Gut microbiota depletion aggravates bile acid-induced liver pathology in mice with a human-like bile acid composition.

Cyp2c70-deficient mice have a human-like bile acid (BA) composition due to their inability to convert chenodeoxycholic acid (CDCA) into rodent-specific muricholic acids (MCAs). However, the hydrophobic BA composition in these animals is associated with liver pathology. Although Cyp2c70-ablation has been shown to alter gut microbiome composition, the impact of gut bacteria on liver pathology in Cyp2c70-/- mice remains to be established. Therefore, we treated young-adult male and female wild-type (WT) and Cyp2c70-/- mice with antibiotics (AB) with broad specificity to deplete the gut microbiota and assessed the consequences on BA metabolism and liver pathology. Female Cyp2c70-/- mice did not tolerate AB treatment, necessitating premature termination of the experiment. Male Cyp2c70-/- mice did tolerate AB but showed markedly augmented liver pathology after 6 weeks of treatment. Dramatic downregulation of hepatic Cyp8b1 expression (-99%) caused a reduction in the proportions of 12α-hydroxylated BAs in the circulating BA pools of AB-treated male Cyp2c70-/- mice. Interestingly, the resulting increased BA hydrophobicity strongly correlated with various indicators of liver pathology. Moreover, genetic inactivation of Cyp8b1 in livers of male Cyp2c70-/- mice increased liver pathology, while addition of ursodeoxycholic acid to the diet prevented weight loss and liver pathology in AB-treated female Cyp2c70-/- mice. In conclusion, depletion of gut microbiota in Cyp2c70-/- mice aggravates liver pathology at least in part by increasing the hydrophobicity of the circulating BA pool. These findings highlight that the potential implications of AB administration to cholestatic patients should be evaluated in a systematic manner.

Bile Acid Sequestration via Colesevelam Reduces Bile Acid Hydrophobicity and Improves Liver Pathology in Cyp2c70-/- Mice with a Human-like Bile Acid Composition.

Bile acids (BAs) and their signaling pathways have been identified as therapeutic targets for liver and metabolic diseases. We generated Cyp2c70-/- (KO) mice that were not able to convert chenodeoxycholic acid into rodent-specific muricholic acids (MCAs) and, hence, possessed a more hydrophobic, human-like BA pool. Recently, we have shown that KO mice display cholangiopathic features with the development of liver fibrosis. The aim of this study was to determine whether BA sequestration modulates liver pathology in Western type-diet (WTD)-fed KO mice. The BA sequestrant colesevelam was mixed into the WTD (2% w/w) of male Cyp2c70+/+ (WT) and KO mice and the effects were evaluated after 3 weeks of treatment. Colesevelam increased fecal BA excretion in WT and KO mice and reduced the hydrophobicity of biliary BAs in KO mice. Colesevelam ameliorated diet-induced hepatic steatosis in WT mice, whereas KO mice were resistant to diet-induced steatosis and BA sequestration had no additional effects on liver fat content. Total cholesterol concentrations in livers of colesevelam-treated WT and KO mice were significantly lower than those of untreated controls. Of particular note, colesevelam treatment normalized plasma levels of liver damage markers in KO mice and markedly decreased hepatic mRNA levels of fibrogenesis-related genes in KO mice. Lastly, colesevelam did not affect glucose excursions and insulin sensitivity in WT or KO mice. Our data show that BA sequestration ameliorates liver pathology in Cyp2c70-/- mice with a human-like bile acid composition without affecting insulin sensitivity.

Perinatal exposure to UDCA prevents neonatal cholestasis in Cyp2c70-/- mice with human-like bile acids.

BACKGROUND: Cyp2c70-/- mice with a human-like bile acid (BA) composition display features of neonatal cholestasis. We assessed whether perinatal ursodeoxycholic acid (UDCA) exposure prevents neonatal cholestasis in Cyp2c70-/- mice and reduces cholangiopathy development later in life. METHODS: Cyp2c70+/- males were crossed with Cyp2c70+/- females fed either a regular chow diet or a 0.1% UDCA-containing diet during breeding, gestation, and suckling. Cholestasis and liver function parameters were assessed in their Cyp2c70-/- and wild-type offspring at 3 and 8 weeks of age. RESULTS: Three-week-old Cyp2c70-/- pups showed features of neonatal cholestasis, including elevated plasma BAs and transaminases, which were completely prevented in Cyp2c70-/- pups upon perinatal UDCA exposure. In addition, UDCA administration to the dams corrected altered hepatic gene expression patterns in Cyp2c70-/- pups, reduced markers of fibrogenesis and inflammation, and prevented cholangiocyte proliferation. Yet, these beneficial effects of perinatal UDCA exposure were not retained into adulthood upon discontinuation of treatment. CONCLUSION: Perinatal exposure of Cyp2c70-/- mice to UDCA has beneficial effects on liver function parameters, supporting a direct role of BA hydrophobicity in the development of neonatal cholestasis in these mice. However, prevention of neonatal cholestasis in Cyp2c70-/- mice has no long-lasting effects on liver pathophysiology. IMPACT: This is the first study showing that perinatal UDCA exposure prevents features of neonatal cholestasis that are observed in mice with a human-like bile acid composition, i.e., Cyp2c70-/- mice. Perinatal UDCA exposure of Cyp2c70-/- pups leads to UDCA enrichment in their circulating bile acid pool and, consequently, to a reduced hydrophobicity of biliary bile acids. Perinatal UDCA exposure of Cyp2c70-/- pups has no long-lasting effects on the development of cholangiopathy after discontinuation of treatment. The results in this study expand current knowledge regarding acute and long-lasting effects of UDCA treatment in early life.

Low production of 12α-hydroxylated bile acids prevents hepatic steatosis in Cyp2c70-/- mice by reducing fat absorption.

Bile acids (BAs) play important roles in lipid homeostasis, and BA signaling pathways serve as therapeutic targets for nonalcoholic fatty liver disease (NAFLD). Recently, we generated cytochrome P450, family 2, subfamily C, polypeptide 70 (Cyp2c70-/-) mice with a human-like BA composition lacking mouse-/rat-specific muricholic acids to accelerate translation from mice to humans. We employed this model to assess the consequences of a human-like BA pool on diet-induced obesity and NAFLD development. Male and female Cyp2c70-/- mice and WT littermates were challenged with a 12-week high-fat Western-type diet (WTD) supplemented with 0.25% cholesterol. Cyp2c70 deficiency induced a hydrophobic BA pool with high abundances of chenodeoxycholic acid, particularly in females, because of sex-dependent suppression of sterol 12α-hydroxylase (Cyp8b1). Plasma transaminases were elevated, and hepatic fibrosis was present in Cyp2c70-/- mice, especially in females. Surprisingly, female Cyp2c70-/- mice were resistant to WTD-induced obesity and hepatic steatosis, whereas male Cyp2c70-/- mice showed similar adiposity and moderately reduced steatosis compared with WT controls. Both intestinal cholesterol and FA absorption were reduced in Cyp2c70-/- mice, the latter more strongly in females, despite unaffected biliary BA secretion rates. Intriguingly, the biliary ratio 12α-/non-12α-hydroxylated BAs significantly correlated with FA absorption and hepatic triglyceride content as well as with specific changes in gut microbiome composition. The hydrophobic human-like BA pool in Cyp2c70-/- mice prevents WTD-induced obesity in female mice and NAFLD development in both genders, primarily because of impaired intestinal fat absorption. Our data point to a key role for 12α-hydroxylated BAs in control of intestinal fat absorption and modulation of gut microbiome composition.

Cholangiopathy and Biliary Fibrosis in Cyp2c70-Deficient Mice Are Fully Reversed by Ursodeoxycholic Acid.

BACKGROUND AND AIMS: Bile acids (BAs) aid intestinal fat absorption and exert systemic actions by receptor-mediated signaling. BA receptors have been identified as drug targets for liver diseases. Yet, differences in BA metabolism between humans and mice hamper translation of pre-clinical outcomes. Cyp2c70-ablation in mice prevents synthesis of mouse/rat-specific muricholic acids (MCAs), but potential (patho)physiological consequences of their absence are unknown. We therefore assessed age- and gender-dependent effects of Cyp2c70-deficiency in mice. METHODS: The consequences of Cyp2c70-deficiency were assessed in male and female mice at different ages. RESULTS: Cyp2c70-/- mice were devoid of MCAs and showed high abundances of chenodeoxycholic and lithocholic acids. Cyp2c70-deficiency profoundly impacted microbiome composition. Bile flow and biliary BA secretion were normal in Cyp2c70-/- mice of both sexes. Yet, the pathophysiological consequences of Cyp2c70-deficiency differed considerably between sexes. Three-week old male Cyp2c70-/- mice showed high plasma BAs and transaminases, which spontaneously decreased thereafter to near-normal levels. Only mild ductular reactions were observed in male Cyp2c70-/- mice up to 8 months of age. In female Cyp2c70-/- mice, plasma BAs and transaminases remained substantially elevated with age, gut barrier function was impaired and bridging fibrosis was observed at advanced age. Addition of 0.1% ursodeoxycholic acid to the diet fully normalized hepatic and intestinal functions in female Cyp2c70-/- mice. CONCLUSION: Cyp2c70-/- mice show transient neonatal cholestasis and develop cholangiopathic features that progress to bridging fibrosis in females only. These consequences of Cyp2c70-deficiency are restored by treatment with UDCA, indicating a role of BA hydrophobicity in disease development.

A human-like bile acid pool induced by deletion of hepatic Cyp2c70 modulates effects of FXR activation in mice.

Bile acids (BAs) facilitate intestinal absorption of lipid-soluble nutrients and modulate various metabolic pathways through the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5. These receptors are targets for therapy in cholestatic and metabolic diseases. However, dissimilarities in BA metabolism between humans and mice complicate translation of preclinical data. Cytochrome P450 family 2 subfamily c polypeptide 70 (CYP2C70) was recently proposed to catalyze the formation of rodent-specific muricholic acids (MCAs). With CRISPR/Cas9-mediated somatic genome editing, we generated an acute hepatic Cyp2c70 knockout mouse model (Cyp2c70ako) to clarify the role of CYP2C70 in BA metabolism in vivo and evaluate whether its activity modulates effects of pharmacologic FXR activation on cholesterol homeostasis. In Cyp2c70ako mice, chenodeoxycholic acid (CDCA) increased at the expense of ßMCA, resulting in a more hydrophobic human-like BA pool. Tracer studies demonstrated that, in vivo, CYP2C70 catalyzes the formation of ßMCA primarily by sequential 6ß-hydroxylation and C7-epimerization of CDCA, generating αMCA as an intermediate metabolite. Physiologically, the humanized BA composition in Cyp2c70ako mice blunted the stimulation of fecal cholesterol disposal in response to FXR activation compared with WT mice, predominantly due to reduced stimulation of transintestinal cholesterol excretion. Thus, deletion of hepatic Cyp2c70 in adult mice translates into a human-like BA pool composition and impacts the response to pharmacologic FXR activation. This Cyp2c70ako mouse model may be a useful tool for future studies of BA signaling and metabolism that informs human disease development and treatment.

Potential of Intestine-Selective FXR Modulation for Treatment of Metabolic Disease.

Farnesoid X receptor controls bile acid metabolism, both in the liver and intestine. This potent nuclear receptor not only maintains homeostasis of its own ligands, i.e., bile acids, but also regulates glucose and lipid metabolism as well as the immune system. These findings have led to substantial interest for FXR as a therapeutic target and to the recent approval of an FXR agonist for treating primary biliary cholangitis as well as ongoing clinical trials for other liver diseases. Given that FXR biology is complex, including moderate expression in tissues outside of the enterohepatic circulation, temporal expression of isoforms, posttranscriptional modifications, and the existence of several other bile acid-responsive receptors such as TGR5, clinical application of FXR modulators warrants thorough understanding of its actions. Recent findings have demonstrated remarkable physiological effects of targeting FXR specifically in the intestine (iFXR), thereby avoiding systemic release of modulators. These include local effects such as improvement of intestinal barrier function and intestinal cholesterol turnover, as well as systemic effects such as improvements in glucose homeostasis, insulin sensitivity, and nonalcoholic fatty liver disease (NAFLD). Intriguingly, metabolic improvements have been observed with both an iFXR agonist that leads to production of enteric Fgf15 and increased energy expenditure in adipose tissues and antagonists by reducing systemic ceramide levels and hepatic glucose production. Here we review the recent findings on the role of intestinal FXR and its targeting in metabolic disease.