Association between Nordic and Mediterranean diets with lipoprotein phenotype assessed by 1HNMR in children with familial hypercholesterolemia.

BACKGROUND AND AIMS: Both Nordic and Mediterranean diets are considered healthy despite notable regional differences. Although these dietary patterns may lower cardiovascular risk, it is unclear if they improve the lipoprotein phenotype in children with familial hypercholesterolemia (FH). The aim is to determine the impact of Nordic and Mediterranean diets on the advanced lipoprotein profile in children with heterozygous FH (HeFH). METHODS: This was a cross-sectional study performed in children with FH recruited from the Lipid Clinics at Sant Joan University Hospital in Reus (Spain) and Oslo University Hospital (Norway). Two-hundred fifty-six children (mean age 10 y/o; 48% girls): 85 Spanish and 29 Norwegian FH children, and 142 non-FH healthy controls (119 from Spain and 23 from Norway) were included in the study. A pathogenic FH-associated genetic variant was present in 81% of Spanish children with FH and all Norwegian children with FH. An 1H NMR based advanced lipoprotein test (Nightingale®) providing information on the particle number, size and lipid composition of 14 lipoprotein subclasses was performed and correlated to the dietary components. RESULTS: Levels of LDL-C, HDL-C and triglycerides were not significantly different between the Nordic and Mediterranean FH groups. Spanish children with FH had more LDL particles, mainly of the large and medium LDL subclasses, than Norwegian FH children. Spanish FH children also had more HDL particles, mainly medium and small, than Norwegian FH children. The mean LDL size of Spanish FH children was larger, while the HDL size was smaller than that of the Norwegian FH children. The HDL particle number and size were the main determinants of differences between the two groups. In Norwegian children with FH, dietary total fat and MUFAs showed a significant correlation with all apolipoprotein B-containing lipoproteins and LDL size, whereas there was no correlation to SFA. A weaker association pattern was observed in the Spanish children. CONCLUSIONS: The lipoprotein profiles of Spanish and Norwegian children showed differences when studied by 1H NMR. These differences were in part associated with differences in dietary patterns.

MCF-7 Drug Resistant Cell Lines Switch Their Lipid Metabolism to Triple Negative Breast Cancer Signature.

Obesity and adipose tissue have been closely related to a poor cancer prognosis, especially in prostate and breast cancer patients. The ability of transferring lipids from the adipose tissue to the tumor cells is actively linked to tumor progression. However, different types of breast tumor seem to use these lipids in different ways and metabolize them in different pathways. In this study we have tracked by mass spectrometry how palmitic acid from the adipocytes is released to media being later incorporated in different breast cancer cell lines (MDA-MB-231, SKBR3, BT474, MCF-7 and its resistant MCF-7 EPIR and MCF-7 TAXR). We have observed that different lines metabolize the palmitic acid in a different way and use their carbons in the synthesis of different new lipid families. Furthermore, we have observed that the lipid synthesis pattern varied according to the cell line. Surprisingly, the metabolic pattern of the resistant cells was more related to the TNBC cell line compared to their sensitive cell line MCF-7. These results allow us to determine a specific lipid pattern in different cell lines that later might be used in breast cancer diagnosis and to find a better treatment according to the cancer molecular type.

Evolution of Serum Acute-Phase Glycoproteins Assessed by 1H-NMR in HIV Elite Controllers.

Elite controllers (ECs) are an exceptional group of people living with HIV (PLWH) who maintain undetectable viral loads (VLs) despite not being on antiretroviral therapy (ART). However, this phenotype is heterogeneous, with some of these subjects losing virological control over time. In this longitudinal retrospective study, serum acute-phase glycoprotein profile assessed by proton nuclear magnetic resonance (1H-NMR) was determined in 11 transient controllers (TCs) who spontaneously lost virological control and 11 persistent controllers (PCs) who persistently maintained virological control over time. Both PCs and TCs showed similar acute-phase glycoprotein profiles, even when TCs lost the virological control (GlycB, p = 0.824 and GlycA, p = 0.710), and the serum acute-phase glycoprotein signature in PCs did not differ from that in HIV-negative subjects (GlycB, p = 0.151 and GlycA, p = 0.243). Differences in serum glycoproteins A and B were significant only in ECs compared to HIV-typical progressors (TPs) with < 100 CD4+ T-cells (p < 0.001). 1H-NMR acute-phase glycoprotein profile does not distinguish TCs form PCs before the loss of viral control. ECs maintain a low-grade inflammatory state compared to TPs. PCs revealed a closer serum signature to HIV-negative subjects, reaffirming this phenotype as a closer model of functional control of HIV.

Altered Serum Metabolic Profile Assessed by Advanced 1H-NMR in Breast Cancer Patients.

BACKGROUND: Altered lipid metabolism has been described in some types of cancer. To analyse in depth the metabolic modifications in breast cancer patients, advanced 1H-nuclear magnetic resonance was performed in these patients. The main objective of this paper was to define a specific lipidomic signature for these cancer patients. MATERIALS AND METHODS: Serum from 240 women (171 breast cancer patients and 69 control women) were studied and analysed by nuclear magnetic resonance. RESULTS: Triglyceride-enriched particles, specifically very low-density lipoprotein triglycerides, intermediate-density lipoprotein triglycerides, low-density lipoprotein triglycerides, and high-density lipoprotein triglycerides, were positively associated with breast cancer. Moreover, alanine, tyrosine, and branched amino acids were also associated with increased risk of breast cancer. CONCLUSIONS: Breast cancer patients showed a modified metabolome, giving a very interesting tool to draw different radar charts between control women and breast cancer patients. To our knowledge, this is the first time that advanced nuclear magnetic resonance profiling has been used to identify relevant and specifically altered lipid or amino acid metabolites in BC serum samples. The altered metabolic signature could be analysed for early and reliable BC patient diagnosis and prognosis.

Serum glycoproteins A and B assessed by 1H-NMR in familial hypercholesterolemia.

BACKGROUND AND AIMS: Inflammation is a pathophysiological mechanism of atherosclerosis, and several mediators have been proposed as biomarkers. Acute-phase serum glycoproteins are upregulated in the liver during systemic inflammation. Because of their unique biochemical characteristics, they can be measured by 1H-NMR, and their role as subclinical inflammation markers is under clinical evaluation. We aimed to assess the clinical value of plasma glycoproteins in familial hypercholesterolemia (FH) patients. METHODS: We recruited 295 FH patients (75.6% with FH-associated genetic variants). At baseline, a full glycoprotein profile, glycoprotein A and B (GlycA and B) concentrations and their height and width ratios (H/W) were analysed by 1H-NMR. A carotid artery ultrasound study was performed at baseline and prospectively at the 5-year follow-up in 144 FH patients. RESULTS: At baseline, the GlycA and GlycB concentrations and their H/W ratios were correlated with lipid profile and adiposity parameters, with the correlation between the GlycA and triglyceride concentrations (r = 0.780; p < 0001) being the strongest. Glycoprotein concentrations were also correlated with inflammation markers, mainly hsCRP. Higher glycoprotein concentrations were observed in patients with higher intima media thickness, arterial rigidity and presence of arteriosclerotic plaques. In the multivariate and random forest analyses, the baseline GlycB concentration showed a significant contribution to the detection of FH individuals prone to develop carotid plaques. CONCLUSIONS: The concentrations of serum glycoproteins as assessed by 1H-NMR are robust markers of subclinical inflammation. In FH patients, they are increased in the presence of subclinical vascular damage and could be considered atherosclerosis risk markers in the long term.

Dietary intake and lipid levels in Norwegian and Spanish children with familial hypercholesterolemia.

BACKGROUND AND AIMS: Both the Nordic and Mediterranean diets claim to have a beneficial effect on lipid metabolism and cardiovascular prevention. The objective of this study was to compare diets consumed by children with FH at the time of diagnosis in Norway and Spain and to study their relationship with the lipid profile. METHODS AND RESULTS: In this cross-sectional study, we appraised the dietary intake in children (4-18 years old) with (n = 114) and without FH (n = 145) from Norway and Spain. We compared Nordic and Mediterranean diet composition differences and determined the association between food groups and lipid profiles. RESULTS: The Spanish FH group had a higher intake of total fats (mainly monounsaturated fatty acids (MUFAs)), cholesterol and fibre, but a lower intake of polyunsaturated fatty acids (PUFAs) compared to the Norwegian FH group. The Norwegian children consumed more rapeseed oil, low-fat margarine and whole grains and less olive oil, eggs, fatty fish, meat, legumes and nuts. In the Norwegian FH group, fat and MUFAs were directly correlated with total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B and inversely correlated with high-density lipoprotein (HDL-C). In Spanish children with FH, the intake of fats (mainly MUFAs) was directly associated with HDL-C and apolipoprotein A1. CONCLUSIONS: Despite a similar lipid phenotype, diets consumed by children with FH in Norway and Spain have significant differences at time of diagnosis. Nutrition advice should be more adapted to local intake patterns than on specific nutrient composition.

Hepatic Lipidomics and Molecular Imaging in a Murine Non-Alcoholic Fatty Liver Disease Model: Insights into Molecular Mechanisms.

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-mass spectrometry (LC-MS) and laser desorption/ionization-mass spectrometry (LDI-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-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-MS tissue imaging. The accumulation of these triglycerides was associated with the upregulation of lipogenesis and inflammatory genes and the downregulation of ß-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.

LDL Receptor Regulates the Reverse Transport of Macrophage-Derived Unesterified Cholesterol via Concerted Action of the HDL-LDL Axis: Insight From Mouse Models.

RATIONALE: The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown. OBJECTIVE: We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice. METHODS AND RESULTS: Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins. CONCLUSIONS: Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.

Efficacy of therapeutic lifestyle changes on lipid profiles assessed by NMR in children with familial and non-familial hypercholesterolemia.

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.

Plasma glucose, triglycerides, VLDL, leptin and resistin levels as potential biomarkers for myocardial fat in mice. / Niveles plasmáticos de glucosa, triglicéridos, VLDL, leptina y resistina como potenciales biomarcadores de la grasa miocárdica en ratones.

INTRODUCTION: The increase in myocardial fat has been proposed as one of the main precursors of myocardial dysfunction due to diabetic aetiology, independently of coronary artery disease. However, biomarkers reflecting the myocardial fat content for the clinical detection of this pathology are currently lacking. METHODS: Correlations between cardiac triglyceride content and plasma levels of major altered molecules 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. The content of cardiac triglycerides was directly associated with plasma levels of glucose, triglycerides, VLDL, resistin and leptin. In addition, an inverse correlation was observed between the content of cardiac triglycerides and the cardiac mRNA levels of Cd36 and Pdk4. CONCLUSIONS: Our data reveal that the cardiac triglyceride content is associated with altered plasma biochemical profile and reprogramming of gene expression aimed to mitigate the impact of ectopic lipid accumulation in the myocardium.

Lipoprotein profile assessed by 2D-1H-NMR and subclinical atherosclerosis in children with familial hypercholesterolaemia.

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) is underdiagnosed in children. In addition to lipid concentrations, lipoprotein particle quantity and quality could influence cardiovascular risk. We aimed to perform a comprehensive plasma lipid study, including lipoprotein particle number and size assessment by two-dimensional nuclear magnetic resonance (2D-1H-NMR), in children with FH compared to non-affected children and to evaluate the clinical value of these factors as subclinical atherosclerosis biomarkers. METHODS: One hundred eighty-three children participating in the broad "Hypercholesterolemia Early Detection Programme" (Decopin Project) were recruited. They were categorized as FH, if they had either a positive genetic test or clinical certainty, or as control children (CCh). Medical history, anthropometry and clinical variables were recorded. Standard biochemical measurements were performed. The lipoprotein profile was studied by 2D-1H-NMR. Carotid intima-media thickness (cIMT) was assessed by sonography in 177 children. RESULTS: FH children had a significant 36% increase in LDL particles. The small LDL fraction was increased by 33% compared to CCh. The relative relationship between large, medium and small LDL and the mean LDL particle size was similar between FH children and CCh. The total and small LDL particle numbers were directly associated with and contributed to the determination of the mean cIMT according to bivariate and multivariate analyses in FH children. CONCLUSIONS: The higher cholesterol levels of FH children are due to an overall increased number of all LDL particle subclasses, including a notable 33% increase in small LDL. Total and small LDL particle number shows a good correlation with cIMT in FH children.

Plasma inducible degrader of the LDLR, soluble low-density lipoprotein receptor, and proprotein convertase subtilisin/kexin type 9 levels as potential biomarkers of familial hypercholesterolemia in children.

BACKGROUND: Familial hypercholesterolemia (FH) in children is under-detected. Plasma biomarkers associated with low-density lipoprotein receptor (LDLR) function could help identifying FH children. OBJECTIVES: We aim to assess the clinical value of inducible degrader of the LDLR (IDOL), soluble LDLR (sLDLR), and proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma concentrations in children with FH compared with control children (CCh). METHODS: This was a cross-sectional study performed in a Lipid Unit from a University hospital. The participants were 177 children distributed into FH (n = 77) and CCh (n = 100). Main outcomes were changes in IDOL, sLDLR, and PCSK9 plasma concentrations between children groups; secondary outcomes were the association between IDOL, sLDLR, and PCSK9 and lipid profile determined by 2-dimensional nuclear magnetic resonance. RESULTS: The IDOL levels were higher in FH compared with CCh (P = .007). The PCSK9 levels were elevated in FH (P < .001). The sLDLR levels had no significant differences between groups. IDOL was significantly positively associated to total and LDL cholesterol and ApoB100 but not to LDL particle number. However, a robust correlation with Lp(a) (P = .001) was observed. PCSK9 had the strongest correlation with LDL-associated parameters including particle number. sLDLR was associated with triglyceride levels (P < .001) and triglyceride-rich particles and inversely to LDL size. CONCLUSIONS: The IDOL and PCSK9 plasma levels are significantly higher in FH children. Interestingly, sLDLR was associated with atherogenic dyslipidemia components. IDOL concentrations show a robust association with Lp(a) levels. To study the role of plasma biomarkers associated with LDLR expression in FH is warranted.

Adipose-Derived Fatty Acid-Binding Proteins Plasma Concentrations Are Increased in Breast Cancer Patients.

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 < .0001) and 11.4% (p < .05) higher blood concentration of FABP4 and FABP5, respectively. Fatty acid-binding protein 4 was positively associated with age, body mass index (BMI), FABP5, very-low-density lipoprotein cholesterol (VLDLc), non-high-density lipoprote in cholesterol (non-HDLc), Apolipoprotein B 100 (ApoB100), triglycerides, glycerol, glucose, and hsCRP (p < .05), and was negatively associated with HDLc (p < .005) in breast cancer patients. Fatty acid-binding protein 5 was positively associated with BMI, FABP4, VLDLc, triglycerides, glycerol, and hsCRP (p < .05), and was negatively associated with HDLc and Apolipoprotein AI (ApoAI) (p < .05) in breast cancer patients. Using a logistic regression analysis and adjusting for age, BMI, hsCRP, non-HDLc, and triglycerides, FABP4 was independently associated with breast cancer (odds ratio [OR]: 1.091 [95% CI: 1.037-1.149]). Moreover, total cholesterol, VLDLc, non-HDLc, ApoB100, triglycerides, and hsCRP were significantly increased in breast cancer patients (p < .005). In contrast, the non-esterified fatty acids concentrations were significantly decreased in breast cancer patients (p < .05). CONCLUSION: Circulating FABP4 and FABP5 levels were increased in breast cancer patients compared with controls. The positive association of FABP4 with breast cancer was maintained after adjusting for important covariates, while the association with FABP5 was lost. Our data reinforce the role of adipose tissue and their adipokines in breast cancer. Despite these data, further studies must be performed to better explain the prognosis or diagnostic value of these blood parameters and their possible role in breast cancer. IMPLICATIONS FOR PRACTICE: We focus on the effect of adipose tissue on cancer, which is increasingly recognized. The association between adipocyte-derived adipokines and breast cancer opens new diagnosis and therapy perspectives. In this study, we provide original data concerning FABP4 and FABP5 plasma concentrations in breast cancer patients. Compared to control group, breast cancer patients show higher FABP4 and FABP5 blood levels. Our data suggest that, particularly, circulating FABP4 levels could be considered a new independent breast cancer biomarker. Our work translates basic science data to clinic linking the relationship between adipose tissue and lipid metabolism to breast cancer.

Exogenous FABP4 increases breast cancer cell proliferation and activates the expression of fatty acid transport proteins.

Adipose tissue plays an important role in tumor progression, because it provides nutrients and adipokines to proliferating cells. Fatty acid binding protein 4 (FABP4) is a key adipokine for fatty acid transport. In metabolic pathologies, plasma levels of FABP4 are increased. However, the role of this circulating protein is unknown. Recent studies have demonstrated that FABP4 might have a role in tumor progression, but the molecular mechanisms involved are still unclear. In this study, we analysed the role of eFABP4 (exogenous FABP4) in breast cancer progression. MCF-7 and MDA-MB-231 breast cancer cells did not express substantial levels of FABP4 protein, but intracellular FABP4 levels increased after eFABP4 incubation. Moreover, eFABP4 enhanced the proliferation of these breast cancer cells but did not have any effect on MCF-7 and MDA-MB-231 cell migration. Additionally, eFABP4 induced the AKT and MAPK signaling cascades in breast cancer cells, and the inhibition of these pathways reduced the eFBAP4-mediated cell proliferation. Interestingly, eFABP4 treatment in MCF-7 cells increased levels of the transcription factor FoxM1 and the fatty acid transport proteins CD36 and FABP5. In summary, we showed that eFABP4 plays a key role in tumor proliferation and activates the expression of fatty acid transport proteins in MCF-7 breast cancer cells. © 2016 Wiley Periodicals, Inc.

Exogenous FABP4 induces endoplasmic reticulum stress in HepG2 liver cells.

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.

New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes.

Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.

Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells.

BACKGROUND: Recent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels are associated with impaired endothelial function in type 2 diabetes (T2D). In this work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production by endothelial cells in vitro. METHODS: In human umbilical vascular endothelial cells (HUVECs), we measured the effects of FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression and activation and on NO production. We also explored the impact of exogenous FABP4 on the insulin-signalling pathway (insulin receptor substrate 1 (IRS1) and Akt). RESULTS: We found that eNOS expression and activation and NO production are significantly inhibited by exogenous FABP4 in HUVECs. FABP4 induced an alteration of the insulin-mediated eNOS pathway by inhibiting IRS1 and Akt activation. These results suggest that FABP4 induces endothelial dysfunction by inhibiting the activation of the insulin-signalling pathway resulting in decreased eNOS activation and NO production. CONCLUSION: These findings provide a mechanistic linkage between FABP4 and impaired endothelial function in diabetes, which leads to an increased cardiovascular risk.

Lifestyle changes lower FABP4 plasma concentration in patients with cardiovascular risk.

INTRODUCTION AND OBJECTIVES: To analyze the impact of lifestyle changes on adipocyte fatty acid-binding protein (FABP4) plasma levels in patients with cardiovascular risk. METHODS: A 1-year prospective study enrolled 140 patients with cardiovascular risk but without previous cardiovascular disease to evaluate the impact of therapeutic lifestyle changes on cardiovascular risk, focusing on tobacco, nutrition education, and physical activity. RESULTS: The FABP4 variation was inversely associated to physical activity changes (MET·h/wk). FABP4 significantly decreased in patients with increased physical activity, whereas it increased with physical activity reduction. These FABP4 changes were also associated with modifications in body mass index and insulin resistance parameters; however, the correlations between physical activity and FABP4 remained after adjusting for these confounding variables. Changes in physical activity were the main predictors of FABP4 modifications. FABP4 reductions were directly associated with low-density lipoprotein-cholesterol and apolipoprotein B reductions. Neither tobacco cessation nor diet composition modified FABP4 concentrations. CONCLUSIONS: Increasing aerobic physical activity can decrease FABP4 plasma levels, independently of weight reduction. If a causal role of FABP4 in metabolic and vascular alterations could be established, our results would add new positive effects on metabolic and cardiovascular risk of both physical activity and avoiding obesity.

Heterozygous familial hypercholesterolaemic patients have increased arterial stiffness, as determined using the augmentation index.

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 (ß= 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.

Effect of the long-term regular intake of virgin olive oil on the phenolic metabolites in human fasting plasma.

The effect of repeated consumption of virgin olive oil on endogenous phenolic metabolites of fasting plasma is unknown. For this reason, we hypothesized that regular long-term virgin olive oil intake could have an indirect protection effect on the endogenous phenols. Thus, the aim of the study was to determine the phenolic profile of human plasma in a fasting state of long-term regular virgin olive oil consumers, using the fasting plasma of non-consumers as a natural control. Forty participants living in the area of Reus (Catalonia, Spain) were selected, 20 life-long regular consumers of virgin olive oil and a natural control of 20 non-consumers, the latter being Rumanians who dislike the taste of olive oil. The diet was obtained from 3-day food records. The results showed similar phenolic composition of fasting plasmas of the two volunteer groups. Of special interest is that more of the compounds quantified showed higher concentration in fasting plasma from habitual virgin olive oil consumers. The compounds were semi-quantified using caffeic acid as the calibration standard. The quantification of fasting consumer's plasma showed higher concentration of a hydroxyflavanone type compound (2.90+/-0.04 microM vs 1.5+/-0.04 microM) and a catecholamine derivative (0.70+/-0.03 microM vs 0.56+/-0.03 microM) than the plasma of non-consumers (P<0.05). The results suggest an indirect protective mechanism of long-term regular virgin olive oil consumption related to the protection of the endogenous antioxidant system.