RESUMEN

An electrochemiluminescence and photothermal immunosensor based on a dual-modality integrated probe was proposed for sensitive and reliable detection of lipolysis stimulated lipoprotein receptor (LSR), a new biomarker of ovarian cancer. Black phosphorous quantum dots (BPQDs) possess fascinating electrochemical property and unique photothermal effect, which could not only enhance ECL signal of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) through accelerating dissolved O2 evolution but also realize temperature signal output by converting laser energy into heat. Furthermore, NiFe2O4 nanotubes (NiFe2O4 NTs) have large specific surface area and favorable adsorption ability, which could increase the immobilized amount of ABEI and BPQDs, further strengthening ECL and temperature signal. As a result, a dual-mode immunosensor was constructed and realized ECL and temperature dual signal to detect LSR, making the results more reliable. This work provided a new thought for the development of sensitive and accurate sensors and was expected to employ for determination of other biomarkers.

Asunto(s)

Biomarcadores de Tumor/análisis , Técnicas Biosensibles , Compuestos Férricos/química , Nanotubos/química , Níquel/química , Neoplasias Ováricas , Fósforo/química , Puntos Cuánticos/química , Receptores de Lipoproteína/análisis , Anticuerpos/química , Biomarcadores de Tumor/inmunología , Técnicas Electroquímicas , Femenino , Humanos , Inmunoensayo , Luz , Mediciones Luminiscentes , Luminol/análogos & derivados , Luminol/química , Receptores de Lipoproteína/inmunología , Temperatura

RESUMEN

BACKGROUND AND AIMS: Epicardial adipose tissue (EAT) is a visceral AT, surrounding myocardium and coronary arteries. Its volume is higher in Type 2 diabetic (DM2) patients, associated with cardiovascular disease risk. Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) from circulating lipoproteins, supplying fatty acids to AT, contributing to its expansion. We aimed to evaluate LPL expression and activity in EAT from DM2 and no DM2 patients, and its regulators ANGPTL4, GPIHBP1 and PPARγ levels, together with VLDLR expression and EAT LPL association with VLDL characteristics. METHODS: We studied patients undergoing coronary by-pass graft (CABG) divided into CABG-DM2 (n = 21) and CABG-noDM2 (n = 29), and patients without CABG (No CABG, n = 30). During surgery, EAT and subcutaneous AT (SAT) were obtained, in which LPL activity, gene and protein expression, its regulators and VLDLR protein levels were determined. Isolated circulating VLDLs were characterized. RESULTS: EAT LPL activity was higher in CABG-DM2 compared to CABG-noDM2 and No CABG (p=0.002 and p<0.001) and in CABG-noDM2 compared to No CABG (p=0.02), without differences in its expression. ANGPTL4 levels were higher in EAT from No CABG compared to CABG-DM2 and CABG-noDM2 (p<0.001). GPIHBP1 levels were higher in EAT from CABG-DM2 and CABG-noDM2 compared to No CABG (p= 0.04). EAT from CABG-DM2 presented higher PPARγ levels than CABG-noDM2 and No CABG (p=0.02 and p=0.03). No differences were observed in VLDL composition between groups, although EAT LPL activity was inversely associated with VLDL-TG and TG/protein index (p<0.05). CONCLUSIONS: EAT LPL regulation would be mainly post-translational. The higher LPL activity in DM2 could be partly responsible for the increase in EAT volume.

Asunto(s)

Proteína 4 Similar a la Angiopoyetina/análisis , Diabetes Mellitus Tipo 2/enzimología , Grasa Intraabdominal/enzimología , Lipoproteína Lipasa/análisis , Receptores de Lipoproteína/análisis , Adiposidad , Anciano , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/fisiopatología , Activación Enzimática , Ácidos Grasos/sangre , Femenino , Humanos , Grasa Intraabdominal/fisiopatología , Lipoproteínas VLDL/sangre , Masculino , Persona de Mediana Edad , PPAR gamma/metabolismo , Pericardio , Receptores de LDL/análisis , Triglicéridos/sangre

RESUMEN

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1Enh/Enh). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia-even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1Enh/- mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

Asunto(s)

Tejido Adiposo Pardo/metabolismo , Lipoproteína Lipasa/metabolismo , Receptores de Lipoproteína/genética , Animales , Sistemas CRISPR-Cas/genética , Cromatina/genética , Corazón , Humanos , Ratones , Ratones Endogámicos , Receptores de Lipoproteína/análisis , Receptores de Lipoproteína/metabolismo , Análisis de Secuencia de ADN , Triglicéridos/sangre , Triglicéridos/metabolismo

RESUMEN

In mammals, GPIHBP1 is absolutely essential for transporting lipoprotein lipase (LPL) to the lumen of capillaries, where it hydrolyzes the triglycerides in triglyceride-rich lipoproteins. In all lower vertebrate species (e.g., birds, amphibians, reptiles, fish), a gene for LPL can be found easily, but a gene for GPIHBP1 has never been found. The obvious question is whether the LPL in lower vertebrates is able to reach the capillary lumen. Using purified antibodies against chicken LPL, we showed that LPL is present on capillary endothelial cells of chicken heart and adipose tissue, colocalizing with von Willebrand factor. When the antibodies against chicken LPL were injected intravenously into chickens, they bound to LPL on the luminal surface of capillaries in heart and adipose tissue. LPL was released rapidly from chicken hearts with an infusion of heparin, consistent with LPL being located inside blood vessels. Remarkably, chicken LPL bound in a specific fashion to mammalian GPIHBP1. However, we could not identify a gene for GPIHBP1 in the chicken genome, nor could we identify a transcript for GPIHBP1 in a large chicken RNA-seq data set. We conclude that LPL reaches the capillary lumen in chickens - as it does in mammals - despite an apparent absence of GPIHBP1.

Asunto(s)

Capilares/metabolismo , Pollos/metabolismo , Lipoproteína Lipasa/metabolismo , Receptores de Lipoproteína/metabolismo , Tejido Adiposo/irrigación sanguínea , Tejido Adiposo/metabolismo , Animales , Anticuerpos , Células Endoteliales/metabolismo , Femenino , Cabras , Corazón , Heparina , Humanos , Inmunoglobulina G , Metabolismo de los Lípidos , Lipoproteína Lipasa/genética , Lipoproteínas/metabolismo , Masculino , Ratones , Receptores de Lipoproteína/análisis , Receptores de Lipoproteína/genética , Triglicéridos/metabolismo

RESUMEN

It is not well-established whether patients with androgenetic alopecia (AGA) show a higher cardiovascular risk and higher prevalence of metabolic syndrome (MS). Therefore, we aimed to analyze the cardiovascular risk and the prevalence of MS by means of a case-control study. We determined lipidic, inflammatory, hormonal and insulin resistance parameters with conventional laboratory methods in 50 male early-onset AGA patients and 50 controls. AGA patients did not show statistical differences for insulin resistance (glucose, insulin, C peptide, HOMA), lipids (total-cholesterol, HDL-cholesterol, tryglicerides) or hormonal parameters (testosterone, free androgen index, sex hormone-binding globulin) P >  0.05, respectively. No differences between groups were observed in prevalence of MS or its components (P >  0.05). AGA patients showed higher levels of fibrinogen, C-reactive protein (CRP) and lipoprotein(a) (Lp(a)) (P = 0.016, P = 0.019 and P = 0.032, respectively). In the unadjusted logistic regression analyses, PCR >4 mg/L, fibrinogen >395 mg/dL and Lp(a) >59 mg/dL increased the risk of AGA, but in the adjusted logistic regression analyses, only PCR >4 mg/L and Lp(a) >59 mg/dL independently increased this risk (OR = 5.83, 95% CI 1.33-25.59 P = 0.020; OR = 3.94 CI 95% 1.08-14.43 P = 0.038). The present study indicates that AGA patients do not show differences in either insulin resistance or prevalence of MS. However, AGA patients show a higher cardiovascular risk characterised by an increase in inflammatory parameters and Lp(a) levels.

Asunto(s)

Alopecia/complicaciones , Biomarcadores/análisis , Enfermedades Cardiovasculares/etiología , Receptores de Lipoproteína/análisis , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Factores de Riesgo

RESUMEN

UNLABELLED: Clostridium difficile is the leading cause of antibiotics-associated diarrhea and pseudomembranous colitis. Hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT (C. difficile transferase), in addition to the Rho-glucosylating toxins A and B. We recently identified the lipolysis-stimulated lipoprotein receptor (LSR) as the host receptor that mediates uptake of CDT into target cells. Here we investigated in H1-HeLa cells, which ectopically express LSR, the influence of CDT on the plasma membrane distribution of the receptor. We found by fluorescence microscopy that the binding component of CDT (CDTb) induces clustering of LSR into subcompartments of the plasma membrane. Detergent extraction of cells treated with CDTb, followed by sucrose gradient fractionation, uncovered accumulation of LSR in detergent-resistant membranes (DRMs) that contained typical marker proteins of lipid rafts. Membrane cholesterol depletion with methyl-ß-cyclodextrin inhibited the association of LSR with DRMs upon addition of CDTb. The receptor-binding domain of CDTb also triggered LSR clustering into DRMs. CDTb-triggered clustering of LSR into DRMs could be confirmed in Caco-2 cells. Our data suggest that CDT forces its receptor to cluster into lipid rafts and that oligomerization of the B component might enhance but is not essential for this process. IMPORTANCE: C. difficile binary toxin CDT is a member of the iota-like, actin ADP-ribosylating toxin family. The mechanism that mediates endocytic uptake of these toxins still remains elusive. Previous studies highlighted the importance of lipid rafts for oligomerization of the binding component of these toxins and for cell entry. Recently, the host cell receptor for this toxin family, namely, the lipolysis-stimulated lipoprotein receptor (LSR), has been identified. Our study now demonstrates that the binding component of CDT (CDTb) induces clustering of LSR into lipid rafts. Importantly, LSR clustering is efficiently induced also by the receptor-binding domain of CDTb, suggesting that oligomerization of the B component of CDT is not the main trigger of this process. The current work extends our knowledge on the cooperative play between iota-like toxins and their receptor.

Asunto(s)

ADP Ribosa Transferasas/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Clostridioides difficile/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Receptores de Lipoproteína/análisis , Células CACO-2 , Análisis Mutacional de ADN , Células HeLa , Humanos , Microscopía Fluorescente , Estructura Terciaria de Proteína

RESUMEN

The lipolytic processing of triglyceride-rich lipoproteins by lipoprotein lipase (LPL) is the central event in plasma lipid metabolism, providing lipids for storage in adipose tissue and fuel for vital organs such as the heart. LPL is synthesized and secreted by myocytes and adipocytes, but then finds its way into the lumen of capillaries, where it hydrolyzes lipoprotein triglycerides. The mechanism by which LPL reaches the lumen of capillaries has remained an unresolved problem of plasma lipid metabolism. Here, we show that GPIHBP1 is responsible for the transport of LPL into capillaries. In Gpihbp1-deficient mice, LPL is mislocalized to the interstitial spaces surrounding myocytes and adipocytes. Also, we show that GPIHBP1 is located at the basolateral surface of capillary endothelial cells and actively transports LPL across endothelial cells. Our experiments define the function of GPIHBP1 in triglyceride metabolism and provide a mechanism for the transport of LPL into capillaries.

Asunto(s)

Capilares/enzimología , Lipoproteína Lipasa/metabolismo , Receptores de Lipoproteína/metabolismo , Tejido Adiposo/irrigación sanguínea , Animales , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Metabolismo de los Lípidos , Lipoproteína Lipasa/análisis , Lipoproteínas/metabolismo , Ratones , Ratones Noqueados , Receptores de Lipoproteína/análisis , Receptores de Lipoproteína/genética , Triglicéridos/metabolismo

RESUMEN

Desde que el NCEP ATP III (National Cholesterol Education Program. Adult Treatment Panel III) aceptó el predominio de partículas LDL (low density lipoproteins "lipoproteínas de baja densidad") pequeñas y densas como factor de riesgo emergente de desarrollo de enfermedad cardiovascular, el interés por los métodos para fraccionar las LDL ha aumentado. Por eso, el presente trabajo pretende valorar la utilidad de un sistema de electroforesis en gel de poliacrilamida (Lipoprint(R)) para separar LDL en nuestra población. Se recogieron 194 muestras de sangre de personas de entre 15 y 94 años (el 49%, hombres) y se calculó la imprecisión del ensayo, así como los valores de referencia por sexo. Además, se realizaron correlaciones entre los distintos parámetros lipídicos. Se obtuvieron resultados aceptables para el estudio de imprecisión mediante el sistema Lipoprint®. Al correlacionar el diámetro medio de las partículas LDL con otros marcadores del metabolismo lipídico, destacamos una asociación inversamente proporcional con la concentración de triglicéridos y apolipoproteína (apo) B100 y directamente proporcional con la de colesterol ligado a lipoproteínas de alta densidad (cHDL). Encontramos diferencias entre sexos en los niveles de triglicéridos y colesterol ligado a LDL (mayores en hombres), y cHDL y diámetro medio de las partículas LDL (mayores en mujeres). Al comparar el diámetro medio de las partículas LDL con los parámetros lipídicos encontramos que está asociado inversamente con la concentración de triglicéridos y apo B100, y directamente con la de cHDL, lo que se asocia a un mayor riesgo cardiovascular. El sistema Lipoprint® es útil para la medida de la concentración y diámetro medio de las partículas LDL debido a su sencillez y rapidez de resultados. Aún así faltan estudios que relacionen los resultados obtenidos con los parámetros clínicos que se emplean en la valoración del riesgo cardiovascular (AU)

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Since NCEP ATP III accepted the small and dense LDL particle as an emergent risk factor of cardiovascular disease, the methods to calculate LDL subfractions have increased. The present report attempts to evaluate the usefulness of a polyacrylamide gel electrophoresis system (LipoprintTM) to separate LDL in our population. 194 blood samples were collected from subjects between 15–94 years old (49% men). Imprecision and lipid parameter study population ranges by sex, and the correlations between them were calculated. Imprecision study results were acceptable. When correlating the average diameter of particle LDL with other lipid markers, we observed an inverse association with triglyceride concentration and Apo B100, and a direct association with HDL-cholesterol. We found differences between sex in triglyceride and LDL-cholesterol levels (greater in men) and HDL-cholesterol and average diameter of LDL particles (greater in women). When comparing the average LDL particle diameter with the lipid parameters, we found that it is inversely associated with the triglyceride and Apo B100 concentration, and directly with HDL-cholesterol, which is associated with a greater cardiovascular risk. We believe that LipoprintTM system is useful for the measurement of the concentration and average diameter of LDL particles, due to its simplicity and speed of results. Nevertheless, studies are needed that can associate the results obtained to the clinical parameters that are used in the evaluation of cardiovascular risk (AU)

Asunto(s)

Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Electroforesis , Lipoproteínas LDL/análisis , Triglicéridos/análisis , Colesterol/análisis , Enfermedades Cardiovasculares/diagnóstico , Metabolismo de los Lípidos , Electroforesis/métodos , Receptores de Lipoproteína/análisis , 28599

RESUMEN

Central nervous system(CNS) has nearly a quarter of total body cholesterol, although its weight is only 2% of the whole body. In the adult brain, cholesterol synthesis is extremely low, and a half life of cholesterol is about 5 years. In the cerebrospinal fluid(CSF), the only lipoprotein is high-density lipoprotein (HDL). CSF-HDL is larger than plasma HDL, and rich in apolipoprotein E (apoE). In CNS, neurons and glia cells express several lipoprotein receptors and ATP-binding cassette (ABC) transporters. These lipoprotein receptors can bind to and take up CSF-HDL for cholesterol recycling. Recent research has focused on investigating the role of CSF-HDL and lipoprotein receptors in the pathogenesis of Alzheimer disease.

Asunto(s)

Lipoproteínas/líquido cefalorraquídeo , Receptores de Lipoproteína/análisis , Sistema Nervioso Central/química

RESUMEN

La participación de la vía Wnt en el metabolismo óseo ha sido objeto de numerosos estudios en los últimos años. Esta vía de señalización es compleja y está integrada por numerosos componentes, incluyendo ligandos, receptores de membrana, efectores intracelulares y antagonistas. Los mecanismos mejor conocidos de transmisión de la señal de los ligandos Wnt se incluyen en la llamada vía canónica, en la cual la b-catenina desempeña un papel central. Sin embargo, existen otras vías alternativas o no canónicas que emplean mediadores diferentes. Los resultados de diversos estudios clínicos y experimentales indican que la vía Wnt modula la diferenciación y la actividad de las células óseas y está implicada en diferentes trastornos esqueléticos, como la osteoporosis, la artrosis, el mieloma o las metástasis. Por tanto, una mejor comprensión de esta vía puede permitir desarrollar nuevas dianas terapéuticas para esos procesos. En este trabajo pretendemos hacer una breve puesta al día de los principales mediadores implicados en la vía Wnt, y en particular de su papel en las enfermedades óseas

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The participation of the Wnt pathway in bone metabolism has been the subject of many studies in recent years. This signal pathway is complex and is made up of many components, including ligands, membrane receptors, intracellular effectors and antagonists. The best known mechanisms of Wnt ligand signal transmission are included in the so-called canonical pathway, in which the b-catenin plays a central role. However, there are other alternative or non-canonical pathways that use different mediators. The results of different clinical and experimental studies indicate that the Wnt pathway modulates the differentiation and activity of the bone cells and is involved in different skeletal disorders, such as osteoporosis, arthrosis, myeloma or metastases. Thus, better understanding of this pathway may make it possible to develop new therapeutic targets for these diseases. In this work, we aim to make a brief up-date of the principal mediators involved in the Wnt pathway and especially in its role in bone diseases

Asunto(s)

Humanos , Osteoporosis/fisiopatología , Transducción de Señal/fisiología , Enfermedades Óseas Metabólicas/fisiopatología , Biomarcadores/análisis , Receptores de Lipoproteína/análisis , Osteoartritis/fisiopatología , Densidad Ósea/fisiología , Neoplasias Óseas/fisiopatología

RESUMEN

Among other factors, fetal growth requires maternal supply of cholesterol. Cellular cholesterol uptake is mainly mediated by the LDL receptor (LDL-R) and the scavenger receptor family. We hypothesized that expression levels of key receptors of these families were regulated differently in placentas from IUGR pregnancies with varying degrees of severity. Third-trimester placentas from IUGR pregnancies with (IUGR-S) and without (IUGR-M) fetal hemodynamic changes and from control (AGA) pregnancies were studied. LDL-R, LDL-R-related protein (LRP-1), and scavenger receptor class B type I (SR-BI) mRNA and protein levels were measured. Cholesterol concentration and composition of lipoproteins were analyzed enzymatically and by lipid electrophoresis, respectively, in maternal and umbilical cord blood. LDL-R mRNA levels in IUGR-M were similar to AGA but lower (P < 0.05) in IUGR-S. In contrast, LDL-R protein was twofold (IUGR-M) and 1.8-fold (IUGR-S) higher (P < 0.05) than in the AGA group. LRP-1 mRNA and protein levels were not altered in the IUGR cases. SR-BI mRNA was unchanged in IUGR, but protein levels were lower (P < 0.05) in IUGR-S than in the other groups. Maternal plasma concentrations of LDL cholesterol were higher (P < 0.05) in the AGA group (188.5 +/- 23.6 mg/dl) than in the IUGR-S group (154.2 +/- 26.1). Electrophoretic mobility of the LDL fraction in maternal plasma demonstrated significant changes in migration toward higher values (AGA 0.95 +/- 0.06, IUGR-M 1.12 +/- 0.11, P < 0.001; IUGR-S 1.28 +/- 0.20, P = 0.002). We conclude that LDL-R and SR-BI levels are altered in IUGR pregnancies. These differences were associated with changes in LDL, but not HDL, mobility and cholesterol concentration in maternal circulation.

Asunto(s)

Retardo del Crecimiento Fetal/etiología , Lipoproteínas/análisis , Placenta/química , Receptores de Lipoproteína/análisis , Adulto , Colesterol/análisis , Femenino , Humanos , Recién Nacido , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/análisis , Intercambio Materno-Fetal , Persona de Mediana Edad , Embarazo , Receptores de LDL/análisis

RESUMEN

The Reelin signaling pathway operates in migrating neurons and is indispensable for their correct positioning during embryonic brain development. Many biochemical and cell biological studies to dissect the Reelin pathway at the molecular level are hampered by the lack of a cell line harboring a functional Reelin signaling pathway. Here we present fibroblast cell lines in which all required functional components of the pathway have been reconstituted. These cells react upon Reelin treatment in the same way as primary neurons. We have subsequently used these cell lines to study the subcellular localization of ApoER2 and the VLDL receptor and could demonstrate that receptor-mediated Dab1 phosphorylation does not depend on lipid rafts and that phosphorylated Dab1 remains bound to the receptor tail when the pathway is activated by Reelin.

Asunto(s)

Moléculas de Adhesión Celular Neuronal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Microdominios de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Receptores de LDL/metabolismo , Receptores de Lipoproteína/metabolismo , Serina Endopeptidasas/fisiología , Animales , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Matriz Extracelular/genética , Fibroblastos/metabolismo , Fibroblastos/fisiología , Proteínas Relacionadas con Receptor de LDL , Microdominios de Membrana/química , Ratones , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Fosforilación , Receptores de LDL/análisis , Receptores de Lipoproteína/análisis , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal

RESUMEN

The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.

Asunto(s)

Antígenos CD36/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de la Membrana , Receptores Inmunológicos , Receptores de Lipoproteína/metabolismo , Animales , Antígenos CD36/análisis , Antígenos CD36/genética , Glicosilación , Metabolismo de los Lípidos , Ratones , Mutagénesis , Receptores de Lipoproteína/análisis , Receptores de Lipoproteína/genética , Receptores Depuradores , Receptores Depuradores de Clase B , Transducción de Señal

RESUMEN

The retinal pigment epithelial (RPE) cells that underlie the retina ingest and metabolize thousands of lipid-rich photoreceptor outer segments (POS) every day. The scavenger receptor (SR) CD36 and integrin alphavbeta5 have been shown to participate in POS binding and internalization by RPE cells. The objective of the current study was to determine whether RPE cells express SRs other than CD36. Primary cultures of human RPE cells express both mRNA and protein for SR-BI and SR- BII. SR-BI and SR-BII mRNAs were detected by reverse transcription-polymerase chain reaction. SR-BI protein was detected by immune precipitation of [(35)S]methionine-labeled crude cellular extracts. SR-BII was detected by Western blotting of immune precipitated crude cellular extracts. SR-BI and SR-BII proteins were also detected by immunofluorescence staining of RPE cells in culture. The results suggest that these SRs may play a role in POS lipid binding and uptake by RPE cells in the eye.

Asunto(s)

Antígenos CD36/biosíntesis , Proteínas de la Membrana , Epitelio Pigmentado Ocular/metabolismo , Receptores de Lipoproteína/biosíntesis , Sialoglicoproteínas , Western Blotting , Antígenos CD36/análisis , Antígenos CD36/genética , Extractos Celulares/química , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de Membrana de los Lisosomas , Epitelio Pigmentado Ocular/química , Epitelio Pigmentado Ocular/citología , Pruebas de Precipitina , Isoformas de Proteínas/análisis , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Lipoproteína/análisis , Receptores de Lipoproteína/genética , Receptores Depuradores , Receptores Depuradores de Clase B

RESUMEN

BACKGROUND: An important aspect of inclusion-body myositis (IBM) vacuolated muscle fibers (VMF) is abnormal accumulation of amyloid-beta precursor protein (AbetaPP) epitopes and its product, amyloid-beta (Abeta), and of phosphorylated tau (p-tau) in the form of paired helical filaments. Lipoprotein receptors and cholesterol are known to play an important role in AbetaPP processing, Abeta production, and tau phosphorylation. METHODS: In 10 IBM and 22 control muscle biopsies the authors immunolocalized low-density lipoprotein receptor (LDLR), very low-density lipoprotein receptor (VLDLR), and low-density lipoprotein receptor-related protein (LRP), and colocalized them with Abeta, p-tau, APOE, and free cholesterol. RESULTS: In each biopsy, virtually all IBM VMF had strong LDLR-immunoreactive inclusions, which colocalized with Abeta, APOE, p-tau, and free cholesterol. VLDLR was increased mainly diffusely, but in approximately 50% of the VMF it was also accumulated in the form of inclusions colocalizing with Abeta, APOE, and free cholesterol, but not with p-tau. LRP inclusions were present in a few VMF. In all myopathies, a subset of regenerating and necrotizing muscle fibers had prominent diffuse accumulation of both LDLR and free cholesterol. At normal neuromuscular junctions (NMJ) postsynaptically, LDLR and VLDLR, but not LRP, were immunoreactive. CONCLUSIONS: 1) Abnormal accumulation of LDLR, VLDLR, LRP, and cholesterol within IBM vacuolated muscle fibers suggests novel roles for them in the IBM pathogenesis. 2) Expression of LDLR and VLDLR at normal NMJ suggests physiologic roles for them in transsynaptic signaling pathways, increased internalization of lipoproteins there, or both. 3) Increased LDLR and free cholesterol in some regenerating and necrotizing muscle fibers suggest a role for them in human muscle fiber growth and repair and necrotic death.

Asunto(s)

Colesterol/análisis , Músculo Esquelético/química , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Receptores de Lipoproteína/análisis , Péptidos beta-Amiloides/análisis , Apolipoproteínas E/análisis , Biopsia , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/análisis , Microscopía Inmunoelectrónica , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/patología , Fosforilación , Receptores de LDL/análisis , Proteínas tau/análisis , Proteínas tau/metabolismo

RESUMEN

Avaliou-se o efeito do GH in vitro sobre a expressão gênica do receptor LDL (RLDL) e da HMG-CoA redutase, bem como sobre a proliferação celular e acúmulo de lípides intracelulares em células mesangiais cultivadas em meio com soro deficiente de lipoproteínas (LPDS) durante 1, 2, 4 e 6 dias. A expressão coordenada entre o RLDL e a HMG-CoA redutase foi observada nas células mesangiais cultivadas em meio com LPDS. O GH aaumentou a proliferação das células mesangiais, dependente da sua concentração. A exposição prolongada ao GH induziu o aumento da expressão de RNAm do RLDL e da HMG-CoA redutase na células mesangiais, bem como o acúmulo de lípides neutros no citoplasma. Nos estudos in vivo...

Asunto(s)

Animales , Bovinos , Ratones , Bioquímica/historia , ADN , Expresión Génica/genética , Expresión Génica/inmunología , Glomeruloesclerosis Focal y Segmentaria , Técnicas In Vitro , Nefrología , Receptores de LDL , ARN , Técnicas de Cultivo de Célula , Técnicas de Laboratorio Clínico , Medios de Cultivo , Ratones , Reacción en Cadena de la Polimerasa , Receptores de Lipoproteína/administración & dosificación , Receptores de Lipoproteína/análisis , Espectrofotometría

RESUMEN

OBJECTIVE: To study the effects of lipid (triglyceride and very low-density lipoprotein) on low-density lipoprotein (LDL) and high-density lipoprotein (HDL) receptors in the hepatic stellate cell (HSC) from the rat liver. METHODS: HSC were isolated and cultured from the liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz. Radioligand conjugation assay with (125)I-LDL and (125)I-HDL(3) was detected for the effects of lipid on LDL and HDL receptor of HSC. RESULTS: LDL and HDL receptors were found on the membrane of the rat HSC. The lipid might increase the binding of LDL to LDL receptor, but decrease the binding of HDL(3) to HDL receptor. CONCLUSION: LDL and HDL receptors on the HSC membrane may have an important role in the metabolism of lipoprotein and the regulation of cholesterol. These results provided the basis of theory and experimentation for the genesis of fatty liver and liver fibrosis.

Asunto(s)

Proteínas Portadoras , Lípidos/farmacología , Lipoproteínas HDL , Hígado/química , Hígado/citología , Proteínas de Unión al ARN , Receptores de LDL/análisis , Receptores de Lipoproteína/análisis , Animales , Membrana Celular/química , Células Cultivadas , Humanos , Masculino , Ratas , Ratas Wistar

RESUMEN

BACKGROUND: Nephrotic syndrome (NS) is a prototype of acquired hypercholesterolemia. Hepatic synthesis and removal of cholesterol play major roles in the regulation of plasma concentration of this sterol. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles are the primary vehicles for cholesterol transport to the liver. We have recently demonstrated that NS results in acquired hepatic LDL receptor deficiency in rats. This study was undertaken to determine the effect of NS on hepatic expression of the newly discovered, long-sought HDL receptor. METHODS: Hepatic HDL receptor and apolipoprotein A-I (apo A-I) expressions were studied in rats with puromycin-induced NS. The results were compared with those obtained in placebo-treated, normal controls. RESULTS: The NS group exhibited a marked reduction in hepatic tissue HDL receptor protein abundance when compared with the control group. In contrast, hepatic HDL receptor mRNA abundance in the NS group was similar to that of the control group. As expected, the NS group showed a marked increase in hepatic apo A-I mRNA abundance. CONCLUSIONS: The study explored the effect of experimental NS on hepatic HDL receptor expression, and the results revealed a marked down-regulation of HDL receptor in rats with NS. In contrast, hepatic expression of Apo A-I, the principal protein constituent of HDL, was markedly increased in NS rats. The HDL receptor deficiency shown here can potentially limit the efficiency of HDL as the primary vehicle for reverse cholesterol transport in NS.

Asunto(s)

Proteínas Portadoras , Regulación hacia Abajo/fisiología , Lipoproteínas HDL , Proteínas de la Membrana , Síndrome Nefrótico/metabolismo , Proteínas de Unión al ARN , Receptores Inmunológicos/genética , Receptores de Lipoproteína/genética , Animales , Apolipoproteína A-I/genética , Western Blotting , Cartilla de ADN , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Masculino , Inhibidores de la Síntesis de la Proteína/farmacología , Proteinuria/genética , Proteinuria/metabolismo , Puromicina/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores Inmunológicos/análisis , Receptores Inmunológicos/metabolismo , Receptores de Lipoproteína/análisis , Receptores de Lipoproteína/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase B

RESUMEN

CLA-1, a human homologue of rodent scavenger receptor class B1 (SR-B1), has been identified as a receptor for high density lipoprotein (HDL) and is highly expressed in the adrenal gland. Several studies have indicated that HDL might be a source of cholesterol for steroidogenesis in the adrenal gland. In this study, we show that ACTH and its second messenger cAMP stimulated CLA-1 protein expression in a human adrenocortical cell line. We also determined whether CLA-1 plays an important role in steroidogenesis by investigating CLA-1 expression levels in various adrenal tumors including the adenomas of Cushing's and Conn's syndrome. Western blot analysis showed that CLA-1 expression was much higher in the tumors of Cushing's syndrome than in non-tumor lesions of Conn's syndrome and pheochromocytoma. We were able to detect a strong CLA-1 signal in tumors of Conn's syndrome, too. On the other hand, much less CLA-1 expression was detected in Cushing's adenoma adjacent adrenal glands. The immunohistochemical analysis showed that CLA-1 was expressed in the outer region of the adrenal cortex mainly in plasma membranes of the cortical cells but not in the medulla. These findings demonstrated for the first time that ACTH increased CLA-1 protein in cultured human adrenocortical cells, and that cortisol- and aldosterone-secreting adenomas had high CLA-1 proteins in their cell surfaces.

Asunto(s)

Neoplasias de la Corteza Suprarrenal/metabolismo , Antígenos CD36/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Inmunológicos , Receptores de Lipoproteína/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenoma/metabolismo , Corteza Suprarrenal/química , Corteza Suprarrenal/metabolismo , Hormona Adrenocorticotrópica/farmacología , Aldosterona/biosíntesis , Western Blotting , Antígenos CD36/análisis , Antígenos CD36/genética , Línea Celular , Síndrome de Cushing/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Feocromocitoma/metabolismo , ARN Mensajero/análisis , Receptores de Lipoproteína/análisis , Receptores de Lipoproteína/genética , Receptores Depuradores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptores Depuradores de Clase B , Distribución Tisular

RESUMEN

A new type of mosaic protein was recently discovered as a new member of the low density lipoprotein receptor (LDLR) family, designated as LR11. The predominant expression of LR11 transcripts in brain tissue and the presence of elements found in neural adhesion molecules suggested a function(s) in the central nervous system (CNS). In order to gain insight about this complex receptor in the CNS, we raised a rabbit polyclonal antibody and examined immunohistochemically rat and human brain tissue. A strong LR11 immunoreactivity was found to be localized mainly in neurons throughout the brain in both species. A detailed mapping in the rat brain showed a distribution of LR11 immunoreactivity in a widespread population of neurons, though the intensity varied between different locations. The most prominent immunoreactivity was observed in neurons of the hippocampus, some nuclei of brain stem and Purkinje cells, whereas neurons of the thalamus and the hypothalamus showed weak staining. Uniquely, the single LR11 immunoreactive cytoplasmic puncta were observed in the proximity of apical dendrites, most conspicuously in the pyramidal neurons of hippocampus. In the human brain, one to four immunoreactive puncta were seen within individual neurons. The neuronal localization of LR11 and its unique association of cytoplasmic structure, presumably botrysome, may suggest the roles of LR11 in both the lipoprotein metabolism and intracellular trafficking in certain neuronal population of the CNS.

Asunto(s)

Química Encefálica , Proteínas de Transporte de Membrana , Neuronas/química , Receptores de LDL/análisis , Receptores de Lipoproteína/análisis , Adulto , Enfermedad de Alzheimer/metabolismo , Animales , Anticuerpos , Western Blotting , Citoplasma/química , Humanos , Técnicas para Inmunoenzimas , Proteínas Relacionadas con Receptor de LDL , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Mosaicismo , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Conejos , Ratas , Ratas Wistar , Receptores de LDL/inmunología , Receptores de Lipoproteína/inmunología