Javier Margareto

Differential Expression of Oxidative Stress and Inflammation Related Genes in Peripheral Blood Mononuclear Cells in Response to a Low-Calorie Diet: A Nutrigenomics Study

Nutrigenomics is a new application of omics technologies in nutritional science. Nutrigenomics aims to identify molecular markers of diet-related diseases and mechanisms of interindividual variability in response to food. The aim of this study was to evaluate peripheral blood mononuclear cells (PBMC) as a model system and readily available source of RNA to discern gene expression signatures in relation to personalized therapy of obesity. PBMC were collected from obese men before and after an 8-week low-calorie diet (LCD) to lose weight. Changes in gene expression before and after the LCD were initially screened using a DNA-microarray platform and validated by qRT-PCR. Global gene expression analysis identified 385 differentially expressed transcripts after the LCD. Further analyses showed a decrease in some specific oxidative stress and inflammation genes. Interestingly, expression of these genes was directly related to body weight, while a lower IL8 gene expression was associated with higher fat mass decrease. Collectively, these observations suggest that PBMCs are a suitable RNA source and model system to perform nutrigenomics studies related to obesity and development of personalized dietary treatments. IL8 gene expression warrant further research as a putative novel biomarker of changes in body fat percentage in response to an LCD.

Klebsiella pneumoniae Capsule Polysaccharide Impedes the Expression of β-Defensins by Airway Epithelial Cells

ABSTRACT Human β-defensins (hBDs) contribute to the protection of the respiratory tract against pathogens. It is reasonable to postulate that pathogens have developed countermeasures to resist them. Klebsiella pneumoniae capsule polysaccharide (CPS), but not the lipopolysaccharide O antigen, mediated resistance against hBD1 and hBD2. hBD3 was the most potent hBD against Klebsiella. We investigated the possibility that as a strategy for survival in the lung, K. pneumoniae may not activate the expression of hBDs. Infection of A549 and normal human bronchial cells with 52145-ΔwcaK2, a CPS mutant, increased the expression of hBD2 and hBD3. Neither the wild type nor the lipopolysaccharide O antigen mutant increased the expression of hBDs. In vivo, 52145-ΔwcaK2 induced higher levels of mBD4 and mBD14, possible mouse orthologues of hBD2 and hBD3, respectively, than the wild type. 52145-ΔwcaK2-dependent upregulation of hBD2 occurred via NF-κB and mitogen-activated protein kinases (MAPKs) p44/42, Jun N-terminal protein kinase (JNK)-dependent pathways. The increase in hBD3 expression was dependent on the MAPK JNK. 52145-ΔwcaK2 engaged Toll-like receptors 2 and 4 (TLR2 and TLR4) to activate hBD2, whereas hBD3 expression was dependent on NOD1. K. pneumoniae induced the expression of CYLD and MKP-1, which act as negative regulators for 52145-ΔwcaK2-induced expression of hBDs. Bacterial engagement of pattern recognition receptors induced CYLD and MKP-1, which may initiate the attenuation of proinflammatory pathways. The results of this study indicate that K. pneumoniae CPS not only protects the pathogen from the bactericidal action of defensins but also impedes their expression. These features of K. pneumoniae CPS may facilitate pathogen survival in the hostile environment of the lung.

Effects of dietary supplementation with epigallocatechin-3-gallate on weight loss, energy homeostasis, cardiometabolic risk factors and liver function in obese women: randomised, double-blind, placebo-controlled clinical trial

The aim of the present study was to examine the effects of green tea epigallocatechin-3-gallate (EGCG) on changes in body composition, energy and substrate metabolism, cardiometabolic risk factors and liver function enzymes after an energy-restricted diet intervention in obese women. In the present randomised, double-blind, placebo-controlled study, eighty-three obese (30 kg/m² > BMI < 40 kg/m²) pre-menopausal women consumed 300 mg/d of EGCG or placebo (lactose). We measured body weight and adiposity (dual-energy X-ray absorptiometry), energy expenditure and fat oxidation rates (indirect calorimetry), blood lipid levels (TAG, total cholesterol, LDL-cholesterol and HDL-cholesterol), insulin resistance, C-reactive protein and liver function markers (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyltransferase, urea, bilirubin and 2-keto[1-¹³C]isocaproate oxidation) before and after the intervention in the EGCG and control groups. We did not find any significant difference in the changes in body weight (-0.3 kg, 95% CI -5.0, 4.3), fat mass (-0.7 kg, 95% CI -3.5, 2.1), energy (0.3 kJ/kg per d, 95% CI -3.1, 2.7) and fat (-0.1 g/min, 95% CI -0.03, 0.01) metabolism, homeostasis assessment model for insulin resistance (0.2, 95% CI -0.2, 0.7), total cholesterol (-0.21 mmol/l, 95% CI -0.55, 0.13), LDL-cholesterol (-0.15 mmol/l, 95% CI -0.50, 0.20), TAG (-0.4 mmol/l, 95% CI -0.56, 0.29) and liver function markers between the EGCG and control groups. In conclusion, the present results suggest that dietary supplementation with 300 mg/d of EGCG for 12 weeks did not enhance energy-restricted diet-induced adiposity reductions, and did not improve weight-loss-induced changes in cardiometabolic risk factors in obese Caucasian women. The intake of 300 mg/d of EGCG for 12 weeks did not cause any adverse effect on liver function biomarkers.

Klebsiella pneumoniae Increases the Levels of Toll-Like Receptors 2 and 4 in Human Airway Epithelial Cells

ABSTRACT Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IκBα-dependent NF-κΒ pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

Up-regulation of a thermogenesis-related gene (UCP1) and down-regulation of PPARgamma and aP2 genes in adipose tissue: possible features of the antiobesity effects of a beta3-adrenergic agonist.

A number of experiments have demonstrated the antiobesity effects of beta(3)-adrenergic receptor stimulation by promoting thermogenesis and/or lipolysis. While many studies have been performed in order to develop beta(3)-adrenergic agonists as a novel strategy in the management of obesity, more information is needed about the mechanisms involved in thermogenesis and the actions of these drugs on adipocyte differentiation. To address this, the possible thermogenic and antiadipogenic properties of Tertatolol, a beta(3)-adrenergic agonist, in a diet-induced obesity model has been tested. Animals fed on a high-fat diet gained more weight and fat mass as compared with control and high-fat fed animals treated with Tertatolol. A RT-PCR was carried out in white adipose tissue specific genes involved in thermogenesis such as uncoupling proteins (UCPs) and adipogenesis such as peroxisome proliferator-activated receptor (PPARgamma2), retinoid receptors (RXRalpha/RARalpha), and fatty acid binding protein (aP2). Levels of UCP1 mRNA were augmented in the Tertatolol-treated group as compared to non-treated high-fat fed animals, while the beta(3)-adrenergic agonist treatment significantly decreased the expression levels of aP2 and transcription factors such as PPARgamma2 and the ratio RXRalpha/RARalpha as compared to obese rats. Altogether these data suggest that the antiobesity effects of beta(3)-adrenergic agonists are not limited to the promotion of thermogenesis and/or lipolysis and support the implication that these beta(3)-adrenergic agonists also affect fat deposition by impairing adipogenesis in white adipose tissue (WAT).

Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner.

Klebsiella pneumoniae is an important cause of community‐acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro‐inflammatory mediators‐induced IL‐8 secretion. Klebsiella antagonizes the activation of NF‐κB via the deubiquitinase CYLD and blocks the phosphorylation of mitogen‐activated protein kinases (MAPKs) via the MAPK phosphatase MKP‐1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti‐inflammatory effect, Klebsiella engages NOD1. In NOD1 knock‐down cells, Klebsiella neither induces the expression of CYLD and MKP‐1 nor blocks the activation of NF‐κB and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1‐mediated CYLD and MKP‐1 expression which in turn attenuates IL‐1β‐induced IL‐8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL‐1β‐induced IL‐8 secretion nor induces the expression of CYLD and MKP‐1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.

Changes in UCP mRNA expression levels in brown adipose tissue and skeletal muscle after feeding a high-energy diet and relationships with leptin, glucose and PPARγ

Brown adipose tissue and skeletal muscle are known to be important sites for nonshivering thermogenesis. In this context, it is accepted that uncoupling proteins (UCPs) are involved in such process, but little is known about the physiological regulation of these proteins as affected by the intake of a high-energy (cafeteria) diet inducing fat deposition. In this study, the UCP messenger RNA (mRNA) expression in interscapular brown adipose tissue (iBAT) and skeletal muscle was assessed to evaluate the influence of a dietary manipulation on energy homeostasis regulation. We report a statistically significant increase in mRNA levels of iBAT UCP1 and UCP3 and a statistical marginal rise in skeletal muscle UCP3 mRNA expression after feeding a high-energy diet, whereas no changes in UCP2 expression were found in either tissue. Furthermore, significant positive associations between iBAT UCP1 and UCP3 mRNA levels with serum leptin were found. Although the expression of the beta(3) adrenoceptor (beta(3)AR) was about 50% in the lean controls compared with the obese group in iBAT, no statistically significant changes were observed concerning peroxisome proliferator-activated receptor gamma2 (PPARgamma2) mRNA levels in muscle or iBAT. We conclude that feeding a diet inducing weight and fat gain produces different outcomes on iBAT and skeletal muscle UCP mRNA expression, revealing a tissue-dependent response for the three UCPs. Results suggest that the regulation of UCP expression in both tissues under these specific dietary conditions may be related to leptin circulating levels.

A new NPY-antagonist strongly stimulates apoptosis and lipolysis on white adipocytes in an obesity model.

Neuropeptide Y (NPY) is a 36 amino acid peptide released in central and peripheral mammalian neurons, which appears to contribute to adiposity regulation by increasing food intake, thus promoting weight gain on animals. Nevertheless, little is known about NPY direct actions on white adipocytes. This trial, which was designed to test the possible effects of a new NPY antagonist, S.A.0204, on white adipose tissue, revealed that the administration of this novel molecule strongly ex vivo stimulates apoptosis and lipolysis in animals fed on a high-fat diet.

Preliminary findings on the role of PLIN1 polymorphisms on body composition and energy metabolism response to energy restriction in obese women

The aim of the present study was to investigate the association of PLIN1 11482G>A (rs894160) and PLIN1 13041A>G (rs2304795) polymorphisms with body composition, energy and substrate metabolism, and the metabolic response to a 12-week energy-restricted diet in obese women. The study comprised a total of seventy-eight obese (BMI 34·0 (SD 2·8) kg/m(2)) women (age 36·7 (SD 7) years). We measured weight, height and waist circumference before and after a 12-week controlled energy-restricted diet intervention. Body fat mass and lean mass were measured by dual-energy X-ray absorptiometry. RMR and lipid oxidation rate were measured by indirect calorimetry. We also analysed fasting plasma glucose, insulin, cholesterol and leptin. Women carrying the 11482A allele had a lower reduction in waist circumference than non-A allele carriers (3·2 (SD 0·5) v. 4·6 (SD 0·6) %, respectively, P = 0·047; P for gene-diet interaction = 0·064). Moreover, women with the 11482A allele had a higher decrease in lipid oxidation rate than non-A allele carriers (58·9 (SD 6·7) v. 31·3 (SD 8·2) %, respectively, P = 0·012; P for gene-diet interaction = 0·004). There was no interaction effect between the 13041A>G polymorphism and diet-induced changes on the outcome variables (all P>0·1). These results confirm and extend previous findings suggesting that the PLIN1 11482G>A polymorphism plays a modulating role on diet-induced changes in body fat and energy metabolism in obese women.

Influence of menopause on adipose tissue clock gene genotype and its relationship with metabolic syndrome in morbidly obese women

Menopausal women exhibit a loss of circadian coordination, a process that runs parallel with a redistribution of adipose tissue. However, the specific genetic mechanisms underlying these alterations have not been studied. Thus, the aim of the present study was to determine whether the development of menopause induces an alteration of the genes that control biological rhythms in human subcutaneous (SAT) and visceral (VAT) adipose tissue, and whether changes in clock gene expression are involved in the increased risk of developing metabolic syndrome (MetS), which is frequently associated with menopause. To this end, VAT and SAT biopsies were taken in pre- (n = 7) and postmenopausal (n = 7) women at similar hours in the morning. RNA was extracted, and a microarray analysis was made. Data were confirmed by quantitative real-time polymerase chain reaction. Western blot and immunohistochemical analysis were also performed. When clock gene expression was compared between both groups of women, data in SAT showed that expression of the core clock gene period3 was significantly higher in postmenopausal women, while casein kinase-1δ, E1A-binding protein and cAMP-responsive element were preferentially expressed in the premenopausal group. In VAT, period2 (PER2) and v-myc myelocytomatosis viral oncogene expressions were significantly higher in the postmenopausal group. Western blot analysis indicated that PER2 and PER3 protein expression was also increased in postmenopausal women. In addition, several genes, including PER2, were differentially expressed depending on whether or not the patient met the MetS criteria. We conclude that menopause transition induces several changes in the genotype of the adipose tissue chronobiological machinery related to an increased risk of developing MetS.