Bile Acid Homeostasis in a Cholesterol 7α-Hydroxylase and Sterol 27-Hydroxylase Double Knockout Mouse Model

Abstract

Bile acids (BAs) are diverse molecules that are synthesized from cholesterol in the liver. The synthesis of BAs has traditionally been shown to occur through two pathways. Cholesterol 7α-hydroxylase (CYP7A1) performs the initial and rate-limiting step in the classical pathway, and sterol 27-hydroxylase (CYP27A1) initiates the hydroxylation of cholesterol in the alternative pathway. While the role of individual BA species as physiological detergents is relatively ubiquitous, their endocrine functions as signaling molecules and roles in disease pathogenesis have been emerging to be BA species-specific. In order to better understand the pharmacologic and toxicologic roles of individual BA species in an in vivo model, we created cholesterol 7α-hydroxylase (Cyp7a1) and sterol 27-hydroxylase (Cyp27a1) double knockout (DKO) mice by cross-breeding single knockout mice (Cyp7a1^-/- and Cyp27a1^-/- ). BA profiling and quantification by liquid chromatography-mass spectrometry of serum, gallbladder, liver, small intestine, and colon of wild-type, Cyp7a1^-/- , Cyp27a1^-/- , and DKO mice showed that DKO mice exhibited a reduction of BAs in the plasma (45.9%), liver (60.2%), gallbladder (76.3%), small intestine (88.7%), and colon (93.6%), while maintaining a similar BA pool composition compared to wild-type mice. The function of the farnesoid X receptor (FXR) in DKO mice was lower, revealed by decreased mRNA expression of well-known FXR target genes, hepatic small heterodimer partner, and ileal fibroblast growth factor 15. However, response to FXR synthetic ligands was maintained in DKO mice as treatment with GW4064 resulted in similar changes in gene expression in all strains of mice. Conclusion: We provide a useful tool for studying the role of individual BAs in vivo; DKO mice have a significantly reduced BA pool, have a similar BA profile, and maintained response to FXR activation.

Fig. 1.. Plasma BA pool size and composition of male WT, Cyp7a1−/−, Cyp27a1−/−, and DKO mice.

(A) Plasma BA pool size was measured with 90μL aliquots of plasma using UPLC-ITMS. Values are displayed in ng/mL plasma ± 1 SD. These data failed Levene’s test therefore Kruskal-Wallis was used for analysis. An asterisk denotes a significant difference from WT (P < 0.05). (B) Plasma concentration of individual BA species ± 1 SD. (C) Percent composition of BA species in plasma. BAs that represent <1% of total BAs in plasma are represented as “other” and denoted alongside the pie charts.

Fig. 2.. Liver BA pool size and composition of male WT, Cyp7a1−/−, Cyp27a1−/−, and DKO mice.

(A) Liver BA pool size was measured using UPLC-ITMS. Values are displayed in ng/mg liver ± 1 SD. These data failed Levene’s test therefore Kruskal-Wallis was used for analysis. An asterisk denotes a significant difference from WT (P < 0.05). (B) Liver concentration of individual BA species ± 1 SD. (C) Percent composition of BA species in plasma. BAs that represent <1% of total BAs in the liver are represented as “other” and denoted alongside the pie charts.

Fig. 3.. Gallbladder BA pool size and composition of male WT, Cyp7a1−/−, Cyp27a1−/−, and DKO mice.

(A) Gallbladder BA pool size was measured using UPLC-ITMS. Values are displayed in μg/100g body weight (x1000) ± 1 SD. An asterisk denotes a significant difference from WT (P < 0.05). (B) Gallbladder concentration of individual BA species (μg/100g body weight) ± 1 SD. (C) Percent composition of BA species in the gallbladder. BAs that represent <1% of total BAs in the gallbladder are represented as “other” and denoted alongside the pie charts.

Fig. 4.. Small intestine BA pool size and composition of male WT, Cyp7a1−/−, Cyp27a1−/−, and DKO mice.

(A) Small intestine BA pool size was measured using UPLC-ITMS and includes luminal content. Values are displayed in μg/100g body weight (x1000) ± 1 SD. An asterisk denotes a significant difference from WT (P < 0.05). (B) Small intestine concentration of individual BA species (μg/100g body weight) ± 1 SD. (C) Percent composition of BA species in the small intestine. BAs that represent <1% of total BAs in the small intestine are represented as “other” and denoted alongside the pie charts.

Fig. 5.. Large intestine BA pool size and composition of male WT, Cyp7a1−/−, Cyp27a1−/−, and DKO mice.

(A) Large intestine BA pool size was measured using UPLC-ITMS and includes luminal content. Values are displayed in μg/100g body weight (x1000) ± 1 SD. An asterisk denotes a significant difference from WT (P < 0.05). (B) Large intestine concentration of individual BA species (μg/100g body weight) ± 1 SD. (C) Percent composition of BA species in the large intestine. BAs that represent <1% of total BAs in the large intestine are represented as “other” and denoted alongside the pie charts.

Fig. 6.. Relative mRNA levels of BA related genes for male WT, Cyp7a1−/−, Cyp27a1−/−, and DKO mice.

Genes were measured using RT q-PCR, normalized to β-actin mRNA levels, and graphed as relative mRNA ± 1 SD. An asterisk denotes a significant difference from WT (P < 0.05). (A) Relative mRNA of hepatic genes involved in classic, alternative, and minor BA synthetic pathways. (B) Relative mRNA of hepatic genes for BA conjugation. (C) Relative mRNA of hepatic FXR, Shp, Fgfr4, Ntcp, and Bsep. (D) Relative mRNA of ileal genes involved in BA regulation and transport.

Fig. 7.. Relative mRNA levels of hepatic enzymes involved in BA synthesis for male WT, Cyp7a1−/−, Cyp27a1−/−, and DKO mice with FXR activation.

Mice were treated with vehicle or a synthetic FXR agonist, GW4064. The expression of genes at mRNA levels was measured using RT q-PCR and normalized to β-actin mRNA levels, and graphed as relative mRNA ± 1 SD. An asterisk denotes a significant difference from WT vehicle treated mice, an octothorpe denotes a significant difference between vehicle and GW4064 treatments (P < 0.05).

Fig. 8.. Relative mRNA levels of ileal enzymes involved in BA transport and regulation for male WT, Cyp7a1−/−. Cyp27a1−/−, and DKO mice with FXR activation.

Mice were treated with vehicle or a synthetic FXR agonist, GW4064. The expression of genes at mRNA levels was measured using RT q-PCR and normalized to β-actin mRNA levels, and graphed as relative mRNA ± 1 SD. An asterisk denotes a significant difference from WT vehicle treated mice, an octothorpe denotes a significant difference between vehicle and GW4064 treatments (P < 0.05).