Valproate and Short-Chain Fatty Acids Activate Transcription of the Human Vitamin D Receptor Gene through a Proximal GC-Rich DNA Region Containing Two Putative Sp1 Binding Sites

The vitamin D receptor (VDR) mediates 1,25-dihydroxyvitamin D3 pleiotropic biological actions through transcription regulation of target genes. The expression levels of this ligand-activated nuclear receptor are regulated by multiple mechanisms both at transcriptional and post-transcriptional levels. Vitamin D3 is the natural VDR activator, but other molecules and signaling pathways have also been reported to regulate VDR expression and activity. In this study, we identify valproic acid (VPA) and natural short-chain fatty acids (SCFAs) as novel transcriptional activators of the human VDR (hVDR) gene. We further report a comprehensive characterization of VPA/SCFA-responsive elements in the 5′ regulatory region of the hVDR gene. Two alternative promoter DNA regions (of 2.4 and 3.8 kb), as well as subsequent deletion fragments, were cloned in pGL4-LUC reporter vector. Transfection of these constructs in HepG2 and human Upcyte hepatocytes followed by reporter assays demonstrated that a region of 107 bp (from −107 to −1) upstream of the transcription start site in exon 1a is responsible for most of the increase in transcriptional activity in response to VPA/SCFAs. This short DNA region is GC-rich, does not contain an apparent TATA box, and includes two bona fide binding sites for the transcription factor Sp1. Our results substantiate the hypothesis that VPA and SCFAs facilitate the activity of Sp1 on novel Sp1 responsive elements in the hVDR gene, thus promoting VDR upregulation and signaling. Elevated hepatic VDR levels have been associated with liver steatosis and, therefore, our results may have clinical relevance in epileptic pediatric patients on VPA therapy. Our results could also be suggestive of VDR upregulation by SCFAs produced by gut microbiota

The Vitamin D Receptor Regulates Glycerolipid and Phospholipid Metabolism in Human Hepatocytes

The vitamin D receptor (VDR) must be relevant to liver lipid metabolism because VDR deficient mice are protected from hepatosteatosis. Therefore, our objective was to define the role of VDR on the overall lipid metabolism in human hepatocytes. We developed an adenoviral vector for human VDR and performed transcriptomic and metabolomic analyses of cultured human hepatocytes upon VDR activation by vitamin D (VitD). Twenty percent of the VDR responsive genes were related to lipid metabolism, including MOGAT1, LPGAT1, AGPAT2, and DGAT1 (glycerolipid metabolism); CDS1, PCTP, and MAT1A (phospholipid metabolism); and FATP2, SLC6A12, and AQP3 (uptake of fatty acids, betaine, and glycerol, respectively). They were rapidly induced (4-6 h) upon VDR activation by 10 nM VitD or 100 µM lithocholic acid (LCA). Most of these genes were also upregulated by VDR/VitD in mouse livers in vivo. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics demonstrated intracellular accumulation of triglycerides, with concomitant decreases in diglycerides and phosphatidates, at 8 and 24 h upon VDR activation. Significant alterations in phosphatidylcholines, increases in lyso-phosphatidylcholines and decreases in phosphatidylethanolamines and phosphatidylethanolamine plasmalogens were also observed. In conclusion, active VitD/VDR signaling in hepatocytes triggers an unanticipated coordinated gene response leading to triglyceride synthesis and to important perturbations in glycerolipids and phospholipids

La educación físico-corporal en el Humanismo médico español: el "Examen de ingenios", de Juan Huarte

P. 55-70El capítulo analiza la obra "Examen de ingenios" de Juan Huarte, considerada un referente importante en la historia de la psicopedagogía y de la medicina. Los autores hacen un rápido recorrido por la vida y obra de Juan Huarte de San Juan para después centrarse en el análisis del contenido de su “Examen de ingenios”

Towards a Rapid Screening of Liver Grafts at the Operating Room Using Mid-Infrared Spectroscopy

The estimation of steatosis in a liver graft is mandatory prior to liver transplantation, as the risk of graft failure increases with the level of infiltrated fat. However, the assessment of liver steatosis before transplantation is typically based on a qualitative or semiquantitative characterization by visual inspection and palpation and histological analysis. Thus, there is an unmet need for transplantation surgeons to have access to a diagnostic tool enabling an in situ fast classification of grafts prior to extraction. In this study, we have assessed an attenuated total reflection−Fourier transform infrared (ATR−FTIR) spectroscopic method compatible with the requirements of an operation room for the evaluation of the lipid content in human livers. A set of human liver biopsies obtained from organs intended for transplantation were analyzed by expert pathologists, ATR−FTIR spectroscopy, lipid biochemical analysis, and UPLC−ESI(+/−)TOFMS for lipidomic profiling. Comparative analysis of multisource data showed strong correlations between ATR−FTIR, clinical, and lipidomic information. Results show that ATR−FTIR captures a global picture of the lipid composition of the liver, along with information for the quantification of the triradylglycerol content in liver biopsies. Although the methodology performance needs to be further validated, results support the applicability of ATR−FTIR for the in situ determination of the grade of liver steatosis at the operation room as a fast, quantitative method, as an alternative to the qualitative and subjective pathological examination

New microRNA biomarkers for drug-induced steatosis and their potential to predict the contribution of drugs to non-alcoholic fatty liver disease

Background and Aims: Drug-induced steatosis is a major reason for drug failure in clinical trials and post-marketing withdrawal; and therefore, predictive biomarkers are essential. These could be particularly relevant in non-alcoholic fatty liver disease (NAFLD), where most patients show features of the metabolic syndrome and are prescribed with combined chronic therapies, which can contribute to fatty liver. However, specific biomarkers to assess the contribution of drugs to NAFLD are lacking. We aimed to find microRNAs (miRNAs) responsive to steatotic drugs and to investigate if they could become circulating biomarkers for drug-induced steatosis. Methods: Human HepG2 cells were treated with drugs and changes in miRNA levels were measured by microarray and qRT-PCR. Drug-induced fat accumulation in HepG2 was analyzed by high-content screening and enzymatic methods. miRNA biomarkers were also analyzed in the sera of 44 biopsy-proven NAFLD patients and in 10 controls. Results: We found a set of 10 miRNAs [miR-22-5p, -3929, -24-2-5p, -663a, -29a-3p, -21 (5p and 3p), -27a-5p, -1260 and -202-3p] that were induced in human HepG2 cells and secreted to the culture medium upon incubation with model steatotic drugs (valproate, doxycycline, cyclosporin A and tamoxifen). Moreover, cell exposure to 17 common drugs for NAFLD patients showed that some of them (e.g., irbesartan, fenofibrate, and omeprazole) also induced these miRNAs and increased intracellular triglycerides, particularly in combinations. Finally, we found that most of these miRNAs (60%) were detected in human serum, and that NAFLD patients under fibrates showed both induction of these miRNAs and a more severe steatosis grade. Conclusion: Steatotic drugs induce a common set of hepatic miRNAs that could be used in drug screening during preclinical development. Moreover, most of these miRNAs are serum circulating biomarkers that could become useful in the diagnosis of iatrogenic steatosis

PTEN Deletion in Adult Mice Induces Hypoinsulinemia With Concomitant Low Glucose Levels

The PI3K/AKT pathway, negatively regulated by PTEN, plays a paramount role in glucose metabolism regulation due to its activation by the insulin receptor signaling pathway. We generated a PTEN-KO mouse to evaluate the systemic effect of the overactivation of the PI3K/AKT pathway in insulin signaling and glucose homeostasis. Our results demonstrate that PTEN-KO mice show very low glucose levels in the fasted state, which poorly respond to glucose and pyruvate administration. Insulinemia decreased without alterations in pancreatic islets. Among the possible reasons, we uncover the deregulation of the expression of proximal tubule glucose transporter and consequent glycosuria. Moreover, we evidence an altered activation of hepatic gluconeogenesis-related genes. In addition, the expression of several genes related to β-oxidation showed a delayed or even absent response to fasting, suggesting that the lack of PTEN not only impairs glucose metabolism but also slows down the use of lipids as a metabolic fuel. We conclude that the inducible full PTEN-KO mice could be a good model to study the metabolic interactions between glycidic and lipidic metabolism in hypoinsulinemic hypoglycemia and that PTEN could be an important mediator in the disease and/or a potential drug target

SOC-V-11 New serum miRNA biomarkers to predict liver steatosis by valproic acid in paediatric epileptic patients

Depakine (Valproate, VPA) has been the first line, most-frequently prescribed, anti-epileptic drug in children for the past 50 years. Idiosyncratic hepatotoxicity (iDILI) by VPA has been demonstrated in several case reports, where microvesicular liver steatosis was the most frequent feature. Moreover, more than half of VPA-treated patients could have silent fatty liver as demonstrated by ultrasounds. Extensive experimental studies support that VPA has a high potential to induce steatosis in hepatocytes. However, there is an apparent lack of significant hepatic problems in the Neuropediatric Units, despite transaminitis is not uncommon. One of the reasons could be that iDILI and liver steatosis diagnosis lack specific biomarkers. Thus, it is likely that a relevant number of children under VPA treatment may have a significant, but sub-clinical, hepatosteatosis

Aplicació d’un test d’esforç intervàlic (Test de Probst) per valorar la qualitat aeròbica en futbolistes de la lliga espanyola

Els tests continus realitzats en ergòmetres estandarditzats són poc específics per al futbol. El test intervàlic de Probst permet de valorar la velocitat màxima aeròbica (VMA) i el LAn (llindar anaeròbic) en el camp de futbol, car no existeixen dades de referència. Es pretenen de valorar aquests paràmetres en futbolistes espanyols per comprovar la sensibilitat a nivell de pràctica i la utilitat per determinar el LAn. Van participar-hi 231 futbolistes de categories juvenil (n = 26), amateur (n= 74), semiprofessional (n = 36) i professional (n = 95); es va registrar la VMA i la freqüència cardíaca (Fc), i es van calcular les velocitats i Fc en el LAn, determinat manualment (LAnI) i matemàticament (LAnM). Va existir inflexió de la Fc en més del 89 % dels tests. Les Fc en el LAn van ser similars a les descrites a la literatura (90-95% respecte a la màxima) i la velocitat en el LAn va obtenir rangs amplis de VMA (72-96%). El test va ser sensible al nivell de pràctica en les variables LAnI i VMA. El protocol intervàlic de Probst va permetre de realitzar un test de camp específic sensible al grau de professionalització dels futbolistes, i es van establir dades de referència per a les categories estudiades. L’anàlisi de la Fc va permetre d’identificar un punt d’inflexió que pogués correspondre’s amb el LAn

Aplicación de un test de esfuerzo interválico (Test de Probst) para valorar la cualidad aeróbica en futbolistas de la liga española

Los tests continuos realizados en ergómetros estandarizados son poco específicos para el fútbol. El test interválico de Probst permite valorar la velocidad máxima aeróbica (VMA) y el UAn en el campo de fútbol, no existiendo datos de referencia. Se pretenden valorar estos parámetros en futbolistas españoles para comprobar la sensibilidad al nivel de práctica y la utilidad para determinar el UAn. Participaron 231 futbolistas de categorías juvenil (n = 26), amateur (n = 74), semiprofesional (n = 36) y profesional (n = 95), registrando la VMA y la frecuencia cardíaca (Fc), y calculando las velocidades y Fc en el UAn determinado manualmente (UAnI) y matemáticamente (UAnM). Existió inflexión de la Fc en más del 89 % de los tests. Las Fc en el UAn fueron similares a las descritas en la literatura (90-95 % respecto a la máxima) y la velocidad en el UAn obtuvo rangos amplios de VMA (72-96 %). El test fue sensible al nivel de práctica en las variables UAnI y VMA. El protocolo interválico de Probst permitió realizar un test de campo específico sensible al grado de profesionalización de los futbolistas, estableciéndose datos de referencia para las categorías estudiadas. El análisis de la Fc permitió identificar un punto de inflexión que pudiera corresponderse con el UAn

Intestinal Microbiota Transplantation From HFD-fed and Quercetin Treated Donors Results in a Complex Metabolic Phenotype Transfer that Modulates Obesity-Related NAFLD in Germ Free Mice

2 p.Intestinal microbiota imbalance and related gut-liver axis activation have been identified as key mechanisms in nonalcoholic fatty liver disease (NAFLD) development. Modulation of intestinal microbiota, through administration of prebiotics or faecal microbiota transplantation, is a promising therapeutic approach for obesity associated diseases including NAFLD. The aim of the present study is to evaluate the benefits of gut microbiota transplantation from donors to germ free mice (GFm) following an experimental treatment with the flavonoid quercetin in a high fat diet (HFD)-based NAFLD model. Resumen de un trabajo resultado del proyecto de investigación financiado por la Consejería de Educación de la Junta de Castilla y León (referencia LE063U16)S