety-four unique articles were included that met the selection criteria. A large heterogeneity was found regarding the characteristics of the animals used, induction methods, treatment protocol, and reporting quality. If categorized by colitis model only a small number of studies used a chronic model (10/194). Almost all use acute chemical models that investigate a response to epithelial damage, rather than chronic colitis. Fifty-six percent used a TNBS model and 20% used a dextran sodium sulfate model. In these models, an ameliorating effect of 5-ASA and corticosteroids was demonstrated and also a difference in outcome when male or female animals are used. CONCLUSIONS: This scope describes a huge heterogeneity in study designs for preclinical drug efficacy. In addition, more than three-quarters of the studies used an acute model irrelevant for testing new treatment options for inflammatory bowel disease.
Asunto(s)
Investigación Biomédica/normas , Colitis/tratamiento farmacológico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Proyectos de Investigación/normas , Corticoesteroides/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Colitis/patología , Mesalamina/uso terapéutico , Factores SexualesRESUMEN
Progressive familial intrahepatic cholestasis (PFIC) types 1 and 3 are severe cholestatic liver diseases caused by deficiency of ATB8B1 and ABCB4, respectively. Mouse models for PFIC display mild phenotypes compared with human patients, and this can be explained by the difference in bile salt pool composition. Mice, unlike humans, have the ability to detoxify hydrophobic bile salts by cytochrome P450-mediated (re)hydroxylation and thus have a less toxic bile salt pool. We have crossed mouse models for PFIC1 and PFIC3 with Hrn mice that have a reduced capacity to (re)hydroxylate bile salts. Double transgenes were obtained by backcrossing Atp8b1(G308V/G308V) and Abcb4(-/-) mice with Hrn mice that have a liver-specific disruption of the cytochrome P450 reductase gene and therefore have markedly reduced P450 activity. In these mice, a more hydrophobic bile salt pool was instilled by cholic acid supplementation of the diet, and bile formation and liver pathology was studied. As opposed to single transgenes, Atp8b1(G308V/G308V)/Hrn and Abcb4(-/-)/Hrn mice rapidly developed strong cholestasis that was evidenced by increased plasma bilirubin and bile salt levels. The bile salt pool was more toxic in both models; Atp8b1(G308V/G308V)/Hrn mice had a more hydrophobic plasma pool compared with the single transgene, whereas Abcb4(-/-)/Hrn mice had a more hydrophobic biliary pool compared with the single transgene. In line with these findings, liver damage was not aggravated in Atp8b1(G308V/G308V)/Hrn but was more severe in Abcb4(-/-)/Hrn mice. These data indicate that bile salt pool composition is a critical determinant in the initiation and progression of cholestasis and liver pathology in PFIC1 and PFIC3. Most importantly, our data suggest that the hydrophobicity of the plasma bile salt pool is an important determinant of the severity of cholestasis, whereas the hydrophobicity of the biliary bile salt pool is an important determinant of the severity of liver pathology.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Adenosina Trifosfatasas/genética , Colestasis Intrahepática , Modelos Animales de Enfermedad , Proteínas de Transferencia de Fosfolípidos/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/sangre , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Animales , Colestasis Intrahepática/sangre , Colestasis Intrahepática/patología , Sistema Enzimático del Citocromo P-450/deficiencia , Hígado/enzimología , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
It has been established that bile salts play a role in the regulation of hepatic lipid metabolism. Accordingly, overt signs of steatosis have been observed in mice with reduced bile salt synthesis. The aim of this study was to identify the mechanism of hepatic steatosis in mice with bile salt deficiency due to a liver specific disruption of cytochrome P450 reductase. In this study mice lacking hepatic cytochrome P450 reductase (Hrn) or wild type (WT) mice were fed a diet supplemented with or without either 0.1% cholic acid (CA) or 0.025% obeticholic acid, a specific FXR-agonist. Feeding a CA-supplemented diet resulted in a significant decrease of plasma ALT in Hrn mice. Histologically, hepatic steatosis ameliorated after CA feeding and this was confirmed by reduced hepatic triglyceride content (115.5±7.3mg/g liver and 47.9±4.6mg/g liver in control- and CA-fed Hrn mice, respectively). The target genes of FXR-signaling were restored to normal levels in Hrn mice when fed cholic acid. VLDL secretion in both control and CA-fed Hrn mice was reduced by 25% compared to that in WT mice. In order to gain insight in the mechanism behind these bile salt effects, the FXR agonist also was administered for 3weeks. This resulted in a similar decrease in liver triglycerides, indicating that the effect seen in bile salt fed Hrn animals is FXR dependent. In conclusion, steatosis in Hrn mice is ameliorated when mice are fed bile salts. This effect is FXR dependent. Triglyceride accumulation in Hrn liver may partly involve impaired VLDL secretion.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Hígado Graso/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Animales , VLDL-Colesterol/metabolismo , Ácidos Cólicos/farmacología , Modelos Animales de Enfermedad , Expresión Génica , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Depuradores de Clase B/metabolismoRESUMEN
UNLABELLED: The difference in bile salt (BS) composition between rodents and humans is mainly caused by formation of muricholate in rodents as well as by efficient rehydroxylation of deoxycholic acid. The aim of this study was to characterize bile formation in a mouse model (Hrn mice) with hepatic disruption of the cytochrome p450 (CYP) oxidoreductase gene, encoding the single electron donor for all CYPs. Bile formation was studied after acute BS infusion or after feeding a BS-supplemented diet for 3 weeks. Fecal BS excretion in Hrn mice was severely reduced to 7.6% ± 1.8% of wild-type (WT), confirming strong reduction of (CYP-mediated) BS synthesis. Hrn bile contained 48% ± 18% dihydroxy BS, whereas WT bile contained only 5% ± 1% dihydroxy BS. Upon tauroursodeoxycholate infusion, biliary BS output was equal in WT versus Hrn, indicating that canalicular secretion capacity was normal. In contrast, taurodeoxycholic acid (TDC) infusion led to markedly impaired bile flow and BS output, suggesting onset of cholestasis. Feeding a cholate-supplemented diet (0.1%) resulted in a completely restored bile salt pool in Hrn mice, with 50% ± 9% TDC and 42% ± 10% taurocholic acid in bile, as opposed to 2% ± 1% and 80% ± 3% in WT mice, respectively. Under these conditions, biliary cholesterol secretion was strongly increased in Hrn mice, whereas serum alanine aminotransferase levels were decreased. CONCLUSION: Hrn mice have strongly impaired bile salt synthesis and (re)hydroxylation capacity and are more susceptible to acute TDC-induced cholestasis. In this mouse model, a more-human BS pool can be instilled by BS feeding, without hepatic damage, which makes Hrn mice an attractive model to study the effects of human BS.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Sistema Enzimático del Citocromo P-450/deficiencia , Hepatopatías/metabolismo , Hepatopatías/patología , Animales , Colesterol/metabolismo , Modelos Animales de Enfermedad , Hidroxilación , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Hepatopatías/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidorreductasas/deficiencia , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Fosfolípidos/metabolismo , Ácido Taurocólico/farmacologíaRESUMEN
Some cephalosporins, such as cefadroxil, are orally available. H(+)-coupled peptide transporter 1 mediates the transport of cephalosporins across the apical membrane of enterocytes. It is not known which mechanism(s) is responsible for the subsequent transport of cephalosporins across the basolateral membrane toward the circulation. We have tested whether ATP-binding cassette (ABC) transporters ABCC3 and/or ABCC4 are involved in the latter process. Transport experiments with plasma membrane vesicles expressing these transporters were used to determine whether ABCC3 and ABCC4 can transport cephalosporins in vitro. The involvement of Abcc3 and Abcc4 in the transport of cefadroxil from enterocytes was subsequently studied using intestinal explants from wild-type, Abcc3(-/-), Abcc4(-/-), and Abcc3(-/-)/Abcc4(-/-) mice in an Ussing chamber setup. Finally, appearance of cefadroxil in portal blood was investigated in vivo after intrajejunal administration of cefadroxil in wild-type, Abcc3(-/-), Abcc4(-/-), and Abcc3(-/-)/Abcc4(-/-) mice. ABCC3- and ABCC4-mediated transport of estradiol-17ß-glucuronide was dose-dependently inhibited by cephalosporins in vesicular transport experiments. Furthermore, transport of cefadroxil by ABCC3 and ABCC4 was saturable with K(m) values of 2.5 ± 0.7 and 0.25 ± 0.07 mM, respectively. Transport of cefadroxil from the apical to the basolateral side of jejunal tissue explants was unchanged in Abcc3(-/-) but significantly reduced (approximately 2-fold) in Abcc4(-/-) and Abcc3(-/-)/Abcc4(-/-) when compared with wild-type tissue. Upon instillation of cefadroxil in the jejunum, portal and peripheral blood concentrations were similar in Abcc3(-/-) and Abcc4(-/-) but approximately 2-fold reduced in Abcc3(-/-)/Abcc4(-/-) compared with wild-type mice. Our data demonstrate that intestinal absorption of cefadroxil depends partly on ABCC3 and ABCC4.
Asunto(s)
Antibacterianos/farmacocinética , Cefadroxilo/farmacocinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Administración Oral , Animales , Antibacterianos/administración & dosificación , Antibacterianos/sangre , Disponibilidad Biológica , Transporte Biológico , Cefadroxilo/administración & dosificación , Cefadroxilo/sangre , Membrana Celular/genética , Membrana Celular/metabolismo , Enterocitos/metabolismo , Estradiol/análogos & derivados , Estradiol/metabolismo , Absorción Intestinal , Mucosa Intestinal/metabolismo , Yeyuno/metabolismo , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genéticaRESUMEN
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
Adeno-associated virus (AAV) liver-directed gene therapy seems a feasible treatment for Crigler-Najjar syndrome type I, an inherited liver disorder characterized by severe unconjugated hyperbilirubinemia. Transient immunosuppression coupled with vector administration seems needed to overcome host immune responses that prevent long-term expression in patients. The immunosuppressive mycophenolate mofetil (MMF), which inhibits de novo synthesis of purines, is a promising candidate. To investigate the potential use of MMF in patients with Crigler-Najjar syndrome, we studied its effect on single-stranded AAV (ssAAV)-mediated correction of hyperbilirubinemia in the relevant preclinical model, the Gunn rat. Although MMF was well tolerated and effective it also impaired the efficacy of ssAAV. Subsequent in vitro studies showed that this effect is not specific for UGT1A deficiency. In fact, clinical relevant concentrations of mycophenolic acid (MPA), the active compound of MMF, also impair the transduction of HEK-293T cells by ssAAV. Because this effect was reversed by guanosine addition, it seems that intracellular levels of this nucleotide become limited, suggesting that MPA impairs second-strand DNA synthesis. This is corroborated by observations that MPA did not impair transduction of 293T cells by a self-complementary AAV (scAAV) vector and that MMF did not reduce the scAAV efficacy in the Gunn rat. In conclusion, MMF impairs ssAAV-mediated liver-directed gene therapy, which is relevant for the use of this immunosuppressive agent with single-stranded vectors. Furthermore, because this effect is due to impaired second-strand synthesis, the use of MMF with scAAV seems warranted.
Asunto(s)
Dependovirus/efectos de los fármacos , Terapia Genética/métodos , Vectores Genéticos/genética , Inmunosupresores/farmacología , Ácido Micofenólico/análogos & derivados , Transducción Genética/métodos , Animales , Bilirrubina/sangre , Línea Celular , Síndrome de Crigler-Najjar/genética , Síndrome de Crigler-Najjar/inmunología , Síndrome de Crigler-Najjar/terapia , Cartilla de ADN/genética , Replicación del ADN/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/efectos de los fármacos , Humanos , Modelos Lineales , Ácido Micofenólico/farmacología , Reacción en Cadena de la Polimerasa , Ratas , Ratas GunnRESUMEN
UNLABELLED: The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH. Male C57Bl/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied. Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor γ (Pparγ), fibroblast growth factor 21 (Fgf21), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice. IN CONCLUSION: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established.
Asunto(s)
Grasas de la Dieta/efectos adversos , Hígado Graso/metabolismo , Hipernutrición/complicaciones , Tejido Adiposo/inmunología , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Progresión de la Enfermedad , Proteínas de Unión a Ácidos Grasos/metabolismo , Hígado Graso/etiología , Hígado Graso/patología , Hiperinsulinismo/etiología , Inflamación/etiología , Lipogénesis , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico , Tamaño de los Órganos , PPAR gamma/metabolismoRESUMEN
Ezetimibe stimulates faecal neutral sterol (FNS) excretion in mice, which cannot be explained by cholesterol absorption inhibition alone. We investigated whether these effects are mediated via the sterol exporter ATP binding cassette transporter G8 (abcg8). Ezetimibe increased FNS excretion 2.7-fold in WT mice and 1.5-fold in abcg8(-/-) mice, without affecting biliary cholesterol secretion. Daily FNS excretion exceeded the sum of dietary cholesterol intake and biliary secretion by about 60%. Ezetimibe enhanced this 'extra' FNS excretion by 3.5-fold and 1.5-fold in wildtype (WT) and abcg8(-/-) mice, respectively. Ezetimibe stimulates fecal sterol excretion of non-biliary and non-dietary origin, probably through stimulation of trans-intestinal cholesterol excretion. We show that this effect depends on intact abcg8 function.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Azetidinas/farmacología , Lipoproteínas/metabolismo , Esteroles/metabolismo , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/genética , Animales , Bilis/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Colesterol/metabolismo , Ezetimiba , Heces/química , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Lipoproteínas/deficiencia , Lipoproteínas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Cholyl-L-lysyl-fluorescein (CLF) is a fluorescent bile salt derivative that is being developed as an agent for determining in vivo liver function. However, the mechanisms of uptake and excretion by hepatocytes have not been rigorously studied. We have directly assessed the transport capacity of various hepatobiliary transporters for CLF. Uptake experiments were performed in Chinese hamster ovary cells transfected with human NTCP, OATP1B1, OATP1B3, and OATP2B1. Conversely, excretory systems were tested with plasma membrane vesicles from Sf21 insect cells expressing human ABCB11, ABCC2, ABCC3, and ABCG2. In addition, plasma clearance and biliary excretion of CLF were examined in wild-type, Abcc2(-/-), and Abcc3(-/-) mice. Human Na(+)-dependent taurocholic-cotransporting polypeptide (NTCP) and ATP-binding cassette B11 (ABCB11) were incapable of transporting CLF. In contrast, high-affinity transport of CLF was observed for organic anion-transporting polypeptide 1B3 (OATP1B3), ABCC2, and ABCC3 with K(m) values of 4.6 +/- 2.7, 3.3 +/- 2.0, and 3.7 +/- 1.0 microM, respectively. In Abcc2(-/-) mice biliary excretion of CLF was strongly reduced compared with wild-type mice. This resulted in a much higher hepatic retention of CLF in Abcc2(-/-) versus wild-type mice: 64 versus 1% of the administered dose (2 h after administration). In mice intestinal uptake of CLF was negligible compared with that of taurocholate. Our conclusion is that human NTCP and ABCB11 are incapable of transporting CLF, whereas OATP1B3 and ABCC2/Abcc2 most likely mediate hepatic uptake and biliary excretion of CLF, respectively. CLF can be transported back into the blood by ABCC3. Enterohepatic circulation of CLF is minimal. This renders CLF suitable as an agent for assessing in vivo liver function.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácidos Cólicos/farmacocinética , Fluoresceínas/farmacocinética , Colorantes Fluorescentes/farmacocinética , Hígado/metabolismo , Animales , Bilis/metabolismo , Transporte Biológico , Células CHO , Proteínas Portadoras/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Humanos , Insectos/citología , Insectos/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Simportadores/metabolismo , TransfecciónRESUMEN
The main endogenous source of glutamine is de novo synthesis in striated muscle via the enzyme glutamine synthetase (GS). The mice in which GS is selectively but completely eliminated from striated muscle with the Cre-loxP strategy (GS-KO/M mice) are, nevertheless, healthy and fertile. Compared with controls, the circulating concentration and net production of glutamine across the hindquarter were not different in fed GS-KO/M mice. Only a approximately 3-fold higher escape of ammonia revealed the absence of GS in muscle. However, after 20 h of fasting, GS-KO/M mice were not able to mount the approximately 4-fold increase in glutamine production across the hindquarter that was observed in control mice. Instead, muscle ammonia production was approximately 5-fold higher than in control mice. The fasting-induced metabolic changes were transient and had returned to fed levels at 36 h of fasting. Glucose consumption and lactate and ketone-body production were similar in GS-KO/M and control mice. Challenging GS-KO/M and control mice with intravenous ammonia in stepwise increments revealed that normal muscle can detoxify approximately 2.5 micromol ammonia/g muscle.h in a muscle GS-dependent manner, with simultaneous accumulation of urea, whereas GS-KO/M mice responded with accumulation of glutamine and other amino acids but not urea. These findings demonstrate that GS in muscle is dispensable in fed mice but plays a key role in mounting the adaptive response to fasting by transiently facilitating the production of glutamine. Furthermore, muscle GS contributes to ammonia detoxification and urea synthesis. These functions are apparently not vital as long as other organs function normally.
Asunto(s)
Amoníaco/química , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/metabolismo , Músculos/enzimología , Alelos , Amoníaco/toxicidad , Animales , Femenino , Privación de Alimentos , Genotipo , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/enzimología , Factores Sexuales , Urea/químicaRESUMEN
Cafestol and kahweol are diterpene compounds present in unfiltered coffees. Cafestol is known as the most potent cholesterol-raising agent that may be present in the human diet. Remarkably, the mechanisms behind this effect have only been partly resolved so far. Even less is known about the metabolic fate of cafestol and kahweol. From the structure of cafestol, carrying a furan moiety, we hypothesized that epoxidation may not only be an important biotransformation route but that this also plays a role in its effects found. In bile duct-cannulated mice, dosed with cafestol, we were able to demonstrate the presence of epoxy-glutathione (GSH) conjugates, GSH conjugates and glucuronide conjugates. In addition, it was shown that cafestol was able to induce an electrophile-responsive element (EpRE). Using a murine hepatoma cell line with a luciferase reporter gene under control of an EpRE from the human NQO1 regulatory region, we also found that metabolic activation by CYP450 enzymes is needed for EpRE induction. Furthermore, raising intracellular GSH resulted in a decrease in EpRE-mediated gene induction, whereas lowering intracellular GSH levels increased EpRE-mediated gene induction. In conclusion, evidence suggests that cafestol induces EpRE, apparently via a bioactivation process that possibly involves epoxidation of the furan ring. The epoxides themselves appear subject to conjugation with GSH. The effects on EpRE can also explain the induction of GSH which seems to be involved in the reported beneficial effects of cafestol, for example, when administered with aflatoxin B1 or other toxic or carcinogenic compounds.
Asunto(s)
Café/química , Diterpenos/farmacología , Compuestos Epoxi/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Animales , Bilis/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Diterpenos/efectos adversos , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BLRESUMEN
Ezetimibe lowers plasma cholesterol levels by inhibiting the uptake of cholesterol in the intestine. Because of the extensive enterohepatic circulation of ezetimibe, relatively low doses are required to be effective. In blood and bile the majority of ezetimibe is present as a glucuronide conjugate, which is formed in the enterocyte. Presently, it is not clear which mechanisms are responsible for this efficient enterohepatic circulation. Abcc2, Abcc3, and Abcg2 are ATP-binding cassette (ABC) transporters that are expressed in both liver and intestine and are capable of transporting glucuronidated compounds. The aim of this study was to investigate the contribution of these transporters in the enterohepatic cycling of ezetimibe glucuronide (Ez-gluc). Transport studies were performed in plasma membrane vesicles from ABCC2-, ABCC3-, and ABCG2-expressing Sf21 insect cells. Furthermore, intestinal explants from wild-type and Abcc3(-/-) mice were used to study vectorial transport in a Ussing chamber setup. Finally, biliary excretion of Ez-gluc was measured in vivo after duodenal delivery of ezetimibe in wild-type, Abcc3(-/-), Abcc2(-/-), Abcg2(-/-), and Abcg2(-/-)/Abcc2(-/-) mice. ABCC3-, ABCC2-, and ABCG2-mediated transport was dose dependently inhibited by Ez-gluc. In the Ussing chamber Ez-gluc recovered from the basolateral side was significantly reduced in duodenal (2.2%), in jejunal (23%), and in ileal (23%) tissue of Abcc3(-/-) mice compared with that in tissues of wild-type mice. Biliary excretion of Ez-gluc was significantly reduced in Abcc3(-/-) (34%), Abcc2(-/-) (56%), and Abcg2(-/-)/Abcc2(-/-) (2.5%) compared with that in wild-type mice. These data demonstrate that the enterohepatic circulation of Ez-gluc strongly depends on the joint function of Abcc3, Abcc2, and Abcg2.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Anticolesterolemiantes/farmacocinética , Azetidinas/farmacocinética , Absorción Intestinal , Intestino Delgado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Administración Oral , Animales , Anticolesterolemiantes/administración & dosificación , Azetidinas/administración & dosificación , Transporte Biológico , Biotransformación , Línea Celular , Circulación Enterohepática , Estradiol/análogos & derivados , Ezetimiba , Glucurónidos/farmacocinética , Intubación Gastrointestinal , Metotrexato/metabolismo , Ratones , Ratones Noqueados , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/deficiencia , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , TransfecciónRESUMEN
Mutations in ATP8B1 cause severe inherited liver disease. The disease is characterized by impaired biliary bile salt excretion (cholestasis), but the mechanism whereby impaired ATP8B1 function results in cholestasis is poorly understood. ATP8B1 is a type 4 P-type ATPase and is a flippase for phosphatidylserine. Atp8b1-deficient mice display a dramatic increase in the biliary extraction of cholesterol from the canalicular (apical) membrane of the hepatocyte. Here we studied the hypothesis that disproportionate cholesterol extraction from the canalicular membrane impairs the activity of the bile salt transporter, ABCB11, and as a consequence causes cholestasis. Using single pass liver perfusions, we show that not only ABCB11-mediated transport but also Abcc2-mediated transport were reduced at least 4-fold in Atp8b1 deficiency. We show that canalicular membranes of cholestatic Atp8b1-deficient mice have a dramatically reduced cholesterol to phospholipid ratio, i.e. 0.75 +/- 0.24 versus 2.03 +/- 0.71 for wild type. In vitro depletion of cholesterol from mouse liver plasma membranes using methyl-beta-cyclodextrin demonstrated a near linear relation between cholesterol content of the membranes and ATP-dependent taurocholate transport. Abcc2-mediated transport activity was not affected up to 30% of membrane cholesterol depletion but declined to negligible levels at 70% of membrane cholesterol depletion. These effects were reversible as cholesterol repletion of the liver membranes completely restored Abcb11- and Abcc2-mediated transport. Our data demonstrate that membrane cholesterol content is a critical determinant of ABCB11/ABCC2 transport activity, provide an explanation for the etiology of ATP8B1 disease, and suggest a novel mechanism protecting the canalicular membrane against luminal bile salt overload.
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Transportadoras de Casetes de Unión a ATP/fisiología , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Canalículos Biliares/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Hígado/metabolismo , Mutación , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Animales , Transporte Biológico , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Perfusión , Proteínas de Transferencia de Fosfolípidos , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Conventional lactate-buffered peritoneal dialysis (PD) fluids containing glucose and glucose degradation products are believed to contribute to the development of fibrosis and angiogenesis in the dialyzed peritoneum. To reduce potential negative effects of lactate, pyruvate was substituted as a buffer and its effects on peritoneal pathological alterations were studied in a chronic peritoneal exposure model in the rat. METHODS: 20 Wistar rats were infused intraperitoneally with pyruvate-buffered (n = 9) or lactate-buffered PD fluid. After 20 weeks of daily infusion, peritoneal function was assessed. In omental peritoneal tissue, the number of blood vessels was analyzed following alpha-smooth muscle actin staining. The degree of fibrosis was quantitated in Picro Sirius Red-stained sections and by assessment of the hydroxyproline content. Plasma lactate/pyruvate and betahydroxybutyrate/acetoacetate (BBA/AA) ratios were determined. Plasma and dialysate vascular endothelial growth factor (VEGF) levels were quantitated by ELISA. RESULTS: The mass transfer area coefficient of creatinine was higher and the dialysate-to-plasma ratio of sodium was lower in pyruvate-treated animals compared to the lactate-treated group (0.11 vs 0.05 mL/min, p < 0.05, and 78% vs 89%, p < 0.05). The BBA/AA ratio tended to be lower in the pyruvate animals (p = 0.07). The number of blood vessels was lower in pyruvate-treated animals (16 vs 37 per field, p < 0.001). Total surface area, luminal area, and wall/total area of the vessels were larger in the pyruvate group. The degree of fibrosis was lower in intersegmental and perivascular areas of pyruvate-exposed animals. Effluent VEGF was higher in the pyruvate group. CONCLUSIONS: Replacement of lactate by pyruvate resulted in changes in peritoneal solute transport, accompanied by a reduction in both peritoneal membrane angiogenesis and fibrosis, suggesting potentially novel mechanisms to reduce glucose-driven alterations to the peritoneal membrane in PD patients.
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Soluciones para Hemodiálisis , Diálisis Peritoneal/métodos , Peritoneo/irrigación sanguínea , Animales , Transporte Biológico , Fibrosis , Inmunohistoquímica , Lactatos , Masculino , Peritoneo/metabolismo , Peritoneo/patología , Piruvatos , Ratas , Ratas Wistar , UltrafiltraciónRESUMEN
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.
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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: Cyclosporin A (CsA) stimulates the development of fibrosis. We investigated whether CsA contributes to peritoneal alterations induced by long-term exposure to dialysis solutions. METHODS: Ten rats received peritoneal infusion of dialysis solution and oral CsA for 8 weeks. Eight received only the dialysis solution (controls). Peritoneal function was assessed at 8 weeks followed by sacrifice. The number of vessels was counted, fibrosis was assessed and hydroxyproline was determined. PCR was performed for vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and transforming growth factor-beta (TGF-beta). RESULTS: Histology revealed more fibrosis, hydroxyproline and vessels (thick walled) in CsA-exposed animals. Peritoneal transport was not different. The mRNA content of TGF-beta, CTGF and VEGF was higher in CsA. CONCLUSION: CsA combined with exposure to dialysis solutions was associated with increased peritoneal fibrosis and angiogenesis.
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Ciclosporina/efectos adversos , Fibrosis/inducido químicamente , Soluciones para Hemodiálisis/efectos adversos , Neovascularización Patológica/inducido químicamente , Diálisis Peritoneal/efectos adversos , Enfermedades Peritoneales/inducido químicamente , Animales , Glucosa/uso terapéutico , Soluciones para Hemodiálisis/química , Ácido Láctico/uso terapéutico , Masculino , Enfermedades Peritoneales/patología , Ratas , Ratas WistarRESUMEN
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.
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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
OBJECTIVE: Several members of the ATP binding cassette (ABC)-transporter super family expressed in macrophages protect against atherosclerosis by promoting macrophage cholesterol and phospholipid efflux. Systemic disruption of ABCB4 in mice results in a virtual absence of phospholipids in bile and a strongly impaired biliary cholesterol secretion, indicating that ABCB4 plays an essential role in cellular lipid efflux. The aim of the current study was to determine the role of bone marrow-derived ABCB4 in atherosclerotic lesion development. METHODS: Chimeras were created that specifically lack ABCB4 in bone marrow-derived cells, including macrophages, by performing a bone marrow transplantation on LDL receptor knockout (LDLr-/-) mice. Atherosclerotic lesion development was induced by feeding a high-cholesterol diet (15% fat and 0.25% cholesterol). RESULTS: Serum cholesterol levels were significantly lower in mice reconstituted with ABCB4 knockout bone marrow as a result of reduced VLDL and LDL cholesterol levels. Despite the lower serum cholesterol levels, ABCB4 deficiency in bone marrow-derived cells resulted in a 1.8-fold (p=0.005) increase in lesion size. In vitro foam cell formation, induced with acetylated LDL (AcLDL) in peritoneal macrophages, was increased in the absence of ABCB4, possibly due to a 2-fold (p<0.05) increased association of AcLDL, while the efflux of cholesterol was unaffected. CONCLUSION: Bone marrow-derived ABCB4 has an important anti-atherosclerotic function, probably by limiting macrophage foam cell formation.