Modulation of CYP3a expression and activity in mice models of type 1 and type 2 diabetes

Abstract

CYP3A4, the most abundant cytochrome P450 enzyme in the human liver and small intestine, is responsible for the metabolism of about 50% of all marketed drugs. Numerous pathophysiological factors, such as diabetes and obesity, were shown to affect CYP3A activity. Evidences suggest that drug disposition is altered in type 1 (T1D) and type 2 diabetes (T2D). The objective was to evaluate the effect of T1D and T2D on hepatic and intestinal CYP3a drug-metabolizing activity/expression in mice. Hepatic and intestinal microsomes were prepared from streptozotocin-induced T1D, db/db T2D and control mice. Domperidone was selected as a probe substrate for CYP3a and formation of five of its metabolites was evaluated using high performance liquid chromatography. Hepatic CYP3a protein and mRNA expression were assessed by Western blot and reverse-transcription quantitative polymerase chain reaction respectively. Hepatic microsomal CYP3a activity was significantly increased in both T1D and T2D groups versus control group. Intestinal CYP3a activity was also significantly increased in both T1D and T2D groups. Moreover, significant increases of both hepatic CYP3a mRNAs and protein expression were observed in both T1D and T2D groups versus control group. Additional experiments with testosterone further validated the increased activity of CYP3a under the effect of both T1D and T2D. Although differences exist in the pathophysiological insults associated with T1D and T2D, our results suggest that these two distinct diseases may have the same modulating effect on the regulation of CYP3a, ultimately leading to variability in drug response, ranging from lack of effect to life-threatening toxicity.

Keywords: Domperidone as a CYP3a substrate; mice models of type 1 and type 2 diabetes; modulation of CYP3a expression & activity.

Figure 1

(A) Hepatic microsomal CYP3a activity: This figure shows peak-height ratios of metabolites of domperidone to internal standard for the three groups. Formation of four of the five metabolites (M1-4) were significantly increased in the T1D group (M1: 0.417 ± 0.071, M2: 0.199 ± 0.011, M3: 0.818 ± 0.117, M4: 0.201 ± 0.019, M5: 0.178 ± 0.018; n = 17) when compared to the control group (M1: 0.158 ± 0.014, M2: 0.090 ± 0.003, M3: 0.384 ± 0.033, M4: 0.109 ± 0.005, M5: 0.152 ± 0.011; n = 14). In the T2D group, M2 and M4 were significantly increased while M5 was significantly decreased (M1: 0.209 ± 0.029, M2: 0.171 ± 0.007, M3: 0.487 ± 0.065, M4: 0.147 ± 0.011, M5: 0.093 ± 0.006; n = 18). Metabolites of domperidone were not formally identified. (B) Intestinal microsomal CYP3a activity: Formation of the four metabolites was significantly increased in both T1D (M1: 0.113 ± 0.013, M2: 0.054 ± 0.007, M3: 0.389 ± 0.040, M4: 0.077 ± 0.007; n = 8) and T2D (M1: 0.093 ± 0.017, M2: 0.032 ± 0.010, M3: 0.338 ± 0.065, M4: 0.075 ± 0.011; n = 6) groups when compared to the control group (M1: 0.004 ± 0.004, M2: 0.002 ± 0.002, M3: 0.039 ± 0.013, M4: 0.015 ± 0.006; n = 8), except for M4 in the T2D group. (C) Hepatic microsomal CYP3a activity: This figure shows peak-height ratios of the CYP3a-catalized metabolite of testosterone to internal standard for the three groups. Formation of 6-β-OH-testosterone was significantly increased in both T1D and T2D groups when compared to control group (0.588 ± 0.063, 0.661 ± 0.098 vs. 0.277 ± 0.018; n = 15, 17, 16, respectively)

Figure 2

(A) Relative steady-state levels of CYP3a mRNAs in mouse livers. A significant increase of CYP3a11 and CYP3a13 was observed for both T1D and T2D groups when compared to the control group (3a11: 3.31 ± 0.24, 1.99 ± 0.22 vs. 1.00 ± 0.10; n = 17, 20, 18 respectively) (3a13: 3.25 ± 0.10, 3.19 ± 0.26 vs. 1.00 ± 0.05; n = 17, 20, 17 respectively), and for CYP3a44 in T1D group (1.68 ± 0.14, 0.95 ± 0.10 vs. 1.00 ± 0.11; n = 17, 20, 17 respectively). No significant difference was observed for CYP3a25 (0.93 ± 0.05, 0.89 ± 0.09 vs. 1.00 ± 0.11; n = 17, 19 respectively). (B) Relative steady-state levels of PXR and CAR mRNAs in mouse livers. A significant increase of PXR and CAR was observed in both T1D and T2D groups when compared to the control group (PXR: 1.67 ± 0.14, 2.04 ± 0.20 vs. 1.00 ± 0.05; n = 17, 19, 17 respectively) (CAR: 3.43 ± 0.16, 2.44 ± 0.24 vs. 1.00 ± 0.08; n = 17, 15 respectively). (C) Relative expression of CYP3a proteins in mouse livers. On the left, a significant increase was observed in both T1D and T2D groups when compared to the control group (1.35 ± 0.106, 1.62 ± 0.11 vs. 1.00 ± 0.11; n = 17, 18, 17 respectively). On the right are the representative images of the ChemiBlot of CYP3a and the total proteins loaded for the three groups.