Marta Benet

The use of stable isotope tracers and mass spectrometry (MS) is the gold standard method for the analysis of fatty acid (FA) metabolism. Yet, current state-of-the-art tools provide limited and difficult-to-interpret information about FA biosynthetic routes. Here we present FAMetA, an R package and a web-based application (www.fameta.es) that uses ¹³C mass isotopologue profiles to estimate FA import, de novo lipogenesis, elongation and desaturation in a user-friendly platform. The FAMetA workflow covers the required functionalities needed for MS data analyses. To illustrate its utility, different in vitro and in vivo experimental settings are used in which FA metabolism is modified. Thanks to the comprehensive characterization of FA biosynthesis and the easy-to-interpret graphical representations compared to previous tools, FAMetA discloses unnoticed insights into how cells reprogram their FA metabolism and, when combined with FASN, SCD1 and FADS2 inhibitors, it enables the identification of new FAs by the metabolic reconstruction of their synthesis route.

The use of stable isotope tracers and mass spectrometry (MS) is the gold standard method for the analysis of fatty acids (FAs) metabolism. Yet current state-of-the-art tools provide limited and difficult to interpret information about FA biosynthetic routes. Here we present FAMetA, an R-package and a web-based application ( www.fameta.es ) that use ¹³ C mass-isotopologue profiles to estimate FA import, de novo lipogenesis, elongation, and desaturation in a user-friendly platform. The FAMetA workflow covers all the functionalities needed for MS data analyses. To illustrate its utility, different in vitro and in vivo experimental settings are used in which FA metabolism is modified. Thanks to the comprehensive characterisation of FA biosynthesis and the easy-to-interpret graphical representations compared to previous tools, FAMetA discloses unnoticed insights into how cells reprogramme their FA metabolism and, when combined with FASN, SCD1 and FADS2 inhibitors, it enables the straightforward identification of new FAs by the metabolic reconstruction of their synthesis route.

The small heterodimer partner (SHP, NR0B2) is an atypical nuclear receptor that lacks 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 non-alcoholic fatty liver disease (NAFLD) on SHP expression, and to investigate the potential mechanisms. SHP was found repressed by steatotic drugs (valproate, doxycycline, tetracycline and cyclosporin A) in cultured hepatic cells, and in 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 co-localize between -340 and -509 bp of the SHP promoter and mutation of a predicted C/EBPα response element at -473 bp 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.

Scope: Flavonoids and related compounds seem to have favorable effects on nonalcoholic fatty liver disease (NAFLD) progression, although the exact mechanisms implicated are poorly understood. In this study, we aimed to investigate the effect of the flanovol quercetin on gene expression deregulation involved in the development of NAFLD, as well as the possible implication of phosphatidylinositol 3‐kinase (PI3K)/AKT pathway modulation.Methods and results: We used an in vivo model based on methionine‐ and choline‐deficient (MCD) diet‐fed mice and an in vitro model consisting of Huh7 cells incubated with MCD medium. MCD‐fed mice showed classical pathophysiological characteristics of nonalcoholic steatohepatitis, associated with altered transcriptional regulation of fatty acid uptake‐ and trafficking‐related gene expression, with increased lipoperoxidation. PI3K/AKT pathway was activated by MCD and triggered gene deregulation causing either activation or inhibition of all studied genes as demonstrated through cell incubation with the PI3K‐inhibitor LY294002. Treatment with quercetin reduced AKT phosphorylation, and oxidative/nitrosative stress, inflammation and lipid metabolism‐related genes displayed a tendency to normalize in both in vivo and in vitro models.Conclusion: These results place quercetin as a potential therapeutic strategy for preventing NAFLD progression by attenuating gene expression deregulation, at least in part through PI3K/AKT pathway inactivation.

Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infecting primary cultured hepatocytes. This recombinant adenovirus induces robust expression of the protein of interest in hepatocytes within a wide range of doses.

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.