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Resumen Introduccion El incremento de grasa miocardica ha sido propuesto como uno de los principales precursores de la disfuncion miocardica de etiologia diabetica independiente de la enfermedad arterial coronaria. Sin embargo, actualmente se carece de biomarcadores que reflejen el contenido de grasa miocardica para la deteccion clinica de esta patologia. Metodos Las correlaciones entre el contenido de trigliceridos cardiacos y los niveles plasmaticos de las principales moleculas alteradas durante la diabetes y los niveles cardiacos de ARNm de genes implicados en el metabolismo cardiaco (Cd36 y Pdk4) han sido exploradas en un modelo murino de resistencia a la insulina inducida por una dieta con alto contenido en grasas. Resultados En ratones resistentes a la insulina, la dieta grasa aumento los niveles de trigliceridos del miocardio, en comparacion con animales controles alimentados con una dieta estandar. El contenido de trigliceridos cardiacos se encontro directamente asociado con los niveles plasmaticos de glucosa, trigliceridos, VLDL, resistina y leptina. Ademas, se observo una correlacion inversa entre el contenido de trigliceridos y los niveles cardiacos de ARNm de Cd36 y Pdk4. Conclusiones Nuestros datos revelan que el contenido cardiaco de trigliceridos se encuentra asociado con un perfil bioquimico plasmatico alterado y con una reprogramacion de la expresion de genes dirigida a atenuar el impacto de la acumulacion ectopica de lipidos en miocardio.

OBJECTIVE To investigate the metabolic and immunologic factors associated with the presence of central arterial stiffness as measured by the augmentation index (AIx). METHODS We conducted a cross-sectional study of 69 female patients with systemic lupus erythematosus (SLE) compared with a control group of 34 healthy women. The anthropometrical variables, the vascular studies, and the analytic data were obtained the same day. The AIx was assessed by peripheral arterial tonometry. The analysis of lipoprotein populations was performed using nuclear magnetic resonance (NMR) spectroscopy. RESULTS Arterial stiffness was increased in patients with SLE compared with control subjects (mean ± SD 20.30 ± 21.54% versus 10.84 ± 11.51%; P = 0.0021). Values for the AIx were correlated with the Framingham risk score (r = 0.481, P < 0.001), carotid intima-media thickness (r = 0.503, P < 0.001), systolic blood pressure (r = 0.270, P < 0.001), and age (r = 0.365, P < 0.001). Patients receiving antimalarial drugs had a lower AIx (mean ± SD 11.74 ± 11.28% versus 24.97 ± 20.63%; P = 0.024). The AIx was correlated with the atherogenic lipoproteins analyzed by NMR. The immunologic variables associated with the AIx were C4 (r = 0.259, P = 0.046) and IgM anti-β2 -glycoprotein I (IgM anti-β2 GPI) (r = 0.284, P = 0.284). In the multivariate analysis, age (β = 0.347, 95% confidence interval [95% CI] 0.020-0.669, P = 0.035), IgM β2 GPI (β = 0.321, 95% CI 0.024-0.618, P = 0.035) and small dense high-density lipoprotein (HDL) particles (β = 1.288, 95% CI 0.246-2.329, P = 0.017) predicted the AIx. CONCLUSION SLE patients had increased arterial stiffness compared with healthy control subjects. Arterial stiffness was decreased in patients treated with antimalarial drugs. Age, IgM β2 GPI, and the number of small dense HDL particles predicted the AIx.

An imbalance between hepatic fatty acid uptake and removal results in ectopic fat accumulation, which leads to non-alcoholic fatty liver disease (NAFLD). The amount and type of accumulated triglycerides seem to play roles in NAFLD progression, however, a complete understanding of how triglycerides contribute to NAFLD evolution is lacking. Our aim was to evaluate triglyceride accumulation in NAFLD in a murine model and its associations with molecular mechanisms involved in liver damage and adipose tissue-liver cross talk by employing lipidomic and molecular imaging techniques. C57BL/6J mice fed a high-fat diet (HFD) for 12 weeks were used as a NAFLD model. Standard-diet (STD)-fed animals were used as controls. Standard liver pathology was assessed using conventional techniques. The liver lipidome was analyzed by liquid chromatography&ndash, mass spectrometry (LC&ndash, MS) and laser desorption/ionization&ndash, mass spectrometry (LDI&ndash, MS) tissue imaging. Liver triglycerides were identified by MS/MS. The transcriptome of genes involved in intracellular lipid metabolism and inflammation was assessed by RT-PCR. Plasma leptin, resistin, adiponectin, and FABP4 levels were determined using commercial kits. HFD-fed mice displayed increased liver lipid content. LC&ndash, MS analyses identified 14 triglyceride types that were upregulated in livers from HFD-fed animals. Among these 14 types, 10 were identified in liver cross sections by LDI&ndash, MS tissue imaging. The accumulation of these triglycerides was associated with the upregulation of lipogenesis and inflammatory genes and the downregulation of &beta, oxidation genes. Interestingly, the levels of plasma FABP4, but not of other adipokines, were positively associated with 8 of these triglycerides in HFD-fed mice but not in STD-fed mice. Our findings suggest a putative role of FABP4 in the liver-adipose tissue cross talk in NAFLD.

The structural similarity among lipid species and the low sensitivity and spectral resolution of nuclear magnetic resonance (NMR) have traditionally hampered the routine use of 1H NMR lipid profiling of complex biological samples in metabolomics, which remains mostly manual and lacks freely available bioinformatics tools. However, 1H NMR lipid profiling provides fast quantitative screening of major lipid classes (fatty acids, glycerolipids, phospholipids, and sterols) and some individual species and has been used in several clinical and nutritional studies, leading to improved risk prediction models. In this Article, we present LipSpin, a free and open-source bioinformatics tool for quantitative 1H NMR lipid profiling. LipSpin implements a constrained line shape fitting algorithm based on voigt profiles and spectral templates from spectra of lipid standards, which automates the analysis of severely overlapped spectral regions and lipid signals with complex coupling patterns. LipSpin provides the most detailed quantification of fatty acid families and choline phospholipids in serum lipid samples by 1H NMR to date. Moreover, analytical and clinical results using LipSpin quantifications conform with other techniques commonly used for lipid analysis.

Background: Glucose-regulated protein 78/Binding immunoglobulin protein (GRP78/BiP) is a protein associated with endoplasmic reticulum stress and is upregulated by metabolic alterations at the tissue-level, such as hypoxia or glucose deprivation, and it is hyper-expressed in fat tissue of obese individuals. Objective: To investigate the role of the GRP78/BiP level as a metabolic and vascular disease biomarker in patients with type 2 diabetes (DM), obesity and metabolic syndrome (MS). Methods: Four hundred and five patients were recruited, of whom 52.5% were obese, 72.8% had DM, and 78.6% had MS. The intimae media thickness (cIMT) was assessed by ultrasonography. The plasma GRP78/BiP concentration was determined, and its association with metabolic and vascular parameters was assessed. Circulating GRP78/BiP was also prospectively measured in 30 DM patients before and after fenofibrate/niacin treatment and 30 healthy controls. Results: In the cross-sectional study, the GRP78/BiP level was significantly higher in the patients with obesity, DM, and MS. Age-, gender- and BMI-adjusted GRP78/BiP was directly associated with LDL-cholesterol, non-HDL-cholesterol, triglycerides, apoB, and cIMT. GRP78/BiP was positively associated to carotid plaque presence in the adjusted model, irrespective of obesity, DM and MS. In the prospective study, nicotinic acid treatment produced a significant reduction in the GRP78/BiP levels that was not observed with fenofibrate. Conclusions: GRP78/BiP plasma concentrations are increased in patients with both metabolic derangements and subclinical atherosclerosis. GRP78/BiP could be a useful marker of metabolic and cardiovascular risk.

ObjectivesTo identify potential biomarkers of disease activity analysing the proteome of high-density lipoprotein (HDL) particles from SLE patients in clinical remission and when they develop a flare compared with a healthy control group.MethodsQuantitative proteomic analyses of purified HDL were performed using Tandem Mass Tag isobaric tag-labelling and nanoLC-Orbitrap (nLC-MS/MS) from nine SLE patients in clinical remission when they developed a flare and from nine healthy controls (9–9-9). We verified the identified proteins by Western blot and ELISA in a cohort of 104 SLE women patients, 46 healthy women and 14 SLE patients when a flare developed.ResultsWe found 17 proteins with a significant fold-change (>1.1) compared with the control group. In lupus patients experiencing a flare compared with those in remission, we identified four proteins with a significant fold-change (C4, Indian Hedgehog protein, S100A8 and gelsolin). Plasma gelsolin (pGSN) levels were decreased in the 104 SLE patients (176.02(74.9) mcg/l) compared with the control group (217.13(86.7) mcg/l); P=0.005 and when they developed a clinical flare (104.84(41.7) mcg/l); P=0.002). pGSN levels were associated with HDL cholesterol levels (r = 0.316, PConclusionDecreased pGSN are associated with clinical disease activity in SLE patients. Antimalarial treatment and HDL cholesterol are associated with higher levels of pGSN.

Background and aims The first line of therapy in children with hypercholesterolaemia is therapeutic lifestyle changes (TLSC). The efficacy of lifestyle intervention in children with familial hypercholesterolaemia (FH), where LDL-C levels are genetically driven, deserves a focused study. Aims To evaluate the impact of a lifestyle education program, focused on food patterns and physical activity, on lipid profiles assessed by nuclear magnetic resonance (NMR) in children with FH vs. non-FH. Methods Phase 1 was a cross-sectional study of baseline characteristics, and phase 2 was a prospective TLSC intervention study. In total, the study included 238 children (4 to 18 years old; 47% girls) attending the lipid unit of our hospital due to high cholesterol levels. Eighty-five were diagnosed with FH (72% genetic positive), and 153 were diagnosed with non-Familial hypercholesterolaemia. A quantitative food frequency questionnaire (FFQ) including 137 items was used. Physical activity (PA) was assessed by the Minnesota questionnaire. The lipid profile was assessed using the 2D-1H-NMR (Liposcale test). A total of 127 children (81 in the FH group) participated in the prospective phase and were re-assessed after 1 year of the TLSC intervention, consisting of education on lifestyle changes delivered by a specialized nutritionist. Results The FH and non-FH groups were similar in anthropometry and clinical data, except that those in the FH were slightly younger than those in the non-FH group. Both the FH and non-FH groups showed a similar diet composition characterized by a high absolute calorie intake and a high percentage of fat, mainly saturated fat. The PA was below the recommended level in both groups. After one year of TLSC, the percentage of total and saturated fats was reduced, and the amount of fiber increased significantly in both groups. The percentage of protein increased slightly. The number of children engaged in at least 1 hour/day of PA increased by 56% in the FH group and by 53% in the non-FH group, and both these increases were significant. The total and small-LDL particle numbers were reduced in both groups, although the absolute change was greater in the FH group than in the non-FH group. Conclusions Educational strategies to implement TLSC in children lead to empowerment, increased adherence, and overall metabolic improvement in children with high blood cholesterol, including those with FH.

While cholesterol content in high-density lipoproteins (HDLs) is a well-established inverse marker of cardiovascular risk, the importance of HDL&ndash, triglyceride (HDL-TG) concentration is not well known. We aim to examine plasma HDL-TG concentrations, assessed by 1H-NMR, in patients with metabolic diseases and their association with classical biomarkers. In this cross-sectional study, we included 502 patients with type 2 diabetes or metabolic syndrome attending the lipid unit of our University Hospital. The presence of arteriosclerotic plaques was assessed by ultrasonography. A complete lipoprotein profile was performed by 1H-NMR (Liposcale test). HDL-TG was strongly positively correlated with total triglycerides, glycerol, and fatty liver index, while a strong negative correlation was observed with HDL-cholesterol (HDL-C) and HDL-particle number (HDL-P). HDL-TG was associated with all triglyceride-rich lipoprotein parameters and had an opposite association with HDL-C and HDL-P. It was also significantly correlated with circulating cholesterol ester transfer protein (CETP). HDL-TG concentrations were higher as metabolic syndrome components increased. HDL-TG was also higher with worsening glucose metabolism. Patients with carotid plaques also showed higher HDL-TG. In contrast to HDL-C, HDL-TG is directly associated with metabolism and arteriosclerotic vascular alterations. HDL-TG should be considered a biomarker of metabolic and cardiovascular risk and could be a marker of HDL dysfunction.

Background: Recent proteomics techniques have demonstrated that high-density lipoprotein (HDL) associated proteins are involved in functions related to systemic inflammatory and immune responses in several pathological conditions, including autoimmune diseases. HDL undergoes structural and functional modifications in systemic lupus erythematous (SLE) patients. Objectives: To identify potential biomarkers of disease activity analyzing the proteome of HDL particles from SLE patients in clinical remission and when they develop a flare compared to a healthy control group. Methods: Quantitative proteomic analyses of purified HDL were performed using Tandem Mass Tag (TMT) isobaric tag-labeling and nanoLC-Orbitrap (nLC-MS/MS) from 9 SLE patients in clinical remission when they developed a flare and from 9 healthy controls (9-9-9). We verified the identified proteins by Western blot and ELISA in a cohort of 104 SLE women patients, 46 healthy women and 14 SLE patients when developed a flare. Results: A total of 83 proteins associated with HDL were identified. We found 17 proteins with a significant fold-change (>1.1) compared with their levels in control patients. In lupus patients experiencing a flare compared with those in remission, we identified 4 proteins with a significant fold-change (C4, Indian Hedgehog protein, S100A8 and gelsolin). Plasma Gelsolin (pGSN) levels were decreased in the 104 SLE patients (176.02(74.9) mcg/l) compared with the control group (217.13(86.7)mcg/l); p =0.005 and when they developed a clinical flare (104.84(41.7)mcg/l); p = 0002). pGSN levels were associated with HDL-c levels (r =0.316, p Conclusion: The proteome cargo from HDL differentiates SLE patients from healthy controls. HDL from SLE patients carries proteins that are involved in the activation of the immune system. Decreased pGSN are associated with clinical disease activity in SLE patients. Antimalarial treatment and HDL-c are associated with higher levels of pGSN in SLE patients. pGSN is a potential biomarker of disease activity in SLE patients. Disclosure of Interests: None declared

Introduction The increase in myocardial fat has been proposed as one of the main precursors of myocardial dysfunction of diabetic aetiology, independent of coronary artery disease. However, biomarkers reflecting the myocardial fat content for the clinical detection of this pathology are currently lacking. Methods Correlations between the content of cardiac triglycerides and plasma levels of the main molecules altered during diabetes and cardiac mRNA levels of genes involved in cardiac metabolism (Cd36 and Pdk4) have been explored in a murine model of insulin resistance induced by a high-fat diet. Results In insulin-resistant mice, the fatty diet increased myocardial triglyceride levels, compared to control animals fed with a standard diet. Cardiac triglyceride content was found to be directly associated with plasma levels of glucose, triglycerides, VLDL, resistin and leptin In addition, an inverse correlation was observed between triglyceride content and cardiac mRNA levels of Cd36 and Pdk4. Conclusions Our data reveal that cardiac triglyceride content is associated with an altered plasma biochemical profile and with a reprogramming of gene expression aimed at attenuating the impact of ectopic lipid accumulation in the myocardium.

Objective: Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone involved in the crosstalk between adipose and peripheral tissues, and it contributes to widespread insulin resistance in cells, including cardiac cells. However, the role of this adipokine in regulating cardiac metabolism and myocardial neutral lipid content in patients with type 2 diabetes has not been elucidated. Methods: The impact of circulating FABP4 on the cardiac neutral lipid content was measured by proton magnetic resonance spectroscopy (H-MRS) in patients with type 2 diabetes. Additionally, circulating FABP4 and the cardiac triglyceride content were analysed in high-fat diet (HFD)-fed mice, and the impact of the exogenous FABP4 was explored in HL-1 cardiac cells. Results: Serum FABP4 levels were higher in type 2 diabetic patients compared to healthy individuals. Circulating FABP4 levels were associated with myocardial neutral lipid content in type 2 diabetic patients. In HFD-fed mice, both serum FABP4 and myocardial triglyceride content were increased. In FABP4-challenged HL-1 cells, extracellular FABP4 increased intracellular lipid accumulation, which led to impairment of the insulin-signalling pathway and reduced insulin-stimulated glucose uptake. However, these effects were partially reversed by FABP4 inhibition with BMS309403, which attenuated the intracellular lipid content and improved insulin signalling and insulin-stimulated glucose uptake. Conclusions: Taken together, our results identify FABP4 as a molecule involved in diabetic/lipid-induced cardiomyopathy and indicate that this molecule may be an emerging biomarker for diabetic cardiomyopathy-related disturbances, such as myocardial neutral lipid accumulation. Additionally, FABP4 inhibition may be a potential therapeutic target for metabolic-related cardiac dysfunctions., This work was supported by funds from Instituto de Salud Carlos III (ISCIII), Madrid, Spain [PI15/00627 to LM and PI14/01729 to VLlC], Fondo Europeo de Desarrollo Regional [FEDER to LM], the CIBER in Diabetes and Associated Metabolic Disorders [CB07/08/0028 to LM and CB07/08/0003 to MV-C] and CIBER Cardiovascular [CB16/11/00403 to DdG-C and VL-C], the Spanish Ministerio de Economía y Competitividad [SAF2015-64146-R to MV-C and TEC2015-69076-P to XC] and Fundació la Marató de TV3 2014 [to MV-C]. DdG-C was a recipient of Sara Borrell grant from the Instituto de Salud Carlos III Grant [CD14/00109].

Background and aims Fatty acid binding protein 4 (FABP4) is an intracellular fatty acid (FA) carrier protein that is, in part, secreted into circulation. Circulating FABP4 levels are increased in obesity, diabetes and other insulin resistance (IR) diseases. FAs contribute to IR by promoting endoplasmic reticulum stress (ER stress) and altering the insulin signaling pathway. The effect of FABP4 on ER stress in the liver is not known. The aim of this study was to investigate whether exogenous FABP4 (eFABP4) is involved in the lipid-induced ER stress in the liver. Methods HepG2 cells were cultured with eFABP4 (40 ng/ml) with or without linoleic acid (LA, 200 μM) for 18 h. The expression of ER stress-related markers was determined by Western blotting (ATF6, EIF2α, IRE1 and ubiquitin) and real-time PCR (ATF6, CHOP, EIF2α and IRE1). Apoptosis was studied by flow cytometry using Annexin V-FITC and propidium iodide staining. Results eFABP4 increased the ER stress markers ATF6 and IRE1 in HepG2 cells. This effect led to insulin resistance mediated by changes in AKT and JNK phosphorylation. Furthermore, eFABP4 significantly induced both apoptosis, as assessed by flow cytometry, and CHOP expression, without affecting necrosis and ubiquitination. The presence of LA increased the ER stress response induced by eFABP4. Conclusions eFABP4, per se , induces ER stress and potentiates the effect of LA in HepG2 cells, suggesting that FABP4 could be a link between obesity-associated metabolic abnormalities and hepatic IR mechanisms.

PCSK9 is a pivotal molecule in the regulation of lipid metabolism. Previous studies have suggested that PCSK9 expression and its function in LDL receptor regulation could be altered in the context of diabetes. The aim was to assess PCSK9 plasma levels in patients with type 2 diabetes (T2DM) and other related metabolic disorders as well as its relation to the metabolomic profile generated by nuclear magnetic resonance (NMR) and glucose homeostasis.There were recruited a total of 457 patients suffering from T2DM and other metabolic disorders (metabolic syndrome (MetS), obesity and atherogenic dyslipidaemia (AD) and other disorders). Anamnesis, anthropometry and physical examinations were conducted, and vascular and abdominal adiposity imaging were carried out. Biochemical studies were performed to determine PCSK9 plasma levels 6 weeks after lipid lowering drug wash-out in treated patients. A complete metabolomic lipid profile was also generated by NMR. The rs505151 and rs11591147 genetic variants of PCSK9 gene were identified in patients.The results showed that PCSK9 levels are increased in patients with T2DM and MetS (14% and 13%; p0.005, respectively). Circulating PCSK9 levels were correlated with an atherogenic lipid profile and with insulin resistance parameters. PCSK9 levels were also positively associated with AD, as defined by lipoprotein particle number and size. The rs11591147 genetic variant resulted in lower levels of circulating PCSK9 and LDL cholesterol (LDL-C).PCSK9 plasma levels are increased in T2DM and MetS patients and are associated with LDL-C and other parameters of AD and glucose metabolism.

Aims The aim of this study is to determine the physical and functional interplay between fatty acid-binding protein 4 (FABP4) and its membrane receptor-like candidate protein, cytokeratin 1 (CK1), and to determine the effect of hindering CK1-mediated FABP4 cellular uptake on non-disturbed or metabolically stressed endothelial cells. Methods We monitored the direct interaction between FABP4 and CK1 using surface plasmon resonance, and the effects of blocking exogenous FABP4 (eFABP4) cellular uptake were determined by using specific siRNA to knock down the expression of CK1 in human umbilical vein endothelial cells (HUVECs). The expression and nuclear translocation of transcription factors involved in oxidative stress (NRF2) and inflammation (p65 subunit of NF-ĸB transcription factor) were determined by Western blotting analysis. Results Our data showed that FABP4 and CK1 bind to each other and that the putative FABP4 binding domain would be within the 151GIQEVTINQSLLQPLNVEID170 CK1 sequence. We determined that in non-disturbed or metabolically stressed endothelial cells, eFABP4 regulates the cellular response to oxidative stress. In addition, we also found that in the presence of palmitate, eFABP4 increases the pro-inflammatory effects induced by palmitate per se, probably due to an increase in the transport of palmitate inside cells, suggesting that these FABP4-mediated pro-oxidative and pro-inflammatory effects are dependent on CK1 expression. Conclusions We demonstrated that CK1 facilitates eFABP4 cellular uptake in endothelial cells. Therefore, the CK1-targeted inhibition of exogenous FABP4 cellular uptake might be a potential therapeutic strategy to protect endothelial cells against FABP4-induced activation of inflammation and oxidative stress.

The structural similarity among lipid species and the low sensitivity and spectral resolution of nuclear magnetic resonance (NMR) have traditionally hampered the routine use of

Aims Fatty acid binding protein 4 (FABP4) inhibitors have been proposed as potential therapeutic approaches against insulin resistance-related inflammation and type 2 diabetes mellitus. However, the underlying molecular mechanisms by which these molecules drive these effects in skeletal muscle remain unknown. Here, we assessed whether the FABP4 inhibitor BMS309403 prevented lipid-induced endoplasmic reticulum (ER) stress-associated inflammation in skeletal muscle. Materials and methods The BMS309403 treatment was assessed both in the skeletal muscle of high-fat diet (HFD)-fed mice and in palmitate-stimulated C2C12 myotubes. Results HFD feeding promoted insulin resistance, which is characterized by increased plasma levels of glucose, insulin, non-esterified fatty acids, triglycerides, resistin, and leptin and reduced plasma levels of adiponectin compared with control mice fed a standard diet. Additionally, insulin-resistant animals showed increased FABP4 plasma levels. In line with this evidence, recombinant FABP4 attenuated the insulin-induced AKT phosphorylation in C2C12 myotubes. Treatment with BMS309403 reduced lipid-induced ER stress and inflammation in both mouse skeletal muscle and C2C12 myotubes. The effects of the FABP4 inhibitor reducing lipid-induced ER stress-associated inflammation were related to the reduction of fatty acid-induced intramyocellular lipid deposits, ROS and nuclear factor-kappaB (NF-κB) nuclear translocation. Accordingly, BMS309403 reduced lipid-induced p38 MAPK phosphorylation, which is upstream of NF-κB activation. Conclusion Overall, these findings indicate that BMS309403 reduces fatty acid-induced ER stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

Determination of lipoprotein particle size and number using advanced lipoprotein tests (ALTs) is of particular importance to improve cardiovascular risk prediction. Here we present the Liposcale test, a novel ALT based on 2D diffusion-ordered 1H NMR spectroscopy. Our method uses diffusion coefficients to provide a direct measure of the mean particle sizes and numbers. Using 177 plasma samples from healthy individuals and the concentration of ApoB and ApoA from isolated lipoprotein fractions, our test showed a stronger correlation between the NMR-derived lipoprotein particle numbers and apolipoprotein concentrations than the LipoProfile ® test commercialized by Liposcience. We also converted LDL particle numbers to ApoB equivalents (milligrams per deciliter) and our test yielded similar values of LDL-ApoB to the LipoProfile ® test (absolute mean bias of 8.5 and 7.4 mg/dl, respectively). In addition, our HDL particle number values were more concordant with the calibrated values determined recently using ion mobility. Finally, principal component analysis distinguished type 2 diabetic patients with and without atherogenic dyslipidemia (AD) on a second cohort of 307 subjects characterized using the Liposcale test (area under the curve = 0.88) and showed concordant relationships between variables explaining AD. Altogether, our method provides reproducible and reliable characterization of lipoprotein particles and it is applicable to pathological states such as AD. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

HDL-increasing drugs such as fenofibrate and niacin have failed to decrease the cardiovascular risk in patients with type 2 diabetes. Drug-mediated quantitative and qualitative HDL modifications could be involved in these negative results. To evaluate the quantitative and qualitative effects of niacin and fenofibrate on HDL in patients with type 2 diabetes, a prospective, randomised controlled intervention trial was conducted. Thirty type 2 diabetic patients with low HDL were randomised to receive either fenofibrate (FFB) or niacin + laropiprant (ERN/LPR) as an add-on to simvastatin treatment for 12 weeks according to a crossover design. At the basal point and after each intervention period, physical examinations and comprehensive standard biochemical determinations and HDL metabolomics were performed. Thirty nondiabetic patients with normal HDL were used as a basal control group. ERN/LRP, but not FFB, significantly increased HDL cholesterol. Neither ERN/LRP nor FFB reversed the HDL particle size or particle number to normal. ERN/LRP increased apoA-I but not apoA-II, whereas FFB produced the opposite effect. FFB significantly increased Preβ1-HDL, whereas ERN/LRP tended to lower Preβ1-HDL. CETP and LCAT activities were significantly decreased only by ERN/LRP. PAF-AH activity in HDL and plasma decreased with the use of both agents. Despite their different actions on antioxidant parameters, none of the treatments induced detectable antioxidant improvements. ERN/LRP and FFB had strikingly different effects on HDL quantity and quality, as well as on HDL cholesterol concentrations. When prescribing HDL cholesterol increasing drugs, this differential action should be considered.

Resumen Introduccion Fatty acid-binding protein 4 (FABP4) es una adipocina secretada por el tejido adiposo implicada en la regulacion del metabolismo energetico y la inflamacion. FABP4 circulante se asocia con obesidad, dislipidemia aterogenica y sindrome metabolico. Estudios recientes muestran una asociacion entre FABP4 circulante y disfuncion endotelial, aunque se desconoce como se produce esta. El objetivo de este trabajo es estudiar la interaccion entre FABP4 con las proteinas de la membrana citoplasmatica en celulas endoteliales. Metodologia Se incubaron celulas HUVEC con y sin FABP4 (100 ng/ml) durante 5 min. La inmunolocalizacion de FABP4 se estudio mediante microscopia confocal. Para estudiar las interacciones de FABP4 con las proteinas de membrana de las celulas HUVEC se diseno una estrategia que combina incubaciones con o sin 6XHistidine-tag FABP4 (FABP4-His) (100 ng/ml), cross-linking con formaldehido, extraccion de proteinas de membrana y western blot. Resultados Los resultados muestran que FABP4 colocaliza con CD31, una proteina utilizada como marcador de membrana citoplasmatica. Ademas se observan diferentes patrones de western blot en funcion de la incubacion con o sin FABP4-His. El inmunoblot revela la existencia de 3 complejos proteicos de aproximadamente 108, 77 y 33 kDa formados por FABP4 exogena y su posible receptor/es. Discusion Los resultados obtenidos apoyan la existencia de un complejo proteico capaz de unir FABP4 a las celulas endoteliales mediante una union especifica. Ademas, nos permiten avanzar en el conocimiento de los efectos moleculares de FABP4 y, en caso de confirmarse, podrian utilizarse como diana terapeutica para prevenir enfermedades cardiovasculares.

While the impact of very low concentrations of low-density lipoprotein cholesterol (LDL-C) on cardiovascular prevention is very reassuring, it is intriguing to know what effect these extremely low LDL-C concentrations have on lipid homoeostasis. The evidence supporting the safety of extremely low LDL levels comes from genetic studies and clinical drug trials. Individuals with lifelong low LDL levels due to mutations in genes associated with increased LDL-LDL receptor (LDLR) activity reveal no safety issues. Patients achieving extremely low LDL levels in the IMPROVE-IT and FOURIER, and the PROFICIO and ODYSSEY programs seem not to have an increased prevalence of adverse effects. The main concern regarding extremely low LDL-C plasma concentrations is the adequacy of the supply of cholesterol, and other molecules, to peripheral tissues. However, LDL proteomic and kinetic studies reaffirm that LDL is the final product of endogenous lipoprotein metabolism. Four of 5 LDL particles are cleared through the LDL-LDLR pathway in the liver. Given that mammalian cells have no enzymatic systems to degrade cholesterol, the LDL-LDLR pathway is the main mechanism for removal of cholesterol from the body. Our focus, therefore, is to review, from a physiological perspective, why such extremely low LDL-C concentrations do not appear to be detrimental. We suggest that extremely low LDL-C levels due to increased LDLR activity may be a surrogate of adequate LDL-LDLR pathway function.

Background Patients affected by Systemic Lupus Erythematosus (SLE) show an increase in cardiovascular mortality and morbidity. The accelerated atherosclerosis observed in patients with SLE cannot be entirely explained by the traditional cardiovascular risk factors. Patients with SLE show increased subclinical atherosclerosis determined by an increased carotid arterial thickness, endothelial dysfunction and central arterial stiffness. Analysis of lipoproteins by magnetic nuclear resonance (MNR) provide information of those lipoproteins associated with sublcinial atherosclerosis in SLE. Objectives To investigate the metabolic and immunological factors associated with the presence of central arterial stiffness determined by the Augmentation Index (AIx) as well as the detailed analysis of the lipid profile performed by magnetic nuclear resonance (MNR) Methods Descriptive cross-sectional study of 69 women with SLE compared with a control group of 34 age matched healthy women. On the same day of the study, blood extraction, physical examination and augmentation index (AIx) obtained by Peripheral Arterial Tonometry were performed. The carotid intima-media thickness (IMTc) was also performed on the same day of the study to correlate the arterial stiffness with another subclinical atherosclerosis marker. Analysis of lipoprotein populations by NMR (Liposcale,Biosfer Teslab) were performed. Results Patients with SLE showed significant increased arterial stiffness respect the control group (20.30 (21.54)% vs 10.84 (11.51)%, P=0.0021)The values of AIx were well correlated with Framingham risk score (r=0.486, P Conclusions SLE patients show increased central arterial stiffness respect healthy population. Patients treated with antamalarial drugs show lower arterial stifness. In multivariate analyses variables that predicted levels of AIx were age, levels of IgM-β2-glycoprotein and the number of small-dense HDL particles. Disclosure of Interest None declared

Background and aims Obesity is associated with increased cardiovascular risk. However, the impact of morbid obesity on vascular structure and function is not well understood. This study was designed to appraise subclinical atherosclerosis markers, including carotid intima media thickness (cIMT), endothelial function, and arterial wall stiffness, and their determinants, in morbidly obese patients. Methods and results In this cross-sectional study 194 overweight and obese patients were distributed in morbid-obese patients (MOP, n = 110), obese (OP, n = 84) and overweight patients (OwP, n = 33) groups. Demography, anthropometry, clinical and standard biochemical data were recorded. cIMT, endothelial function, defined as the small artery reactivity index (saRHI), and artery wall rigidity, studied by the augmentation index, were determined. More than 50% of the MOP, OP and OwP had a cIMT above the 75th percentile per age and gender. No differences in cIMT or saRHI were observed, although overweight and obese patients (OOP) had higher arterial rigidity compared with the morbid-obese patients. In a multivariate regression test, while cholesterol was the main determinant of cIMT in overweight and obese patients, glucose metabolism was the determinant in MOP. Conclusion More than half of the population have a cIMT above general population ranges. OwP, OP and MOP have similar cIMT and saRHI. However, OOP have greater arterial wall rigidity. Dysglycemia is the main factor associated with subclinical atherosclerosis in MOP.

Background Low carbohydrate diets have become increasingly popular for weight loss. Although they may improve some metabolic markers, particularly in type 2 diabetes mellitus (T2D) or metabolic syndrome (MS), their net effect on vascular function remains unclear. Objective Evaluate the relation between dietary macronutrient composition and the small artery reactive hyperaemia index (saRHI), a marker of small artery vascular function, in a cohort of MS patients. Design This cross-sectional study included 160 MS patients. Diet was evaluated by a 3-day food-intake register and reduced to a novel low-carbohydrate diet score (LCDS). Physical examination, demographic, biochemical and anthropometry parameters were recorded, and saRHI was measured in each patient. Results Individuals in the lowest LCDS quartile (Q1; 45% carbohydrate, 19% protein, 31% fat) had higher saRHI values than those in the top quartile (Q4; 30% carbohydrate, 25% protein, 43% fat) (1.84 ± 0.42 vs. 1.55 ± 0.25, P = .012). These results were similar in T2D patients (Q1 = 1.779 ± 0.311 vs. Q4 = 1.618 ± 0.352, P = .011) and also in all of the MS components, except for low HDLc. Multivariate analysis demonstrated that individuals in the highest LCDS quartile, that is, consuming less carbohydrates, had a significantly negative coefficient of saRHI which was independent of confounders (HR: −0.747; 95%CI: 0.201, 0.882; P = .029). Conclusions These data suggest that a dietary pattern characterized by a low amount of carbohydrate, but reciprocally higher amounts of fat and protein, is associated with poorer vascular reactivity in patients with MS and T2D.

Background PCSK9 inhibition is a new powerful cholesterol-lowering strategy. Recently, it was reported that CETP inhibitors influence PCSK9 levels as an off-target effect. We explored the relationship between circulating PCSK9 levels and CETP activity in patients with metabolic disease who were not on lipid-lowering therapy. Methods Plasma CETP activity and PCSK9 levels were measured in 450 participants (median age, 58 years; 49 % women) who attended the metabolism unit because of metabolic syndrome (MetS) (78 %), atherogenic dyslipidemia (32 %), obesity (50 %), type 2 diabetes mellitus (72 %), and other risk factors (13 %). A 6 week lipid-lowering drug wash-out period was established in treated patients. Results Both PCSK9 levels and CETP activity were higher in patients with an increasing number of MetS components. PCSK9 levels were positively correlated with CETP activity in the entire cohort (r = 0.256, P

Recent studies have shown that pharmacological increases in HDL cholesterol concentrations do not necessarily translate into clinical benefits for patients, raising concerns about its predictive value for cardiovascular events. Here we hypothesize that the size-modulated lipid distribution within HDL particles is compromised in metabolic disorders that have abnormal HDL particle sizes, such as type 2 diabetes mellitus (DM2). By using NMR spectroscopy combined with a biochemical volumetric model we determined the size and spatial lipid distribution of HDL subclasses in a cohort of 26 controls and 29 DM2 patients before and after two drug treatments, one with niacin plus laropiprant and another with fenofibrate as an add-on to simvastatin. We further characterized the HDL surface properties using atomic force microscopy and fluorescent probes to show an abnormal lipid distribution within smaller HDL particles, a subclass particularly enriched in the DM2 patients. The reduction in the size, force cholesterol esters and triglycerides to emerge from the HDL core to the surface, making the outer surface of HDL more hydrophobic. Interestingly, pharmacological interventions had no effect on this undesired configuration, which may explain the lack of clinical benefits in DM2 subjects.

Resumen Introduccion La hipercolesterolemia familiar (HF) heterocigota es un trastorno genetico del metabolismo de las lipoproteinas que conlleva un riesgo cardiovascular (RCV) muy elevado. El estudio de la pared arterial y su funcion son de especial interes en pacientes con HF. La rigidez arterial esta asociada a un incremento del RCV. El objetivo del estudio fue determinar la rigidez arterial en pacientes con HF y su asociacion con parametros bioquimicos y vasculares. Metodos Se incluyeron 125 pacientes con HF y 59 controles sanos (GC). Se obtuvieron datos clinicos, antropometricos y parametros bioquimicos. Se determino la rigidez arterial basada en el indice de aumento ajustado por 75 latidos por minuto (IAx@75), mediante tonometria arterial periferica, el grosor intima-media carotideo (GIMc) y el indice tobillo-brazo (ITB). Resultados Los pacientes con HF presentaron un incremento significativo en el IAx@75 con respecto a GC (9,8 ± 18,3 vs. 2,4 ± 11,1%; p = 0,011), tambien un mayor GIMc (0,758 ± 0,280 vs. 0,635 ± 0,160 mm; p Conclusiones Los pacientes con HF presentan rigidez arterial incrementada. El IAx@75 esta asociado con las concentraciones de Apo-B100 y es un determinante del GIMc. El IAx@75 podria utilizarse como marcador precoz de RCV en pacientes con HF.

Low-carbohydrate diets have become increasingly popular for weight loss. Although they may improve some metabolic markers, particularly in type 2 diabetes mellitus (T2D) or the metabolic syndrome (MS), their net effect on arterial wall function remains unclear. The objective was to evaluate the relation between dietary macronutrient composition and the small artery reactive hyperaemia index (saRHI), a marker of small artery endothelial function, in a cohort of patients at increased cardiovascular (CV) risk. The present cross-sectional study included 247 patients. Diet was evaluated by a 3-d food-intake register and reduced to a novel low-carbohydrate diet score (LCDS). Physical examination, demographic, biochemical and anthropometry parameters were recorded, and the saRHI was measured in each patient. Individuals in the lowest LCDS quartile (Q1, 45 % carbohydrate; 20 % protein; 32 % fat) had higher saRHI values than those in the top quartile (Q4, 29 % carbohydrate, 24 % protein, 40 % fat; 1·66 (sd 0·41) v. 1·52 (sd 0·22), P= 0·037). These results were particularly strong in patients with the MS (Q1 = 1·82 (sd 0·32) v. Q4 = 1·61 (sd 027); P= 0·021) and T2D (Q1 = 1·78 (sd 0·31) v. Q4 = 1·62 (sd 0·35); P= 0·011). Multivariate analysis demonstrated that individuals in the highest LCDS quartile had a significantly negative coefficient of saRHI, which was independent of confounders (OR − 0·85; 95 % CI 0·19, 0·92; P= 0·031). These findings suggest that a dietary pattern characterised by a low amount of carbohydrate, but high amounts of protein and fat, is associated with a poorer small artery vascular reactivity in patients with increased CV risk.

The sizes of certain types of lipoprotein particles have been associated with an increased risk of cardiovascular disease. However, there is currently no gold standard technique for the determination of this parameter. Here, we propose an analytical procedure to measure lipoprotein particles sizes using diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY). The method was tested on six lipoprotein fractions, VLDL, IDL, LDL1, LDL2, HDL2, and HDL3, which were obtained by sequential ultracentrifugation from four patients. We performed a pulsed-field gradient experiment on each fraction to obtain a mean diffusion coefficient, and then determined the apparent hydrodynamic radius using the Stokes–Einstein equation. To validate the hydrodynamic radii obtained, the particle size distribution of these lipoprotein fractions was also measured using transmission electron microscopy (TEM). The standard errors of duplicate measurements of diffusion coefficient ranged from 0.5% to 1.3%, confirming the repeatability of the technique. The coefficient of determination between the hydrodynamic radii and the TEM-derived mean particle size was r2 = 0.96, and the agreement between the two techniques was 85%. Thus, DOSY experiments have proved to be accurate and reliable for estimating lipoprotein particle sizes.

Background and aims Abdominal obesity (AO) is associated with endothelial function (EF) alteration and increased global cardiovascular (CV) risk. Therapeutic lifestyle changes (TLSC) reduce CV risk, but the impact on EF assessed by peripheral artery tonometry (PAT) is unknown. In this study, we aimed to prospectively assess the effects of TLSC on EF measured by PAT in increased CV risk patients with AO. Methods and results 150 patients with AO and moderate CV risk were randomized to groups receiving a one-year intervention of either conventional medical care (control group, CG) or an intensive TLSC program (intervention group, IG). Vascular studies (EF by PAT, intima-media thickness (IMT)) and lifestyle (LS) assessment were performed before and after intervention. The PAT ratio improved in the IG and worsened in the CG. The global CV risk was reduced ( P = 0.017) in the IG due to a significant decrease in systolic blood pressure ( P P = 0.013). More individuals in the IG than in the CG quit smoking ( P = 0.001) and increased their physical activity ( P = 0.014). The improvement in at least two LS components was associated with a PAT ratio increase (2.44 IC: 95% 0.99–6.00, P = 0.051). The PAT ratio increase determined less IMT progression (−1.1 IC: 95% 0.91–1.00, P = 0.053). Conclusions Good adherence to a TLSC program reduces global CV risk and determines PAT ratio improvement. The PAT ratio increase is the main determinant of lower IMT progression.

Resumen Introduccion y objetivos Analizar el impacto de cambios de estilo de vida en la concentracion plasmatica de adipocyte fatty acid-binding protein (FABP4) en pacientes con riesgo cardiovascular. Metodos Se incluyo a 140 pacientes con riesgo cardiovascular sin enfermedad cardiovascular previa en un estudio prospectivo de 1 ano de duracion para evaluar el impacto de cambios terapeuticos de estilo de vida (tabaco, educacion nutricional y actividad fisica) en el riesgo cardiovascular. Resultados Las variaciones de la FABP4 se asociaron inversamente con cambios en la actividad fisica (MET·h/sem). La FABP4 se redujo significativamente en los pacientes que aumentaron su actividad fisica, mientras que aumento en quienes la disminuyeron. Los cambios observados en la FABP4 tambien se asociaron con modificaciones en el indice de masa corporal y la resistencia a la insulina; sin embargo, las correlaciones entre actividad fisica y FABP4 permanecieron significativas tras ajustarlas por variables de confusion. Los cambios en la actividad fisica fueron los principales predictores de las modificaciones en la FABP4. La disminucion de FABP4 se asocio directamente con reducciones de colesterol ligado a las lipoproteinas de baja densidad y apolipoproteina B. Dejar de fumar y la composicion de la dieta no modificaron las concentraciones de FABP4. Conclusiones La concentracion plasmatica de FABP4 puede reducirse aumentando la actividad fisica aerobica. La actividad fisica actua sobre las concentraciones de FABP4 independientemente de la reduccion de peso. Si se pudiera establecer un papel causal de la FABP4 en las alteraciones metabolicas y vasculares, nuestros resultados anadirian un nuevo efecto positivo tanto de la actividad fisica como de la reduccion de la obesidad en el riesgo metabolico y cardiovascular.

Background: In endothelial dysfunction, vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression (collectively termed cell adhesion molecules; CAMs) increase at sites of atherosclerosis and are stimulated by proinflammatory cytokines such as tumor necrosis factor-α (TNF-α). Methods: We evaluated the effect of alpha-tocopherol (AT; 10–150 µM) and BAY 11-7082 (BAY; 0.1 or 1 µM) on CAMs mRNA expression as well as their protein in soluble release form (sCAMs) in human aortic endothelial cells (HAECs) activated by TNF-α (1 or 10 ng/ml). Also, we determined the extent of lymphocyte adhesion to activated HAECs. Results: BAY reduced VCAM-1, E-selectin and ICAM-1 mRNA expression by 30, 30 and 10%, respectively. Furthermore, protein reduction of sVCAM-1 by 70%, sE-selectin by 51% and sICAM-1 by 25% compared to HAECs stimulated by TNF-α was observed (p < 0.05). AT (50, 75 and 150 µM) decreased VCAM-1 mRNA expression by 30% and sVCAM-1 protein by 33% compared to HAECs stimulated by TNF-α (p < 0.05). TNF-α-activated HAEC adhesion to human Jurkat T lymphocytes was higher compared to nonactivated HAECs (p < 0.05). BAY (2 and 5 µM) reduced this lymphocyte adhesion (p < 0.05). Conclusion: BAY reduces all the CAMs studied as well as cell adhesion, while AT selectively inhibits VCAM-1; both induce endothelial dysfunction improvement.

Eur J Clin Invest 2012; 42 (1): 34–41 Abstract Background The use of methods based on reactive hyperaemia of small distal arteries to assess endothelial function (EF) is increasing; however, the mechanisms regulating vascular function in large and small arteries are probably different. We studied the correlations between the hyperaemia reactivity of small peripheral arteries determined by peripheral artery tonometry (PAT) and the levels of serum biomarkers of EF, inflammation and oxidation in patients with cardiovascular (CV) risk factors. Methods Four hundred and seven patients with intermediate CV risk were recruited into a cross-sectional study to examine whether soluble endothelial, inflammatory and lipid oxidative biomarkers correlate with small artery reactive hyperaemia index (saRHI) values, which were measured by PAT. Results A significant correlation was found between saRHI values and the concentrations of soluble E-selectin (sE-selectin) and soluble vascular cell adhesion molecule 1 (sVCAM-1). These correlations were stronger when only non-metabolic syndrome patients (46%) were analysed (r = −0·310, P

Aim: In heterozygous familial hypercholesterolaemic (FH) patients, study of the arterial wall and its function are of particular interest. Arterial stiffness has been shown to be associated with increased cardiovascular risk (CVR). In this study, we examined arterial stiffness in FH patients and its association with biochemical and vascular parameters.Methods: In this cross-sectional study, we included 125 FH patients (20-60 years old) and 59 gender- and age-matched healthy controls (CG). Clinical, anthropometry and biochemical data were obtained. Arterial stiffness determined based on the augmentation index (AIx) was assessed with peripheral artery tonometry. Carotid intima-media thickness (cIMT) and ankle-brachial index (ABI) were also assessed.Results: FH patients displayed a significant increase in AIx with respect to CG subjects (9.6±17.2 vs. 2.6±10.3%, P= 0.011). FH patients also had a thicker cIMT (0.758±0.280 vs. 0.635±0.160 mm, P< 0.001), while their ABIs were not different from CG subjects. AIx values were positively correlated with LDLc, non-HDLc, apolipoprotein B100, triglyceride and sE-selectin levels. Moreover, apolipoprotein B100-rich particles, along with systolic blood pressure and glucose levels, were the main determinants of AIx. In addition, we found that AIx (β= 0.224, P= 0.014) was an independent determinant of cIMT.Conclusions: FH patients have increased arterial stiffness, despite advancements in typical clinical management. AIx is clearly associated with apolipoprotein B100 concentrations, and it is a determinant of cIMT. AIx can be utilised as a vascular risk marker in FH patients.

Circulating FABP4 and FABP5 may be a biomarker for breast cancer. This article focuses on the association of FABP4 and FABP5 plasma levels with the presence of breast cancer., Background. Adipose tissue is an endocrine organ that could play a role in tumor progression via its secreted adipokines. The role of adipose‐derived fatty acid‐binding protein (FABP) 4 and FABP5 in breast cancer is presently under study, but their circulating levels in this pathology are poorly known. We analyzed the blood concentrations of FABP4 and FABP5 in breast cancer patients to determine whether there is an association between them and breast cancer. Materials and Methods. We studied 294 women in the oncology department with a family history of breast cancer; 198 of the women had breast cancer, and 96 were healthy controls. The levels of FABP4, FABP5, lipid profile, standard biochemical parameter, and high‐sensitivity C‐reactive protein (hsCRP) were determined. We analyzed the association of FABP4 and FABP5 with breast cancer, while adjusting for demographic, anthropometric, and biochemical parameters. Results. Breast cancer patients had a 24.8% (p