In vivo mouse models to study bile acid synthesis and signaling.

The synthesis of bile acids (BAs) is carried out by complex pathways characterized by sequential chemical reactions in the liver through various cytochromes P450 (CYP) and other enzymes. Maintaining the integrity of these pathways is crucial for normal physiological function in mammals, encompassing hepatic and neurological processes. Studying on the deficiencies in BA synthesis genes offers valuable insights into the significance of BAs in modulating farnesoid X receptor (FXR) signaling and metabolic homeostasis. By creating mouse knockout (KO) models, researchers can manipulate deficiencies in genes involved in BA synthesis, which can be used to study human diseases with BA dysregulation. These KO mouse models allow for a more profound understanding of the functions and regulations of genes responsible for BA synthesis. Furthermore, KO mouse models shed light on the distinct characteristics of individual BA and their roles in nuclear receptor signaling. Notably, alterations of BA synthesis genes in mouse models have distinct differences when compared to human diseases caused by the same BA synthesis gene deficiencies. This review summarizes several mouse KO models used to study BA synthesis and related human diseases, including mice deficient in Cyp7a1, Cyp27a1, Cyp7a1/Cyp27a1, Cyp8b1, Cyp7b1, Cyp2c70, Cyp2a12, and Cyp2c70/Cyp2a12, as well as germ-free mice.

Environmental Chemical Contribution to the Modulation of Bile Acid Homeostasis and Farnesoid X Receptor Signaling.

Maintaining bile acid (BA) homeostasis is important and regulated by BA activated receptors and signaling pathways. Farnesoid X receptor (FXR) and its regulated target networks in both the liver and the intestines are critical in suppressing BA synthesis and promoting BA transport and enterohepatic circulation. In addition, FXR is critical in regulating lipid metabolism and reducing inflammation, processes critical in the development of cholestasis and fatty liver diseases. BAs are modulated by, but also control, gut microflora. Environmental chemical exposure could affect liver disease development. However, the effects and the mechanisms by which environmental chemicals interact with FXR to affect BA homeostasis are only emerging. In this minireview, our focus is to provide evidence from reports that determine the effects of environmental or therapeutic exposure on altering homeostasis and functions of BAs and FXR. Understanding these effects will help to determine liver disease pathogenesis and provide better prevention and treatment in the future. SIGNIFICANCE STATEMENT: Environmental chemical exposure significantly contributes to the development of cholestasis and nonalcoholic steatohepatitis (NASH). The impact of exposures on bile acid (BA) signaling and Farnesoid X receptor-mediated gut-liver crosstalk is emerging. However, there is still a huge gap in understanding how these chemicals contribute to the dysregulation of BA homeostasis and how this dysregulation may promote NASH development.

Characterization of individual bile acids in vivo utilizing a novel low bile acid mouse model.

Bile acids (BAs) are signaling molecules synthesized in the liver initially by CYP7A1 and CYP27A1 in the classical and alternative pathways, respectively. BAs are essential for cholesterol clearance, intestinal absorption of lipids, and endogenous modulators of farnesoid x receptor (FXR). FXR is critical in maintaining BA homeostasis and gut-liver crosstalk. Complex reactions in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs. In this study, we characterized the in vivo effects of three-day feeding of cholic acid (CA), deoxycholic acid (DCA), or ursodeoxycholic acid (UDCA) at physiological/non-hepatotoxic concentrations in a novel low-BA mouse model (Cyp7a1-/-/Cyp27a1-/-, DKO). Liver injury, BA levels and composition and BA signaling by the FXR-fibroblast growth factor 15 (FGF15) axis were determined. Overall, higher basal inflammation and altered lipid metabolism in DKO mice might be associated with low BAs. CA, DCA, and UDCA feeding activated FXR signals with tissue specificity. Dietary CA and DCA similarly altered tissue BA profiles to be less hydrophobic, while UDCA promoted a more hydrophobic tissue BA pool with the profiles shifted toward non-12α-OH BAs and secondary BAs. However, UDCA did not offer any overt protective effects as expected. These findings allow us to determine the precise effects of individual BAs in vivo on BA-FXR signaling and overall BA homeostasis in liver physiology and pathologies.

Effects of Therapeutically Approved Individual Bile Acids on The Development of Metabolic Dysfunction-Associated Steatohepatitis a Low Bile Acid Mouse Model.

Bile acid (BA) signaling dysregulation is an important etiology for the development of Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD). As diverse signaling molecules synthesized in the liver by pathways initiated with CYP7A1 and CYP27A1, BAs are endogenous modulators of farnesoid x receptor (FXR). FXR activation is crucial in maintaining BA homeostasis, regulating lipid metabolism, and suppressing inflammation. Additionally, BAs interact with membrane receptors and gut microbiota to regulate energy expenditure and intestinal health. Complex modulation of BAs in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs, especially during MASLD development. Previously, we determined that acute feeding of individual BAs differentially affects lipid, inflammation, and oxidative stress pathways in a low-BA mouse model, Cyp7a1/Cyp27a1 double knockout (DKO) mice. Currently, we investigated to what degree that cholic acid (CA), deoxycholic acid (DCA) or ursodeoxycholic acid (UDCA) at physiological concentrations impact MASLD development in DKO mice. The results showed that these three BAs varied in ability to activate hepatic and intestinal FXR, disrupt lipid homeostasis, and modulate inflammation and fibrosis. Additionally, UDCA activated intestinal FXR in these low-BA mice. Significant alterations in lipid uptake and metabolism in DKO mice following CA and DCA feeding indicate differences in cholesterol and lipid handling across genotypes. Overall, the DKO were less susceptible to weight gain, but more susceptible to MASH diet induced inflammation and fibrosis on CA and DCA supplement, while WT mice were more vulnerable to CA-induced fibrosis on control diet.

Effects of intestine-specific deletion of FGF15 on the development of fatty liver disease with vertical sleeve gastrectomy.

BACKGROUND: Vertical sleeve gastrectomy (SGx) is a type of bariatric surgery to treat morbid obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). The molecular mechanisms of SGx to improve MASLD are unclear, but increased bile acids (BAs) and FGF19 (mouse FGF15) were observed. FGF15/19 is expressed in the ileum in response to BAs and is critical in not only suppressing BA synthesis in the liver but also promoting energy expenditure. We hypothesized the reduction of obesity and resolution of MASLD by SGx may be mediated by FGF15/19. METHODS: First, we conducted hepatic gene expression analysis in obese patients undergoing SGx, with the results showing increased expression of FGF19 in obese patients' livers. Next, we used wild-type and intestine-specific Fgf15 knockout mice (Fgf15ile-/-) to determine the effects of FGF15 deficiency on improving the metabolic effects. RESULTS: SGx improved metabolic endpoints in both genotypes, evidenced by decreased obesity, improved glucose tolerance, and reduced MASLD progression. However, Fgf15ile-/- mice showed better improvement compared to wild-type mice after SGx, suggesting that other mediators than FGF15 are also responsible for the beneficial effects of FGF15 deficiency. Further gene expression analysis in brown adipose tissue suggests increased thermogenesis. CONCLUSIONS: FGF15 deficiency, the larger BA pool and higher levels of secondary BAs may increase energy expenditure in extrahepatic tissues, which may be responsible for improved metabolic functions following SGx.

Myclobutanil-mediated alteration of liver-gut FXR signaling in mice.

The effects of exposure to Myclobutanil, a triazole fungicide, on the development and progression of nonalcoholic fatty liver disease (NAFLD) are unclear, but activation of nuclear receptors (NRs) is a known mechanism of azole-induced liver toxicity. Farnesoid X receptor (FXR) is a NR and is highly expressed in the liver and intestine. Activation of FXR tightly regulates bile acid (BA), lipid and glucose homeostasis, and inflammation partly through the induction of fibroblast growth factor 15 (FGF15; human ortholog FGF19). FXR activation is downregulated during NAFLD and agonists are currently being explored as potential therapeutic strategy. In this study, we aimed to clarify the effects of Myclobutanil exposure on FXR activation and NAFLD development. Reporter assay showed Myclobutanil treatment, following FXR activation with potent FXR agonist (GW4064), resulted in a dose-dependent decrease of FXR activity. Furthermore, a 10-day study in male mice demonstrated that cotreatment with Myclobutanil led to an 80% reduction of GW4064-induced ileal expression of Fgf15. In a diet-induced NAFLD study, low-fat diet (LFD) fed mice administered myclobutanil displayed decreased FXR activity in the liver and ileum, while high-fat-high-sugar-diet (HFHSD) fed mice showed an increase in hepatic FXR activity and an induction of target genes regulated by constitutive androstane receptor and/or pregnane X receptor. Our work demonstrates Myclobutanil inhibits FXR activity and modulates FXR activity differentially in mice fed LFD or HFHSD. Our studies suggest the importance of understanding how Myclobutanil could contribute to BA dysregulation in disease states such as NAFLD.

Bile Acid Profiling in Mouse Biofluids and Tissues.

Bile acids (BAs) serve as important signaling molecules and are endogenous ligands of nuclear and cell membrane receptors to regulate physiological and pathological processes. BA synthesis and metabolism have been impaired in NASH patients because of liver injury, inflammation or obstruction of bile ducts. On the other hand, the changes in BA composition might alter the activation status of various cell signaling pathways and contribute to NASH pathogenesis. Due to the rapidly increasing interests in the roles of individual BA in disease development, this chapter will focus on the method for analyzing individual BA profile in mouse biofluids and tissues by high-performance liquid chromatography coupled with ion trap mass spectrometry (HPLC-MS).

Bile acid homeostasis in female mice deficient in Cyp7a1 and Cyp27a1.

Bile acids (BAs) are amphipathic molecules important for metabolism of cholesterol, absorption of lipids and lipid soluble vitamins, bile flow, and regulation of gut microbiome. There are over 30 different BA species known to exist in humans and mice, which are endogenous modulators of at least 6 different membrane or nuclear receptors. This diversity of ligands and receptors play important roles in health and disease; however, the full functions of each individual BA in vivo remain unclear. We generated a mouse model lacking the initiating enzymes, CYP7A1 and CYP27A1, in the two main pathways of BA synthesis. Because females are more susceptible to BA related diseases, such as intrahepatic cholestasis of pregnancy, we expanded this model into female mice. The null mice of Cyp7a1 and Cyp27a1 were crossbred to create double knockout (DKO) mice. BA concentrations in female DKO mice had reductions in serum (63%), liver (83%), gallbladder (94%), and small intestine (85%), as compared to WT mice. Despite low BA levels, DKO mice had a similar expression pattern to that of WT mice for genes involved in BA regulation, synthesis, conjugation, and transport. Additionally, through treatment with a synthetic FXR agonist, GW4064, female DKO mice responded to FXR activation similarly to WT mice.