RESUMEN

5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-alpha). In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CLp) after intravenous administration was low in Sprague-Dawley rats (3.2 +/- 1.4 ml min(-1) kg(-1)) and cynomolgus monkeys (6.1 +/- 1.6 ml min(-1) kg(-1)) resulting in plasma half-lives of 7.1 +/- 0.7 h and 9.4 +/- 0.8 h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50 mg kg(-1)). In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC50's > 30 microM) was discerned with 1. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.

Asunto(s)

Benzoatos/farmacocinética , Sistema Enzimático del Citocromo P-450/metabolismo , PPAR alfa/agonistas , Administración Oral , Animales , Benzoatos/química , Benzoatos/farmacología , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450 , Evaluación Preclínica de Medicamentos , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Inyecciones Intravenosas , Macaca fascicularis , Tasa de Depuración Metabólica , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Ratas , Ratas Sprague-Dawley , Triglicéridos/antagonistas & inhibidores , Triglicéridos/sangre

RESUMEN

Excess accumulation of cholesterol in macrophages results in foam cell production and lesion development. Recent studies have demonstrated that ATP-binding cassette protein A1 (ABCA1) is highly regulated in macrophages and mediates the efflux of cholesterol and phospholipids to apolipoproteins, a process necessary for HDL formation. The goal of this study was to determine the contribution of monocyte/macrophage ABCA1 to HDL formation in vivo. We generated mice expressing ABCA1 in macrophages and mice with selected inactivation of ABCA1 in macrophages by bone marrow transplantation in ABCA1-deficient (ABC1(-/-)) and wild-type (WT) mice. At all times, the level of HDL in ABC1(-/-) recipient mice remained low relative to WT recipient mice irrespective of the genotype of the donor macrophage ABCA1 or high-fat feeding. Expression of WT macrophage ABCA1 in ABC1(-/-) mice resulted in a small but significant increase in apoA-I levels starting 2 weeks after transplantation. No further increase in apoAI was observed up to 14 weeks after transplantation. The increase in apoAI was accompanied by a small but significant increase in HDL cholesterol 6 weeks after transplantation. The HDL formed as a consequence of the expression of WT macrophage ABCA1 migrated to the alpha position in a two-dimensional gel electrophoresis. These results demonstrate that monocyte/macrophage ABCA1 contributes to HDL formation; however, the contribution to the overall plasma HDL levels is minimal.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/biosíntesis , Lipoproteínas HDL/sangre , Macrófagos/metabolismo , Monocitos/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/genética , Animales , Trasplante de Médula Ósea , Cruzamientos Genéticos , Electroforesis en Gel Bidimensional , Humanos , Macrófagos Peritoneales/metabolismo , Ratones , Unión Proteica , Enfermedad de Tangier , Factores de Tiempo

RESUMEN

Studies show that lipid-free apoA-I stimulates release of cholesterol and phospholipid from fibroblasts and macrophages. ATP-binding cassette 1 (ABC1) is implicated in this release and has been identified as the genetic defect in Tangier disease, evidence that ABC1 is critical to the biogenesis of high density lipoprotein. We quantified levels of ABC1 mRNA, protein, and cholesterol efflux from J774 mouse macrophages +/- exposure to a cAMP analog. Up-regulating ABC1 mRNA correlated to increased cholesterol efflux in a dose- and time-dependent manner. mRNA levels rose after 15 min of exposure while protein levels rose after 1 h, with increased efflux 2-4 h post-treatment. In contrast to cells from wild-type mice, peritoneal macrophages from the Abc1 -/- mouse showed a lower level of basal efflux and no increase with cAMP treatment. The stimulation of efflux exhibits specificity for apoA-I, high density lipoprotein, and other apolipoproteins as cholesterol acceptors, but not for small unilamellar vesicles, bile acid micelles, or cyclodextrin. We have studied a number of cell types and found that while other cell lines express ABC1 constitutively, only J774 and elicited mouse macrophages show a substantial increase of mRNA and efflux with cAMP treatment. ApoA-I-stimulated efflux was detected from the majority of cell lines examined, independent of treatment.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Colesterol/metabolismo , Glicoproteínas/genética , ARN Mensajero/análisis , Transportador 1 de Casete de Unión a ATP , Animales , Línea Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Humanos , Lipoproteínas HDL/biosíntesis , Macrófagos/metabolismo , Ratones , Tionucleótidos/farmacología

RESUMEN

Both in vitro and in vivo studies of scavenger receptor class B type I (SR-BI) have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate the effect of SR-BI on atherogenesis, we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) SR-BI expression in an inbred mouse background hemizygous for a human apolipoprotein (apo) B transgene. Unlike non-HDL cholesterol levels that minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol, whereas the high expression SR-BI transgene was associated with 2-fold decreases in HDL cholesterol as well as dramatic alterations in HDL composition and size including the near absence of alpha-migrating particles as determined by two-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a 2-fold decrease in the development of diet-induced fatty streak lesions compared with the apo B transgenics (4448 +/- 1908 micrometer(2)/aorta to 10133 +/- 4035 micrometer (2)/aorta; p < 0.001), whereas the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692 +/- 7238 micrometer(2)/aorta) but 3-fold greater than the low SR-BI/apo B mice (p < 0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences, illustrating the complexity of the relationship between SR-BI and atherogenesis.

Asunto(s)

Arteriosclerosis/genética , Antígenos CD36/genética , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína , Animales , Aorta/patología , Apolipoproteínas B/sangre , Apolipoproteínas B/genética , Arteriosclerosis/sangre , Antígenos CD36/sangre , HDL-Colesterol/sangre , Dieta Aterogénica , Electroforesis en Gel Bidimensional , Femenino , Regulación de la Expresión Génica/genética , Histocitoquímica , Humanos , Lípidos/sangre , Lipoproteínas HDL/sangre , Hígado/metabolismo , Ratones , Ratones Transgénicos , Miocardio/patología , Receptores de LDL/genética , Receptores Depuradores , Ribonucleasas/metabolismo , Receptores Depuradores de Clase B

RESUMEN

Recently, the human ATP-binding cassette transporter-1 (ABC1) gene has been demonstrated to be mutated in patients with Tangier disease. To investigate the role of the ABC1 protein in an experimental in vivo model, we used gene targeting in DBA-1J embryonic stem cells to produce an ABC1-deficient mouse. Expression of the murine Abc1 gene was ablated by using a nonisogenic targeting construct that deletes six exons coding for the first nucleotide-binding fold. Lipid profiles from Abc1 knockout (-/-) mice revealed an approximately 70% reduction in cholesterol, markedly reduced plasma phospholipids, and an almost complete lack of high density lipoproteins (HDL) when compared with wild-type littermates (+/+). Fractionation of lipoproteins by FPLC demonstrated dramatic alterations in HDL cholesterol (HDL-C), including the near absence of apolipoprotein AI. Low density lipoprotein (LDL) cholesterol (LDL-C) and apolipoprotein B were also significantly reduced in +/- and -/- compared with their littermate controls. The inactivation of the Abc1 gene led to an increase in the absorption of cholesterol in mice fed a chow or a high-fat and -cholesterol diet. Histopathologic examination of Abc1-/- mice at ages 7, 12, and 18 mo demonstrated a striking accumulation of lipid-laden macrophages and type II pneumocytes in the lungs. Taken together, these findings demonstrate that Abc1-/- mice display pathophysiologic hallmarks similar to human Tangier disease and highlight the capacity of ABC1 transporters to participate in the regulation of dietary cholesterol absorption.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Células Espumosas/citología , Glicoproteínas/genética , Lipoproteínas HDL/deficiencia , Mutación , Transportador 1 de Casete de Unión a ATP , Animales , Secuencia de Bases , Colesterol/sangre , Cartilla de ADN , Humanos , Lipoproteínas HDL/sangre , Ratones , Ratones Noqueados

RESUMEN

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI. These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.

Asunto(s)

Antígenos CD36/metabolismo , Proteínas Portadoras/farmacología , Ésteres del Colesterol/metabolismo , Glicoproteínas , Lipasa/metabolismo , Lipoproteínas HDL/metabolismo , Hígado/enzimología , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína/metabolismo , Animales , Antígenos CD36/genética , Células CHO , Proteínas Portadoras/genética , Proteínas de Transferencia de Ésteres de Colesterol , Cricetinae , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Ratones , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B

RESUMEN

It has been proposed that the plasma phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids and cholesterol from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). To evaluate the in vivo role of PLTP in lipoprotein metabolism, we used homologous recombination in embryonic stem cells and produced mice with no PLTP gene expression. Analysis of plasma of F2 homozygous PLTP-/- mice showed complete loss of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sphingomyelin, and partial loss of free cholesterol transfer activities. Moreover, the in vivo transfer of [3H]phosphatidylcholine ether from very-low-density proteins (VLDL) to HDL was abolished in PLTP-/- mice. On a chow diet, PLTP-/- mice showed marked decreases in HDL phospholipid (60%), cholesterol (65%), and apo AI (85%), but no significant change in non-HDL lipid or apo B levels, compared with wild-type littermates. On a high-fat diet, HDL levels were similarly decreased, but there was also an increase in VLDL and LDL phospholipids (210%), free cholesterol (60%), and cholesteryl ester (40%) without change in apo B levels, suggesting accumulation of surface components of TRL. Vesicular lipoproteins were shown by negative-stain electron microscopy of the free cholesterol- and phospholipid-enriched IDL/LDL fraction. Thus, PLTP is the major factor facilitating transfer of VLDL phospholipid into HDL. Reduced plasma PLTP activity causes markedly decreased HDL lipid and apoprotein, demonstrating the importance of transfer of surface components of TRL in the maintenance of HDL levels. Vesicular lipoproteins accumulating in PLTP-/- mice on a high-fat diet could influence the development of atherosclerosis.

Asunto(s)

Proteínas Sanguíneas/genética , Proteínas Portadoras/genética , Lipoproteínas LDL/sangre , Proteínas de la Membrana/genética , Proteínas de Transferencia de Fosfolípidos , Animales , Apolipoproteína A-I/sangre , Apolipoproteínas B/sangre , Colesterol/sangre , Ésteres del Colesterol/sangre , Heterocigoto , Homocigoto , Lipoproteínas/ultraestructura , Lipoproteínas VLDL/metabolismo , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfolípidos/sangre , ARN Mensajero/análisis

RESUMEN

Human lecithin:cholesterol acyltransferase (LCAT) circulates in plasma bound to high density lipoproteins (HDL) and modulates the rate by which cholesteryl ester is transported to the liver. So far, little is known about the regulation of the expression of the LCAT gene. In this study we have defined the cis-elements, identified the trans-acting factors and demonstrated their functional effects and significance in determining transcriptional activity of the proximal LCAT promoter. Using deletion mutants having 5' variable ends (from nucleotides -72 to -27), we have identified the presence of two non-consensus GC-rich regions that stimulate transcription in HepG2 and HeLa cells. These regions designated sites A (-29 to -47) and B (-49 to -65) contain the CCTCC core sequence which in electromobility shift analysis is critical for the formation of two DNA-protein complexes designated I and II. Site-directed mutagenesis suggests that both sites are equally important in promoter activity, and that cooperative interactions between both sites are not required for activity. Electromobility shift and supershift experiments using oligonucleotides spanning sites A and B identified Sp1 and Sp3 as the transcription factors interacting at these sites. To determine the significance and functional effects that Sp1 and Sp3 have in regulating LCAT promoter activity, we performed transfection experiments in Drosophila SL-2 cells as they lack endogenous Sp1 and Sp3. Sp1 but not Sp3 activates the human LCAT promoter and when Sp1 is co-transfected along with Sp3, Sp3 functions as a dose-dependent repressor of Sp1-mediated activation. These findings indicate that Sp1 is capable of transactivating a reporter gene linked to the LCAT promoter containing Sp binding sites and suggests that the levels of Sp3 or the nuclear Sp1/Sp3 ratio may play an important role in determining the transcriptional activity of the LCAT promoter in vivo.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Secuencia de Bases , Células Cultivadas , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Eliminación de Secuencia , Factor de Transcripción Sp3 , Supresión Genética , Transcripción Genética , Activación Transcripcional

RESUMEN

Lecithin:cholesterol acyltransferase (LCAT) is the major determinant of the cholesteryl ester (CE) content of high density lipoprotein (HDL) in plasma. The selective uptake of HDL-CE is postulated to participate in delivery of tissue-derived cholesterol both to the liver and steroidogenic tissues. Recent studies comparing mice with similarly low levels of HDL, due to the absence of either of the two major HDL-associated apolipoproteins apoA-I and apoA-II, suggest that apoA-I is crucial in modulating this process, possibly through interaction with scavenger receptor class B type I (SR-BI). Because of the central role of LCAT in determining the size, lipid composition, and plasma concentration of HDL, we have created LCAT-deficient mice by gene targeting to examine the effect of LCAT deficiency on HDL structure and composition and adrenal cholesterol delivery. The HDL in the LCAT-deficient mice was reduced in its plasma concentration (92%) and CE content (96%). The HDL particles were heterogeneous in size and morphology and included numerous discoidal particles, mimicking those observed in LCAT-deficient humans. The adrenals of the male Lcat (-/-) mice were severely depleted of lipid stores, which was associated with a 2-fold up-regulation of the adrenal SR-BI mRNA. These studies demonstrate that LCAT deficiency, similar to apoA-I deficiency, is associated with a marked decrease in adrenal cholesterol delivery and supports the hypothesis that adrenal SR-BI expression is regulated by the adrenal cholesterol.

Asunto(s)

Glándulas Suprarrenales/metabolismo , Antígenos CD36/metabolismo , Deficiencia de la Lecitina Colesterol Aciltransferasa/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Receptores Inmunológicos , Receptores de Lipoproteína , Regulación hacia Arriba , Glándulas Suprarrenales/patología , Animales , Apolipoproteína A-I/sangre , Southern Blotting , Antígenos CD36/genética , Ésteres del Colesterol/sangre , Marcación de Gen , Lípidos/sangre , Lipoproteínas/sangre , Lipoproteínas HDL/sangre , Masculino , Ratones , Microscopía Electrónica , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B

RESUMEN

Human lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol and is postulated to participate in a physiologic process called reverse cholesterol transport. We have used transgenic mice expressing the human LCAT transgene to study the effect of increased plasma levels of LCAT in each of the proposed steps involved in the reverse cholesterol transport pathway. High density lipoprotein (HDL) from LCAT transgenic mice was 44% more efficient than control mouse HDL in the efflux of cholesterol from human skin fibroblasts. Esterification of cell-derived cholesterol was also markedly increased in mice expressing the human LCAT transgene. The rate of plasma clearance of HDL cholesteryl ester was virtually the same in both types of animals whereas the HDL cholesteryl ester transport rate was significantly increased in mice expressing the human LCAT transgene (152.3 +/- 16.9 micrograms/h vs. 203.1 +/- 30.9 micrograms/h in control and transgenic mice, respectively). Liver cholesteryl ester uptake was significantly increased in mice expressing human LCAT (58.0 +/- 1.4 micrograms/h/g liver vs. 77.9 +/- 1.7 micrograms/h/g liver in control and transgenic mice, respectively). These studies indicate that LCAT modulates the rate by which cholesterol is effluxed from cell membranes onto HDL, esterified, and transported to the liver.

Asunto(s)

Ésteres del Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Animales , Transporte Biológico , Cromatografía en Agarosa , Densitometría , Esterificación , Femenino , Humanos , Lipoproteínas/sangre , Lipoproteínas HDL/química , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Factores de Tiempo

RESUMEN

Familial combined hyperlipidemia (FCHL) is a common inherited lipid disorder, affecting 1 to 2 percent of the population in Westernized societies. Individuals with FCHL have large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) and develop premature coronary heart disease. A mouse model displaying some of the features of FCHL was created by crossing mice carrying the human apolipoprotein C-III (APOC3) transgene with mice deficient in the LDL receptor. A synergistic interaction between the apolipoprotein C-III and the LDL receptor defects produced large quantities of VLDL and LDL and enhanced the development of atherosclerosis. This mouse model may provide clues to the origin of human FCHL.

Asunto(s)

Apolipoproteínas C/genética , Modelos Animales de Enfermedad , Glicoproteínas , Hiperlipidemia Familiar Combinada , Ratones Transgénicos , Receptores de LDL/genética , Animales , Apolipoproteína C-III , Apolipoproteínas B/sangre , Apolipoproteínas E/sangre , Arteriosclerosis/etiología , Proteínas Portadoras/genética , Colesterol/sangre , Proteínas de Transferencia de Ésteres de Colesterol , HDL-Colesterol/sangre , LDL-Colesterol/sangre , VLDL-Colesterol/sangre , Dieta , Susceptibilidad a Enfermedades , Femenino , Humanos , Hiperlipidemia Familiar Combinada/sangre , Hiperlipidemia Familiar Combinada/genética , Hiperlipoproteinemia Tipo IV/genética , Lipoproteínas/sangre , Lipoproteínas VLDL/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de LDL/metabolismo , Transgenes , Triglicéridos/sangre

RESUMEN

Human plasma phospholipid transfer protein (PLTP) circulates bound to high density lipoprotein (HDL) and mediates both net transfer and exchange of phospholipids between different lipoproteins. However, its overall function in lipoprotein metabolism is unknown. To assess the effects of increased plasma levels of PLTP, human PLTP transgenic mice were established using the human PLTP gene driven by its natural promoter. One line of PLTP transgenic mice with moderate expression of PLTP mRNA and protein was obtained. The order of human PLTP mRNA expression in tissues was: liver, kidney, brain, small intestine > lung > spleen > heart, adipose tissue. Western blotting using a human PLTP monoclonal antibody revealed authentic human PLTP (Mr 80 kD) in plasma. Plasma PLTP activity was increased by 29% in PLTP transgenic mice. However, plasma lipoprotein analysis, comparing PLTP transgenic mice to control littermates, revealed no significant changes in the plasma lipoprotein lipids or apolipoproteins. Since previous studies have shown that human cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase only function optimally in human apoAI transgenic mice, the human PLTP transgenic mice were cross-bred with human apoAI transgenic mice. In the human apoAI transgenic background, PLTP expression resulted in increased PLTP activity (47%), HDL phospholipid (26%), cholesteryl ester (24%), free cholesterol (37%), and apoAI (22%). There was a major increase of apoAI in prebeta-HDL (56%) and a small increase in alpha-HDL (14%). The size distribution of HDL particles within alpha- and prebeta-migrating species was not changed. The results suggest that PLTP increases the influx of phospholipid and secondarily cholesterol into HDL, leading to an increase in potentially antiatherogenic prebeta-HDL particles.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Transgénicos/genética , Ratones Transgénicos/metabolismo , Proteínas de Transferencia de Fosfolípidos , Animales , Anticuerpos Monoclonales/inmunología , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Apolipoproteínas/análisis , Apolipoproteínas/sangre , Western Blotting , Encéfalo/metabolismo , Proteínas Portadoras/sangre , Proteínas Portadoras/inmunología , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Clonación Molecular , ADN/análisis , Femenino , Humanos , Intestino Delgado/metabolismo , Riñón/metabolismo , Lipoproteínas/análisis , Lipoproteínas/sangre , Lipoproteínas HDL/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Proteínas de la Membrana/sangre , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Músculos/metabolismo , Miocardio/metabolismo , Hibridación de Ácido Nucleico , ARN/análisis , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Bazo/metabolismo

RESUMEN

Mutagenesis was carried out in human lecithin:cholesterol acyltransferase (LCAT) to generate mutants with stop codons at positions corresponding to amino acids 315, 341, 359, 375, 388, 394, and 398 of the 416-amino acid sequence of the mature enzyme protein. Deletion of the 18 terminal amino acids of the protein was without effect on LCAT phospholipase or acyltransferase activity, or the stability of the protein to denaturation at 37 degrees C. Further deletion led to loss of most of the activity, associated with a 10-fold increase in the rate of denaturation at 37 degrees C. These data indicate that the proline-rich C-terminus of LCAT is not required for effective enzyme activity. The loss of activity that accompanied deletion of residues 394-398 suggests a structural role for these residues, part of a series of predicted beta-sheet sequences in the C-terminal third of the LCAT primary sequence.

Asunto(s)

Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Animales , Apolipoproteína A-I/farmacología , Secuencia de Bases , Células CHO/metabolismo , Colesterol/metabolismo , Ésteres del Colesterol/biosíntesis , Cricetinae , ADN Complementario/genética , Estabilidad de Enzimas , Humanos , Liposomas/metabolismo , Mutagénesis Sitio-Dirigida , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfatidilcolinas/metabolismo , Fosfolipasas/metabolismo , Estructura Secundaria de Proteína , Eliminación de Secuencia

RESUMEN

The effects of cholesteryl ester transfer protein (CETP) on the distribution of apolipoprotein (apo) A-I between high density lipoprotein (HDL) subspecies and its impact on efflux and esterification of cell-derived cholesterol was studied in transgenic mice expressing either the human apoA-I (HuAITg) or both the human apoA-I and CETP (HuAICETPTg) transgenes. The simultaneous expression of the human CETP and apoA-I transgenes induced a 2-fold increase in the proportion of human apoA-I in the prebeta-HDL fraction and 1.4- and 2.2-fold increases in the HDL3a and HDL3c fractions, respectively, at the expense of the larger HDL2b population. HuAICETPTg mouse plasma has a greater ability to cause efflux of cholesterol from 3H-labeled fibroblasts than plasma from HuAITg mice. Furthermore, the LCAT-mediated esterification of cell-derived cholesterol is increased 1.7-fold in mice expressing the human apoA-I and CETP transgenes compared to HuAITg mouse plasma. LCAT activity (measured with an exogenous substrate) was increased 1.4-fold and LCAT mRNA levels were increased 1.3-fold as a result of CETP expression. Taken together, these data indicate that in vivo, the expression of CETP resulted in an increase in the proportion of apoA-I in the prebeta-HDL fraction and a stimulation of the efflux and esterification of cell-derived cholesterol.

Asunto(s)

Apolipoproteína A-I/biosíntesis , Proteínas Portadoras/biosíntesis , Colesterol/metabolismo , Glicoproteínas , Lipoproteínas HDL/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Animales , Apolipoproteína A-I/sangre , Apolipoproteína A-I/genética , Proteínas Portadoras/sangre , Proteínas Portadoras/genética , Línea Celular , Proteínas de Transferencia de Ésteres de Colesterol , Fibroblastos , Lipoproteínas de Alta Densidad Pre-beta , Humanos , Cinética , Ratones , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , ARN Mensajero/biosíntesis , Piel/metabolismo , Transcripción Genética

RESUMEN

Human (Hu) lecithin-cholesterol acyltransferase (LCAT) is a key enzyme in the plasma metabolism of cholesterol. To assess the effects of increased plasma levels of LCAT, four lines of transgenic mice were created expressing a Hu LCAT gene driven by either its natural or the mouse albumin enhancer promoter. Plasma LCAT activity increased from 1.2- to 1.6-fold higher than that found in control mouse plasma. Lipid profiles, upon comparing Hu LCAT transgenics to control animals, revealed a 20 t0 60% increase in total and cholesteryl esters that were mainly present in HDL. The in vivo substrate specificity of Hu LCAT was assessed by creating animals expressing Hu apo AI + Hu LCAT (HuAI/ LCAT), Hu apo AI + Hu apo AII + Hu LCAT (HuAI/ AII/LCAT), and Hu apo AII + Hu LCAT (HuAII/LCAT). Plasma cholesterol was increased up to 4.2-fold in HuAI/ LCAT transgenic mice and twofold in the HuAI/AII/LCAT transgenic mice, compared with HuAI and HuAI/AII transgenic mice. HDL cholesteryl ester levels were increased more than twofold in both the HuAI/LCAT and HuAI/AII/LCAT mice compared with the HuAI, HuAI/AII, and HuLCAT animals. The HDL particles were predominantly larger in the HuAI/LCAT and the HuAI/AII/LCAT mice compared with those in HuAI, HuAII/LCAT, and HuLCAT animals. The increase in LCAT activity in the HuAI/LCAT and HuAI/AII/LCAT mice was associated with 62 and 27% reductions respectively, in the proportion of Hu apo AI in the pre beta-HDL fraction, when compared with HuAI and HuAI/AII transgenic mice. These data demonstrate that moderate increases in LCAT activity are associated with significant changes in lipoprotein cholesterol levels and that Hu LCAT has a significant preference for HDL containing Hu apo AI.

Asunto(s)

Apolipoproteína A-II/metabolismo , Apolipoproteína A-I/metabolismo , Expresión Génica , Fosfatidilcolina-Esterol O-Aciltransferasa/biosíntesis , Animales , Apolipoproteína A-I/biosíntesis , Apolipoproteína A-II/biosíntesis , Cloranfenicol O-Acetiltransferasa/biosíntesis , Colesterol/sangre , Elementos de Facilitación Genéticos , Femenino , Humanos , Cinética , Lipoproteínas/sangre , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Fosfolípidos/sangre , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Triglicéridos/sangre

RESUMEN

Endotoxin (LPS) administration, which mimics infection, stimulates the production of many cytokines, including TNF, that are thought to mediate the alterations in lipid metabolism that occur during infection. The aims of this study were to determine the effect of LPS or TNF administration on plasma LCAT activity and hepatic LCAT mRNA levels in Syrian hamsters. Plasma LCAT activity was decreased 8 h after LPS administration, reached a maximum level of inhibition at 16 h which persisted for at least 24 h, at which time the activity was 53% of control values. The decrease in plasma LCAT activity was first seen at an LPS dose of 0.01 microgram/100 g body weight and reached a maximum at 50-100 micrograms/100 g body weight. The ratio of free to esterified cholesterol in the plasma increased in the LPS-treated animals. Moreover, LPS administration decreased LCAT mRNA levels in the liver. The decrease in hepatic LCAT mRNA levels preceded the decrease in plasma LCAT activity. Additionally, TNF treatment (16.7 micrograms/100 g body weight) decreased plasma LCAT activity by 35% and LCAT mRNA levels in the liver by 60% 16 h after administration. Lastly, in cultured rat H35 hepatocytes, TNF decreased LCAT mRNA levels in the liver by 60% 16 h after administration. Lastly, in cultured rat H35 hepatocytes, TNF decreased LCAT mRNA levels by 50% with a 1/2 maximal dose of approximately 1 ng/ml. Thus, plasma LCAT activity and hepatic mRNA levels are decreased by LPS or TNF treatment. LCAT is a member of a group of proteins that affect lipid and lipoprotein metabolism whose levels are altered during the host's acute phase response.

Asunto(s)

Lipopolisacáridos/farmacología , Hígado/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/sangre , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Cricetinae , Humanos , Cinética , Lípidos/sangre , Lipopolisacáridos/administración & dosificación , Masculino , Mesocricetus , Ratas

RESUMEN

Site-directed mutagenesis was used to generate lecithin-cholesterol acyltransferase (LCAT) species in which individual attachment sites for N-linked oligosaccharide residues were replaced with residues that prevent the attachment of carbohydrate. Mutants at three of four sites retained significant acyltransferase activity, and phospholipase activity in the absence of cholesterol. Mutation at one site (asn272) converted LCAT to a phospholipase generating fatty acids not cholesteryl esters.

Asunto(s)

Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Secuencia de Aminoácidos , Sitios de Unión , Ésteres del Colesterol/metabolismo , ADN/aislamiento & purificación , Escherichia coli/genética , Ácidos Grasos/metabolismo , Glicosilación , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Oligosacáridos/metabolismo , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Fosfolipasas/metabolismo , Reacción en Cadena de la Polimerasa , Especificidad por Sustrato , Transfección

RESUMEN

Triacylglycerol (TG) stored in cytoplasmic lipid droplets of hepatocytes was labeled by in vivo [1-(14)C]oleic acid injection to study the effect of a high-fat diet on its incorporation into very-low-density lipoproteins (VLDL). Compared with the control diet, hepatocytes of fat-fed rats 1) contained 7.6 times more cytoplasmic (floating fat) TG and 1.9 times more endoplasmic reticulum (microsomal) TG; 2) had 8 and 6 times lower TG specific activities in cytoplasm and endoplasmic reticulum, respectively; 3) incorporated 22% less 14C label into hepatocyte esterified lipids (TG, cholesterol, phospholipid); 4) secreted 48 and 33% less radioactive and total VLDL-TG, respectively; 5) oxidized more cytoplasmic TG-fatty acid (FA); and 6) showed a 50% decreased total utilization of stored TG-FA. With both diets, the lysosomal inhibitor chloroquine concomitantly decreased productions of labeled VLDL-TG, CO2, and acid-soluble oxidation products. The decreased incorporation of stored TG into VLDL-TG appreciably contributes to the overall inhibition of hepatic VLDL secretion by fat feeding. It appears to be related to the decreased mobilization rate of stored TG and its increased channelling toward oxidation.

Asunto(s)

Grasas de la Dieta/farmacología , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Triglicéridos/metabolismo , Animales , Radioisótopos de Carbono , Medios de Cultivo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Hígado/citología , Masculino , Oxidación-Reducción , Proteínas/química , Proteínas/metabolismo , Ratas , Ratas Wistar

RESUMEN

The functions of serine residues at positions 181 and 216 of human plasma lecithin:cholesterol acyltransferase have been studied by site-directed mutagenesis. The serine residue at either site was replaced by alanine, glycine, or threonine in LCAT secreted from stably transfected CHO cells. All substitutions at position 181 gave rise to an enzyme product that was normally secreted but had no detectable catalytic activity. On the other hand, all substitutions at position 216 gave active products, whose activity was fully inhibitable by the serine esterase inhibitor diisopropyl fluorophosphate (DFP). A secondary (although not direct) role for serine-216 was indicated by a 14-fold increase in catalytic rate when this residue was substituted by alanine. Sequence comparison with other lipases suggests that serine-216 may be at or near the hinge of a helical flap displaced following substrate binding. These data strengthen the structural-functional relationship between LCAT and other lipases.

Asunto(s)

Mutagénesis Sitio-Dirigida , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Serina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Línea Celular , Cricetinae , Cricetulus , Femenino , Humanos , Isoflurofato , Cinética , Datos de Secuencia Molecular , Peso Molecular , Ovario , Fosfatidilcolina-Esterol O-Aciltransferasa/antagonistas & inhibidores , Relación Estructura-Actividad

RESUMEN

Native lecithin-cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol acyltransferase; phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) protein, and LCAT in which either or both of the enzyme free cysteines had been replaced with glycine residues by site-directed mutagenesis, has been expressed in cultured Chinese hamster ovary cells stably transfected with the human LCAT gene. The mass of LCAT secreted, determined by immunoassay, did not differ in the native and mutant species. LCAT specific activity was also unchanged in the mutant species. In particular, the cysteine-free double mutant, in which Cys-31 and Cys-184 had both been replaced, was fully active in the synthesis of cholesteryl esters. This result is not consistent with a catalytic role for LCAT free cysteine residues. The classical inhibitor of LCAT activity, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), which strongly (89%) inhibited the native enzyme, had partial (45%) inhibitory activity with mutant enzyme species containing a single -SH residue, while the double mutant was not significantly inhibited by DTNB. These data are interpreted to suggest that Cys-31 and Cys-184 are vicinal both to each other and to the "interfacial binding site" at residues 177-182, and that DTNB exerts its effect by steric inhibition.

Asunto(s)

Cisteína , Mutagénesis Sitio-Dirigida , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carcinoma Hepatocelular , Línea Celular , Humanos , Cinética , Neoplasias Hepáticas , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Plásmidos , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Mapeo Restrictivo , Transfección