Over the past decade, it has become apparent that bile acids are involved in a host of activities beyond their classic functions in bile formation and fat absorption. The identification of the farnesoid X receptor (FXR) as a nuclear receptor directly activated by bile acids has opened new avenues of research.
FXR regulates various elements of glucose, lipid and energy metabolism. Consequently, a picture has emerged of bile acids acting as modulators of (postprandial) metabolism. Therefore, strategies that interfere with either bile acid metabolism or signaling cascades mediated by bile acids represent novel therapeutic approaches for metabolic diseases, in addition to more evident applications in (cholestatic) liver disease. Synthetic modulators of FXR have been designed and the first, i.e., obeticholic acid (OCALIVA) has been FDA-approved in 2016 for the treatment of the rare cholestatic liver disease Primary Biliary Cholangitis.
Disorders associated with disrupted nutrient/energy homeostasis, e.g., obesity, metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD), are increasing worldwide. Susceptibility to develop these diseases may originate from early life (programming). In NAFLD, a progressive subtype exists, designated as non-alcoholic steatohepatitis (NASH), that is recognized as major cause liver cirrhosis and hepatocellular carcinoma. Disturbed signaling of FXR appears to contribute to the pathogenesis of NAFLD. Standard therapeutic interventions have not been established for NAFLD, but some new agents that activate FXR signaling have shown promise as possible therapeutics. Yet, many steps, involving mechanistic studies in relevant animal models, are still required for tailoring pharmacotherapy to the dominant pathogenic pathways in a given patient, possibly with use of combination therapy.
The current project will contribute to the future direction in (personalized) treatment of patients with NASH, through application of a newly developed mouse model with humanized bile acid metabolism to allow rapid and more accurate translation to the human situation.
Evaluation of novel FXR modulators in mice for potential application in humans is hampered by the presence of mouse-specific pathways in bile acid metabolism. We have recently generated a mouse model with humanized bile acid metabolism.
Overall aims:
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.
Obesity, metabolic syndrome, and non-alcoholic fatty liver disease (NAFLD) are increasing worldwide. Standard therapeutic interventions have not been established for NAFLD and its sequel NASH. Apart from genetic components, environmental factors – e.g., by exposure during early life - such as dietary intake, diet composition but also composition of the microbiome have been implicated in the etiologies of NAFLD/NASH. Specific metabolic and/or inflammatory pathways may be involved in development and progress of the disease, hence, a one-size-fits-all preventive/therapeutic approach is deemed to be unsuccessful. New agents that activate FXR signaling have shown promise, yet, are likely to be more effective in combination with dietary recommendations and/or other pharmacological means (e.g., inhibitors of lipogenesis, bile acid sequestrants) depending on the individual’s specific features. This project will contribute to our understanding of the etiology of (humanized) NAFLD/NASH and may contribute identification of biomarkers to allow personalized tailoring of therapies for NAFLD/NASH in humans.
