Pilot Study to Estimate Dietary Fiber Intake in Adults Residing in Chile.

Dietary fiber has been associated with health benefits; therefore, the availability of validated tools to assess food consumption associated with high-fiber foods would allow the quantification of the intake of this functional nutrient, the identification of risk groups and target populations, and the development of public policies and/or programs aimed at improving the health of the population. In this study, a fiber intake short food frequency questionnaire (FFQ) was translated into Spanish, and its content validity was determined by a group of experts, to subsequently conduct a pilot test including 198 subjects aged 36 ± 12.5 years, residing in Chile (46 men and 150 women), with the purpose of quantifying dietary fiber intake. The global assessment of the FFQ revealed a validity coefficient of 0.98 ± 0.02; after the application of the pilot, the mean dietary fiber intake in adult Chilean residents was 12.3 g per day, with similar results to those found in the National Food Consumption Survey 2010 (12.5 g per day in men, and 11.5 g in women). The FFQ is a quick and valid tool to classify people on the basis of their habitual dietary fiber intake.

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.

Elimination of Vitamin D Signaling Causes Increased Mortality in a Model of Overactivation of the Insulin Receptor: Role of Lipid Metabolism.

Vitamin D (VD) deficiency has been associated with cancer and diabetes. Insulin signaling through the insulin receptor (IR) stimulates cellular responses by activating the PI3K/AKT pathway. PTEN is a tumor suppressor and a negative regulator of the pathway. Its absence enhances insulin signaling leading to hypoglycemia, a dangerous complication found after insulin overdose. We analyzed the effect of VD signaling in a model of overactivation of the IR. We generated inducible double KO (DKO) mice for the VD receptor (VDR) and PTEN. DKO mice showed severe hypoglycemia, lower total cholesterol and increased mortality. No macroscopic tumors were detected. Analysis of the glucose metabolism did not show clear differences that would explain the increased mortality. Glucose supplementation, either systemically or directly into the brain, did not enhance DKO survival. Lipidic liver metabolism was altered as there was a delay in the activation of genes related to ß-oxidation and a decrease in lipogenesis in DKO mice. High-fat diet administration in DKO significantly improved its life span. Lack of vitamin D signaling increases mortality in a model of overactivation of the IR by impairing lipid metabolism. Clinically, these results reveal the importance of adequate Vitamin D levels in T1D patients.

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 ß-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.

Efectos de la COVID-19 en la lactancia materna en Chile durante el 2020 / Effects of COVID-19 on breastfeeding in Chile during 2020

RESUMEN Actualmente en Chile y en gran parte del planeta se está viviendo una infección respiratoria que ha causado un gran impacto, conocida como coronavirus o COVID-19. La presente revisión tuvo como objetivo describir la influencia del COVID-19 en la lactancia materna en Chile durante el año 2020. Hasta el momento no existe evidencia científica que revele la presencia de COVID-19 en la leche materna. Se recomienda seguir con la lactancia en ausencia o presencia de COVID-19, pero con todas las medidas higiénicas y de seguridad orientadas por la autoridad sanitaria. Se ciaron un total de 34 artículos.

ABSTRACT Currently in Chile and in much of the planet a respiratory infection that has caused a great impact, known as coronavirus or COVID-19, is being experienced. The present review aimed to describe the influence of COVID-19 on breastfeeding in Chile during 2020. To date, there is no scientific evidence that reveals the presence of COVID-19 in breast milk. It is recommended to continue breastfeeding in the absence or presence of COVID-19, but with all hygiene and safety measures directed by the health authority. A total of 34 articles were published.

Harnessing of geothermal energy for a greenhouse in Ecuador employing a heat pump: design, construction, and feasibility assessment.

Globally, the greenhouses' farming area comprises 500 000 ha, and they efficiently produce more than half of the vegetables consumed around the world. Nevertheless, high-yield crops tend to be incredibly energy-intensive. This study proposes designing and building a coupled geothermal heat pump for a 470 m2 greenhouse in the Andean zone conditions addressing a requirement of 15 °C at night and 30 °C during the day. Firstly, the study determined the energy potential of the solar and geothermal sources employing actual measurements and contrasting the results with theoretical models. Then, it developed an energy balance in the greenhouse to size the geothermal heat pump using the vapor compression cycle. Finally, the comprehensive system was built and evaluated through the Leveled Cost of Heat (LCOH). The operation requires a potential of 29.56 and 65.76 kW for heating and cooling; this is technically feasible when running the system with a heating flow driven by an optimized temperature ramp of 1.64 °C h-1. Also, the capacity factor (CF) shows that a lifespan between 12 to 14 years is required to reach acceptable LCOH when CF is as low as 0.45. Financially, it is necessary to foster customs exemptions to make it competitive versus more traditional sources such as electricity and LPG since the main components of the heat pump and the geothermal exchanger are not produced locally and represent nearly 70 % of the upfront costs.

Efectos de una dieta con alto contenido de grasas sobre parámetros alimentarios y tejido adiposo blanco visceral de ratas Sprague Dawley / Effects of a high fat diet on dietary parameters and visceral white adipose tissue of Sprague Dawley rats

El incremento en las cifras de obesidad se debe esencialmente a factores de carácter ambiental asociados al consumo de alimentos con alto contenido de grasas saturadas. El objetivo del trabajo fue evaluar el efecto de una dieta alta en grasas sobre parámetros alimentarios y tejido adiposo blanco visceral. Se utilizaron ratas macho Sprague Dawley (n=10), divididas en dos grupos experimentales, el grupo control recibió dieta convencional (DC) y el grupo experimental una dieta alta en grasas (HFD), durante 10 semanas. Se determinó peso corporal, ingesta alimentaria, conversión alimenticia y características de tejido adiposo. El análisis de datos se realizó utilizando software IBM SPSS versión 21; tras evaluación de la normalidad de los datos, se aplicaron pruebas paramétricas T para muestras independientes y ANOVA de dos vías para medidas repetidas en uno de los factores, con ajuste Bonferroni. Se observó que el promedio de peso fue mayor en los animales alimentados con HFD, sin diferencia estadística respecto a DC, no obstante, existen diferencias significativas en el peso de las ratas alimentadas con HFD en distintos tiempos del protocolo, específicamente semanas 1, 5 y 10 (p<0,001). La ingesta alimentaria fue mayor en los animales alimentados con DC (p<0,005), sin embargo el consumo de energía fue mayor en aquellos alimentados con HFD (p=0,016), lo que derivó en una mayor conversión alimenticia (p<0,005). El promedio de diámetro teórico calculado de los adipocitos es estadísticamente mayor en grupo HFD (p<0,005), lo que se relaciona a la hipertrofia clásica generada tras un período de alimentación con elevado contenido de grasas. Conclusión: El protocolo permite establecer que efectivamente, dado la mayor densidad energética, HFD induce hipertrofia de los adipocitos, proceso característico de la obesidad.

The continued increase in obesity statistics is the result of environmental factors associated with the consumption of foods high in saturated fat. The objective of the work was to evaluate the effect of a high fat diet on food parameters and visceral white adipose tissue. in Male Sprague Dawley rats (n = 10) were used, divided into two experimental groups, the control group received conventional diet (DC) and the experimental group a high fat diet (HFD), for 10 weeks. Body weight, food intake, food conversion and adipose tissue characteristics were determined. Data analysis was performed using IBM SPSS version 21 software; after evaluating the normality of the data, parametric T tests were applied for independent samples and two-way ANOVA for repeated measurements in one of the factors, with Bonferroni adjustment. It was observed that the average weight was higher in animals fed with HFD, without statistical difference with respect to DC, however, there were significant differences in the weight of rats fed with HFD at different times of the protocol, specifically weeks 1.5 and 10 (p <0.001). Food intake was higher in animals fed DC (p <0.005), however the energy consumption was higher in those fed with HFD (p=0.016), which resulted in a higher feed conversion (p <0.005). The average theoretical diameter calculated for adipocytes is statistically higher in the HFD group (p <0.005), which is related to the classical hypertrophy generated after a period of feeding with high fat content. In conclusion, the protocol allows us to establish that, given the higher energy density, HFD induces adipocyte hypertrophy, a characteristic in the obesity process.

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.

Epistane, an anabolic steroid used for recreational purposes, causes cholestasis with elevated levels of cholic acid conjugates, by upregulating bile acid synthesis (CYP8B1) and cross-talking with nuclear receptors in human hepatocytes.

Anabolic-androgenic steroids are testosterone derivatives, used by body-builders to increase muscle mass. Epistane (EPI) is an orally administered 17α-alkylated testosterone derivative with 2a-3a epithio ring. We identified four individuals who, after EPI consumption, developed long-lasting cholestasis. The bile acid (BA) profile of three patients was characterized, as well the molecular mechanisms involved in this pathology. The serum BA pool was increased from 14 to 61-fold, basically on account of primary conjugated BA (cholic acid (CA) conjugates), whereas secondary BA were very low. In in vitro experiments with cultured human hepatocytes, EPI caused the accumulation of glycoCA in the medium. Moreover, as low as 0.01 µM EPI upregulated the expression of key BA synthesis genes (CYP7A1, by 65% and CYP8B1, by 67%) and BA transporters (NTCP, OSTA and BSEP), and downregulated FGF19. EPI increased the uptake/accumulation of a fluorescent BA analogue in hepatocytes by 50-70%. Results also evidenced, that 40 µM EPI trans-activated the nuclear receptors LXR and PXR. More importantly, 0.01 µM EPI activated AR in hepatocytes, leading to an increase in the expression of CYP8B1. In samples from a human liver bank, we proved that the expression of AR was positively correlated with that of CYP8B1 in men. Taken together, we conclude that EPI could cause cholestasis by inducing BA synthesis and favouring BA accumulation in hepatocytes, at least in part by AR activation. We anticipate that the large phenotypic variability of BA synthesis enzymes and transport genes in man provide a putative explanation for the idiosyncratic nature of EPI-induced cholestasis.

The relationship between chronic inflammation associated with obesity and vitamin D deficiency / Relación entre inflamación crónica asociada a obesidad y déficit de vitamina D

Obesity is characterized by an abnormal production of adipocytokines, generating chronic inflammation associated in turn with endothelial dysfunction, atherosclerosis and insulin resistance. On the other hand, it is a risk factor for vitamin D deficiency, thus establishing an inverse relationship between the plasma levels of this nutrient and acute phase proteins with low vitamin D levels, being able to boost the inflammatory response in obesity. In this context, the correction of poor vitamin D status could be an effective addition to the treatment of obesity; however, evidence of future trials that can support the regulatory effects of supplementation is required. The objective of this review is to analyze the existing evidence and establish the relationship between plasma levels of vitamin D and chronic inflammation associated with obesity. The methodology consists of a sensitive search in the PubMed and Trip Database, limiting the search to articles in English and Spanish published through January 2019. Priority was given to clinical trials, original articles and systematic reviews, from which other relevant research was identified.

La obesidad se caracteriza por la producción anormal de adipocitocinas, generando inflamación crónica asociada a su vez a disfunción endotelial, aterosclerosis y resistencia a insulina. Por otra parte, es un factor de riesgo de déficit de vitamina D, estableciéndose una relación inversa entre los niveles plasmáticos de dicho nutriente y proteínas de fase aguda, pudiendo potenciar la respuesta inflamatoria en obesidad. En este contexto la corrección del mal estado de vitamina D podría ser una adición efectiva al tratamiento de la obesidad, sin embargo se requiere evidencia de futuros ensayos que se puedan respaldar los efectos reguladores de la suplementación. El objetivo de esta revisión es analizar la evidencia existente y establecer la relación entre los niveles plasmáticos de vitamina D y la inflamación crónica asociada con la obesidad. La metodología consiste en una búsqueda sensible en las bases de datos PubMed y Trip Database, limitándose la búsqueda a artículos en inglés y español hasta enero 2019. Se priorizó por ensayos clínicos, artículos originales y revisiones sistemáticas, a partir de los cuales se identificaron otras investigaciones relevantes.

A Network Involving Gut Microbiota, Circulating Bile Acids, and Hepatic Metabolism Genes That Protects Against Non-Alcoholic Fatty Liver Disease.

SCOPE: Gut microbiota contributes to non-alcoholic fatty liver disease (NAFLD) pathogenesis by multiple mechanisms not yet completely understood. Novel differential features between germ-free mice (GFm) transplanted with protective or non-protective cecal microbiota against NAFLD are investigated. METHODS AND RESULTS: Gut microbiota composition, plasma, and fecal bile acids (BAs) and liver mRNAs are quantified in GFm recipients from four donor mice differing in NAFLD severity (control diet, high-fat diet [HFD]-responder, HFD-non-responder, and quercetin-supplemented HFD). Transplanted GFm are on control or HFD for 16-weeks. Multivariate analysis shows that GFm colonized with microbiota from HFD-non-responder and quercetin supplemented-HFD donors (protected against NAFLD) clusters together, whereas GFm colonized with microbiota from control and HFD-responder mice (non-protected against NAFLD) establishes another cluster. Protected phenotype is associated with increased gut Desulfovibrio and Oscillospira, reduced gut Bacteroides and Oribacterium, lower primary and higher secondary BAs in plasma and feces, induction of hepatic BA transporters, and repression of hepatic lipogenic and BA synthesis genes. CONCLUSION: Protective gut microbiota associates with increased specific secondary BAs, which likely inhibit lipogenic pathways and enhance bile flow in the liver. This novel cross-talk between gut and liver, via plasma BAs, that promotes protection against NAFLD may have clinical and nutritional relevance.

Angiopoietin-Like Protein 8 Is a Novel Vitamin D Receptor Target Gene Involved in Nonalcoholic Fatty Liver Pathogenesis.

Hepatic vitamin D receptor (VDR) expression is increased in patients with nonalcoholic fatty liver (NAFL) and is required for liver steatosis in an NAFL mouse model. However, how hepatocyte VDR is involved in setting up steatosis remains unclear. The authors transduced human hepatocyte-derived cells with an adenoviral vector encoding human VDR and found that angiopoietin-like protein 8 (ANGPTL8) expression was increased upon VDR activation by vitamin D or lithocholic acid. The mRNA levels of hepatic VDR- and vitamin D-related genes [cytochrome P450 (CYP) 2R1, CYP27A1, and CYP3A4] were higher in NAFL patients compared with normal liver subjects. Noteworthy, hepatic ANGPTL8 mRNA and protein levels were elevated in NAFL patients, and its mRNA correlated with VDR mRNA and with the steatosis grade. Moreover, increases in serum conjugated bile acids, including the VDR agonist glycine-lithocholic acid, were observed in NAFL patients. Additionally, free fatty acids and insulin were able to up-regulate both VDR and ANGPTL8 mRNA in human hepatocytes, whereas ANGPTL8 gene knockdown attenuated free fatty acids-induced triglyceride accumulation in these cells. In conclusion, activated VDR up-regulates ANGPTL8 expression, contributing to triglyceride accumulation in human hepatocytes. Moreover, hepatic ANGPTL8 mRNA positively correlates with VDR mRNA content and the grade of steatosis in NAFL patients, suggesting that this novel pathway may play a key role in the pathogenesis of hepatosteatosis.

Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis.

BACKGROUND & AIMS: The pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD) is still incompletely understood. Several nuclear receptors play a role in liver lipid metabolism and can promote hepatosteatosis, but the possible role of vitamin D receptor (VDR) in NAFLD has not been investigated. METHODS: The expression of liver VDR was investigated in apolipoprotein E knockout (apoE(-/-)) mice on a high fat diet, in wild-type mice on methionine and choline deficient diet and in NAFLD patients with hepatosteatosis and non-alcoholic steatohepatitis. The relevance of VDR was assessed in apoE(-/-) mice by deletion of VDR or paricalcitol treatment and in human HepG2 cells by VDR transfection or silencing. The role of VDR in fibrosis was also determined in VDR knockout mice (VDR(-/-)) treated with thioacetamide. RESULTS: Expression of liver VDR was markedly induced in two mouse models of NAFLD, as well as in patients with hepatosteatosis, but decreased in non-alcoholic steatohepatitis. VDR deletion in high fat diet-fed apoE(-/-) mice protected against fatty liver, dyslipidemia and insulin resistance, and caused a decrease in taurine-conjugated bile acids, but did not influence fibrosis by thioacetamide. apoE(-/-)VDR(-/-) mouse livers showed decreased gene expression of CD36, DGAT2, C/EBPα and FGF21, and increased expression of PNPLA2, LIPIN1 and PGC1α. Treatment of apoE(-/-) mice on high fat diet with paricalcitol had modest opposite effects on steatosis and gene expression. Finally, this set of genes showed concordant responses when VDR was overexpressed or silenced in HepG2 cells. CONCLUSIONS: Induced hepatocyte VDR in NAFLD regulates key hepatic lipid metabolism genes and promotes high fat diet-associated liver steatosis. Therapeutic inhibition of liver VDR may reverse steatosis in early NAFLD. LAY SUMMARY: The amount of vitamin D receptor is induced early in the livers of mice and humans when they develop non-alcoholic fatty liver disease. If the gene for the vitamin D receptor is deleted, hepatic lipid metabolism changes and mice do not accumulate fat in the liver. We conclude that the vitamin D receptor can contribute to the fatty liver disease promoted by a high fat diet.

Human Upcyte Hepatocytes: Characterization of the Hepatic Phenotype and Evaluation for Acute and Long-Term Hepatotoxicity Routine Testing.

The capacity of human hepatic cell-based models to predict hepatotoxicity depends on the functional performance of cells. The major limitations of human hepatocytes include the scarce availability and rapid loss of the hepatic phenotype. Hepatoma cells are readily available and easy to handle, but are metabolically poor compared with hepatocytes. Recently developed human upcyte hepatocytes offer the advantage of combining many features of primary hepatocytes with the unlimited availability of hepatoma cells. We analyzed the phenotype of upcyte hepatocytes comparatively with HepG2 cells and adult primary human hepatocytes to characterize their functional features as a differentiated hepatic cell model. The transcriptomic analysis of liver characteristic genes confirmed that the upcyte hepatocytes expression profile comes closer to human hepatocytes than HepG2 cells. CYP activities were measurable and showed a similar response to prototypical CYP inducers than primary human hepatocytes. Upcyte hepatocytes also retained conjugating activities and key hepatic functions, e.g. albumin, urea, lipid and glycogen synthesis, at levels close to hepatocytes. We also investigated the suitability of this cell model for preclinical hepatotoxicity risk assessments using multiparametric high-content screening, as well as transcriptomics and targeted metabolomic analysis. Compounds with well-documented in vivo hepatotoxicity were screened after acute and repeated doses up to 1 week. The evaluation of complex mechanisms of cell toxicity, drug-induced steatosis and oxidative stress biomarkers demonstrated that, by combining the phenotype of primary human hepatocytes and the ease of handling of HepG2 cells, upcyte hepatocytes offer suitable properties to be potentially used for toxicological assessments during drug development.

Repression of the nuclear receptor small heterodimer partner by steatotic drugs and in advanced nonalcoholic fatty liver disease.

The small heterodimer partner (SHP) (NR0B2) is an atypical nuclear receptor that lacks a DNA-binding domain. It interacts with and inhibits many transcription factors, affecting key metabolic processes, including bile acid, cholesterol, fatty acid, and drug metabolism. Our aim was to determine the influence of steatotic drugs and nonalcoholic fatty liver disease (NAFLD) on SHP expression and investigate the potential mechanisms. SHP was found to be repressed by steatotic drugs (valproate, doxycycline, tetracycline, and cyclosporin A) in cultured hepatic cells and the livers of different animal models of NAFLD: iatrogenic (tetracycline-treated rats), genetic (glycine N-methyltransferase-deficient mice), and nutritional (mice fed a methionine- and choline-deficient diet). Among the different transcription factors investigated, CCAAT-enhancer-binding protein α (C/EBPα) showed the strongest dominant-repressive effect on SHP expression in HepG2 and human hepatocytes. Reporter assays revealed that the inhibitory effect of C/EBPα and steatotic drugs colocalize between -340 and -509 base pair of the SHP promoter, and mutation of a predicted C/EBPα response element at -473 base pair abolished SHP repression by both C/EBPα and drugs. Moreover, inhibition of major stress signaling pathways demonstrated that the mitogen-activated protein kinase kinase 1/2 pathway activates, while the phosphatidylinositol 3 kinase pathway represses SHP in a C/EBP-dependent manner. We conclude that SHP is downregulated by several steatotic drugs and in advanced NAFLD. These conditions can activate signals that target C/EBPα and consequently repress SHP, thus favoring the progression and severity of NAFLD.

A simple transcriptomic signature able to predict drug-induced hepatic steatosis.

It is estimated that only a few marketed drugs are able to directly induce liver steatosis. However, many other drugs may exacerbate or precipitate fatty liver in the presence of other risk factors or in patients prone to non-alcoholic fatty liver disease. On the other hand, current in vitro tests for drug-induced steatosis in preclinical research are scarce and not very sensitive or reproducible. In the present study, we have investigated the effect of well-characterized steatotic drugs on the expression profile of 47 transcription factors (TFs) in human hepatoma HepG2 cells and found that these drugs are able to up- and down-regulate a substantial number of these factors. Multivariate data analysis revealed a common TF signature for steatotic drugs, which consistently and significantly repressed FOXA1, HEX and SREBP1C in cultured cells. This signature was also observed in the livers of rats and in cultured human hepatocytes. Therefore, we selected these three TFs as predictive biomarkers for iatrogenic steatosis. With these biomarkers, a logistic regression analysis yielded a predictive model, which was able to correctly classify 92 % of drugs. The developed algorithm also predicted that ibuprofen, nifedipine and irinotecan are potential steatotic drugs, whereas troglitazone is not. In summary, this is a sensitive, specific and simple RT-PCR test that can be easily implemented in preclinical drug development to predict drug-induced steatosis. Our results also indicate that steatotic drugs affect expression of both common and specific subsets of TF and lipid metabolism genes, thus generating complex transcriptomic responses that cause or contribute to steatosis in hepatocytes.

The human liver fatty acid binding protein (FABP1) gene is activated by FOXA1 and PPARα; and repressed by C/EBPα: Implications in FABP1 down-regulation in nonalcoholic fatty liver disease.

Liver fatty acid binding protein (FABP1) prevents lipotoxicity of free fatty acids and regulates fatty acid trafficking and partition. Our objective is to investigate the transcription factors controlling the human FABP1 gene and their regulation in nonalcoholic fatty liver disease (NAFLD). Adenovirus-mediated expression of multiple transcription factors in HepG2 cells and cultured human hepatocytes demonstrated that FOXA1 and PPARα are among the most effective activators of human FABP1, whereas C/EBPα is a major dominant repressor. Moreover, FOXA1 and PPARα induced re-distribution of FABP1 protein and increased cytoplasmic expression. Reporter assays demonstrated that the major basal activity of the human FABP1 promoter locates between -96 and -229bp, where C/EBPα binds to a composite DR1-C/EBP element. Mutation of this element at -123bp diminished basal reporter activity, abolished repression by C/EBPα and reduced transactivation by HNF4α. Moreover, HNF4α gene silencing by shRNA in HepG2 cells caused a significant down-regulation of FABP1 mRNA expression. FOXA1 activated the FABP1 promoter through binding to a cluster of elements between -229 and -592bp, whereas PPARα operated through a conserved proximal element at -59bp. Finally, FABP1, FOXA1 and PPARα were concomitantly repressed in animal models of NAFLD and in human nonalcoholic fatty livers, whereas C/EBPα was induced or did not change. We conclude that human FABP1 has a complex mechanism of regulation where C/EBPα displaces HNF4α and hampers activation by FOXA1 and PPARα. Alteration of expression of these transcription factors in NAFLD leads to FABP1 gen repression and could exacerbate lipotoxicity and disease progression.

Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver.

Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid ß-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha) synergistically cooperate with constitutive androstane receptor to transactivate the human cytochrome P450 2B6 (CYP2B6) gene: application to the development of a metabolically competent human hepatic cell model.

The transcription of tissue-specific and inducible genes is usually subject to the dynamic control of multiple activators. Dedifferentiated hepatic cell lines lose the expression of tissue-specific activators and many characteristic hepatic genes, such as drug-metabolizing cytochrome P450. Here we demonstrate that by combining adenoviral vectors for CCAAT/enhancer-binding protein alpha (C/EBPalpha), hepatocyte nuclear factor 4alpha (HNF4alpha), and constitutive androstane receptor, the CYP2B6 expression and inducibility by CITCO are restored in human hepatoma HepG2 cells at levels similar to those in cultured human hepatocytes. Moreover, several other phase I and II genes are simultaneously activated, which suggests that this is an effective approach to endow dedifferentiated human hepatoma cells with a particular metabolic competence and response to inducers. In order to gain insight into the molecular mechanism, we examined the cooperation of these three transcription factors on the CYP2B6 5'-flanking region. We show new CYP2B6-responsive sequences for C/EBPalpha and HNF4alpha and a novel synergistic regulatory mechanism whereby C/EBPalpha, HNF4alpha, and constitutive androstane receptor bind and cooperate through proximal and distal response elements to confer a maximal level of expression. The results obtained from human liver also suggest that important differences in the expression and binding of C/EBPalpha and HNF4alpha could account for the large interindividual variability of the hepatic CYP2B6 enzyme, which metabolizes commonly used drugs.