RESUMEN
Supplementation with antioxidant carotenoids is a therapeutic strategy to protect against age-related macular degeneration (AMD); however, the transport mechanism of carotenoids from the liver to the retina is still not fully understood. Here, we investigate if HDL serves as the primary transporter for the macular carotenoids. ApoA-I, the key apolipoprotein of HDL, was genetically deleted from BCO2 knockout (Bco2-/-) mice, a macular pigment mouse model capable of accumulating carotenoids in the retina. We then conducted a feeding experiment with a mixed carotenoid chow (lutein:zeaxanthin:ß-carotene = 1:1:1) for one month. HPLC data demonstrated that the total carotenoids were increased in the livers but decreased in the serum, retinal pigment epithelium (RPE)/choroids, and retinas of ApoA-I-/-/Bco2-/- mice compared to Bco2-/- mice. In detail, ApoA-I deficiency caused a significant increase of ß-carotene but not lutein and zeaxanthin in the liver, decreased all three carotenoids in the serum, blocked the majority of zeaxanthin and ß-carotene transport to the RPE/choroid, and dramatically reduced ß-carotene and zeaxanthin but not lutein in the retina. Furthermore, surface plasmon resonance spectroscopy (SPR) data showed that the binding affinity between ApoA-I and ß-carotene â« zeaxanthin > lutein. Our results show that carotenoids are transported from the liver to the eye mainly by HDL, and ApoA-I may be involved in the selective delivery of macular carotenoids to the RPE.
Asunto(s)
Apolipoproteína A-I/genética , Carotenoides/metabolismo , Dioxigenasas/genética , Lipoproteínas HDL2/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Carotenoides/sangre , Modelos Animales de Enfermedad , Humanos , Hígado , Luteína/metabolismo , Degeneración Macular/metabolismo , Ratones , Ratones Noqueados , Retina , Zeaxantinas/metabolismo , beta Caroteno/metabolismoRESUMEN
Triglyceride hydrolysis by lipoprotein lipase (LPL), regulated by apolipoproteins C-II (apoC-II) and C-III (apoC-III), is essential for maintaining normal lipid homeostasis. During triglyceride lipolysis, the apoCs are known to be transferred from very low-density lipoprotein (VLDL) to high-density lipoprotein (HDL), but the detailed mechanisms of this transfer remain unclear. In this study, we investigated the extent of the apoC transfers and their distribution in HDL subfractions, HDL2 and HDL3. Each HDL subfraction was incubated with VLDL or biotin-labeled VLDL, and apolipoproteins and lipids in the re-isolated HDL were quantified using western blotting and high-performance liquid chromatography (HPLC). In consequence, incubation with VLDL showed the increase of net amount of apoC-II and apoC-III in the HDL. HPLC analysis revealed that the biotin-labeled apolipoproteins, including apoCs and apolipoprotein E, were preferably transferred to the larger HDL3. No effect of cholesteryl ester transfer protein inhibitor on the apoC transfers was observed. Quantification of apoCs levels in HDL2 and HDL3 from healthy subjects (n = 8) showed large individual differences between apoC-II and apoC-III levels. These results suggest that both apoC-II and apoC-III transfer disproportionately from VLDL to HDL2 and the larger HDL3, and these transfers might be involved in individual triglyceride metabolism.
Asunto(s)
Apolipoproteína C-III/metabolismo , Apolipoproteína C-II/metabolismo , Lipoproteínas HDL2/metabolismo , Lipoproteínas HDL3/metabolismo , Lipoproteínas LDL/metabolismo , Voluntarios Sanos , HumanosRESUMEN
Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.
Asunto(s)
Bioensayo/métodos , HDL-Colesterol/análisis , HDL-Colesterol/sangre , Pruebas de Enzimas/métodos , Lipoproteínas HDL3/análisis , Lipoproteínas HDL3/sangre , Calibración , Colesterol Oxidasa/química , Colesterol Oxidasa/metabolismo , HDL-Colesterol/metabolismo , Sulfato de Dextran/química , Humanos , Lipoproteínas HDL2/análisis , Lipoproteínas HDL2/sangre , Lipoproteínas HDL2/metabolismo , Lipoproteínas HDL3/metabolismo , Lipoproteínas LDL/análisis , Lipoproteínas LDL/sangre , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/análisis , Lipoproteínas VLDL/sangre , Lipoproteínas VLDL/metabolismo , Compuestos de Magnesio/química , Nitratos/química , Tamaño de la Partícula , Polietilenglicoles/química , Reproducibilidad de los Resultados , Esterol Esterasa/química , Esterol Esterasa/metabolismo , UltracentrifugaciónRESUMEN
BACKGROUND & AIMS: High-density lipoprotein cholesterol (HDL-C) levels are reduced in patients with chronic liver disease and inversely correlate with disease severity. During acute conditions such as sepsis, HDL-C levels decrease rapidly and HDL particles undergo profound changes in their composition and function. We aimed to determine whether indices of HDL quantity and quality associate with progression and survival in patients with advanced liver disease. METHODS: HDL-related biomarkers were studied in 508 patients with compensated or decompensated cirrhosis (including acute-on-chronic liver failure [ACLF]) and 40 age- and gender-matched controls. Specifically, we studied levels of HDL-C, its subclasses HDL2-C and HDL3-C, and apolipoprotein A1 (apoA-I), as well as HDL cholesterol efflux capacity as a metric of HDL functionality. RESULTS: Baseline levels of HDL-C and apoA-I were significantly lower in patients with stable cirrhosis compared to controls and were further decreased in patients with acute decompensation (AD) and ACLF. In stable cirrhosis (n = 228), both HDL-C and apoA-I predicted the development of liver-related complications independently of model for end-stage liver disease (MELD) score. In patients with AD, with or without ACLF (n = 280), both HDL-C and apoA-I were MELD-independent predictors of 90-day mortality. On ROC analysis, both HDL-C and apoA-I had high diagnostic accuracy for 90-day mortality in patients with AD (AUROCs of 0.79 and 0.80, respectively, similar to that of MELD 0.81). On Kaplan-Meier analysis, HDL-C <17 mg/dl and apoA-I <50 mg/dl indicated poor short-term survival. The prognostic accuracy of HDL-C was validated in a large external validation cohort of 985 patients with portal hypertension due to advanced chronic liver disease (AUROCs HDL-C: 0.81 vs. MELD: 0.77). CONCLUSION: HDL-related biomarkers are robust predictors of disease progression and survival in chronic liver failure. LAY SUMMARY: People who suffer from cirrhosis (scarring of the liver) have low levels of cholesterol carried by high-density lipoproteins (HDL-C). These alterations are connected to inflammation, which is a problem in severe liver disease. Herein, we show that reduced levels of HDL-C and apolipoprotein A-I (apoA-I, the main protein carried by HDL) are closely linked to the severity of liver failure, its complications and survival. Both HDL-C and apoA-I can be easily measured in clinical laboratories and are as good as currently used prognostic scores calculated from several laboratory values by complex formulas.
Asunto(s)
Insuficiencia Hepática Crónica Agudizada , Apolipoproteína A-I , HDL-Colesterol , Lipoproteínas HDL2 , Lipoproteínas HDL3 , Cirrosis Hepática , Insuficiencia Hepática Crónica Agudizada/sangre , Insuficiencia Hepática Crónica Agudizada/diagnóstico , Insuficiencia Hepática Crónica Agudizada/epidemiología , Insuficiencia Hepática Crónica Agudizada/metabolismo , Apolipoproteína A-I/sangre , Apolipoproteína A-I/metabolismo , Biomarcadores , HDL-Colesterol/sangre , HDL-Colesterol/metabolismo , Estudios Transversales , Progresión de la Enfermedad , Europa (Continente)/epidemiología , Femenino , Humanos , Lipoproteínas HDL2/sangre , Lipoproteínas HDL2/metabolismo , Lipoproteínas HDL3/sangre , Lipoproteínas HDL3/metabolismo , Cirrosis Hepática/sangre , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/epidemiología , Masculino , Persona de Mediana Edad , Puntuaciones en la Disfunción de Órganos , Valor Predictivo de las Pruebas , Pronóstico , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: In order to identify putative biomarkers in lipoprotein, we compared lipid and lipoprotein properties between rheumatoid arthritis (RA) patients and control with similar age. METHODS: We analyzed four classes of lipoproteins (VLDL, LDL, HDL2, HDL3) from both male (n = 8, 69±4 year-old) and female (n = 25, 53±7 year-old) rheumatoid arthritis (RA) patients as well as controls with similar age (n = 13). RESULTS: Although RA group showed normal levels of total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol, and glucose, however, the RA group showed significantly reduced high-density lipoprotein (HDL)-C level and ratio of HDL-C/TC. The RA group showed significantly elevated levels of blood triglyceride (TG), uric acid, and cholesteryl ester transfer protein (CETP) activity. The RA group also showed elevated levels of advanced glycated end (AGE) products in all lipoproteins and severe aggregation of apoA-I in HDL. As CETP activity and TG contents were 2-fold increased in HDL from RA group, paraoxonase activity was reduced upto 20%. Electron microscopy revealed that RA group showed much less HDL2 particle number than control. LDL from the RA group was severely oxidized and glycated with greater fragmentation of apo-B, especially in female group, it was more atherogenic via phagocytosis. CONCLUSION: Lipoproteins from the RA patients showed severely altered structure with impaired functionality, which is very similar to that observed in coronary heart patients. These dysfunctional properties in lipoproteins from the RA patients might be associated with high incidence of cardiovascular events in RA patients.
Asunto(s)
Artritis Reumatoide/inmunología , Lipoproteínas HDL2/metabolismo , Lipoproteínas HDL2/ultraestructura , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/ultraestructura , Anciano , Artritis Reumatoide/metabolismo , Línea Celular , Femenino , Humanos , Lípidos/análisis , Lipoproteínas HDL/inmunología , Lipoproteínas HDL2/inmunología , Macrófagos/metabolismo , Macrófagos/ultraestructura , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Oxidación-ReducciónRESUMEN
OBJECTIVE: Oral mitotane (o,p'-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC). AIM: Serum mitotane concentrations >14 âmg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p'-dichlorodiphenylacetic acid (o,p'-DDA) and o,p'-dichlorodiphenyldichloroethane (o,p'-DDE). DESIGN: Lipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI-H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins. RESULTS: Chyle of the index patient contained 197 âmg/ml mitotane, 53 âmg/ml o,p'-DDA, and 51â mg/l o,p'-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction. DISCUSSION: Mitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p'-DDE was similarly distributed, but 87.9±4.2% of o,p'-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI-H295 cells and reduced ER stress marker gene expression. CONCLUSION: Mitotane absorption involves chylomicron binding. High concentrations of o,p'-DDA and o,p'-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/tratamiento farmacológico , Carcinoma Corticosuprarrenal/tratamiento farmacológico , Antineoplásicos Hormonales/metabolismo , Quilomicrones/metabolismo , Lipoproteínas/metabolismo , Mitotano/metabolismo , Adulto , Anciano , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Quilo/química , Estudios de Cohortes , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Absorción Gastrointestinal , Humanos , Lipoproteínas/farmacología , Lipoproteínas HDL2/metabolismo , Lipoproteínas IDL/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Masculino , Persona de Mediana Edad , Mitotano/análogos & derivados , Mitotano/farmacología , Mitotano/uso terapéuticoRESUMEN
OBJECTIVES: Cholesterol efflux has been thought to be the main and basic mechanism by which free cholesterol is transferred from extra hepatic cells to the liver or intestine for excretion. Salvianolic acid B (Sal B) has been widely used for the prevention and treatment of atherosclerotic diseases. Here, we sought to investigate the effects of Sal B on the cholesterol efflux in THP-1 macrophages. METHODS: After PMA-stimulated THP-1 cells were exposed to 50 mg/L of oxLDL and [(3)H] cholesterol (1.0 µCi/mL) for another 24 h, the effect of Sal B on cholesterol efflux was evaluated in the presence of apoA-1, HDL2 or HDL3. The expression of ATP binding cassette transporter A1 (ABCA1), peroxisome proliferator-activated receptor-gamma (PPAR-γ), and liver X receptor-alpha (LXRα) was detected both at protein and mRNA levels in THP-1 cells after the stimulation of Sal B. Meanwhile, specific inhibition of PPAR-γ and LXRα were performed to investigate the mechanism. RESULTS: The results showed that Sal B significantly accelerated apoA-I- and HDL-mediated cholesterol efflux in both dose- and time-dependent manners. Meanwhile, Sal B treatment also enhanced the expression of ABCA1 at both mRNA and protein levels. Then the data demonstrated that Sal B increased the expression of PPAR-γ and LXRα. And the application of specific agonists and inhibitors of further confirmed that Sal exert the function through PPAR-γ and LXRα. CONCLUSION: These results demonstrate that Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRα pathway.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Benzofuranos/farmacología , Colesterol/metabolismo , Receptores Nucleares Huérfanos/metabolismo , PPAR gamma/metabolismo , Transportador 1 de Casete de Unión a ATP/genética , Apolipoproteína A-I/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Medicamentos Herbarios Chinos/farmacología , Humanos , Lipoproteínas HDL2/metabolismo , Lipoproteínas HDL3/metabolismo , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/antagonistas & inhibidores , Receptores Nucleares Huérfanos/genética , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
RATIONALE: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied. OBJECTIVE: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. METHODS AND RESULTS: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. CONCLUSIONS: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/fisiología , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Transportador 1 de Casete de Unión a ATP/antagonistas & inhibidores , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador 1 de Casete de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células CHO , Línea Celular , Cricetinae , Cricetulus , Células Espumosas/metabolismo , Silenciador del Gen , Humanos , Lipoproteínas/deficiencia , Lipoproteínas/fisiología , Lipoproteínas HDL2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de la Partícula , Proteínas Recombinantes de Fusión/metabolismo , Enfermedad de Tangier/enzimología , Enfermedad de Tangier/genéticaRESUMEN
Liver X Receptors (LXRs) α and ß are oxysterol-activated nuclear receptors involved in the control of lipid metabolism and inflammation. Pharmacological activation of LXR is promising in the treatment of atherosclerosis since it can promote cholesterol efflux from macrophages and prevent foam cell formation. However, the development of LXR agonists has been limited by undesirable side-effects such as hepatic steatosis mediated by LXRα activation. Therefore, it has been proposed that targeting LXRα activators to extrahepatic tissues or using LXRß-specific activators could be used as alternative strategies. It is not clear whether these molecules will retain the full atheroprotective potential of non-selective agonists. Our aim was therefore to determine the contribution of LXRα and LXRß to the control of cholesterol efflux in human macrophages. LXRα and/or LXRß expression was suppressed by small interfering RNAs in human primary macrophages treated or not with synthetic LXRα/ß dual agonists T0901317 and GW3965. We observed that LXRß silencing had no detectable impact on the expression of LXR-target genes such as ABCA1 and ABCG1. Moreover it did not affect cholesterol efflux. In contrast, LXRα silencing reduced the response of these LXR-target genes to LXR agonist and inhibited cholesterol efflux to ApoA-I, HDL2 or to endogenous ApoE. Importantly, no differences were observed between LXRα and LXRα/ß knockdown conditions. Altogether, our data demonstrate that LXRß activation is unable to maintain maximal cholesterol efflux capacities in human primary macrophages when LXRα expression is impaired. In contrast to earlier mouse studies, LXRα levels appear as a limiting factor for macrophage cholesterol efflux in humans.
Asunto(s)
Colesterol/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Benzoatos/farmacología , Bencilaminas/farmacología , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Hidrocarburos Fluorados/farmacología , Lipoproteínas HDL2/metabolismo , Receptores X del Hígado , Macrófagos/efectos de los fármacos , Receptores Nucleares Huérfanos/agonistas , Receptores Nucleares Huérfanos/genética , Cultivo Primario de Células , ARN Interferente Pequeño/genética , Sulfonamidas/farmacologíaRESUMEN
OBJECTIVE: In the deep microenvironments of advanced human atherosclerotic lesions, the intimal fluid becomes acidic. We examined the effect of an acidic extracellular pH on cholesterol removal (efflux) from primary human macrophages. METHODS AND RESULTS: When cholesterol efflux from acetyl-low-density lipoprotein-loaded macrophages to various cholesterol acceptors was evaluated at pH 7.5, 6.5, or 5.5, the lower the pH the more was cholesterol efflux reduced. The reduction of efflux to lipid-free apolipoprotein A-I was stronger than to high-density lipoprotein(2) or to plasma. Cholesterol efflux to every acceptor was severely compromised also at neutral pH when the macrophages had been loaded with cholesterol at acidic pH, or when both loading and efflux were carried out at acidic pH. Compatible with these observations, the typical upregulation of ABCA1 and ABCG1 mRNA levels in macrophages loaded with cholesterol at neutral pH was rapidly attenuated in acidic medium. The secondary structure of apolipoprotein A-I did not changed over the pH range studied, supporting the notion that the inhibitory effect of acidic pH on cholesterol efflux rather impaired the ability of the foam cells to facilitate ABCA1-mediated cholesterol release. Secretion of apolipoprotein E from the foam cells was fully inhibited when the pH was 5.5, which further reduced cholesterol efflux. CONCLUSIONS: An acidic pH reduces cholesterol efflux via different pathways and particularly impairs the function of the ABCA1 transporter. The pH-sensitive function of human macrophage foam cells in releasing cholesterol may accelerate lipid accumulation in deep areas of advanced atherosclerotic plaques where the intimal fluid is acidic.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol/metabolismo , Células Espumosas/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células Cultivadas , Regulación hacia Abajo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Lipoproteínas HDL2/metabolismo , Lipoproteínas LDL/metabolismo , Estructura Secundaria de Proteína , ARN Mensajero/metabolismoRESUMEN
OBJECTIVES: In this study we examined the relationship of oxidative stress and hyperglycaemia to antioxidative capacity of high-density cholesterol (HDL-C) particles in type 2 diabetes mellitus (DM). DESIGN AND METHODS: Oxidative stress status parameters (superoxide anion (O2(-)), superoxide dismutase (SOD) activity and paraoxonase (PON1) status were assessed in 114 patients with type 2 DM and 91 healthy subjects. HDL particle diameters were determined by non-denaturing polyacrylamide gradient (3-31%) gel electrophoresis. RESULTS: Patients had significantly higher concentrations of oxidative stress parameter O2(-)(p<0.001) and antioxidative defence, SOD activity (p<0.001). Paraoxonase activity was significantly lower in diabetics (p<0.001). The PON1(192) phenotype distribution among study groups was not significantly different. HDL 3 phenotype was significantly prevalent among patients (p<0.001). Paraoxonase activity was significantly lower in patients with predominantly HDL 2 particles than in controls. CONCLUSIONS: The results of our current study indicate that the diabetic HDL 2 phenotype is associated with hyperglycaemia, lower PON1 activity and elevated oxidative stress.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/enzimología , Hiperglucemia/complicaciones , Hiperglucemia/enzimología , Lipoproteínas HDL2/metabolismo , Estrés Oxidativo , Glucemia/metabolismo , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Femenino , Humanos , Hiperglucemia/sangre , Hiperglucemia/patología , Masculino , Persona de Mediana Edad , Tamaño de la Partícula , Fenotipo , Estadísticas no ParamétricasRESUMEN
Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodeling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular free cholesterol (FC) efflux, cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer, and selective hepatic CE uptake during the postprandial phase in subjects displaying type IIB hyperlipidemia (n = 16). Postprandial, large HDL2 displayed an enhanced capacity to mediate FC efflux via both scavenger receptor class B type I (SR-BI)-dependent (+12%; P < 0.02) and ATP binding cassette transporter G1 (ABCG1)-dependent (+31%; P < 0.008) pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; P < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB lipoproteins and with attenuated capacity (-17%; P < 0.02) of total HDL to deliver CE to hepatic cells. Postprandial lipemia enhanced SR-BI and ABCG1-dependent efflux to large HDL2 particles. However, postprandial lipemia is equally associated with deleterious features by enhancing formation of CE-enriched, triglyceride-rich lipoprotein particles through the action of CETP and by reducing the direct return of HDL-CE to the liver.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antígenos CD36/metabolismo , Colesterol/metabolismo , Hiperlipidemias/metabolismo , Lipoproteínas HDL2/metabolismo , Tamaño de la Partícula , Periodo Posprandial , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Adulto , Transporte Biológico , Ésteres del Colesterol/metabolismo , Femenino , Humanos , Hiperlipidemias/sangre , Hiperlipidemias/patología , Hiperlipidemias/fisiopatología , Cinética , Lipoproteínas HDL2/sangre , Lipoproteínas HDL2/química , Hígado/citología , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Triglicéridos/sangreRESUMEN
Krüppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-containing transcription factor. In the present study, peripheral blood mononuclear cells and phorbol 12-myristate 13-acetate-differentiated THP-1 cells were treated with oxidized low-density lipoproteins and high-density lipoproteins to determine the expression of KLF4 and scavenger receptor class B type I (SR-BI). A full-length cDNA of KLF4 or short interference RNA against KLF4 was transfected into THP-1 cells, and the subsequent expressions of SR-BI were analysed by real-time PCR and western blot. The binding and transcriptional activities of KLF4 to the SR-BI promoter were detected by electrophoretic mobility shift assay, chromatin immunoprecipitation assay and luciferase reporter assay. The results showed that induction of KLF4 by high-density lipoproteins could promote the expression of SR-BI, resulting from the binding to putative KLF4 binding element on the promoter of SR-BI. All results indicate a potential function of KLF4 in the pathogenesis of atherosclerosis through the regulation effect on atherosclerotic-related genes.
Asunto(s)
Antígenos CD36/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Lipoproteínas HDL3/metabolismo , Macrófagos/metabolismo , Regulación hacia Arriba , Aterosclerosis/fisiopatología , Antígenos CD36/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Leucocitos Mononucleares/metabolismo , Lipoproteínas HDL2/metabolismo , Lipoproteínas LDL/metabolismo , Monocitos/efectos de los fármacos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Although HDL-mediated cholesterol transport to the liver is well studied, cholesterol efflux from hepatocytes back to HDL is less well understood. Real-time imaging of efflux of 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), which is poorly esterified, and [(3)H]cholesterol, which is extensively esterified, from cultured primary hepatocytes of wild-type and sterol carrier protein-2 (SCP-2) gene-ablated mice showed that 1) NBD-cholesterol efflux was affected by the type of lipoprotein acceptor, i.e., HDL3 over HDL2; 2) NBD-cholesterol efflux was rapid (detected in 1-2 min) and resolved into fast [half time (t((1/2))) = 2.4 min, 6% of total] and slow (t((1/2)) = 26.5 min, 94% of total) pools, consistent with protein- and vesicle-mediated cholesterol transfer, respectively; 3) SCP-2 gene ablation increased efflux of NBD-cholesterol, as well as [(3)H]cholesterol, albeit less so due to competition by esterification of [(3)H]cholesterol, but not NBD-cholesterol; and 4) SCP-2 gene ablation increased initial rate (2.3-fold) and size (9.7-fold) of rapid effluxing sterol, suggesting an increased contribution of molecular cholesterol transfer. In addition, colocalization, double-immunolabeling fluorescence resonance energy transfer, and electron microscopy, as well as cross-linking coimmunoprecipitation, indicated that SCP-2 directly interacted with the HDL receptor, scavenger receptor class B type 1 (SRB1), in hepatocytes. Other membrane proteins in cholesterol efflux [SRB1 and ATP-binding cassettes (ABC) A-1, ABCG-1, ABCG-5, and ABCG-8] and several soluble/vesicle-associated proteins facilitating intracellular cholesterol trafficking (StARDs, NPCs, ORPs) were not upregulated. However, loss of SCP-2 elicited twofold upregulation of liver fatty acid-binding protein (L-FABP), a protein with lower affinity for cholesterol but higher cytosolic concentration than SCP-2. Ablation of SCP-2 and L-FABP decreased HDL-mediated NBD-cholesterol efflux. These results indicate that SCP-2 expression plays a significant role in HDL-mediated cholesterol efflux by regulating the size of rapid vs. slow cholesterol efflux pools and/or eliciting concomitant upregulation of L-FABP in cultured primary hepatocytes.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Proteínas Portadoras/metabolismo , Colesterol/análogos & derivados , Hepatocitos/metabolismo , Lipoproteínas HDL3/metabolismo , 4-Cloro-7-nitrobenzofurazano/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/genética , Técnicas de Cultivo de Célula , Células Cultivadas , Colesterol/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Inactivación de Genes , Inmunoprecipitación , Cinética , Lipoproteínas HDL2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Fosfoproteínas/metabolismo , Unión Proteica , Receptores Depuradores de Clase B/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
BACKGROUND: Alcohol consumption is associated with increased serum high density lipoprotein (HDL) cholesterol levels and a decreased risk for the development of atherosclerosis. However, the effects of heavy alcohol intake on reverse cholesterol transport, one of the key anti-atherogenic processes related to HDL, are poorly known. METHODS: The ability of total HDL as well as HDL(2) and HDL(3) subclasses to promote cholesterol efflux from (3)H-cholesterol-labeled RAW 264.7 macrophages was studied among 6 heavy alcohol drinkers and 6 controls. Distribution of HDL subclasses was analyzed by 4 to 30% native gradient gels. Serum phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) activities were analyzed among several other biochemical measures. RESULTS: Cholesterol efflux to HDL(2) of heavy drinkers was 22% (p = 0.025) higher relative to controls. The increase in HDL(2) phospholipids, with a concomitant 2-fold (p = 0.055) increase in large HDL(2b) particles, was associated with enhanced cholesterol efflux to HDL(2). Interestingly, the cholesterol efflux to HDL(3) did not differ between the 2 study groups. These findings may be partially explained by a decreased CETP activity (-26%, p = 0.037) and an increased PLTP activity (39%, p = 0.045) in heavy drinkers. CONCLUSIONS: The increased cholesterol efflux potential of HDL(2) is most likely an anti-atherogenic feature linked to heavy alcohol consumption. The cholesterol efflux and HDL phospholipids also associated strongly within the whole study group (r(s) = 0.910, p Asunto(s)
Consumo de Bebidas Alcohólicas/sangre
, Colesterol/metabolismo
, Lipoproteínas HDL2/metabolismo
, Macrófagos/metabolismo
, Adulto
, Alcoholismo/sangre
, Animales
, Línea Celular
, Proteínas de Transferencia de Ésteres de Colesterol/sangre
, HDL-Colesterol/sangre
, Lipoproteínas de Alta Densidad Pre-beta/sangre
, Humanos
, Lipoproteínas HDL/sangre
, Lipoproteínas HDL2/sangre
, Lipoproteínas HDL3/sangre
, Masculino
, Ratones
, Persona de Mediana Edad
, Fosfatidilcolina-Esterol O-Aciltransferasa/sangre
, Proteínas de Transferencia de Fosfolípidos/sangre
RESUMEN
Humans have two major high density lipoprotein (HDL) sub-fractions, HDL(2) and HDL(3), whereas mice have a monodisperse HDL profile. Epidemiological evidence has suggested that HDL(2) is more atheroprotective; however, currently there is no direct experimental evidence to support this postulate. The amino acid sequence of apoA-I is a primary determinant of HDL subclass formation. The majority of the alpha-helical repeats in human apoA-I are proline-punctuated. A notable exception is the boundary between helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain and the C-terminal hydrophobic domain. In this study we ask whether the substitution of a proline-containing sequence (PCS) separating other helices in human apoA-I for the non-proline-containing sequence (NPCS) between helices 7 and 8 (residues 184-190) influences HDL subclass association. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL(2). In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL(3). Thus, the specific amino acid sequence between helices 7 and 8 influences HDL subclass association. The wild-type and mutant proteins exhibited similar physicochemical properties except that the two mutants displayed greater lipid-associated stability versus wild-type human apoA-I. These results focus new attention on the influence of the boundary between helices 7 and 8 on the properties of apoA-I. The expression of these mutants in mice may result in the preferential generation of HDL(2) or HDL(3) and allow us to examine experimentally the anti-atherogenicity of the HDL subclasses.
Asunto(s)
Apolipoproteína A-I/química , Lipoproteínas HDL2/química , Lipoproteínas HDL3/química , Secuencia de Aminoácidos/genética , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Humanos , Lipoproteínas HDL2/genética , Lipoproteínas HDL2/metabolismo , Lipoproteínas HDL3/genética , Lipoproteínas HDL3/metabolismo , Ratones , Mutación , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Especificidad de la EspecieRESUMEN
We evaluated the impact of gender differences in both the quantitative and qualitative features of HDL subspecies on cellular free cholesterol efflux through the scavenger receptor class B type I (SR-BI), ABCA1, and ABCG1 pathways. For that purpose, healthy subjects (30 men and 26 women) matched for age, body mass index, triglyceride, apolipoprotein A-I, and high density lipoprotein-cholesterol (HDL-C) levels were recruited. We observed a significant increase (+14%; P < 0.03) in the capacity of whole sera from women to mediate cellular free cholesterol efflux via the SR-BI-dependent pathway compared with sera from men. Such enhanced efflux capacity resulted from a significant increase in plasma levels of large cholesteryl ester-rich HDL2 particles (+20%; P < 0.04) as well as from an enhanced capacity (+14%; P < 0.03) of these particles to mediate cellular free cholesterol efflux via SR-BI. By contrast, plasma from men displayed an enhanced free cholesterol efflux capacity (+31%; P < 0.001) via the ABCA1 transporter pathway compared with that from women, which resulted from a 2.4-fold increase in the plasma level of prebeta particles (P < 0.008). Moreover, in women, SR-BI-mediated cellular free cholesterol efflux was significantly correlated with plasma HDL-C (r = 0.72, P < 0.0001), whereas this relationship was not observed in men. In conclusion, HDL-C level may not represent the absolute indicator of the efficiency of the initial step of the reverse cholesterol transport.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol/metabolismo , Receptores Depuradores de Clase B/metabolismo , Transportador 1 de Casete de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Femenino , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL2/metabolismo , Masculino , Factores SexualesRESUMEN
OBJECTIVE: The present study is a comparative investigation of cellular lipid mobilization and efflux to lipid-free human apoA-I and apoA-I(Milano), reconstituted high-density lipoprotein (rHDL) particles containing these proteins and serum isolated from mice expressing human apoA-I or apoA-I(Milano). METHODS AND RESULTS: Cholesterol and phospholipid efflux to these acceptors was measured in cell systems designed to assess the contributions of ATP-binding cassette A1 (ABCA1), scavenger receptor type BI (SRBI), and cellular lipid content to cholesterol and phospholipid efflux. Acceptors containing the Milano variant of apoA-I showed no functional increase in lipid efflux in all assays when compared with wild-type apoA-I. In fact, in some systems, acceptors containing the Milano variant of apoA-I promoted significantly less efflux than the acceptors containing wild-type apoA-I (apoA-I(wt)). Additionally, intracellular cholesteryl ester hydrolysis in macrophage foam cells was not different in the presence of either apoA-I(Milano) or apoA-I(wt). CONCLUSION: Collectively these studies suggest that if the Milano variant of apoA-I offers greater atheroprotection than wild-type apoA-I, it is not attributable to greater cellular lipid mobilization.
Asunto(s)
Apolipoproteína A-I/fisiología , Movilización Lipídica/fisiología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Células Cultivadas , Ésteres del Colesterol/metabolismo , Humanos , Lipoproteínas HDL2/metabolismo , Macrófagos Peritoneales/fisiología , Ratones , Ratones Transgénicos , Receptores Depuradores de Clase B/metabolismoRESUMEN
Cholesteryl ester transfer protein (CETP) regulates human lipoprotein metabolism. Because reducing CETP increases plasma HDL, CETP inhibitors are currently being investigated for their pharmacologic value. However, complete CETP deficiency may have undesirable consequences. In contrast, based on previous studies with purified components, we hypothesized that partial CETP inhibition, which will still elevate HDL, may induce beneficial changes in plasma lipid metabolism. To address this, CETP activity in human plasma was variably inhibited with monoclonal antibody. In control plasma, VLDL to LDL lipid transfer was >2-fold higher than to HDL(3) with lipid transfer to HDL(2) intermediate. However, individual lipid transfer events were uniquely sensitive to CETP suppression such that when CETP activity was inhibited by 60%, lipid transfer from VLDL to LDL, HDL(2) and HDL(3) were equal. The ratio of lipid transfers to LDL versus HDL declined linearly with CETP inhibition. In mass lipid transfer experiments, 25-50% inhibition of CETP significantly reduced lipid flux between VLDL and LDL but minimally affected cholesteryl ester (CE) loss from HDL. Complete CETP inhibition did not reduce cholesterol esterification rates but completely blocked the delivery of new CE to VLDL, whereas, 50% inhibition of CETP reduced this CE flux to VLDL by <20%. Thus, inhibition of CETP by Asunto(s)
Anticuerpos Bloqueadores/farmacología
, Transporte Biológico Activo/efectos de los fármacos
, Proteínas de Transferencia de Ésteres de Colesterol/efectos de los fármacos
, Metabolismo de los Lípidos/efectos de los fármacos
, Lipoproteínas HDL2/metabolismo
, Lipoproteínas HDL3/metabolismo
, Línea Celular
, Esterificación/efectos de los fármacos
, Humanos
, Lipoproteínas HDL2/efectos de los fármacos
, Lipoproteínas HDL3/efectos de los fármacos
, Lipoproteínas LDL/efectos de los fármacos
, Lipoproteínas LDL/metabolismo
, Lipoproteínas VLDL/metabolismo
, Macrófagos/metabolismo
, Fosfatidilcolina-Esterol O-Aciltransferasa
, Plasma/enzimología