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Background/Objectives Non-alcoholic fatty liver disease (NAFLD) causes a wide spectrum of liver damage, from simple steatosis (SS) to cirrhosis. SS and non-alcoholic steatohepatitis (NASH) cannot be distinguished by clinical or laboratory features. Dysregulation of the gut microbiota is involved in NASH pathogenesis. The aim of this study was to assess the relationship between microbiota-derived metabolites and the degrees of NAFLD; also, to investigate whether these metabolites could be included in a panel of NASH biomarkers. Subjects/Methods We used liquid chromatography coupled to triple-quadrupole-mass spectrometry (LC-QqQ) analysis to quantify choline and its derivatives, betaine, endogenous ethanol, bile acids, short-chain fatty acids and soluble TLR4 in serum from women with normal weight (n = 29) and women with morbid obesity (MO) (n = 82) with or without NAFLD. We used real-time polymerase chain reaction (RT-PCR) analysis to evaluate the hepatic and intestinal expression level of all genes studied (TLR2, TLR4, TLR9, LXRα, SREBP1C, ACC1, FAS, PPARα, CPT1α, CROT, SREBP2, ABCA1, ABCG1 and FXR in the liver; TLR2, TLR4, TLR5, TLR9, GLP-1R, DPP-4, FXR and PPARɣ in the jejunum) in 82 women with MO with normal liver histology (NL, n = 29), SS (n = 32), and NASH (n = 21). Results Hepatic FAS, TLR2, and TLR4 expression were overexpressed in NAFLD patients. TLR2 was overexpressed in NASH patients. In women with MO with NAFLD, we found upregulation of intestinal TLR9 expression and downregulation of intestinal FXR expression in women with NASH. Circulating TMAO, glycocholic acid and deoxycholic acid levels were significantly increased in NAFLD patients. Endogenous circulating ethanol levels were increased in NASH patients in comparison to those in SS patients. Conclusions These findings suggest that the intestine participates in the progression of NAFLD. Moreover, levels of certain circulating microbiota-related metabolites are associated with NAFLD severity and could be used as a “liquid biopsy” in the noninvasive diagnosis of NASH.

Nonalcoholic fatty liver disease (NAFLD) is a common, multifactorial, and poorly understood liver disease whose incidence is globally rising. During the past decade, several lines of evidence suggest that dysbiosis of intestinal microbiome represents an important factor contributing to NAFLD occurrence and its progression into NASH. The mechanisms that associate dysbiosis with NAFLD include changes in microbiota-derived mediators, deregulation of the gut endothelial barrier, translocation of mediators of dysbiosis, and hepatic inflammation. Changes in short chain fatty acids, bile acids, bacterial components, choline, and ethanol are the result of altered intestinal microbiota. We perform a narrative review of the previously published evidence and discuss the use of gut microbiota-derived mediators as potential markers in NAFLD.

Objective This study was performed to investigate neurotensin plasma levels in patients with nonalcoholic fatty liver disease (NAFLD) associated with severe obesity. Methods The plasma levels of neurotensin in 20 women with normal weight and 51 women with morbid obesity (MO) were measured, and women were subclassified according to their hepatic histology as having MO without NAFLD (n = 18) or MO with NAFLD (n = 33). The NAFLD group included 15 women with simple steatosis (SS) and 18 women with nonalcoholic steatohepatitis (NASH). To quantify neurotensin in plasma, a multiplex sandwich immunoassay with a Luminex magnetic bead-based platform was used. Results Neurotensin levels were significantly decreased (P = 0.001) in women with MO and NAFLD (3.62 ± 0.85 ng/mL), compared with women with MO and normal liver function (11.65 ± 1.95 ng/mL; P = 0.001) and women with normal weight (13.68 ± 2.58 ng/mL; P = 0.001). There was no difference in levels between women with SS and women with NASH (P = 0.415). Conclusions Circulating levels of neurotensin were decreased in women with NAFLD associated with MO.

Este artículo se encuentra disponible en la siguiente URL: https://www.mdpi.com/2076-3921/9/11/1062 Este artículo pertenece al número especial "Oxidative stress in diabetic retinopathy". Hyperglycemia, a defining characteristic of diabetes, combined with oxidative stress, results in the formation of advanced glycation end products (AGEs). AGEs are toxic compounds that have adverse e ects on many tissues including the retina and lens. AGEs promote the formation of reactive oxygen species (ROS), which, in turn, boost the production of AGEs, resulting in positive feedback loops, a vicious cycle that compromises tissue fitness. Oxidative stress and the accumulation of AGEs are etiologically associated with the pathogenesis of multiple diseases including diabetic retinopathy (DR). DR is a devastating microvascular complication of diabetes mellitus and the leading cause of blindness in working-age adults. The onset and development of DR is multifactorial. Lowering AGEs accumulation may represent a potential therapeutic approach to slow this sight-threatening diabetic complication. To set DR in a physiological context, in this review we first describe relations between oxidative stress, formation of AGEs, and aging in several tissues of the eye, each of which is associated with a major age-related eye pathology. We summarize mechanisms of AGEs generation and anti-AGEs detoxifying systems. We specifically feature the potential of the glyoxalase system in the retina in the prevention of AGEs-associated damage linked to DR. We provide a comparative analysis of glyoxalase activity in di erent tissues from wild-type mice, supporting a major role for the glyoxalase system in the detoxification of AGEs in the retina, and present the manipulation of this system as a therapeutic strategy to prevent the onset of DR.

Diabetes and metabolic syndrome are associated with the typical American high glycemia diet and result in accumulation of high levels of advanced glycation end products (AGEs), particularly upon aging. AGEs form when sugars or their metabolites react with proteins. Associated with a myriad of age‐related diseases, AGEs accumulate in many tissues and are cytotoxic. To date, efforts to limit glycation pharmacologically have failed in human trials. Thus, it is crucial to identify systems that remove AGEs, but such research is scanty. Here, we determined if and how AGEs might be cleared by autophagy. Our in vivo mouse and C. elegans models, in which we altered proteolysis or glycative burden, as well as experiments in five types of cells, revealed more than six criteria indicating that p62‐dependent autophagy is a conserved pathway that plays a critical role in the removal of AGEs. Activation of autophagic removal of AGEs requires p62, and blocking this pathway results in accumulation of AGEs and compromised viability. Deficiency of p62 accelerates accumulation of AGEs in soluble and insoluble fractions. p62 itself is subject to glycative inactivation and accumulates as high mass species. Accumulation of p62 in retinal pigment epithelium is reversed by switching to a lower glycemia diet. Since diminution of glycative damage is associated with reduced risk for age‐related diseases, including age‐related macular degeneration, cardiovascular disease, diabetes, Alzheimer's, and Parkinson's, discovery of methods to limit AGEs or enhance p62‐dependent autophagy offers novel potential therapeutic targets to treat AGEs‐related pathologies., AGEs are toxic compounds formed by non‐enzymatic reactions between sugars and proteins. AGEs are prone to aggregate. Insoluble AGEs are efficiently removed via p62‐selective autophagy. The autophagic removal of AGEs is a conserved pathway, and the lack of p62 leads to accumulation of toxic AGEs in mouse and worms. Enhanced autophagy is protective against glycation‐derived damage. p62‐dependent autophagy offers novel potential therapeutic targets to treat AGEs‐related pathologies.

Because of the clinical significance of carotid atherosclerosis, the search for novel biomarkers has become a priority. The aim of the present study was to compare the protein secretion profile of the carotid atherosclerotic plaque (CAP, n = 12) and nonatherosclerotic mammary artery (MA, n = 10) secretomes. We used a nontargeted proteomic approach that incorporated tandem immunoaffinity depletion, iTRAQ labeling, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 162 proteins were quantified, of which 25 showed statistically significant differences in secretome levels between carotid atherosclerotic plaque and nondiseased mammary artery. We found increased levels of neutrophil defensin 1, apolipoprotein E, clusterin, and zinc-alpha-2-glycoprotein in CAP secretomes. Results were validated by ELISA assays. Also, differentially secreted proteins are involved in pathways such as focal adhesion and leukocyte transendothelial migration. In conclusion, this study provides a subset of identified proteins that are differently expressed in secretomes of clinical significance.

Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in developed countries, covering a spectrum of pathological conditions ranging from single steatosis to non-alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. Its pathogenesis has been often interpreted by the "double-hit" hypothesis, where the lipid accumulation in the liver is followed by proinflammatory mediators inducing inflammation, hepatocellular injury and fibrosis. Nowadays, a more complex model suggests that free fatty acids and their metabolites could be the true lipotoxic agents that contribute to the development of NAFLD and hepatic insulin resistance, suggesting a central role for metabolic lipases in that process.

Data from recent studies conducted in rodent models and humans suggest that interleukin-17A (IL-17A) plays a role in the induction of inflammation in adipose tissue during obesity. The aim of this study was to assess the gene expression of IL-17A in adipose tissue of morbidly obese patients. We used RT-PCR to evaluate the expression of IL-17A and several adipo/cytokines in the visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) of 10 normal-weight control women (BMI &lt, 25 kg/m2) and 30 morbidly obese women (MO, BMI &gt, 40 kg/m2). We measured serum levels of IL-17A and adipo/cytokines in MO and normal weight women. IL-17A expression was significantly higher in VAT than in SAT in MO patients (p = 0.0127). It was very low in normal-weight controls in both VAT and SAT tissues. We found positive correlations between IL-17A and IL-6, lipocalin-2 and resistin in VAT of MO patients. The circulating level of IL-17A was higher in the normal-weight group than the MO patients (p = 0.032), and it was significantly related to adiponectin and TNFRII levels. In conclusion, IL-17A expression in VAT is increased in morbidly obese women, which suggests a link between obesity and innate immunity in low-grade chronic inflammation in morbidly obese women.

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.

Background Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in Western countries. Both iron and lipid metabolism seem to be involved in its pathogenesis. We aimed to assess the relationship between levels of hepcidin, the master iron-regulatory protein, in plasma and the presence of NAFLD in morbidly obese (MO) patients, and to investigate the association between the hepatic expression of the main iron and lipid metabolism -related genes. Materials and methods Enzyme-linked immunosorbent assay was used to measure plasma hepcidin levels in 49 normal-weight control women, 23 MO women with normal liver (NL) histology and 46 MO women with NAFLD. The mRNA expression of hepcidin, the main iron metabolism-related genes, and the main lipid-metabolism genes was quantified by qRT-PCR in liver biopsies from members of the MO group undergoing bariatric surgery. Results Circulating hepcidin levels were significantly greater in MO than in normal-weight control women. However, there were no significant differences between MO women with NL and those with NAFLD. PCR analysis showed increased expression of hepcidin, FPN1, TfR1 and TfR2 in the liver of MO NAFLD women compared to those with NL. Moreover, a positive association of hepatic hepcidin mRNA expression and the iron metabolism-related genes was found with some key genes involved in the lipid metabolism. Conclusion These findings suggest that circulating hepcidin levels are associated with obesity but not with the presence of NAFLD. However, the hepatic expression of hepcidin and the iron metabolism-related genes seem to play a role in regulating lipid metabolism pathways in liver, which has implications for NAFLD pathogenesis.

This study was performed to investigate neurotensin plasma levels in patients with nonalcoholic fatty liver disease (NAFLD) associated with severe obesity.The plasma levels of neurotensin in 20 women with normal weight and 51 women with morbid obesity (MO) were measured, and women were subclassified according to their hepatic histology as having MO without NAFLD (n = 18) or MO with NAFLD (n = 33). The NAFLD group included 15 women with simple steatosis (SS) and 18 women with nonalcoholic steatohepatitis (NASH). To quantify neurotensin in plasma, a multiplex sandwich immunoassay with a Luminex magnetic bead-based platform was used.Neurotensin levels were significantly decreased (P = 0.001) in women with MO and NAFLD (3.62 ± 0.85 ng/mL), compared with women with MO and normal liver function (11.65 ± 1.95 ng/mL; P = 0.001) and women with normal weight (13.68 ± 2.58 ng/mL; P = 0.001). There was no difference in levels between women with SS and women with NASH (P = 0.415).Circulating levels of neurotensin were decreased in women with NAFLD associated with MO.

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 Novel pro-inflammatory and anti-inflammatory derivatives from adipose tissue, known as adipokines, act as metabolic factors. The aim of this study was to analyse the secreted expression of different adipo/cytokines in secretomes of unstable carotid atherosclerotic plaque versus non-atherosclerotic mammary artery. Methods We evaluated the secretion levels of adiponectin, visfatin, lipocalin-2, resistin, IL-6 and TNFR2 by ELISA in human secretomes from cultured unstable carotid atherosclerotic plaque (n = 18) and non-atherosclerotic mammary artery (n = 13). We also measured visfatin serum levels in patients suffering from atherosclerosis and in a serum cohort of healthy subjects (n = 16). Results We found that visfatin levels were significantly increased in unstable carotid atherosclerotic plaque secretome than in non-atherosclerotic mammary artery secretome. No differences were found with regard the other adipo/cytokines studied. Regarding visfatin circulating levels, there were no differences between unstable carotid atherosclerotic plaque and non-atherosclerotic mammary artery group. However, these visfatin levels were increased in comparison to serum cohort of healthy subjects. Conclusions Of all the adipo/cytokines analysed, only visfatin showed increased levels in secretomes of unstable carotid atherosclerotic plaque. Additional human studies are needed to clarify the possible role of visfatin as prognostic factor of unstable carotid atherosclerotic plaque.

Recent reports suggest a role for the Patatin-like phospholipase domain-containing protein 3 (PNPLA3) in the pathology of non-alcoholic fatty liver disease (NAFLD). Lipid deposition in the liver seems to be a critical process in the pathogenesis of NAFLD. The aim of the present work was to evaluate the association between the liver PNPLA3 expression, key genes of lipid metabolism, and the presence of NAFLD in morbidly obese women. We used real-time polymerase chain reaction (PCR) analysis to analyze the hepatic expression of PNPLA3 and lipid metabolism-related genes in 55 morbidly obese subjects with normal liver histology (NL, n = 18), simple steatosis (SS, n = 20), and non-alcoholic steatohepatitis (NASH, n = 17). Liver biopsies were collected during bariatric surgery. We observed that liver PNPLA3 expression was increased in NAFLD than in NL. It was also upregulated in SS than in NL. Interestingly, we found that the expression of PNPLA3 was significantly higher in severe than mild SS group. In addition, the expression of the transcription factors LXRα, PPARα, and SREBP2 was positively correlated with PNPLA3 liver expression. Regarding rs738409 polymorphism, GG genotype was positive correlated with the presence of NASH. In conclusion, our results show that PNPLA3 could be related to lipid accumulation in liver, mainly in the development and progression of simple steatosis.

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.

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.

Resumen Introduccion Los cambios terapeuticos sobre el estilo de vida (CTEV) reducen el riesgo cardiovascular (RCV), pero el efecto sobre la funcion endotelial (FE) es desconocido. El objetivo de este estudio fue evaluar prospectivamente el efecto de los CTEV sobre la FE en pacientes con RCV incrementado y obesidad abdominal (OA). Metodos Ciento cincuenta pacientes con OA y RCV incrementado fueron aleatorizados a una intervencion de un ano de control medico convencional (grupo control, GC) o a un programa intensivo de CTEV (grupo intervencion, GI). El estudio vascular, bioquimico y de valoracion del estilo de vida se realizo antes y despues del periodo de intervencion. Resultados El indice de hiperemia reactiva (RHI) mejoro en el GI y empeoro en el GC. El RCV se redujo (p = 0,017) en el GI debido a una disminucion significativa en la presion arterial sistolica (p Conclusiones La buena adherencia a los CTEV redujo el RCV y determino una mejoria en el RHI. El incremento del RHI fue el principal determinante de la menor progresion de cIMT.

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.

Resumen Introduccion Los niveles plasmaticos de la proteina FABP4 son superiores en pacientes con diabetes tipo 2 (DT2), obesidad y sindrome metabolico. La disfuncion endotelial es una caracteristica comun en el desarrollo de las enfermedades vasculares asociadas a las alteraciones metabolicas. El objetivo del trabajo es estudiar la relacion entre FABP4 circulante y la funcion endotelial en pacientes con elevado riesgo cardiovascular (RCV). Metodos Doscientos cincuenta y siete pacientes (57,29 ± 10,66 anos) con RCV incrementado. Se determino FABP4 plasmatica. En el estudio vascular se valoro la funcion endotelial mediante el indice de la hiperemia reactiva (IHR) por tonometria arterial periferica, el grosor de la intima-media carotideo (GIM) y el indice tobillo-brazo (ITB). Las caracteristicas antropometricas y los biomarcadores endoteliales, de inflamacion y de oxidacion fueron determinados. Resultados FABP4 se asocio negativamente con la funcion endotelial medida por IHR (r = -0,226; p = 0,027) en los pacientes DT2. No se observo ninguna asociacion significativa con GIM e ITB en este grupo. Se observo una asociacion inversa y significativa de IHR a traves de los terciles de FABP4 (p = 0,023). En el modelo de regresion lineal, FABP4 fue el unico predictor de IHR en DT2 (p Conclusiones Los niveles aumentados de FABP4 circulante se asocian a una peor funcion endotelial, medida por IHR en pacientes DT2. Nuestro trabajo sugiere un efecto directo de FABP4 plasmatica en el endotelio vascular en pacientes DT2.

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.

The purpose of this work was to evaluate the expression of fatty acid metabolism-related genes in human adipose tissue from moderately obese women. We used qRT-PCR and Western Blot to analyze visceral (VAT) and subcutaneous (SAT) adipose tissue mRNA expression involved in de novo fatty acid synthesis (ACC1, FAS), fatty acid oxidation (PPARα, PPARδ) and inflammation (IL6, TNFα), in normal weight control women (BMI &lt, 25 kg/m2, n = 35) and moderately obese women (BMI 30–38 kg/m2, n = 55). In SAT, ACC1, FAS and PPARα mRNA expression were significantly decreased in moderately obese women compared to controls. The downregulation reported in SAT was more pronounced when BMI increased. In VAT, lipogenic-related genes and PPARα were similar in both groups. Only PPARδ gene expression was significantly increased in moderately obese women. As far as inflammation is concerned, TNFα and IL6 were significantly increased in moderate obesity in both tissues. Our results indicate that there is a progressive downregulation in lipogenesis in SAT as BMI increases, which suggests that SAT decreases the synthesis of fatty acid de novo during the development of obesity, whereas in VAT lipogenesis remains active regardless of the degree of obesity.

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.

Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in developed countries, covering a spectrum of pathological conditions ranging from single steatosis to non-alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. Its pathogenesis has been often interpreted by the "double-hit" hypothesis, where the lipid accumulation in the liver is followed by proinflammatory mediators inducing inflammation, hepatocellular injury and fibrosis. Nowadays, a more complex model suggests that free fatty acids and their metabolites could be the true lipotoxic agents that contribute to the development of NAFLD and hepatic insulin resistance, suggesting a central role for metabolic lipases in that process.

SummaryBackground Infants born small-for-gestational-age (SGA) who develop post-natal weight catch-up are at risk for insulin resistance, central adiposity and cardiovascular disease in later life, even in the absence of overweight. Objective In young (age 3–6 years) non-obese SGA children, we assessed arterial health (as judged by intima-media thickness [IMT]) and abdominal fat distribution (subcutaneous, visceral, preperitoneal and hepatic components by magnetic resonance imaging [MRI] and/or ultrasound [US]) besides a selection of endocrine markers. Methods Comparisons of measures in SGA (n = 27) vs. appropriate-for-GA (AGA) children (n = 19) of similar height, weight and body mass index. Longitudinal outcomes (age 3–6 years) were carotid IMT (cIMT); fasting glucose, circulating insulin, IGF-I and high-molecular-weight (HMW) adiponectin; abdominal fat partitioning by US. Cross-sectional outcomes (age 6 years) were aortic IMT (aIMT) and abdominal fat partitioning by MRI. Results At 3 and 6 years, cIMT and IGF-I results were higher and HMW adiponectin lower in SGA than AGA children; at 6 years, SGA subjects had also a thicker aIMT and more pre-peritoneal and hepatic fat, and were less insulin sensitive (all P values between

Lipid accumulation in the human liver seems to be a crucial mechanism in the pathogenesis and the progression of non-alcoholic fatty liver disease (NAFLD). We aimed to evaluate gene expression of different fatty acid (FA) metabolism-related genes in morbidly obese (MO) women with NAFLD. Liver expression of key genes related to de novo FA synthesis (LXRα, SREBP1c, ACC1, FAS), FA uptake and transport (PPARγ, CD36, FABP4), FA oxidation (PPARα), and inflammation (IL6, TNFα, CRP, PPARδ) were assessed by RT-qPCR in 127 MO women with normal liver histology (NL, n = 13), simple steatosis (SS, n = 47) and non-alcoholic steatohepatitis (NASH, n = 67). Liver FAS mRNA expression was significantly higher in MO NAFLD women with both SS and NASH compared to those with NL (p = 0.003, p = 0.010, respectively). Hepatic IL6 and TNFα mRNA expression was higher in NASH than in SS subjects (p = 0.033, p = 0.050, respectively). Interestingly, LXRα, ACC1 and FAS expression had an inverse relation with the grade of steatosis. These results were confirmed by western blot analysis. In conclusion, our results indicate that lipogenesis seems to be downregulated in advanced stages of SS, suggesting that, in this type of extreme obesity, the deregulation of the lipogenic pathway might be associated with the severity of steatosis.

BACKGROUND/OBJECTIVE: Fibroblast growth factor 19 (FGF19) and 21 (FGF21) have been linked to obesity and type 2 diabetes in adults. We assessed the circulating concentrations of these factors in human neonates and infants, and their association with the endocrine-metabolic changes associated to prenatal growth restraint. SUBJECTS/METHODS: Circulating FGF19 and FGF21, selected hormones (insulin, insulin-like growth factor I and high- molecular-weight (HMW) adiponectin) and body composition (absorptiometry) were assessed longitudinally in 44 infants born appropriate( AGA) or small-for-gestational-age (SGA). Measurements were performed at 0, 4 and 12 months in AGA infants; at 0 and 4 months in SGA infants; and cross-sectionally in 11 first-week AGA newborns. RESULTS: Circulating FGF19 and FGF21 surged >10-fold in early infancy from infra- to supra-adult concentrations, the FGF19 surge appearing slower and more pronounced than the FGF21 surge. Whereas the FGF21 surge was of similar magnitude in AGA and SGA infants, FGF19 induction was significantly reduced in SGA infants. In AGA and SGA infants, cord-blood FGF21 and serum FGF19 at 4 months showed a positive correlation with HMW adiponectin (r = 0.49, P = 0.013; r = 0.43, P = 0.019, respectively). CONCLUSIONS: Our results suggest that these early FGF19 and FGF21 surges are of a physiological relevance that warrants further delineation and that may extend beyond infancy.

Background: Prenatal growth restraint is associated with increased oxidative stress - as judged by mitochondrial dysfunction - in pregnancies complicated by preeclampsia or diabetes, but it is uncertain whether this is also the case in uncomplicated pregnancies. We assessed the link between fetal growth restraint and placental mitochondrial dysfunction, as reflected by changes in mitochondrial DNA (mtDNA) content and superoxide dismutase (SOD) activity. Methods: After uncomplicated pregnancies, placentas (n = 48) were collected at term delivery of singleton infants who were appropriate for gestational age (AGA) or small for gestational age (SGA) (n = 24 in each subgroup). Placental mtDNA content was assessed by real-time PCR, placental SOD activity by colorimetry, and citrate synthase activity - to determine mitochondrial mass - by the spectrophotometric method. Results: Placentas of SGA infants had a lower mtDNA content (p = 0.015) and a higher SOD activity (p = 0.001) than those of AGA controls. These differences were maintained after normalization of the mtDNA content by citrate synthase activity. In placentas of SGA infants, there was a negative association between mtDNA content and SOD activity (r = -0.58, p = 0.008). Conclusions: Fetal growth restraint is accompanied by adaptive changes in the mitochondrial function of the placenta, also in uncomplicated pregnancies.

Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells.HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs.The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor.The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular.

10.1007/s11695-012-0729-6 Background: Endothelial dysfunction is a major underlying mechanism for the elevated cardiovascular risk associated with increased body weight. We aimed to assess the impact of weight loss induced by an intensive very-low-calorie diet (VLCD) on arterial wall function in severely obese patients (SOP). Methods: Thirty-four SOP were admitted to the metabolic ward of the hospital for a 3-week period. A VLCD characterized by a liquid diet providing 800 kcal/day was administered. The small artery reactivity to postischemic hyperemia index (saRHI), a surrogate marker of endothelial function, was assessed before and 1 week after hospital discharge. Anthropometry and biochemical parameters were also measured. Obese and non-obese age- and gender-matched groups were recruited for baseline comparisons. Results: SOP had significantly lower saRHI compared with obese and non-obese individuals. SaRHI significantly increased after the intervention in SOP (1.595 ± 0.236 vs. 1.737 ± 0.417, p = 0.015). A significant improvement in glucose (p = 0.026), systolic blood pressure (p = 0.049), LDLc (p < 0.001), and inflammatory parameters was observed. Body weight loss was associated with a higher saRHI (r = -0.385, p = 0.033), and it was the main determinant of saRHI variation independently of confounders (ß -0.049, IC 95 % -0.091-0.008, p = 0.021). Conclusions: Weight loss induced by a VLCD in SOP improved small artery reactivity, and it was associated with the amelioration of metabolic and inflammation markers. Endothelial dysfunction may be softened by body weight loss interventions and useful in the management of cardiovascular risk factors in SOP.

Background Preadipocyte factor-1 (Pref-1) is a key regulator of adipocyte differentiation acting as an inhibitor of adipogenesis; Pref-1 is highly expressed in embryonic tissues and placenta supporting a role in embryonic and fetal growth. The potential impact of placental Pref-1 expression in human pre- and postnatal development is unclear. Objective and hypotheses To assess the contribution of placental Pref-1 to fetal and postnatal growth. Population and methods Placentas ( N = 99) were collected at term delivery from singleton infants, who were born either appropriate (AGA; n = 59) or small-for-gestational-age (SGA; n = 40). Auxological data of all subjects were obtained at birth. In a subset of subjects ( n = 31) we also obtained weight data at 4 mo and at 1 yr, together with body composition assessment (by DXA) at the age of 1 yr. Placental expression of Pref-1 was quantified by real-time PCR; the housekeeping gene GAPDH was used for comparisons. Results Pref-1 was significantly downregulated in the placentas from SGA babies as compared to AGA controls ( P = 0.005). In SGA infants placental Pref-1 expression associated positively to body weight at 4 and 12 mo ( r = 0.44, P = 0.05; r = 0.66, P = 0.001 respectively); at age 12 mo, placental Pref-1 expression was inversely related to total fat mass and positively correlated with total lean mass ( r = −0.59, P = 0.01; and r = −0.59, P = 0.01,respectively). Conclusion Placental Pref-1 expression in SGA fetuses was decreased and associated with postnatal body weight, suggesting a role of Pref-1 in the regulation of postnatal development.

A B S T R A C T 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 (METh/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.

Objective Reactive hyperaemia after shear stress is a surrogate marker of endothelial function. However, the mechanisms controlling the dilation capacity of small peripheral resistance arteries are not well characterised. We evaluated reactive hyperaemia by peripheral artery tonometry (PAT) in the acral arteries and studied their clinical and biochemical determinants. Methods Eight hundred sixteen subjects at intermediate to high cardiovascular risk were recruited. The reactive hyperaemia index (RHI) of small digital arteries was measured by PAT. Clinical history data, anthropometry and biochemical parameters were also analysed. We studied the associations between clinical and biochemical factors and small artery RHI. Results HDL cholesterol and apolipoprotein A1 levels were strongly and directly correlated with an increased dilation response. Metabolic syndrome components, such as increased waist circumference, hypertriglyceridaemia and smoking, were inversely associated with RHI as were serum markers of inflammation. The predictors of small peripheral artery RHI were HDL cholesterol, which had a protective effect, and smoking, which had a negative impact. Conclusion HDL cholesterol and apolipoprotein A1 levels had a strong, positive correlation with small artery reactive hyperaemia, whereas smoking, waist circumference and triglyceride levels were inversely associated. HDL cholesterol was the main determinant of RHI in small peripheral resistance arteries.

Objective Adipocyte fatty acid-binding protein (FABP4) plasma levels are higher in type 2 diabetes (T2D). Endothelial dysfunction is also common in T2D. We have investigated the relationship between circulating FABP4 levels and endothelial function in diabetic patients. Methods In 257 patients (105 diabetic and 152 non-diabetic) at increased risk of cardiovascular disease, we measured circulating FABP4, reactive hyperemia index (RHI) by peripheral artery tonometry, intima-media thickness, and biomarkers of inflammation, oxidation and endothelial function. Results In T2D subjects, FABP4 was negatively associated with endothelial function, as measured by RHI ( r =−0.226, P =0.05). In a stepwise multivariate linear regression model, FABP4 was a predictor of RHI in T2D patients ( P =0.04). Conclusion Circulating levels of FABP4 are inversely associated with endothelial function in T2D patients, as measured by RHI. We suggest a direct effect of plasma FABP4 on the vascular endothelium in those with T2D.

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.