Ghrelin induces clock gene expression in the liver of goldfish in vitro via protein kinase C and protein kinase A pathways

The liver is the most important link between the circadian system and metabolism. As a food-entrainable oscillator, the hepatic clock needs to be entrained by food-related signals. The objective of the present study was to investigate the possible role of ghrelin (an orexigenic peptide mainly synthesized in the gastrointestinal tract) as an endogenous synchronizer of the liver oscillator in teleosts. To achieve this aim, we first examined the presence of ghrelin receptors in the liver of goldfish. Then, the ghrelin regulation of clock gene expression in the goldfish liver was studied. Finally, the possible involvement of the phospholipase C/protein kinase C (PLC/PKC) and adenylate cyclase/protein kinase A (AC/PKA) intracellular signalling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of goldfish hepatic cells. Ghrelin induced the mRNA expression of the positive (gbmal1a, gclock1a) and negative (gper genes) elements of the main loop of the molecular clock machinery, as well as grev-erbα (auxiliary loop) in cultured liver. These effects were blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a PLC inhibitor (U73122), a PKC activator (phorbol 12-myristate 13-acetate) and a PKC inhibitor (chelerythrine chloride) demonstrated that the PLC/PKC pathway mediates such ghrelin actions. Experiments with an AC activator (forskolin) and a PKA inhibitor (H89) showed that grev-erbα regulation could be due to activation of PKA. Taken together, the present results show for the first time in vertebrates a direct action of ghrelin on hepatic clock genes and support a role for this hormone as a temporal messenger in the entrainment of liver circadian functions

Characterization of Ghrelin O-Acyltransferase (GOAT) in goldfish (Carassius auratus)

Ghrelin is the only known hormone posttranslationally modified with an acylation. This modification is crucial for most of ghrelin’s physiological effects and is catalyzed by the polytopic enzyme ghrelin O-acyltransferase (GOAT). The aim of this study was to characterize GOAT in a teleost model, goldfish (Carassius auratus). First, the full-length cDNA sequence was obtained by RT-PCR and rapid amplification of cDNA ends methods. Two highly homologous cDNAs of 1491 and 1413 bp, respectively, named goat-V1 and goat-V2 were identified. Deduced protein sequences (393 and 367 amino acids, respectively) are predicted to present 11 and 9 transmembrane regions, respectively, and both contain two conserved key residues proposed to be involved in catalysis: asparagine 273 and histidine 304. RT-qPCR revealed that both forms of goat mRNAs show a similar widespread tissue distribution, with the highest expression in the gastrointestinal tract and gonads and less but considerable expression in brain, pituitary, liver and adipose tissue. Immunostaining of intestinal sections showed the presence of GOAT immunoreactive cells in the intestinal mucosa, some of which colocalize with ghrelin. Using an in vitro approach, we observed that acylated ghrelin downregulates GOAT gene and protein levels in cultured intestine in a time-dependent manner. Finally, we found a rhythmic oscillation of goat mRNA expression in the hypothalamus, pituitary and intestinal bulb of goldfish fed at midday, but not at midnight. Together, these findings report novel data characterizing GOAT, and offer new information about the ghrelinergic system in fish

Compuestos perfluorados en equinodermos marinos: metodología analítica para su determinación y monitorización

Los disruptores endocrinos están en el punto de mira de estudios ambientales y ecotoxicológicos. Entre ellos destacan los compuestos perfluorados, tanto por su amplio uso industrial y doméstico como por su elevada actividad estrogénica. Se descargan al medio ambiente a través de efluentes industriales y de estaciones depuradoras de aguas residuales afectando principalmente a la biota donde pueden bioacumularse. En este trabajo se propone un método analítico para la determinación de seis compuestos perfluorados en organismos marinos (Holothuria tubulosa) y se lleva a cabo un programa de monitorización ambiental para el seguimiento de estos compuestos. El análisis de las muestras se realizó mediante extracción con disolventes, limpieza del extracto por extracción en fase sólida dispersiva y posterior determinación mediante cromatografía de líquidos con detección de espectrometría de masas en tándem. Las recuperaciones obtenidas se situaron en el rango de 84 a 101 % y límites de cuantificación inferior a 0,03 ng/g (peso seco (ps)). Todas las muestras dieron positivas en el análisis de los contaminantes con concentraciones entre 667 ng/g (ps) para el ácido perfluorooctanoico y 0,81 ng/g (ps) para el ácido perfluoropentanoico. Además, se observaron concentraciones más elevadas para los compuestos perfluorados de mayor cadena fluorocarbonada.In recent years endocrine disruptors have come into the spotlight of environmental and ecotoxicological studies. Among them, perfluorinated compounds stand out, both, for their wide industrial and domestic use and for their elevated estrogenic activity, showing adverse effects at trace level. They are released into the environment through industrial waste and wastewater discharges affecting to marine organisms, where they can accumulate. To contribute to this goal, this work proposes an analytical method for the simultaneous determination of six perfluorinated compounds in marine organisms (Holothuria tubulosa) and an environmental monitoring program is carried out in these organisms. The sample treatment involve steps of solvent extraction and clean-up of the extracts with dispersive sorbents prior to liquid chromatography–tandem mass spectrometry analysis. Recoveries between 84 and 101 %, precision (RSD < 9 %) and limits of quantification below 0.03 ng/g dry weight (d.w.) were achieved. All tested samples were positive in the analysis of contaminants with concentrations between 667 ng/g (dw) for perflorooctanoic acid (PFOA) and 0.81 ng/g (dw) for perfloropentanoic acid (PFPeA). In general, perfluorinated compounds of larger fluorocarbonated chain were quantified at higher concentration levels than those of shorter one.Plan Propio de la Universidad de Sevilla Proyecto: 2017/0000096

Mediterranean alcohol-drinking pattern, low to moderate alcohol intake and risk of atrial fibrillation in the PREDIMED study

[Background and aims] There is ongoing controversy about the effect of a low to moderate alcohol consumption on atrial fibrillation (AF). Our aim is to assess the association between adherence to a Mediterranean alcohol drinking pattern and AF incidence.[Methods and results] A total 6527 out of the 7447 participants in the PREDIMED trial met our inclusion criteria. A validated frequency food questionnaire was used to measure alcohol consumption. Participants were classified as non-drinkers, Mediterranean alcohol drinking pattern (MADP) (10–30 g/d in men and 5–15 g/day in women, preferably red wine consumption with low spirits consumption), low-moderate drinking (<30 g/day men y and < 15 g/day women), and heavy drinking. We performed multivariable Cox regression models to estimate hazard ratios (HR) with 95% confidence intervals (95% CI) of incident AF according to alcohol drinking patterns. After a mean follow up of 4.4 years, 241 new incident AF cases were confirmed. Alcohol consumption was not associated to AF incidence among low-moderate drinkers (HR: 0.96; 95%CI: 0.67–1.37), adherents to MADP (HR: 1.15 95%CI: 0.75–1.75), or heavy drinkers (HR: 0.92; 95%CI: 0.53–1.58), compared with non-drinkers.[Conclusions] In a high cardiovascular risk adult population, a Mediterranean alcohol consumption pattern (low to moderate red wine consumption) was not associated with an increased incidence of AF.[Clinical trials] URL: http://www.controlled-trials.com. Unique identifier: ISRCTN35739639.PREDIMED trial was supported by the official funding agency for biomedical research of the Spanish government (Instituto de Salud Carlos III) RTIC G03/140 (Coordinator: Dr Estruch) and RTIC RD 06/0045 (Coordinator: Dr Martínez-González). We also acknowledge grants from the National Institutes of Health, United States (1R01HL118264-01); Fondo de Investigación Sanitaria– Fondo Europeo de Desarrollo Regional (PI04/0233, PI05/0976, PI07/0240, PI10/01407, PI10/02658, PI11/00049, PI11/02505 and AGL2010-22319-C03-03); Consejería de Salud de la Junta de Andalucía (PI0105/2007), and by the Generalitat Valenciana, Spain (ACOMP/2013/165 and ACOMP/2013/159)

Effects of a Mediterranean Eating Plan on the Need for Glucose-Lowering Medications in Participants With Type 2 Diabetes: A Subgroup Analysis of the PREDIMED Trial

[Objective]: To examine the effects of two Mediterranean eating plans (Med-EatPlans) versus a low-fat eating plan on the need for glucose-lowering medications. [Research design and methods]: From the Prevención con Dieta Mediterránea (PREDIMED) trial, we selected 3,230 participants with type 2 diabetes at baseline. These participants were randomly assigned to one of three eating plans: Med-EatPlan supplemented with extra-virgin olive oil (EVOO), Med-EatPlan supplemented with mixed nuts, or a low-fat eating plan (control). In a subgroup (15%), the allocation was done in small clusters instead of using individual randomization, and the clustering effect was taken into account in the statistical analysis. In multivariable time-to-event survival models, we assessed two outcomes: 1) introduction of the first glucose-lowering medication (oral or injectable) among participants on lifestyle management at enrollment and 2) insulin initiation. [Results]: After a median follow-up of 3.2 years, in multivariable analyses adjusting for baseline characteristics and propensity scores, the hazard ratios (HRs) of starting a first glucose-lowering medication were 0.78 (95% CI 0.62–0.98) for Med-EatPlan + EVOO and 0.89 (0.71–1.12) for Med-EatPlan + nuts, compared with the control eating plan. After a median follow-up of 5.1 years, the adjusted HRs of starting insulin treatment were 0.87 (0.68–1.11) for Med-EatPlan + EVOO and 0.89 (0.69–1.14) for Med-EatPlan + nuts compared with the control eating plan. [Conclusions]: Among participants with type 2 diabetes, a Med-EatPlan + EVOO may delay the introduction of new-onset glucose-lowering medications. The Med-EatPlan did not result in a significantly lower need for insulin

Quality of dietary fat intake and body weight and obesity in a Mediterranean population: Secondary analyses within the PREDIMED trial

A moderately high-fat Mediterranean diet does not promote weight gain. This study aimed to investigate the association between dietary intake of specific types of fat and obesity and body weight. A prospective cohort study was performed using data of 6942 participants in the PREDIMED trial, with yearly repeated validated food-frequency questionnaires, and anthropometric outcomes (median follow-up: 4.8 years). The effects of replacing dietary fat subtypes for one another, proteins or carbohydrates were estimated using generalized estimating equations substitution models. Replacement of 5% energy from saturated fatty acids (SFA) with monounsaturated fatty acids (MUFA) or polyunsaturated fatty acids (PUFA) resulted in weight changes of −0.38 kg (95% Confidece Iinterval (CI): −0.69, −0.07), and −0.51 kg (95% CI: −0.81, −0.20), respectively. Replacing proteins with MUFA or PUFA decreased the odds of becoming obese. Estimates for the daily substitution of one portion of red meat with white meat, oily fish or white fish showed weight changes up to −0.87 kg. Increasing the intake of unsaturated fatty acids at the expense of SFA, proteins, and carbohydrates showed beneficial effects on body weight and obesity. It may therefore be desirable to encourage high-quality fat diets like the Mediterranean diet instead of restricting total fat intake

Straightforward purification method for the determination of the activity of glucose oxidase and catalase in honey by extracting polyphenols with a film-shaped polymer

Glucose oxidase (GOX) and catalase (CAT) regulate the amount of H2O2 in honey, by generating or consuming it, so they are related to the antibacterial and antioxidant activity of honey. However, their activities are hardly analysed, since the process requires a previous dialysis that is non-selective, very time-consuming (>24 h), eco-unfriendly (>6L of buffer) and expensive. This research shows the design and performance of a material that selectively removes the actual interferents. The film-shaped-polymer is immersed for 90́ within a honey solution (12.5 mL of buffer), where it interacts exclusively with 1,2-dihydroxybenzenes, which we proved to be the real interferents (the material contains motifs derived from phenylboronic acid to interact with 1,2-diols). Polymeric chains favour condensation to occur exclusively with 1,2-dihydroxybenzenes, excluding monosaccharides. The interferents’ removal using our designed polymer is selective, low cost (1.42€ per test), rapid and eco-friendly (saves 6L of buffer and 20.5 h of experimental workout per sample).We gratefully acknowledge the financial support provided by all funders. Author Jose Miguel García received grant PID2020-113264RB-I00 / AEI / 10.13039/501100011033 funded by MCIN/AEI/ 10.13039/501100011033 and by “ERDF A way of making Europe”. Ana Arnaiz received funding from Ministerio de Universidades-European Union in the frame of NextGenerationEU RD 289/2021 (Universidad Politécnica de Madrid). We also gratefully acknowledge European Regional Development Fund (ERDF). Gianluca Utzeri thanks Fundação para a Ciência e a Tecnologia (FCT, Portugal) for PhD grant (SFR/BD/146358/2019). The Coimbra Chemistry Centre is supported by the FCT, through Projects UIDB/00313/2020 and UIDP/00313/2020. To all the beekeepers who provided a sample of honey for this study

Ratones knock-out del receptor lpa1 de ácido lisofosfatídico presentan un acusado déficit de la isoenzima glutaminasa KGA (GLS) y una morfología alterada en las espinas dendríticas de hipocampo y corteza

Objectives: The objective of the present study was to utilize mice with knocked-down lysophosphatidic acid 1 (LPA1) receptor to ascertain changes in glutamatergic transmission that may help to explain part of the cognitive and memory deficits shown by these KO-LPA1 mice. Material & methods: A well characterized KO-LPA1 mouse strain was used as animal model and compared with wild-type (WT) and heterozygous animals. Expression studies were implemented by immunohistochemistry and Western analysis of mouse brain regions, real-time quantitative RT-PCR of GA isoforms, enzymatic analysis of regional GA activity and Golgi staining to assess dendritic spine morphology and density. Results: A strong reduction of KGA immunoreactivity was mostly revealed in cerebral cortex and hippocampus of KO-LPA1 mice versus WT and heterozygous animals. In contrast, neither mRNA levels nor enzyme activity were significantly altered in KO mice suggesting compensatory mechanisms for neurotransmitter Glu synthesis. Interestingly, Golgi staining of hippocampal and cortical neurons revealed a clear morphology change toward a less-mature undifferentiated spine phenotype, without changes in the total number of spines. Conclusions: The molecular mechanisms underlying KGA downregulation in null LPA1 mutant mice are unknown. However, LPA increases neuronal differentiation, arborization and neurite outgrowth of developing neurons, while Gln-derived Glu, through GA reaction, has been also involved in neuronal growth and differentiation. It is tempting to speculate that downregulation of KGA protein in KO-LPA1 mice induce morphological changes in dendritic spines of cortical and hippocampal neurons which, in turn, may account for memory and cognitive deficits shown by KO-LPA1 mice.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Acknowledgements: Red de Trastornos Adictivos, RTA, (RD12/0028/0013/) RETICS, ISCIII, y Consejería Innovación, Ciencia y Empresa, Junta de Andalucía (Proyecto de Excelencia CVI-6656)

Mediterranean diet and atrial fibrillation: results from the predimed trial

Resumen del trabajo presentado en el X Congreso Internacional de la Dieta Mediterránea, celebrado en Barcelona (España) del 02 al 03 de abril de 2014.[Background]: It has been estimated that over 25 million Europeans will have atrial fibrillation by 2050. Even though pathophysiological mechanisms are being disentangled, there are hardly preventive strategies.[Objective]: To assess the effect of a Mediterranean diet (MedDiet) supplemented either with extra virgin olive oil (EVOO) or mixed nuts on the incidence of atrial fibrillation.[Methods]: The PREDIMED study (Prevención con Dieta Mediterránea) is a randomized, singleblind, and controlled trial conducted in Spanish primary healthcare centers. Participants were 6705 men (55-80 years) and women (60-80 years) initially free of atrial fibrillation who were randomly assigned to one of three dietary interventions: a MedDiet supplemented with EVOO, a MedDiet supplemented with nuts, or advice to follow a low-fat diet (control group). Incident atrial fibrillations were identified from inpatient and outpatient charts and adjudicated by a committee blind to treatment assignment. Analyses were performed on an intention-to-treat basis.[Results]: Over 4.7 years of follow-up (median), we observed 72 incident cases of atrial fibrillation in the MedDiet with EVOO group, 82 in the MedDiet with nuts group and 92 in the control group. Participants allocated to the MedDiet supplemented with EVOO group had a significantly lower risk of atrial fibrillation (HR: 0.62; 95%CI: 0.45-0.85), compared to those in the control group. No effect was observed for the MedDiet supplemented with nuts group (HR: 0.89; 95%CI 0.65-1.20).[Conclusions]: These results from the PREDIMED trial support a potential beneficial protection of a MedDiet supplemented with EVOO on the incidence of atrial fibrillation

Endothelial Progenitor Cells as a Potential Biomarker in Interstitial Lung Disease Associated with Rheumatoid Arthritis

Interstitial lung disease (ILD) increases morbidity and mortality in patients with rheumatoid arthritis (RA). Although the pathogenesis of ILD associated with RA (RA-ILD+) remains poorly defined, vascular tissue is crucial in lung physiology. In this context, endothelial progenitor cells (EPC) are involved in endothelial tissue repair. However, little is known about their implication in RA-ILD+. Accordingly, we aimed to investigate the potential role of EPC related to endothelial damage in RA-ILD+. EPC quantification in peripheral blood from 80 individuals (20 RA-ILD+ patients, 25 RA-ILD? patients, 21 idiopathic pulmonary fibrosis (IPF) patients, and 14 healthy controls) was performed by flow cytometry. EPC were considered as CD34+, CD45low, CD309+ and CD133+. A significant increase in EPC frequency in RA-ILD+ patients, as well as in RA-ILD? and IPF patients, was found when compared with controls (p < 0.001, p = 0.02 and p < 0.001, respectively). RA-ILD+ patients exhibited a higher EPC frequency than the RA-ILD? ones (p = 0.003), but lower than IPF patients (p < 0.001). Our results suggest that EPC increase may represent a reparative compensatory mechanism in patients with RA-ILD+. The degree of EPC frequency may help to identify the presence of ILD in RA patients and to discriminate RA-ILD+ from IPFThis work was partially supported by the European Regional Development Fund (ERDF) and ‘Fondo de Investigación Sanitaria’ [PI18/00043] from Instituto de Salud Carlos III (ISCIII), Health Ministry, Spain. VP-C is supported by a pre-doctoral grant from IDIVAL [PREVAL 18/01]. SR-M is supported by funds of RETICS Program [RD16/0012/0009, ISCIII, co-funded by ERDF]. BA-M is a recipient of a ‘López Albo’ Post-Residency Programme funded by Servicio Cántabro de Salud. LL-G is supported by funds of ISCIII, co-funded by ERDF [PI18/00042]. OG is beneficiary of a grant funded by Xunta de Galicia, Consellería de Educación, Universidade Formación Profesional and Consellería de Economía, Emprego e Industria (GAIN), GPC IN607B2019/10. RL-M is a recipient of a Miguel Servet type I fellowship [ISCIII, co-funded by European Social Fund—ESF, CP16/00033]