P-glycoprotein (P-gp) is one of the ATP-binding cassette transporters and acts as an efflux pump for cytotoxic substances. P-gp mRNA expression and transporting activity show the daily rhythm and contribute to the chrono-pharmacokinetic profiles of many drugs. It is reported that the daily rhythm of abcb1a mRNA is regulated by a circadian clock-controlled output pathway. Time-restricted feeding is well known to shift the peripheral circadian phase of clock gene expression without changing the central clock function. This study was undertaken to examine the influence of a time-restricted feeding procedure during the light phase on the daily rhythms of abcb1a mRNA expression and P-gp activity. The abcb1a mRNA and P-gp activity showed a daily rhythm with a peak early in the dark phase in rat intestine under ad libitum feeding. Time-restricted feeding during the light phase shifted these rhythms to 12-h advance. The mRNA expression of clock genes (DBP and HLF, the transcript activators of abcb1a) also showed daily rhythms, and their phases were shifted by the time-restricted feeding procedure. The peak time of DBP mRNA expression was similar to that of abcb1a mRNA expression under ad libitum feeding and time-restricted feeding conditions. These results indicate that a time-restricted feeding procedure changes DBP mRNA expression, which in turn influences abcb1a mRNA expression and P-gp activity.
In mammals, daily rhythms in behavior and physiology are regulated by the circadian timing system, which includes a master pacemaker in the suprachiasmatic nuclei (SCN) (Ripperger and Schibler, 2001; Reppert and Weaver, 2002). A circadian oscillatory mechanism, which is constituted by “clock gene” products, is based on self-sustained transcriptional/translational feedback loops (Mitsui et al., 2001; Ueda et al., 2005; Ukai-Tadenuma et al., 2008). This systematic mechanism interconnects the positive and negative limbs of circadian clockwork circuitry and controls the 24-h variation in output physiology (Jin et al., 1999; Oishi et al., 2003). For example, the circadian-controlled output pathway regulates the expression of many enzymes, which are involved in xenobiotic detoxification, such as cytochrome P450 enzymes, carboxyl esterase, and xenobiotic transporters (Gachon et al., 2006).
Many studies have shown that changing a feeding schedule can alter biological rhythms regardless of lighting conditions (Boulos and Terman, 1980). Although the SCN clock is entrained mainly by the light/dark cycle, peripheral clock-gene oscillators are strongly influenced by daily feeding cycles (Damiola et al., 2000; Le Minh et al., 2001; Stokkan et al., 2001). It has also been reported that time-restricted feeding modifies drug toxicity by changing the rhythms of kinetics and metabolic enzyme activities (Song et al., 1993; Matsunaga et al., 2004). Therefore, food-intake schedule is considered as a dominant Zeitgeber for peripheral circadian oscillators, but not for SCN pacemakers (Damiola et al., 2000; Stokkan et al., 2001).
It is well known that the pharmacokinetics of many drugs are influenced by their dosing time (Lévi, 2002; Hermida and Smolensky, 2004). These are caused by the daily changes in absorption, distribution, metabolism, and elimination of drugs (Ohdo, 2007). P-glycoprotein (P-gp), one of the ATP-binding cassette transporters, is expressed in epithelial cells of several organs including intestine, liver, and kidney (Schinkel et al., 1994) and acts as an energy-dependent efflux pump by expelling cytotoxic substances (Gottesman et al., 2002). We have observed previously that abcb1a mRNA expression in mouse intestine shows a 24-h rhythmicity (Ando et al., 2005). The daily change in the activity of intestinal P-gp, which followed the daily rhythm in mRNA expression, was also detected. Murakami et al. (2008) reported that mouse abcb1a gene expression was regulated by a circadian clock-controlled output pathway. In addition, they showed that the circadian rhythm of abcb1a expression in mouse intestine was dampened in clock-mutant mice (Murakami et al., 2008). Based on these observations, it is speculated that an alteration in clock-gene oscillations could modify the daily rhythm in abcb1a expression.
To address the issue, the influences of the time-restricted feeding procedure on the daily rhythm of abcb1a mRNA expression in intestine and its transporting activity were determined in this study. Because glucocorticoid is one of the major circadian oscillators in the peripheral clock (Balsalobre et al., 2000), the influence of endogenous glucocorticoid on the daily change in abcb1a mRNA expression was also evaluated in adrenalectomized (ADx) rats.
Materials and Methods
Six-week-old male Wistar rats were purchased from Japan SLC (Shizuoka, Japan). They were housed under a 12:12-h light/dark cycle [lights on at 7:00 AM (Zeitgeber time, ZT0) and lights off at 7:00 PM (ZT12)] with a temperature of 24 ± 1°C and humidity of 60 ± 10%. All animals were exposed to the light/dark cycle for at least 2 weeks before the experiment. Unless otherwise specified, they had free access to food and water (ad libitum feeding). The experiment was performed in accordance with the Use and Care of Experimental Animals Committee of Jichi Medical University (Tochigi, Japan).
After the acclimatization period, time-restricted feeding was performed for 2 weeks. Rats were restricted to food between ZT3 and ZT9, but water was available throughout the day.
Experiment 1: Intestine Sample Collection.
Rats (n = 32) were randomly assigned to ad libitum feeding or time-restricted feeding groups. Animals in each group were randomly divided into four subgroups (n = 4 in each). The proximal segment of jejunum was collected at ZT0, ZT6, ZT12, or ZT18. Samples for real-time PCR experiments were stored in RNAlater RNA stabilization reagent (QIAGEN, Valencia, CA) at −80°C until assay.
Experiment 2: Perfusion Study.
Rats (n = 48) were randomly assigned to ad libitum feeding or time-restricted feeding groups. They were randomly divided into four subgroups (n = 6 in each). A proximal segment of jejunum (length 3.5 cm) was isolated at ZT0, ZT6, ZT12, or ZT18. The perfusion study described below was immediately started after isolation.
Adrenalectomy and Corticosterone Replacement
Adrenal glands were removed via a dorsal approach under sodium pentobarbital [5-ethyl-5-(1-methylbutyl)-2,4,6-trioxohexahydropyrimidine] anesthesia (50 mg/kg i.p.). In a preliminary study, all animals died within 2 weeks after adrenalectomy alone. Therefore, corticosterone (11β,21-dihydroxy-4-pregnene-3,20-dione; 10 μg/h) (Makino et al., 1995) was continuously infused in ADx rats by an osmotic mini pump (Alzet, Cupertino, CA), and drinking water was replaced by 0.9% NaCl solution. The experiment was performed 2 weeks after the operation. Sham adrenalectomy was conducted by the same procedure without removal of adrenal glands.
Experiment 3: Tissue and Serum Collection.
Rats (n = 20) were randomly assigned to sham or ADx groups. A proximal segment of jejunum for real-time PCR and blood for corticosterone were collected at ZT0 or ZT12 (n = 5 at each point).
RNA Extraction and Real-Time PCR
Isolation of total RNA was carried out by using the RNeasy Mini kit according to the manufacturer’s instructions (QIAGEN). Reverse transcription was done with a AffinityScript QPCR cDNA synthesis kit (Agilent Technologies, Santa Clara, CA). Real-time PCR was performed with the Stratagene Mx3005P system (Agilent Technologies). Specific sets of primers and TaqMan probes (for abcb1a, dbp, hlf, e4bp4, and gapdh; TaqMan Gene Expression Assays) were obtained from Applied Biosystems (Foster City, CA). To control the variation in the amount of cDNA available for PCR in different samples, mRNA expression levels of the target sequences were normalized to the expression of an endogenous control, glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by the comparative threshold cycle method.
Everted Intestinal Perfusion Study for Assessment of P-gp Function
P-gp function was assessed by serosal (donor) to mucosal (receptor) efflux of digoxin, one of the P-gp substrates, using an everted intestinal perfusion system. Each segment was everted, and a stainless tube was cannulated at both ends of the intestine, which was tied by 5-0 silk. The excised jejunum was mounted in a chamber containing 45 ml of Dulbecco’s modified Eagle’s medium/F12 culture medium (Invitrogen, Carlsbad, CA) and preincubated at 37°C. After incubation for 15 min, serosal perfusate (Dulbecco’s modified Eagle’s medium/F12; 5 ml) containing 10 μg/ml digoxin (12β-hydroxydigitoxin, digosin injection; Chugai Pharmaceutical Co., Ltd, Tokyo, Japan) was circulated at a flow rate of 0.5 ml/min during the study. Subsequently, 0.2 ml of sample was withdrawn from the mucosal side every 30 min for 2 h and replaced with the same volume of blank medium. Digoxin concentration was measured with an automatic fluorescence polarization immunoassay analyzer (TDx; Abbott Japan Co., Tokyo, Japan). Throughout the study period, the mucosal side solution was continuously aerated with 95% O2 and 5% CO2 and warmed to 37°C in a water bath. As for the performance of the assay, the range of reliable response was 0.2 to 5.0 ng/ml, accuracy (calculated by observed/expected concentration of standards) was 94 to 104%, and precision was 0.5 to 1.9% (intraday) and 1.9 to 6.2% (interday).
Serum corticosterone concentration was measured by using a commercially available radioimmunoassay kit (RPA548; GE Healthcare, Little Chalfont, Buckinghamshire, UK). The assay was performed according to the instruction manual. Each sample was assayed twice. As for the performance of the assay, the range of reliable response was 0.78 to 200 ng/ml, accuracy (calculated by observed/expected concentration of standards) was 93 to 109%, and precision was 0.9 to 3.2% (intraday) and 1.5 to 7.0% (interday).
Groups were compared by one-way analysis of variance, and the difference between the two groups was determined by using Bonferroni-Dunn test. In the analysis of the digoxin concentration profile in the intestine perfusion study, groups were compared by repeated measure analysis of variance. P < 0.05 was considered to be significant.
Daily Rhythm in Rat Intestinal abcb1a mRNA Expression Level.
Rat intestinal abcb1a mRNA expression levels showed a significant daily rhythm with a peak at the early dark phase in the ad libitum feeding group (P < 0.01; Fig. 1A). In contrast, the parameter in the time-restricted feeding group had a significant daily rhythm with a trough in the early dark phase (P < 0.01; Fig. 1B).
Daily Rhythm in the P-gp Transporting Function in the Reversed Intestinal Perfusion System.
Digoxin concentration in the serosal fluid time-dependently increased in both the ad libitum and time-restricted feeding groups (Figs. 2 and 3). In the ad libitum feeding group, the increase of digoxin concentration was significantly larger at ZT18 than at ZT0 and ZT6 (P < 0.05). In addition, the AUC value in the concentration-time curve was significantly higher at ZT18 than at ZT0 and ZT6 (P < 0.05 and < 0.01, respectively). On the other hand, the increase of digoxin concentration was significantly larger at ZT6 than at ZT12 and ZT18 (P < 0.05) in the time-restricted feeding group. The AUC value was also significantly higher at ZT6 than at ZT12 and ZT18 (P < 0.01).
Daily Rhythm in DBP, HLF, and E4BP4 mRNA Expression.
DBP, HLF, and E4BP4 mRNA expressions in rat intestine showed the significant daily rhythms in both the ad libitum and time-restricted feeding groups. DBP mRNA peaked at ZT12 in the ad libitum feeding group and at ZT0 in the time-restricted feeding group (Fig. 4A). HLF mRNA peaked at ZT12 in the ad libitum feeding group and at ZT6 in the time-restricted feeding group (Fig. 4B). E4BP4 mRNA showed a similar daily rhythm with a peak at ZT18 in the ad libitum and time-restricted feeding groups (Fig. 4C).
Serum Corticosterone Concentration and Intestinal Gene Expression in ADx Rats.
Serum corticosterone concentration in the sham group was significantly higher at ZT12 than at ZT0 (P < 0.01; Fig. 5A). A lower level of the hormone was detected in the ADx group.
The intestinal abcb1a mRNA expression level was significantly higher at ZT12 than at ZT0 in both the sham and ADx groups (P < 0.01, sham; P < 0.05, ADx; Fig. 5B). Compared with the sham group, in the ADx group the expression level was significantly (P < 0.05) higher at ZT0 and tended to be higher at ZT12. On the other hand, DBP, HLF, and E4BP4 mRNA expression levels in the intestine did not significantly differ between the two groups at any observation points (Fig. 5, C–E).
In this study, rat intestinal abcb1a mRNA expression and P-gp activity showed a daily rhythm with a peak in the dark phase in the ad libitum feeding group, which is similar to our previous finding in mice (Ando et al., 2005). The time-restricted feeding procedure during the light period (ZT3–ZT9) shifted the circadian phase of abcb1a mRNA expression to 12-h advance. In addition, the peak of P-gp activity shifted to the light phase and synchronized with the daily rhythm of abcb1a mRNA expression in the time-restricted feeding group. Therefore, changing the feeding schedule could modify the daily rhythm of rat intestinal abcb1a mRNA expression and its activity. Some of the effects of time-restricted feeding on the daily rhythms in other functions have already been reported (Le Minh et al., 2001; Matsunaga et al., 2004; Koyanagi et al., 2006). In a previous report (Murakami et al., 2008), P-gp transporting activity was decreased, and subsequently its circadian rhythmicity was blunted in the clock-mutant mice. In this study, although the daily rhythm of abcb1a mRNA expression in rat intestine was altered under the time-restricted feeding condition, the P-gp activity in the time-restricted feeding group was similar to that in the ad libitum feeding group. These observations support the idea that oscillatory expressions in clock genes are important for composing a circadian expression in abcb1a mRNA expression and its activity.
Transcriptional regulation of clock genes via the D-box is reported to be involved in the regulation of circadian expression of abcb1a mRNA in mouse intestine (Murakami et al., 2008). The consensus sequence of the D-box (RTTAYGTAAY) existed in the 5′-flanking region of the rat abcb1a gene. DBP and HLF are the transcript activators of the D-box, and the peak times of their mRNA were identical to that of abcb1a mRNA in the ad libitum feeding group in this study. In addition, the peak time of mRNA expression of E4BP4, which is a transcript repressor of D-box, was ZT18, and 6 h later (ZT0), abcb1a mRNA expression showed the trough level in the ad libitum feeding group. These results suggest that the daily expression of intestinal abcb1a mRNA in these nocturnal rodents is at least in part modulated by the transcriptional regulation of clock genes via the D-box.
Time-restricted feeding is well known to alter the phase of gene expression rhythm in peripheral tissues (Damiola et al., 2000; Le Minh et al., 2001; Stokkan et al., 2001; Koyanagi et al., 2006). In this study, the time-restricted feeding procedure shifted the circadian phases of expression in not only abcb1a mRNA, but also clock genes. The circadian phases of DBP and HLF mRNA expression were advanced by 12 and 6 h, respectively, whereas the peak time of E4BP4 mRNA expression was not altered during the time-restricted feeding procedure under the present conditions. Therefore, the influence of the time-restricted feeding procedure on clock genes might be diverse. Murakami et al. (2008) reported that HLF, which is one of the proline and acidic amino acid-rich basic leucine zipper proteins, plays a role in the circadian regulation of abcb1a mRNA expression in mouse intestine. In this study, the peak time of HLF mRNA expression in the time-restricted feeding group was 6 h backward compared with that of abcb1a mRNA expression. However, the peak time (ZT0) of DBP mRNA expression was similar to that of abcb1a mRNA expression in the time-restricted feeding group. Therefore, it is probable that the role of DBP is greater than that of HLF in the circadian regulation of the rat intestinal abcb1a gene.
Glucocorticoid is one of the circadian oscillators in the peripheral clock (Balsalobre et al., 2000). In addition, it is well known that the manipulation of a feeding schedule has influenced the rhythm of endogenous glucocorticoid secretion. The corticosterone concentration showed that a significant daily rhythm peaked at ZT12 in the ad libitum feeding group. However, its rhythm was dampened in the time-restricted feeding group (Supplemental Fig. 1). Based on these data, endogenous glucocorticoids did not contribute to modulating the rhythm of abcb1a expression by the time-restricted feeding procedure.
In the ADx experiment, the abcb1a mRNA expression level of the ADx group was significantly higher than that of the sham group at ZT0 and ZT12. In addition, adrenalectomy did not change the expression level and the time-dependent change of DBP, HLF, or E4BP4 mRNA. Therefore, it is speculated that the transcription of the abcb1a gene in rat intestine is partially suppressed by endogenous corticosterone via DBP-, HLF-, or E4BP4-unrelated mechanisms. Several in vitro studies have shown that the effect of glucocorticoid on P-gp expression differs among the cell types (Fardel et al., 1993; Zhao et al., 1993; Schuetz et al., 1995; Sérée et al., 1998). For example, dexamethasone, a potent glucorticoid, decreased (kidney), increased (liver), or did not affect (testis, heart, muscle, spleen, and stomach) P-gp expression in rat (Demeule et al., 1999). In addition, the possibility remains that the binding of DBP and HLF to D-box in the abcb1a gene promoter is enhanced by adrenalectomy. Thus, the influence of glucocorticoid on P-gp expression is quite complex, and a mechanism responsible for the elevation of rat intestinal abcb1a mRNA expression by adrenalectomy observed in this study remains to be determined.
In this study, we showed the daily rhythms in abcb1a mRNA expression and P-gp transporting activity in rat intestine in an ad libitum feeding group, which were shifted by the time-restricted feeding procedure. These results indicate that time-restricted feeding might alter the pharmacokinetic profiles of drugs, which are substrates of P-gp, and subsequently lead to the intraindividual variety in pharmacokinetics. Further study is needed to evaluate the influence of irregular meal timing on the pharmacokinetics and pharmacodymanics of drugs and clinical outcomes of patients.
This study was supported by a Grant-in-Aid for Young Scientists (B) [Grant K.U. 22790166] and subsidized by the Japan Keirin Association through promotion funds from Keirin Race [Grants 901811, 902006].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- D-site binding protein
- E4 promoter binding protein-4
- hepatic leukemia factor
- suprachiasmatic nuclei
- Zeitgeber time
- polymerase chain reaction
- area under the curve.
- Received June 6, 2010.
- Accepted July 20, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics