Regulation of Intestinal UDP-Glucuronosyltransferase 1A1 by the Farnesoid X Receptor Agonist Obeticholic Acid Is Controlled by Constitutive Androstane Receptor through Intestinal Maturation

Abstract

UDP-glucuronosyltransferase (UGT) 1A1 is the only transferase capable of conjugating serum bilirubin. However, temporal delay in the development of the UGT1A1 gene leads to an accumulation of serum bilirubin in newborn children. Neonatal humanized UGT1 (hUGT1) mice, which accumulate severe levels of total serum bilirubin (TSB), were treated by oral gavage with obeticholic acid (OCA), a potent FXR agonist. OCA treatment led to dramatic reduction in TSB levels. Analysis of UGT1A1 expression confirmed that OCA induced intestinal and not hepatic UGT1A1. Interestingly, Cyp2b10, a target gene of the nuclear receptor CAR, was also induced by OCA in intestinal tissue. In neonatal hUGT1/Car ^-/- mice, OCA was unable to induce CYP2B10 and UGT1A1, confirming that CAR and not FXR is involved in the induction of intestinal UGT1A1. However, OCA did induce FXR target genes, such as Shp, in both intestines and liver with induction of Fgf15 in intestinal tissue. Circulating FGF15 activates hepatic FXR and, together with hepatic Shp, blocks Cyp7a1 and Cyp7b1 gene expression, key enzymes in bile acid metabolism. Importantly, the administration of OCA in neonatal hUGT1 mice accelerates intestinal epithelial cell maturation, which directly impacts on induction of the UGT1A1 gene and the reduction in TSB levels. Accelerated intestinal maturation is directly controlled by CAR, since induction of enterocyte marker genes sucrase-isomaltase, alkaline phosphatase 3, and keratin 20 by OCA does not occur in hUGT1/Car ^-/- mice. Thus, new findings link an important role for CAR in intestinal UGT1A1 induction and its role in the intestinal maturation pathway. SIGNIFICANCE STATEMENT: Obeticholic acid (OCA) activates FXR target genes in both liver and intestinal tissues while inducing intestinal UGT1A1, which leads to the elimination of serum bilirubin in humanized UGT1 mice. However, the induction of intestinal UGT1A1 and the elimination of bilirubin by OCA is driven entirely by activation of intestinal CAR and not FXR. The elimination of serum bilirubin is based on a CAR-dependent mechanism that facilitates the acceleration of intestinal epithelium cell differentiation, an event that underlies the induction of intestinal UGT1A1.

Fig. 1.

Induction of UGT1A1 by OCA in neonatal hUGT1 mice. The 10-day-old neonatal hUGT1 mice were treated with 50 mg/kg of OCA (divided in two doses of 25 mg/kg) or vehicle by oral administration for 5 consecutive days. (A) After the treatment, TSB levels were measured. (B) Liver and SI were used for gene and protein analysis. UGT1A1 gene expression was determined by RT-qPCR and expressed as fold induction. (C) Western blots were performed from liver and SI tissue to examine UGT1A1 expression. The bands have been cropped, and the full-length blots are in Supplemental Material. Values are means ± S.D. (n ≥ 3). Statistically significant differences between vehicle (Veh) and OCA are indicated by asterisks (Student’s t test: **P < 0.01). RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Fig. 2.

Oral OCA effect in liver of hUGT1 mice. Litters were bred to produce hUGT1 mice, and 10-day-old neonatal mice were treated with OCA or vehicle on day 10, followed by tissue preparation after treatment. (A) RNA was isolated from liver and used to conduct RT-qPCR for Cyp2b10, Shp, and Cyp7a1 genes.Supplemental Material (B) Total RNA was prepared from liver, and RT-qPCR was performed to measure expression of Cyp7a1, Cyp8b1, Cyp27a1, and Cyp7b1. (C) Western blots were performed using antibodies toward mouse CYP2B10 and mouse CYP7A1. The bands have been cropped, and the full-length blots are in Supplemental Material. Values are means ± S.D. (n ≥ 3). Statistically significant differences between vehicle (Veh) and OCA are indicated by asterisks (Student’s t test: *P < 0.05, ***P < 0.001). RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Fig. 3.

Oral OCA treatment and effects in intestines of hUGT1 and hUGT1/Car^−/− mice. Litters were bred to produce hUGT1 and hUGT1/Car^−/− mice, and 10-day-old neonatal mice were treated with OCA or vehicle on day 10, followed by tissue preparation after treatment. (A) Intestinal hUGT1 Cyp2b10 was analyzed by RT-qPCR and Western Blot analysis. (B) After treatment, serum bilirubin levels were determined. (C) Intestinal UGT1A1 and Cyp2b10 gene expression was examined by RT-qPCR from OCA-treated hUGT1 and hUGT1/Car^−/− mice. From those same tissues, total cell extracts were prepared for Western blot analysis using anti-UGT1A1 and anti-CYP2B10 antibodies. The bands have been cropped, and the full-length blots are in Supplemental Material. Values are means ± S.D. (n ≥ 3). Statistically significant differences between vehicle (Veh) and OCA are indicated by asterisks in A (Student’s t test: *P < 0.05) and statistical differences between groups are indicated by asterisks (Student’s t test: *P < 0.05; ** P < 0.01) and between treatments are indicated as # in B and C.RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Fig. 4.

OCA treatment promotes FXR target gene induction. (A) Total RNA and cellular extracts were prepared from small intestine and liver of hUGT1 and hUGT1/Car^−/− neonatal mice. (A) Shp and Fgf15 gene expression from small intestines was determined by RT-qPCR. Cellular extracts were prepared for Western blot analysis to examine FGF15 protein expression. (B) Liver Shp and Cyp7a1 gene expression was determined by RT-qPCR, and Cyp7a1 protein expression was examined by Western blot analysis. The bands have been cropped, and the full-length blots are in Supplemental Material. Values are means ± S.D. (n ≥ 3). Statistically significant differences between vehicle (Veh) and OCA are indicated by asterisks (Student’s t test: ^#P < 0.05; ^##P < 0.01; between treatments). RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Fig. 5.

OCA treatment promotes intestinal maturation. (A) After OCA treatment, total RNA was prepared from small intestine of hUGT1 neonatal mice. Sis, Krt20, Akp3, Glb1, Nox4, and Lrp2 gene expression was determined by RT-qPCR. (B) After OCA treatment to neonatal hUGT1 and hUGT1/Car^−/− mice, small intestine RNA was used to examine Sis, Akp3, Krt20, Nox4, and Glb1 gene expression by RT-qPCR. From total cell extracts, Western blot analysis was performed using antibodies toward mouse SIS. Values are means ± S.D. (n ≥ 3). Statistically significant differences between vehicle (Veh) and OCA are indicated by asterisks (Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001). Nox4, NADPH Oxidase 4: Glb1, galactosidase β1; Lrp2, low-density lipoprotein-related protein 2; M, mature; P, precursor; RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Fig. 6.

Treatment of intestinal organoids with OCA. Intestinal organoids cultured from neonatal hUGT1 intestinal tissue were treated with OCA and DAPT for 24 hours. (A) Gene expression of FXR target genes Fgf15 and Shp by RT-qPCR. (B) UGT1A1 gene expression and (C) Cy2b10 gene expression. (D) Gene expression analysis of maturation marker genes Sis, Akp3, and Krt20. (E) Western blot analysis of UGT1A1 and FGF15 after vehicle (Veh) or OCA treatment. The bands have been cropped, and the full-length blots are in Supplemental Material. Values are means ± S.D. (n ≥ 3). Statistically significant differences between Veh and OCA are indicated by asterisks (Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001). RT-qPCR, reverse-transcription quantitative polymerase chain reaction.

Fig. 7.

Pathways involved with OCA treatment. Administration of OCA activates intestinal CAR and FXR and their respective target genes. In small intestines, FXR activation leads to induction of FGF15 and Shp, whereas OCA is capable of inducing liver FXR target genes such as Shp gene expression. Induction of FGF15 results in inhibition of liver Cyp7a1 gene expression. After oral OCA treatment, CAR becomes activated, leading to an increase in intestinal epithelial cell maturation, as displayed by activation of specific marker genes. Activation of intestinal maturation results in induction of intestinal UGT1A1 that drives the metabolism of serum bilirubin.