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

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

Recientes avances en la hepatotoxicidad de fármacos

Conferencias: M. Teresa Donato. Hepatotoxicidad Idiosincrásica por Fármacos: Promesas y Limitaciones de las Estrategias in vitro María José Gómez-Lechón Moliner. Modelos celulares hepáticos avanzados para el estudio preclínico del metabolismo y la hepatotoxicidad de fármacos Ramiro Jover Atienza. Esteatosis hepática y colestasis inducida por medicamentos: nuevos mecanismos y biomarcadore

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

Metabolic Phenotype Transfer Through Gut Microbiota Transplantation from HFD-Fed and Quercetin Treated Donors Modulates Obesity-Related NAFLD in Germ-Free Mice

1 p.Gut microbiota is involved in obesity, metabolic syndrome and nonalcoholic fatty liver disease (NAFLD). Strategies to modulate it, including faecal transplantation and administration of prebiotics as quercetin, are actively being examined. The present study aims to investigate benefits of experimental gut microbiota transfer from donors to germ-free mice (GFm) 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

Akkermansia spp. mediates protection from obesity-associated NAFLD development in germ free mice following intestinal microbiota transplantation from high fat diet and quercetin treated donors

1 p.Dysbiosis and gut-liver axis alteration have been pointed as important contributors to obesity and non-alcoholic fatty liver disease (NAFLD) development. Modulation of intestinal microbiota (IM) emerge as a promising therapeutic strategy for obesity-associated NAFLD. This study aims to determine the effect of IM transplantation and quercetin supplementation in a high fat diet (HFD)-based NAFLD model in germ free mice (GFm). 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

Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice is mediated by modulating intestinal microbiota imbalance and related gut-liver axis activation

60 p.Gut microbiota is involved in obesity, metabolic syndrome and the progression of nonalcoholic fatty liver disease (NAFLD). It has been recently suggested that the flavonoid quercetin may have the ability to modulate the intestinal microbiota composition, suggesting a prebiotic capacity which highlights a great therapeutic potential in NAFLD. The present study aims to investigate benefits of experimental treatment with quercetin on gut microbial balance and related gut-liver axis activation in a nutritional animal model of NAFLD associated to obesity. C57BL/6J mice were challenged with high fat diet (HFD) supplemented or not with quercetin for 16 weeks. HFD induced obesity, metabolic syndrome and the development of hepatic steatosis as main hepatic histological finding. Increased accumulation of intrahepatic lipids was associated with altered gene expression related to lipid metabolism, as a result of deregulation of their major modulators. Quercetin supplementation decreased insulin resistance and NAFLD activity score, by reducing the intrahepatic lipid accumulation through its ability to modulate lipid metabolism gene expression, cytochrome P450 2E1 (CYP2E1)-dependent lipoperoxidation and related lipotoxicity. Microbiota composition was determined via 16S ribosomal RNA Illumina next-generation sequencing. Metagenomic studies revealed HFD-dependent differences at phylum, class and genus levels leading to dysbiosis, characterized by an increase in Firmicutes/Bacteroidetes ratio and in Gram-negative bacteria, and a dramatically increased detection of Helicobacter genus. Dysbiosis was accompanied by endotoxemia, intestinal barrier dysfunction and gut-liver axis alteration and subsequent inflammatory gene overexpression. Dysbiosis-mediated toll-like receptor 4 (TLR-4)-NF-B signaling pathway activation was associated with inflammasome initiation response and reticulum stress pathway induction. Quercetin reverted gut microbiota imbalance and related endotoxemia-mediated TLR-4 pathway induction, with subsequent inhibition of inflammasome response and reticulum stress pathway activation, leading to the blockage of lipid metabolism gene expression deregulation. Our results support the suitability of quercetin as a therapeutic approach for obesity-associated NAFLD via its anti-inflammatory, antioxidant and prebiotic integrative response.Gut microbiota is involved in obesity, metabolic syndrome and the progression of nonalcoholic fatty liver disease (NAFLD). It has been recently suggested that the flavonoid quercetin may have the ability to modulate the intestinal microbiota composition, suggesting a prebiotic capacity which highlights a great therapeutic potential in NAFLD. The present study aims to investigate benefits of experimental treatment with quercetin on gut microbial balance and related gut-liver axis activation in a nutritional animal model of NAFLD associated to obesity. C57BL/6J mice were challenged with high fat diet (HFD) supplemented or not with quercetin for 16 weeks. HFD induced obesity, metabolic syndrome and the development of hepatic steatosis as main hepatic histological finding. Increased accumulation of intrahepatic lipids was associated with altered gene expression related to lipid metabolism, as a result of deregulation of their major modulators. Quercetin supplementation decreased insulin resistance and NAFLD activity score, by reducing the intrahepatic lipid accumulation through its ability to modulate lipid metabolism gene expression, cytochrome P450 2E1 (CYP2E1)-dependent lipoperoxidation and related lipotoxicity. Microbiota composition was determined via 16S ribosomal RNA Illumina next-generation sequencing. Metagenomic studies revealed HFD-dependent differences at phylum, class and genus levels leading to dysbiosis, characterized by an increase in Firmicutes/Bacteroidetes ratio and in Gram-negative bacteria, and a dramatically increased detection of Helicobacter genus. Dysbiosis was accompanied by endotoxemia, intestinal barrier dysfunction and gut-liver axis alteration and subsequent inflammatory gene overexpression. Dysbiosis-mediated toll-like receptor 4 (TLR-4)-NF-B signaling pathway activation was associated with inflammasome initiation response and reticulum stress pathway induction. Quercetin reverted gut microbiota imbalance and related endotoxemia-mediated TLR-4 pathway induction, with subsequent inhibition of inflammasome response and reticulum stress pathway activation, leading to the blockage of lipid metabolism gene expression deregulation. Our results support the suitability of quercetin as a therapeutic approach for obesity-associated NAFLD via its anti-inflammatory, antioxidant and prebiotic integrative respons