Objective We evaluated the influence of sex on the pathophysiology of non-alcoholic fatty liver disease (NAFLD). We investigated diet-induced phenotypic responses to define sex-specific regulation between healthy liver and NAFLD to identify influential pathways in different preclinical murine models and their relevance in humans. Design Different models of diet-induced NAFLD (high-fat diet, choline-deficient high-fat diet, Western diet or Western diet supplemented with fructose and glucose in drinking water) were compared with a control diet in male and female mice. We performed metabolic phenotyping, including plasma biochemistry and liver histology, untargeted large-scale approaches (liver metabolome, lipidome and transcriptome), gene expression profiling and network analysis to identify sex-specific pathways in the mouse liver. Results The different diets induced sex-specific responses that illustrated an increased susceptibility to NAFLD in male mice. The most severe lipid accumulation and inflammation/fibrosis occurred in males receiving the high-fat diet and Western diet, respectively. Sex-biased hepatic gene signatures were identified for these different dietary challenges. The peroxisome proliferator-activated receptor α (PPARα) co-expression network was identified as sexually dimorphic, and in vivo experiments in mice demonstrated that hepatocyte PPARα determines a sex-specific response to fasting and treatment with pemafibrate, a selective PPARα agonist. Liver molecular signatures in humans also provided evidence of sexually dimorphic gene expression profiles and the sex-specific co-expression network for PPARα. Conclusions These findings underscore the sex specificity of NAFLD pathophysiology in preclinical studies and identify PPARα as a pivotal, sexually dimorphic, pharmacological target. Trial registration number NCT02390232 .

Liver physiology is circadian and sensitive to feeding and insulin. Food intake regulates insulin secretion and is a dominant signal for the liver clock. However, how much insulin contributes to the effect of feeding on the liver clock and rhythmic gene expression remains to be investigated. Insulin action partly depends on changes in insulin receptor (IR)-dependent gene expression. Here, we use hepatocyte-restricted gene deletion of IR to evaluate its role in the regulation and oscillation of gene expression as well as in the programming of the circadian clock in the adult mouse liver. We find that, in the absence of IR, the rhythmicity of core-clock gene expression is altered in response to day-restricted feeding. This change in core-clock gene expression is associated with defective reprogramming of liver gene expression. Our data show that an intact hepatocyte insulin receptor is required to program the liver clock and associated rhythmic gene expression.

Background: We recently demonstrated that chronic dietary exposure to a mixture of pesticides at low-doses induced sexually dimorphic obesogenic and diabetogenic effects in adult mice. Perinatal pesticide exposure may also be a factor in metabolic disease etiology. However, the long-term consequences of perinatal pesticide exposure remain controversial and largely unexplored. Objectives: Here we assessed how perinatal exposure to the same low-dose pesticide cocktail impacted metabolic homeostasis in adult mice. Methods: Six pesticides (boscalid, captan, chlopyrifos, thiachloprid, thiophanate, and ziram) were incorporated in food pellets. During the gestation and lactation periods, female (F0) mice were fed either a pesticide-free or a pesticide-enriched diet at doses exposing them to the tolerable daily intake (TDI) level for each compound, using a 1:1 body weight scaling from humans to mice. All male and female offsprings (F1) were then fed the pesticide-free diet until 18 weeks of age, followed by challenge with a pesticide-free high-fat diet (HFD) for 6 weeks. Metabolic parameters, including body weight, food and water consumption, glucose tolerance, and urinary and fecal metabolomes, were assessed over time. At the end of the experiment, we evaluated energetic metabolism and microbiota activity using biochemical assays, gene expression profiling, and H-1 NMR-based metabolomics in the liver, urine, and feces. Results: Perinatal pesticide exposure did not affect body weight or energy homeostasis in 6- and 14-week-old mice. As expected, HFD increased body weight and induced metabolic disorders as compared to a low-fat diet. However, HFD-induced metabolic perturbations were similar between mice with and without perinatal pesticide exposure. Interestingly, perinatal pesticide exposure induced time-specific and sex-specific alterations in the urinary and fecal metabolomes of adult mice, suggesting long-lasting changes in gut microbiota. Conclusions: Perinatal pesticide exposure induced sustained sexually dimorphic perturbations of the urinary and fecal metabolic fingerprints, but did not significantly influence the development of HFD-induced metabolic diseases.

Metabolomics has found numerous applications in the study of liver metabolism in health and disease. Metabolomics studies can be conducted in a variety of biological matrices ranging from easily accessible biofluids such as urine, blood or feces, to organs, tissues or even cells. Sample collection and storage are critical steps for which standard operating procedures must be followed. Inappropriate sample collection or storage can indeed result in high variability, interferences with instrumentation or degradation of metabolites. In this review, we will first highlight important general factors that should be considered when planning sample collection in the study design of metabolomic studies, such as nutritional status and circadian rhythm. Then, we will discuss in more detail the specific procedures that have been described for optimal pre-analytical handling of the most commonly used matrices (urine, blood, feces, tissues and cells).

The pregnane X receptor (PXR) is the main nuclear receptor regulating the expression of xenobiotic-metabolizing enzymes and is highly expressed in the liver and intestine. Recent studies have highlighted its additional role in lipid homeostasis, however, the mechanisms of these regulations are not fully elucidated. We investigated the transcriptomic signature of PXR activation in the liver of adult wild-type vs. Pxr-/- C57Bl6/J male mice treated with the rodent specific ligand pregnenolone 16α-carbonitrile (PCN). PXR activation increased liver triglyceride accumulation and significantly regulated the expression of 1215 genes, mostly xenobiotic-metabolizing enzymes. Among the down-regulated genes, we identified a strong peroxisome proliferator-activated receptor α (PPARα) signature. Comparison of this signature with a list of fasting-induced PPARα target genes confirmed that PXR activation decreased the expression of more than 25 PPARα target genes, among which was the hepatokine fibroblast growth factor 21 (Fgf21). PXR activation abolished plasmatic levels of FGF21. We provide a comprehensive signature of PXR activation in the liver and identify new PXR target genes that might be involved in the steatogenic effect of PXR. Moreover, we show that PXR activation down-regulates hepatic PPARα activity and FGF21 circulation, which could participate in the pleiotropic role of PXR in energy homeostasis.

Current fructose consumption levels often overwhelm the intestinal capacity to absorb fructose. We investigated the impact of fructose malabsorption on intestinal endocrine function and addressed the role of the microbiota in this process. To answer this question, a mouse model of moderate fructose malabsorption [ketohexokinase mutant (KHK)-/-] and wild-type (WT) littermate mice were used and received a 20%-fructose (KHK-F and WT-F) or 20%-glucose diet. Cholecystokinin (Cck) mRNA and protein expression in the ileum and cecum, as well as preproglucagon (Gcg) and neurotensin (Nts) mRNA expression in the cecum, increased in KHK-F mice. In KHK-F mice, triple-label immunohistochemistry showed major up-regulation of CCK in enteroendocrine cells (EECs) that were glucagon-like peptide-1 (GLP-1)+/Peptide YY (PYY-) in the ileum and colon and GLP-1-/PYY- in the cecum. The cecal microbiota composition was drastically modified in the KHK-F in association with an increase in glucose, propionate, succinate, and lactate concentrations. Antibiotic treatment abolished fructose malabsorption-dependent induction of cecal Cck mRNA expression and, in mouse GLUTag and human NCI-H716 cells, Cck mRNA expression levels increased in response to propionate, both suggesting a microbiota-dependent process. Fructose reaching the lower intestine can modify the composition and metabolism of the microbiota, thereby stimulating the production of CCK from the EECs possibly in response to propionate.-Zhang, X., Grosfeld, A., Williams, E., Vasiliauskas, D., Barretto, S., Smith, L., Mariadassou, M., Philippe, C., Devime, F., Melchior, C., Gourcerol, G., Dourmap, N., Lapaque, N., Larraufie, P., Blottière, H. M., Herberden, C., Gerard, P., Rehfeld, J. F., Ferraris, R. P., Fritton, J. C., Ellero-Simatos, S., Douard, V. Fructose malabsorption induces cholecystokinin expression in the ileum and cecum by changing microbiota composition and metabolism.