Gelsolin: a new biomarker of disease activity in SLE patients associated with HDL-c.

OBJECTIVES: To 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. METHODS: Quantitative 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. RESULTS: We 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, P<0.001). Antimalarial treated patients showed significant higher levels of pGSN (214.56(88.94) mcg/l regarding 170.35(66.36) mcg/l); P = 0.017. CONCLUSION: Decreased pGSN are associated with clinical disease activity in SLE patients. Antimalarial treatment and HDL cholesterol are associated with higher levels of pGSN.

HDL Triglycerides: A New Marker of Metabolic and Cardiovascular Risk.

While cholesterol content in high-density lipoproteins (HDLs) is a well-established inverse marker of cardiovascular risk, the importance of HDL-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.

LipSpin: A New Bioinformatics Tool for Quantitative 1H NMR Lipid Profiling.

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.

FABP4 inhibitor BMS309403 decreases saturated-fatty-acid-induced endoplasmic reticulum stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation.

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.

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.

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.

Circulating PCSK9 levels and CETP plasma activity are independently associated in patients with metabolic diseases.

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 < 0.0001) independent of age, gender, body mass index (BMI), systolic blood pressure (SBP), LDL cholesterol (LDL-C), triglycerides and glucose. Individuals with the loss-of-function PCSK9 genetic variant rs11591147 (R46L) had lower levels of PCSK9 (36.5 %, P < 0.0001) and LDL-C (17.8 %, P = 0.010) as well as lower CETP activity (10.31 %, P = 0.009). This association remained significant in the multiple regression analysis even after adjusting for gender, age, BMI, LDL-C, triglycerides, glucose, lecithin-cholesterol acyltransferase, SBP and MetS (P = 0.003). CONCLUSIONS: Our data suggest a metabolic association between PCSK9 and CETP independent of lipid-lowering treatment. The clinical implications of this metabolic relationship could be relevant for explaining the effect of PCSK9 and CETP inhibition on overall lipid profiles.

Liposcale: a novel advanced lipoprotein test based on 2D diffusion-ordered 1H NMR spectroscopy.

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 (1)H 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.

Negative effect of a low-carbohydrate, high-protein, high-fat diet on small peripheral artery reactivity in patients with increased cardiovascular risk.

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.

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.

Alpha-tocopherol and BAY 11-7082 reduce vascular cell adhesion molecule in human aortic endothelial cells.

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.

Small artery dilation and endothelial markers in cardiovascular risk patients.

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 < 0·0001; r = -0·264, P < 0·0001, respectively). In this subgroup, the oxidised low-density lipoprotein/LDL (oxLDL/LDL) was also correlated with saRHI (r = -0·193, P = 0·009). A stepwise regression study showed that sE-selectin was the only biomarker significantly correlated with saRHI values (P < 0·0001). In multivariate linear regression analysis, this relationship was still strong when the main confounding covariates were taken into consideration. CONCLUSIONS: Elevated levels of sE-selectin and, to a smaller degree, sVCAM-1 and oxLDL/LDL are associated with lower postischemic reactivity in the small distal arteries. sE-selectin is the main determinant biomarker of saRHI as assessed by regression analysis. The presence of multiple risk factors weakens this association.

Particle size measurement of lipoprotein fractions using diffusion-ordered NMR spectroscopy.

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 Stokes­Einstein 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.

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.

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.

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.

The Circulating GRP78/BiP Is a Marker of Metabolic Diseases and Atherosclerosis: Bringing Endoplasmic Reticulum Stress into the Clinical Scenario.

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.

Patients With Systemic Lupus Erythematosus Show an Increased Arterial Stiffness That is Predicted by IgM Anti-ß2 -Glycoprotein I and Small Dense High-Density Lipoprotein Particles.

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-ß2 -glycoprotein I (IgM anti-ß2 GPI) (r = 0.284, P = 0.284). In the multivariate analysis, age (ß = 0.347, 95% confidence interval [95% CI] 0.020-0.669, P = 0.035), IgM ß2 GPI (ß = 0.321, 95% CI 0.024-0.618, P = 0.035) and small dense high-density lipoprotein (HDL) particles (ß = 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 ß2 GPI, and the number of small dense HDL particles predicted the AIx.

Fatty acid binding protein 4 (FABP4) as a potential biomarker reflecting myocardial lipid storage in type 2 diabetes.

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 (1H-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.

Oxidized to non-oxidized lipoprotein ratios are associated with arteriosclerosis and the metabolic syndrome in diabetic patients.

BACKGROUND AND AIM: Type 2 diabetic patients have a greater prevalence of the metabolic syndrome, oxidative stress and accelerated atherosclerosis, compared to non-diabetics. We examined the association between biomarkers of lipid peroxidation and the presence of atherosclerosis and the metabolic syndrome in diabetic patients. METHODS AND RESULTS: We studied oxidized LDL (OxLDL), OxLDL/LDL, OxLDL/HDL, lipoperoxides, autoantibodies against OxLDL (OxLDL-Ab), diene formation of LDL (lag phase), vitamin E, vitamin E/cholesterol and PON1 polymorphisms (-108C>T, 55T>A, and 192A>G) in 166 non-smoking type 2 diabetic patients, 119 fulfilling the criteria for the metabolic syndrome, 73 with atherosclerosis and 93 without atherosclerosis. Patients with macrovascular disease had higher values of OxLDL/LDL (11%; P=0.016), OxLDL/HDL (18%; P=0.024) and OxLDL-Ab (12%; P=0.046). OxLDL/LDL and OxLDL/HDL were correlated with the number of components of the metabolic syndrome (P<0.001). PON1 polymorphisms were not associated to LDL oxidation markers, only PON1 (-108TT) was weakly associated with higher OxLDL-Ab concentrations (22%; P=0.040) in patients with atherosclerosis. CONCLUSION: OxLDL/LDL, OxLDL/HDL and OxLDL-Ab are the most useful clinical parameters of lipoprotein oxidation for discriminating the presence of macrovascular disease in diabetic patients. The presence of the metabolic syndrome in these patients is also associated with an increase in the oxidized lipoprotein ratios.