The hepatic circadian clock modulates xenobiotic metabolism in mice

Abstract

The circadian clock generates daily cycles of gene expression that regulate physiological processes. The liver plays an important role in xenobiotic metabolism and also has been shown to possess its own cell-based clock. The liver clock is synchronized by the master clock in the brain, and a portion of rhythmic gene expression can be driven by behavior of the organism as a whole even when the hepatic clock is suppressed. So far, however, there is relatively little evidence indicating whether the liver clock is functionally important in modulating xenobiotic metabolism. Thus, mice lacking circadian clock function in the whole body or specifically in liver were challenged with pentobarbital and acetaminophen, and pentobarbital sleep time (PBST) and acetaminophen toxicity, respectively, was assessed at different times of day in mutant and control mice. The results suggest that the liver clock is essential for rhythmic changes in xenobiotic detoxification. Surprisingly, it seems that the way in which the clock is disrupted determines the rate of xenobiotic metabolism in the liver. CLOCK-deficient mice are remarkably resistant to acetaminophen and exhibit a longer PBST, while PERIOD-deficient mice have a short PBST. These results indicate an essential role of the tissue-intrinsic peripheral circadian oscillator in the liver in regulating xenobiotic metabolism.

Keywords: Bmal1; Clock; Period; acetaminophen; circadian rhythms; cytochrome P450; mouse; pentobarbital; xenobiotic metabolism.

Figure 1. PBST is time-of-day dependent in wild-type mice

Relative pentobarbital sleep time (PBST) of wild-type mice injected i.p. with 50 mg/kg PB at indicated circadian times (n = 4-5 per group) as percent of PBST at CT2 (100% ≙ 125.8 ± 7.1 min). Statistically significant differences vs. CT2 in Bonferroni’s post hoc test are given as *** p < 0.001. Open bars = subjective day (CT2, 8), filled bars = subjective night (CT14, 20).

Figure 2. PBST is not dependent on duration of prior fasting

The influence of fasting on PBST in B6 mice was tested by i.p. injecting 40 mg/kg PB at CT2 or CT14 with prior fasting of 12, 24, or 36 hours. (See Supplemental Figure S1B). PBST was determined for n = 5 animals per group. Values are given as relative PBST (percent of average PBST after 12 hours of fasting at CT2 (control, 100 % ≙ 60.4 ± 2.8 min). Statistically significant differences in Bonferroni’s post-hoc test indicated as * p < 0.05, n.s. not significant.

Figure 3. PB clearance from serum is dependent on time of day

PB (40 mg/kg i.p.) was injected at CT2 or CT14 in wild-type mice (n = 5-7 per group), and PB concentration in serum was measured 30 minutes after injection (left), or (in a second independent groups of animals) after the mice regained their righting reflex (“awake”, right). In post-hoc testing of 2-way ANOVA, PB levels at 30 minutes after injection differed significantly between CTs (*, p = 0.01, Bonferroni’s t-test), but not at the time of recovery from anesthesia (n.s., p = 0.49).

Figure 4

Effect of i.p. saline (Saline, n = 2 per group) or 250 mg/kg acetaminophen (APAP, n = 6 per group) injection at two circadian times in wild-type animals. Statistically significant differences vs. CT2 in Bonferroni’s post hoc test are given as ** p < 0.01. Black bars = saline controls, open bars = subjective day (CT2), filled bars = subjective night (CT14).

Figure 5. Rhythm in PBST is disrupted in mice with whole-body disruption of circadian clock genes

PBST in Clock^−/− (n = 6 per group, left, diagonally hatched), Bmal1^−/− (n = 4-5 per group, middle, horizontally hatched) and Per123^−/− (n = 6 per group, right, dotted) compared to their respective control wild-type mice after injection of 50 mg/kg PB. In order to facilitate easy comparison between experiments, PBST is normalized to CT2 of the wild-type control value for each experiment (100% values of controls at CT2: Clock-WT 107.6 ± 7.3 min, Bmal1-WT 62.8 ± 6.4 min, Per123-WT 50.2 ± 5.4 min). Statistical differences in Bonferroni’s post-hoc test between CTs are indicated as n.s. not significant, ** p < 0.01 or *** p < 0.001. Open bars = subjective day (CT2), filled bars = subjective night (CT14).

Figure 6. Clock^−/− mice are resistant to APAP toxicity

(A) ALT levels in wild-type and Clock^−/− (hatched) mice following APAP (250 mg/kg) administration at CT2 or CT14 (n=5 per group). (B) Dose range experiment with indicated doses of APAP in wild-type (closed circles) and Clock^−/− (open squares) mice at CT 14 (n = 3 per group). (C) Twelve hours of food deprivation before APAP (250 mg/kg) injection increases APAP toxicity in wild-type but not in Clock^−/− (hatched) mice at CT2 and CT14 (note the difference in values between Panel A and Panel C). Statistically significant differences between genotypes in Bonferroni’s post-hoc test after 2-way ANOVA are indicated as n.s. not significant, * p < 0.05, *** p < 0.001.

Figure 7. Rhythms in PBST and APAP toxicity are absent in liver-specific CLOCK deficient mice

(A) PBST in Alb-Cre-;Clock^flox/flox mice (Cre-) and Alb-Cre+;Clock^flox/flox mice (Cre+, hatched) (n = 6 per group) after 50 mg/kg PB injection. PBST is normalized to CT2 of the wild-type control value (100% ≙ 128.7 ± 11.0 min). (B) APAP toxicity measured by ALT levels in Alb-Cre-;Clock^flox/flox mice (Cre-) and Alb-Cre+;Clock^flox/flox mice (Cre+) (n = 6 per group) after injection of 250 mg/kg APAP. Results of Bonferroni’s post-hoc comparison of CTs within each genotype are given as n.s. not significant and *** p < 0.001.