Dietary regulation of mouse intestinal P450 expression and drug metabolism

Abstract

The study was originally designed to test the hypothesis that the compensatory increase in intestinal P450 (cytochrome P450) expression in the intestinal epithelium-specific P450 reductase (CPR) knockout (IE-Cpr-null) mice was attributable to decreased metabolism of putative P450 inducers present in the diet. Thus, we determined the impact of a dietary change from regular rodent chow to a synthetic diet devoid of phytochemicals on the expression of P450 enzymes in the small intestine (SI) and liver of wild-type (WT) and IE-Cpr-null mice. The dietary change diminished expression of CYP1A, 2B, 2C, and 3A in SI and CYP2B, 2C, and 3A in liver of both WT and IE-Cpr-null mice. However, the compensatory increase in SI P450 expression still occurred in IE-Cpr-null, compared with WT, mice, on the synthetic diet. The diet change-induced decrease in P450 expression was accompanied by decreases in microsomal midazolam-hydroxylase activity in vitro and first-pass clearance of midazolam in vivo in WT mice. Further studies showed that the dietary change, but not Cpr deletion, caused large decreases in bile acid (BA) levels in plasma, liver, SI, and intestinal content and that treatment of WT mice on the synthetic diet with GW4064, a farnesoid-X-receptor agonist, restored the levels of CYP3A expression in both liver and SI to those seen in mice fed with regular chow. Taken together, these results highlight the vital role of diet in maintaining adequate expression of major drug-metabolizing P450s and their associated drug-metabolizing activities in the digestive tract and suggest potential involvement of BA signaling in the regulatory mechanisms.

Fig. 1.

Effects of dietary change on P450 expression and microsomal MDZ metabolism. Microsomes were prepared from adult, male WT and IE-Cpr-null mice that were either always receiving a regular laboratory chow diet (Prolab RMH 3500) or were switched from the regular diet to a synthetic diet (AIN-93G) for 3 weeks. (A–D) Immunoblot analysis of P450 expression. Each microsomal sample was pooled from 3 mice and analyzed in duplicate. Antibodies used are described in Materials and Methods. The results represent 2–3 independent experiments. (A) Expression of CYP1A, 2B, 2C, and 3A proteins in mouse SI. Each lane contained 20 (Synthetic/SI samples) or 10 (all others) μg total protein. Calnexin was also analyzed as a loading control. (B) Densitometry analysis of relative SI CYP2B, 2C, and 3A protein levels in WT and IE-Cpr-null mice. WT values from regular laboratory chow group were set as 1 (mean ± S.D. [n = 3]; **P < 0.01), compared with the corresponding regular diet group. (C) Expression of CYP2B, 2C, and 3A proteins in mouse liver. Each lane contained 10 μg total protein. (D) Densitometry analysis of relative hepatic CYP2B, 2C, and 3A protein levels in WT and IE-Cpr-null mice. WT values from regular laboratory chow group were set as 1 (mean ± S.D. [n = 3]; **P < 0.01; *P < 0.05), compared with the corresponding regular diet group. (E) In vitro metabolism of MDZ by liver or SI microsomes. Rates of formation of 1′-OH-MDZ and 4-OH-MDZ were determined. Reaction mixtures contained phosphate buffer (pH, 7.4), 3 μM MDZ, and 0.05 mg/ml liver microsomes or 0.5 mg/ml SI microsomes, in a total volume of 200 μl. The values reported are means ± S.D. (n = 6). **P < 0.01, compared with the corresponding regular diet group; #P < 0.01, compared with the WT SI in the regular diet group (Student's t test).

Fig. 2.

BA levels in plasma, liver, SI epithelium, and intestinal contents of adult male WT mice fed a regular or synthetic diet. Five different BAs were determined using LC-MS/MS as described in Materials and Methods. Results are shown as means ± S.D (n = 6). *P < 0.05; **P < 0.01, compared with the corresponding regular diet group. Corresponding data for IE-Cpr-null mice are shown in Supplemental Fig. 3. CA, cholic acid.

Fig. 3.

Effect of GW4064 treatment on liver and SI CYP3A expression in mice fed synthetic diet. Adult male WT mice fed the synthetic diet for 3 weeks were treated once with GW4064 by oral gavage or with the vehicle alone, and tissues were obtained 18 hours later for microsome preparation. Age-matched male WT mice fed with regular chow were also included for comparison. (A) Immunoblot analysis of CYP3A expression was performed as described in Fig. 1. Microsomes were prepared from pooled enterocytes of 2 mice in each group and analyzed in duplicates. Calnexin was analyzed as a loading control. Each lane contained 10 μg total protein. The results represent three independent experiments. (B) Results of densitometry analysis for relative CYP3A protein levels (mean ± S.D. [n = 3]; **P < 0.01; *P < 0.05, compared with the regular diet group.