RESUMEN
Metabolic syndrome (MS) is a widespread disease in developed countries, accompanied, among others, by decreased adiponectin serum levels and perturbed lipoprotein metabolism. The associations between the serum levels of adiponectin and lipoproteins have been extensively studied in the past under healthy conditions, yet it remains unexplored whether the observed associations also exist in patients with MS. Therefore, in the present study, we analyzed the serum levels of lipoprotein subclasses using nuclear magnetic resonance spectroscopy and examined their associations with the serum levels of adiponectin in patients with MS in comparison with healthy volunteers (HVs). In the HVs, the serum levels of adiponectin were significantly negatively correlated with the serum levels of large buoyant-, very-low-density lipoprotein, and intermediate-density lipoprotein, as well as small dense low-density lipoprotein (LDL) and significantly positively correlated with large buoyant high-density lipoprotein (HDL). In patients with MS, however, adiponectin was only significantly correlated with the serum levels of phospholipids in total HDL and large buoyant LDL. As revealed through logistic regression and orthogonal partial least-squares discriminant analyses, high adiponectin serum levels were associated with low levels of small dense LDL and high levels of large buoyant HDL in the HVs as well as high levels of large buoyant LDL and total HDL in patients with MS. We conclude that the presence of MS weakens or abolishes the strong associations between adiponectin and the lipoprotein parameters observed in HVs and disturbs the complex interplay between adiponectin and lipoprotein metabolism.
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Adiponectina , Lipoproteínas , Síndrome Metabólico , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adiponectina/sangre , Estudios de Casos y Controles , Voluntarios Sanos , Lipoproteínas/sangre , Lipoproteínas HDL/sangre , Lipoproteínas LDL/sangre , Espectroscopía de Resonancia Magnética , Síndrome Metabólico/sangreRESUMEN
PURPOSE OF REVIEW: Lp(a) is one of the most atherogenic lipoproteins, and significant progress has been made to understand its pathophysiology over the last 20âyears. There are now selective therapies in late-stage clinical trials to lower Lp(a). Yet there are many outstanding questions about Lp(a). This review outlines 10 of the most burning questions and tries to answer some of them. RECENT FINDINGS: Antisense oligonucleotide (ASO) treatment is currently the most advanced therapy to lower plasma Lp(a) by 60-80%. There are, however, also two small molecule medications in early stage of development with similar efficacy. SUMMARY: This review aims to answer important preclinical and clinical questions about the metabolism and physiological role of Lp(a) and also outlines possible therapeutic approaches with nutraceuticals, currently available lipid-lowering therapies and new medications. In addition, ways are illustrated to use Lp(a) as a marker to better predict cardiovascular risk.
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Aterosclerosis , Enfermedades Cardiovasculares , Lipoproteína(a) , Humanos , Aterosclerosis/tratamiento farmacológico , Lipoproteína(a)/antagonistas & inhibidores , Lipoproteína(a)/metabolismo , Oligonucleótidos Antisentido/uso terapéutico , Factores de Riesgo , AnimalesRESUMEN
BACKGROUND: Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. METHODS: A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. RESULTS: The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. CONCLUSIONS: A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a).
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Péptidos , Suero , Humanos , Apoproteína(a) , Espectrometría de Masas , Estándares de Referencia , CalibraciónRESUMEN
BACKGROUND: Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. METHODS: The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. RESULTS: Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. CONCLUSIONS: The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System.
Asunto(s)
Química Clínica , Lipoproteína(a) , Humanos , Inmunoensayo , Espectrometría de Masas , Estándares de ReferenciaRESUMEN
Lipoprotein(a) (Lp(a)) is one of the strongest causal risk factors of atherosclerotic disease. It is rich in cholesteryl ester and composed of apolipoprotein B and apo(a). Plasma Lp(a) levels are determined by apo(a) transcriptional activity driven by a direct repeat (DR) response element in the apo(a) promoter under the control of (HNF)4α Farnesoid-X receptor (FXR) ligands play a key role in the downregulation of APOA expression. In vitro studies on the catabolism of Lp(a) have revealed that Lp(a) binds to several specific lipoprotein receptors; however, their in vivo role remains elusive. There are more than 1000 publications on the role of diabetes mellitus (DM) in Lp(a) metabolism; however, the data is often inconsistent and confusing. In patients suffering from Type-I diabetes mellitus (T1DM), provided they are metabolically well-controlled, Lp(a) plasma concentrations are directly comparable to healthy individuals. In contrast, there exists a paradox in T2DM patients, as many of these patients have reduced Lp(a) levels; however, they are still at an increased cardiovascular risk. The Lp(a) lowering mechanism observed in T2DM patients is most probably caused by mutations in the mature-onset diabetes of the young (MODY) gene and possibly other polymorphisms in key transcription factors of the apolipoprotein (a) gene (APOA).
Asunto(s)
Enfermedades Cardiovasculares , Diabetes Mellitus Tipo 2 , Apolipoproteínas A , Apoproteína(a) , Enfermedades Cardiovasculares/genética , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Humanos , Lipoproteína(a)/genéticaRESUMEN
Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.
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Apolipoproteínas/sangre , Enfermedades Cardiovasculares/diagnóstico , Dislipidemias/diagnóstico , Proteómica/métodos , Apolipoproteínas/normas , Enfermedades Cardiovasculares/complicaciones , Conducta Cooperativa , Dislipidemias/complicaciones , Humanos , Espectrometría de Masas/métodos , Estándares de ReferenciaRESUMEN
NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.
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Lesión Renal Aguda/inmunología , Apolipoproteína C-III/inmunología , Caspasa 8/metabolismo , Enfermedades Renales/inmunología , Monocitos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Lesión Renal Aguda/patología , Proteínas Adaptadoras Transductoras de Señales , Animales , Apolipoproteína C-III/genética , Línea Celular , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Inflamasomas/inmunología , Inflamación/genética , Inflamación/inmunología , Enfermedades Renales/patología , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
BACKGROUND AND AIMS: Lipoprotein (a) [Lp(a)] is an established causal risk factor for cardiovascular disease (CVD), independently of low-density lipoproteins (LDL) and other risk factors. The recognition of Lp(a) as an atherogenic molecule has raised the demand for reliable quantification methods in the clinical laboratory. The aim of this work is to compare commercial immunochemical assays. METHODS: We measured Lp(a) serum concentrations using six different assays, providing Lp(a) in mg/dl (Denka Seiken, Abbott Quantia, Beckman, Diasys 21FS, and Siemens N Latex) or in nmol/l (Roche TinaQuant, Diasys 21 FS) in 144 serum samples covering the clinically relevant range of Lp(a) concentrations. All assays relied on five-point calibrations using calibrators provided by the manufacturers. Apolipoprotein(a) phenotyping was performed by sodium dodecyl sulfate-agarose gel electrophoresis (SDS-agarose) followed by immunoblotting. RESULTS: Most bivariate correlation coefficients were greater than 0.90. Compared to an established IFCC-proposed reference material, the results of the different assays diverged from the target values (43.3 mg/dl or 96.6 nmol/l) by -8% (Siemens N Latex) and +22% (Abbott Quantia). Stratification of the samples into five groups with increasing Lp(a) concentrations and difference plots showed that the differences among assays were concentration-dependent. Some assays overestimated Lp(a) at high concentrations compared to the Denka Seiken assay. CONCLUSIONS: Current commercial immunological assays for measuring Lp(a) concentrations are differently calibrated. Their biases differ significantly across the clinically relevant concentration range in a non-linear manner. This is not conclusively explained by apolipoprotein (a) phenotypes. Further international efforts to harmonize assays for Lp(a) are needed.
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Enfermedades Cardiovasculares/sangre , Técnicas de Laboratorio Clínico/normas , Inmunoensayo/métodos , Lipoproteína(a)/sangre , Juego de Reactivos para Diagnóstico/normas , Calibración , Técnicas de Laboratorio Clínico/instrumentación , Humanos , Inmunoturbidimetría , Análisis de los Mínimos Cuadrados , Infarto del Miocardio/sangre , Nefelometría y Turbidimetría , Fenotipo , Reproducibilidad de los Resultados , Factores de RiesgoRESUMEN
Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-ß HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [3H]-cholesterol efflux (by 50%) but did not reduce [3H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Apolipoproteínas/metabolismo , Barrera Hematoencefálica/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Modelos Biológicos , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/genética , Animales , Apolipoproteínas/genética , Transporte Biológico Activo/fisiología , Membrana Celular/genética , Colesterol/genética , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , PorcinosRESUMEN
BACKGROUND AND AIMS: Homozygous familial hypercholesterolemia (hoFH) is a rare genetic disorder with potential severe atherosclerosis in the pediatric age. METHODS: We report on 9 patients with hoFH, who had been diagnosed within the last 30 years and who were consequently treated with apheresis and drugs. RESULTS: Two deaths occurred: one at age 36 years and the other at age four and a half years before effective treatment was commenced. All other patients are still in good clinical condition today, although four of them have proven aortic stenosis or arterial plaques. CONCLUSIONS: Our case report highlights that adequate treatment should start as early as possible to delay the onset of clinical manifestations of atherosclerosis. It can be assumed that the introduction of new drugs can improve the outcome and possibly lengthen the life expectancy of patients affected by hoFH.
Asunto(s)
Anticolesterolemiantes/uso terapéutico , Eliminación de Componentes Sanguíneos , Colesterol/sangre , Enfermedad de la Arteria Coronaria/prevención & control , Homocigoto , Hiperlipoproteinemia Tipo II/terapia , Mutación , Receptores de LDL/genética , Adolescente , Adulto , Austria , Biomarcadores/sangre , Niño , Preescolar , Terapia Combinada , Enfermedad de la Arteria Coronaria/genética , Diagnóstico Precoz , Resultado Fatal , Femenino , Predisposición Genética a la Enfermedad , Herencia , Humanos , Hiperlipoproteinemia Tipo II/sangre , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Lactante , Masculino , Linaje , Fenotipo , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Glucose homeostasis is a complex indispensable process, and its dysregulation causes hyperglycemia and type 2 diabetes mellitus. Glucokinase (GK) takes a central role in these pathways and is thus rate limiting for glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Several reports have described the transcriptional regulation of Gck mRNA, whereas its posttranscriptional mechanisms of regulation, especially those involving microRNAs (miR), are poorly understood. In this study, we investigated the role of miR-206 as a posttranscriptional regulator of Gck In addition, we examined the effects of miR-206 on glucose tolerance, GSIS, and gene expression in control and germ line miR-206 knockout (KO) mice fed either with chow or high-fat diet (HFD). MiR-206 was found in Gck-expressing tissues and was differentially altered in response to HFD feeding. Pancreatic islets showed the most profound induction in the expression of miR-206 in response to HFD. Chow- and HFD-fed miR-206KO mice have improved glucose tolerance and GSIS but unaltered insulin sensitivity. In silico analysis of Gck mRNA revealed a conserved 8-mer miR-206 binding site. Hence, the predicted regulation of Gck by miR-206 was confirmed in reporter and GK activity assays. Concomitant with increased GK activity, miR-206KO mice had elevated liver glycogen content and plasma lactate concentrations. Our findings revealed a novel mechanism of posttranscriptional regulation of Gck by miR-206 and underline the crucial role of pancreatic islet miR-206 in the regulation of whole body glucose homeostasis in a murine model that mimics the metabolic syndrome.
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Glucoquinasa/genética , Islotes Pancreáticos/metabolismo , MicroARNs/genética , ARN Mensajero/metabolismo , Animales , Simulación por Computador , Dieta Alta en Grasa , Glucoquinasa/metabolismo , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Glucógeno/metabolismo , Insulina/metabolismo , Secreción de Insulina , Ácido Láctico/metabolismo , Hígado/metabolismo , Masculino , Síndrome Metabólico , Ratones , Ratones Noqueados , Procesamiento Postranscripcional del ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
Liver X receptors (LXRα and LXRß) are key transcription factors in cholesterol metabolism that regulate cholesterol biosynthesis/efflux and bile acid metabolism/excretion in the liver and numerous organs. In macrophages, LXR signaling modulates cholesterol handling and the inflammatory response, pathways involved in atherosclerosis. Since regulatory pathways of LXR transcription control are well understood, in the present study we aimed at identifying post-transcriptional regulators of LXR activity. MicroRNAs (miRs) are such post-transcriptional regulators of genes that in the canonical pathway mediate mRNA inactivation. In silico analysis identified miR-206 as a putative regulator of LXRα but not LXRß. Indeed, as recently shown, we found that miR-206 represses LXRα activity and expression of LXRα and its target genes in hepatic cells. Interestingly, miR-206 regulates LXRα differently in macrophages. Stably overexpressing miR-206 in THP-1 human macrophages revealed an up-regulation and miR-206 knockdown led to a down-regulation of LXRα and its target genes. In support of these results, bone marrow-derived macrophages (BMDMs) from miR-206 KO mice also exhibited lower expression of LXRα target genes. The physiological relevance of these findings was proven by gain- and loss-of-function of miR-206; overexpression of miR-206 enhanced cholesterol efflux in human macrophages and knocking out miR-206 decreased cholesterol efflux from MPMs. Moreover, we show that miR-206 expression in macrophages is repressed by LXRα activation, while oxidized LDL and inflammatory stimuli profoundly induced miR-206 expression. We therefore propose a feed-back loop between miR-206 and LXRα that might be part of an LXR auto-regulatory mechanism to fine tune LXR activity.
Asunto(s)
Colesterol/metabolismo , Metabolismo de los Lípidos/genética , MicroARNs/genética , Receptores Nucleares Huérfanos/metabolismo , Animales , Aterosclerosis/genética , Aterosclerosis/patología , Colesterol/genética , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Receptores X del Hígado , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Receptores Nucleares Huérfanos/genética , Transducción de SeñalRESUMEN
Recently published epidemiological and genetic studies strongly suggest a causal relationship of elevated concentrations of lipoprotein (a) [Lp(a)] with cardiovascular disease (CVD), independent of low-density lipoproteins (LDLs), reduced high density lipoproteins (HDL), and other traditional CVD risk factors. The atherogenicity of Lp(a) at a molecular and cellular level is caused by interference with the fibrinolytic system, the affinity to secretory phospholipase A2, the interaction with extracellular matrix glycoproteins, and the binding to scavenger receptors on macrophages. Lipoprotein (a) plasma concentrations correlate significantly with the synthetic rate of apo(a) and recent studies demonstrate that apo(a) expression is inhibited by ligands for farnesoid X receptor. Numerous gaps in our knowledge on Lp(a) function, biosynthesis, and the site of catabolism still exist. Nevertheless, new classes of therapeutic agents that have a significant Lp(a)-lowering effect such as apoB antisense oligonucleotides, microsomal triglyceride transfer protein inhibitors, cholesterol ester transfer protein inhibitors, and PCSK-9 inhibitors are currently in trials. Consensus reports of scientific societies are still prudent in recommending the measurement of Lp(a) routinely for assessing CVD risk. This is mainly caused by the lack of definite intervention studies demonstrating that lowering Lp(a) reduces hard CVD endpoints, a lack of effective medications for lowering Lp(a), the highly variable Lp(a) concentrations among different ethnic groups and the challenges associated with Lp(a) measurement. Here, we present our view on when to measure Lp(a) and how to deal with elevated Lp(a) levels in moderate and high-risk individuals.
Asunto(s)
Enfermedades Cardiovasculares/prevención & control , Lipoproteína(a)/metabolismo , Apoproteína(a)/química , Recolección de Muestras de Sangre/métodos , Diabetes Mellitus/fisiopatología , Hormonas/fisiología , Humanos , Hipolipemiantes/farmacología , Fallo Renal Crónico/fisiopatología , Lipoproteína(a)/química , Lipoproteína(a)/genética , Hepatopatías/fisiopatología , Guías de Práctica Clínica como Asunto , Valores de Referencia , Medición de Riesgo/métodosRESUMEN
In chronic lymphocytic leukaemia (CLL), lipoprotein lipase (LPL) mRNA overexpression is an established poor prognostic marker, its function, however, is poorly understood. Measuring extracellular LPL enzymatic activity and protein, we found no difference between levels in CLL patients and those of controls, both before and after heparin treatment in vivo and in vitro. Investigating LPL knock down effects, we determined five potential downstream targets, of which one gene, STXBP3, reportedly is involved in fatty acid metabolism. While possibly reflecting an epigenetic switch towards an incorrect transcriptional program, LPL overexpression by itself does not appear to significantly influence CLL cell survival.
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Biomarcadores de Tumor , Leucemia Linfocítica Crónica de Células B/diagnóstico , Lipoproteína Lipasa/fisiología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Estudios de Cohortes , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Lipoproteína Lipasa/antagonistas & inhibidores , Lipoproteína Lipasa/sangre , Lipoproteína Lipasa/genética , Análisis por Micromatrices , Valor Predictivo de las Pruebas , Pronóstico , ARN Interferente Pequeño/farmacología , Estudios RetrospectivosRESUMEN
Elevated plasma lipoprotein(a) (LPA) levels are recognized as an independent risk factor for cardiovascular diseases. Our knowledge on LPA metabolism is incomplete, which makes it difficult to develop LPA-lowering medications. Nicotinic acid (NA) is the main drug recommended for the treatment of patients with increased plasma LPA concentrations. The mechanism of NA in lowering LPA is virtually unknown. To study this mechanism, we treated transgenic (tg) APOA mice with NA and measured plasma APOA and hepatic mRNA levels. In addition, mouse and human primary hepatocytes were incubated with NA, and the expression of APOA was followed. Feeding 1% NA reduced plasma APOA and hepatic expression of APOA in tg-APOA mice. Experiments with cultured human and mouse primary hepatocytes in addition to reporter assays performed in HepG2 cells revealed that NA suppresses APOA transcription. The region between -1446 and -857 of the human APOA promoter harboring several cAMP response element binding sites conferred the negative effect of NA. In accordance, cAMP stimulated APOA transcription, and NA reduced hepatic cAMP levels. It is suggested that cAMP signaling might be involved in reducing APOA transcription, which leads to the lowering of plasma LPA.
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Hígado/metabolismo , Niacina/farmacología , Animales , Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Aterosclerosis/genética , Aterosclerosis/metabolismo , Células Cultivadas , AMP Cíclico/farmacología , Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/efectos de los fármacos , Ratones , Ratones Transgénicos , ARN MensajeroRESUMEN
The liver X receptors (LXRs) are key regulators of genes involved in cholesterol homeostasis. Natural ligands and activators of LXRs are oxysterols. Numerous steroidal and non-steroidal synthetic LXR ligands are under development as potential drugs for individuals suffering from lipid disorders. N,N-dimethyl-3ß-hydroxycholenamide (DMHCA) is a steroidal ligand of LXRs that exerts anti-atherogenic effects in apolipoprotein E-deficient mice without causing negative side effects such as liver steatosis or hypertriglyceridemia. In this report, we investigated the consequences of DMHCA treatment on cholesterol homeostasis in vivo and in vitro. Despite its hydrophobicity, DMHCA is readily absorbed by C57BL/6 mice and taken up by intestinal cells, the lung, heart and kidneys, but is undetectable in the brain. DMHCA significantly reduces cholesterol absorption and uptake in duodenum and jejunum of the small intestine and in turn leads to a reduction of plasma cholesterol by 24%. The most striking finding of this study is that DMHCA inhibited the enzyme 3ß-hydroxysterol-Δ24-reductase resulting in an accumulation of desmosterol in the plasma and in feces. Thus, the reduction of plasma cholesterol was due to a block in the final step of cholesterol biosynthesis. Taken together, DMHCA is an interesting compound with properties distinct from other LXR ligands and might be used to study desmosterol-mediated effects in cells and tissues.
Asunto(s)
Anticolesterolemiantes/farmacología , Colesterol/biosíntesis , Ácidos Cólicos/farmacología , Inhibidores Enzimáticos/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lipogénesis/efectos de los fármacos , Receptores Nucleares Huérfanos/agonistas , Androstenos/farmacocinética , Androstenos/farmacología , Animales , Anticolesterolemiantes/farmacocinética , Anticolesterolemiantes/toxicidad , Supervivencia Celular/efectos de los fármacos , Colesterol/sangre , Colesterol/metabolismo , Ácidos Cólicos/farmacocinética , Desmosterol/metabolismo , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/toxicidad , Hígado Graso/inducido químicamente , Heces , Células Hep G2 , Humanos , Intestinos/efectos de los fármacos , Receptores X del Hígado , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidoresRESUMEN
Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363(-/-) and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL(-/-) mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363(-/-) but unchanged in HSL(-/-) mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL(-/-) macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.
Asunto(s)
Ésteres del Colesterol/metabolismo , Macrófagos/enzimología , Serina Proteasas/genética , Esterol Esterasa/genética , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Células COS , Chlorocebus aethiops , Diglicéridos/metabolismo , Hidrólisis , Macrófagos/metabolismo , Ratones , Ratones Endogámicos , Microscopía Fluorescente , Serina Proteasas/metabolismo , Esterol Esterasa/metabolismo , TransfecciónRESUMEN
The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.
Asunto(s)
Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , MicroARNs/farmacocinética , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Transporte Biológico Activo/fisiología , Línea Celular , HumanosRESUMEN
Lipotoxicity occurs as a consequence of chronic exposure of non-adipose tissue and cells to elevated concentrations of fatty acids, triglycerides and/or cholesterol. The contribution of mitochondria to lipotoxic cell dysfunction, damage and death is associated with elevated production of reactive oxygen species and initiation of apoptosis. Although there is a broad consensus on the involvement of these phenomena with lipotoxicity, the molecular mechanisms that initiate, mediate and trigger mitochondrial dysfunction in response to substrate overload remain unclear. Here, we focus on protein phosphorylation as an important phenomenon in lipotoxicity that harms mitochondria-related signal transduction and integration in cellular metabolism. Moreover, the degradation of mitochondria by mitophagy is discussed as an important landmark that leads to cellular apoptosis in lipotoxicity.