Genetically-modified mouse models have greatly contributed to our understanding of the physiological functions of FXR. However, the presence of mouse-specific pathways in bile acid metabolism, leading to the formation of very hydrophilic muricholic acids with FXR antagonistic rather than agonistic actions, compromizes the use of mouse models for translational studies in the FXR field. Using CRISPR/Cas9-mediated somatic gene editing, we silenced the Cyp2c70 gene specifically in hepatocytes, resulting in a novel mouse model with a human-like bile acid metabolism that overcomes these drawbacks while still allowing methods developed in our laboratory for in vivo evaluation of NAFLD/NASH development.
To silence Cyp2c70 in liver, young and adult mice expressing Cas9 specifically in hepatocytes will be injected with adenovirus containing sgRNA targeting the Cyp2c70gene. Three weeks after virus injection the bile acid composition will analyzed by LC-MS to evaluate effectivity of the procedure. In subgroups of mice, hepatic Fxr and Cyp2c70will be deleted simultaneously. Mice will be fed chow diet or high-fat/high-fat + cholesterol (to induce inflammation)/fructose (to induce lipogenesis) diets up to 12 weeks to induce varying degrees of NAFLD/NASH.
At specific points in time, relevant physiological pathways will be quantified by stable isotope-based methods established in our laboratory (lipogenesis, lipolysis, beta-oxidation, cholesterogenesis and turnover, bile acid synthesis, glucose metabolism). Upon termination, liver tissue will be harvested for histological evaluation (with prof Alain de Bruin, Utrecht/UMCG), as well as for gene expression and lipid analyses. Simultaneously, other relevant organs (intestine, fat depots) will be harvested for evaluation.
Chow-fed mice will, upon deletion of Cyp2c70, be treated with available (GW6046, PX-20606) and/or novel (collaboration industrial partners) FXR agonist for periods up to 2 weeks to establish their effects on bile formation, bile acid metabolism and cholesterol turnover, as described (De Boer et al. Gastroenterology, 2017).
Next, the metabolic effects of FXR agonists will be evaluated in ‘humanized mice’ with diet-induced NAFLD, chosen on the basis of outcome Aim 1.