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Expression of Hepatic UDP-Glucuronosyltransferase 1A1 and 1A6 Correlated with Increased Expression of the Nuclear Constitutive Androstane Receptor and Peroxisome Proliferator-Activated Receptor in Male Rats Fed a High-Fat and High-Sucrose Diet

Department of Pharmaco-Biochemistry and Global Center of Excellence for Innovation in Human Health Sciences, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan (M.O., J.S., T.F., A.I., M.M.); and Biotechnology Research Center, Toyama Prefectual University, Toyama, Japan (S.I.)

(Received July 24, 2007; Accepted October 26, 2007)

	Abstract

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Rats that consumed a high-fat and high-sucrose (HF1) diet or a high-fat (HF2) diet developed hepatic steatosis. The alteration in nutritional status affected hepatic cytochrome P450 and UDP-glucuronosyltransferase (UGT) levels. Messenger RNA and protein levels of UGT1A1 and UGT1A6 in the liver but not the jejunum were increased in male rats fed the HF1 diet. These protein levels did not increase in HF2-fed male rats or HF1-fed female rats. In contrast, the CYP1A2 protein level was decreased in the HF1 but not HF2 diet group, whereas CYP2E1 and CYP4A protein levels were elevated in the HF2 but not HF1 diet group. No significant difference in the organic anion transporter polypeptide (Oatp) 1, Oatp2, multidrug resistance-associated protein (Mrp) 2, or Mrp3 protein levels was found between the standard and HF1 diet groups of male rats. Consumption of the HF1 diet affected the in vivo metabolism of acetaminophen (APAP) such that the area under the APAP-glucuronide plasma concentration-time curve was elevated 2.1-fold in male rats but not female rats. In liver cell nuclei of male rats but not female rats, constitutive androstane receptor (CAR) and proliferator-activated receptor (PPAR) protein levels were significantly enhanced by intake of the HF1 diet. Additionally, administration of the PPAR agonist clofibrate to male rats up-regulated UGT1A1 and UGT1A6 and down-regulated CYP1A2 in the liver. Taken together, these results indicate that nutritional status may gender-specifically influence the expression and activation of CAR and PPAR in liver cell nuclei, and this effect appears to be associated with alterations in UGT1A1 and UGT1A6 expression.

Acetaminophen (APAP) at therapeutic doses is mainly eliminated through glucuronidation and sulfation, although a small fraction is oxidized by CYP2E1, CYP3A, and CYP1A2 to N-acetyl-p-benzoquinoneimine (NAPQI), a toxic metabolite. Therefore, in this study, we investigated whether elevated levels of UDP-glucuronosyltransferase (UGT) 1A1 and UGT1A6 proteins influence the in vivo metabolism of APAP. Furthermore, to examine whether there is a gender-specific difference in the metabolism of APAP, we characterized the plasma concentration-time profiles of APAP and APAP glucuronide (AG) in male and female rats fed standard (SD) and HF1 diets.

There have been several studies on the regulation of UGT gene expression by transcriptional factors (Mackenzie et al., 2003). We have localized the PB response enhancer activity of human UGT1A1 to the 290-base pair PB responsive enhancer module (gtPBREM) located at –3499/–3210 and have identified the nuclear receptor constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glucocorticoid receptor as transcription factors that regulate gtPBREM (Sugatani et al., 2001, 2005). In addition, an aryl hydrocarbon receptor (AhR) response element and peroxisome proliferator-activated receptor (PPAR) response element have also been reported to be localized within the 290-base pair gtPBREM (Yueh et al., 2003; Senekeo-Effenberger et al., 2007). Previous studies on UGT1A6 gene expression documented that AhR and nuclear factor E2-related factor 2 (Nrf2) binding sites are located in the human UGT1A6 5'-regulatory region (–2052/–1), and hepatocyte nuclear factor 1 (HNF1), CAR, and PXR binding sites were identified by computer-based homology analysis of the regulatory region (Krishnaswamy et al., 2005). Moreover, HNF4 is a regulator of coordinated nuclear receptor (such as CAR and PXR)-mediated responses to xenobiotics (Triona et al., 2003), and peroxisome proliferator-activated receptor-coactivator 1 (PGC1) modulates the function of nuclear receptors such as CAR as a common coactivator (Puigserver and Spiegelman, 2003). To characterize gender-specific differences, we further examined the effect of the HF1 diet on the expression of transcription factors associated with sugar and lipid metabolism, including PPAR and PPAR, in liver cell nuclei.

	Materials and Methods

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Animals and Experimental Protocol. All the studies followed protocols approved by the Institutional Animal Care and Life Committee, University of Shizuoka. Male and female Wistar rats were obtained from Japan SLC (Hamamatsu, Japan) at 4 or 6 weeks of age and were acclimatized for 1 week before the experiment. Animals were housed in stainless steel hanging cages with free access to food and water and maintained on a 12-h light/dark cycle. All the animals were randomly assigned to SD diet, HF1 diet, or high-fat diet (HF2 diet) for 2, 4, and 8 weeks. The HF1 diet consisted of 19.7% casein, 1% soybean oil, 10% lard, 4% mineral mixture, 1% vitamin mixture, 0.15% choline chloride, 0.5% cholesterol, 0.25% sodium cholate, 3.4% cellulose, and 60% sucrose [23.6% lipid, 57.4% carbohydrate, and 18.9% protein (kJ)]; the HF2 diet consisted of 23% casein, 19.8% corn oil, 19.8% lard, 6.7% mineral mixture, 1.4% vitamin mixture, 0.2% choline chloride, 5% cellulose, and 24.1% sucrose [65.4% lipid, 17.7% carbohydrate, and 16.9% protein (kJ)]; and the SD diet consisted of 23.8% crude protein, 5.1% crude fat, 3.2% crude fiber, 6.1% ash, 54% nitrogen-free extract, and 7.8% water [12.9% lipid, 60.4% carbohydrate, and 26.7% protein (kJ)]. Three times per week, all the rats were weighed, and food intake in grams was monitored.

Administration of acetaminophen: at 11 weeks of age after being fed the SD or HF1 diet for 4 weeks, animals were randomly assigned to treatment (APAP) or vehicle groups. APAP was dissolved in 37°C warm 0.9% saline solution (100 mg/ml) with the pH adjusted to 10 with 0.1 N NaOH and injected i.p. at 500 mg/kg b.wt. between 8:00 AM and 9:00 AM. At different time points after APAP administration (0, 1, 2, 4, 8, and 24 h), blood was taken for measuring the plasma concentrations of APAP and AG.

Administration of 1,7-phenanthroline (PH): at 13 weeks of age after being fed the SD or HF1 diet for 8 weeks, male rats were randomly assigned to treatment (PH) (Sigma-Aldrich, St. Louis, MO) or vehicle groups. PH was dispersed in 0.5% methylcellulose containing 0.1 M citric acid (75 mg/ml) and injected p.o. at 75 mg/kg b.wt. once daily for 3 days. The experiments were done 24 h after the last administration of PH.

Administration of PB: the SD-fed male rats at 11 weeks of age were administered PB (Wako Pure Chemicals, Osaka, Japan) dissolved in 0.9% sodium chloride (80 mg/kg) or vehicle by i.p. injection once daily for 4 days between 8:00 AM and 9:00 AM. The experiments were done 4 h after the last administration of PB.

Administration of clofibrate (CFB): the SD-fed rats at 11 weeks of age were administered CFB (Sigma-Aldrich) dissolved in corn oil (300 mg/ml/kg) or vehicle by i.p. injection once daily for 5 days between 8:00 AM and 9:00 AM. The experiments were performed 4 h after the last administration of CFB.

Blood and Tissue Sampling. Rats were anesthetized with diethyl ether, and then the abdominal cavity was rapidly opened, and blood was rapidly and simultaneously drawn from the abdominal vena cava into syringes. Plasma samples were separated from blood collected into heparinized tubes by centrifugation, and serum samples were separated from blood by centrifugation after standing for 30 min at room temperature. The resulting plasma/serum was stored at –30°C until analysis. Livers were rapidly excised and immediately placed in liquid nitrogen. The median lobe of the liver was excised for RNA extraction and for the preparation of microsomes, plasma membranes, and nuclear extracts. Aliquots were snap-frozen and stored at –80°C until analysis.

Preparation of Microsomes. Liver microsomes were prepared by differential centrifugation, first at 900g for 10 min and then at 9000g for 15 min, followed by 105,000g for 60 min at 4°C and stored at –80°C. The microsomal protein concentration was determined with a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL) using bovine serum albumin as a standard.

Membrane Preparation. Crude liver membranes were prepared as described previously (Ogawa et al., 2000). Liver (about 1.6 g) was homogenized in 5 volumes of 0.1 M Tris-HCl buffer, pH. 7.4, containing 1 µg/ml leupeptin and pepstatin A and 50 µg/ml phenylmethylsulfonyl fluoride with 20 strokes of a Dounce homogenizer. After centrifugation at 1500g for 10 min, the supernatant was centrifuged at 100,000g for 30 min. The precipitate was suspended in 6 ml of the buffer and again centrifuged at 100,000g for 30 min. The crude membrane fraction was resuspended in 0.1 M Tris-HCl buffer, pH 7.4, containing the above proteinase inhibitors using five strokes of a Potter homogenizer.

Preparation of Nuclear Extracts. Liver nuclear extracts were prepared as described previously (Sugatani et al., 2006). Livers (about 1 g) were homogenized in 5 volumes of 10 mM HEPES-NaOH buffer, pH 7.6, containing 25 mM KCl, 2 M sucrose, 10% glycerol, 0.15 M spermine, 0.5 mM spermidine, 1 mM EDTA, 1 mM dithiothreitol, 1 µg of leupeptin/ml, 1 µg of pepstatin/ml, 1 µg of aprotinin/ml, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄ with three strokes of a Teflon-glass homogenizer. The homogenate was carefully layered onto 5 ml of the same buffer in a centrifuge tube, and this was followed by centrifugation at 25,000 rpm and 4°C for 60 min with an SW28 rotor (Himac CP56GII, Hitachi Koki, Hitachinaka, Japan). The precipitate was homogenized in 1 ml of lysis buffer (10 mM HEPES-NaOH buffer, pH 7.6, containing 0.1 M KCl, 3 mM MgCl₂, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 1 µg of leupeptin/ml, 1 µg of pepstatin/ml, 1 µgof aprotinin/ml, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄) with 10 strokes of a Dounce homogenizer. Sodium chloride solution was added to the homogenate to bring the final concentration to 0.4 M, and the homogenate was incubated at 4°C for 30 min with gentle shaking. The supernatant after centrifugation at 105,000g for 20 min was dialyzed against 1 l of dialysis buffer (20 mM HEPES-NaOH buffer, pH 7.6, containing 0.1 M NaCl, 20% glycerol, 0.2 mM EDTA, 1 mM DTT, 1 µg of leupeptin/ml, 1 µg of pepstatin/ml, 1 µg of aprotinin/ml, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄) at 4°C overnight and stored at –80°C. The protein concentration was determined by the Bradford assay (Bio-Rad protein assay kit, Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as a standard.

Immunoblot Analysis. Microsomal proteins or nuclear extracts (20 µg) were resolved by electrophoresis on an SDS-12.5% polyacrylamide gel, and plasma membrane fractions (50 µg) were resolved on an SDS-7.5% polyacrylamide gel and electroblotted onto a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA). The immunoblots were incubated with the following primary antibodies: rat CYP1A1: the antibody recognizes rat CYP1A1 and CYP1A2 (the molecular weight of rat CYP1A2 is lower than that of rat CYP1A1), rat CYP2B1, rat CYP2E1, rat CYP3A2, rat CYP4A1, and rat NADPH-cytochrome P450 (P450) reductase (Daiichi Pure Chemicals Co., Tokyo, Japan), human multidrug resistance-associated protein (MRP) 2 (sc-5770), human MRP3 (sc-5776), mouse CAR (sc-13065), mouse PXR (sc-7737), human retinoid X receptor (RXR) (sc-553), human PPAR (sc-9000), human PPAR (sc-7273), human AhR (sc-8088), human AhR nuclear translocator (ARNT) (sc-8076), human HNF1 (sc-6547), human HNF4 (sc-6556), human Nrf2 (sc-13032), human PGC1 (sc-13067), and human histone H1 (sc-8030) (Santa Cruz Biotechnology, Santa Cruz, CA), rat organic anion transporter polypeptide (Oatp) 1 and rat Oatp2 (Alpha Diagnostic International, San Antonio, TX), chicken -tubulin (Oncogen Research Products, Boston, MA), and rabbit antibodies against rat UGT1A1-, UGT1A6-, UGT1A7-, and UGT2B1-specific peptides (Ikushiro et al., 1995, 1997). Antigen-antibody complexes were detected using the appropriate secondary antibody conjugated with horseradish peroxidase and visualized with an enhanced chemiluminescence system (GE Healthcare Bio-Sciences, Piscataway, NJ). The signal intensities were determined with a Fujix BAS-2000 bioimage analyzer (Fuji Photo Film, Tokyo, Japan).

Determination of mRNA Levels. Total RNA was prepared from the liver or jejunum using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). Samples were quantitated by spectrophotometry, and 1 µg of total RNA was used to generate cDNA by reverse transcription using a Prime Script reverse transcription reagent kit (Takara, Ohtsu, Japan) according to the manufacturer's protocol. cDNA synthesized from 50 ng of total RNA was subjected to quantitative real-time polymerase chain reaction as described previously (Sugatani et al., 2006) with an ABI PRISM 7000 Sequence Detector instrument (Applied Biosystems, Foster City, CA) using Premix Ex Taq reagent (Takara) for the TaqMan probe method or SYBR Premix Ex Taq reagent (Takara) for the intercalation reaction with SYBR Green I according to the manufacturer's specifications. The TaqMan probes and primers for rat CAR (NM_022941 [GenBank] ) (Hartley et al., 2004) and rat UGT1A1 (NM_012683 [GenBank] ), UGT1A6 (NM_057105 [GenBank] ), and UGT2B1 (NM_173295 [GenBank] ) (Wang et al., 2003) were as reported previously, and those for rat PPAR (NM_013196 [GenBank] ) (assay identification number Rn00566193_m1) and rat β-actin (NM_031144 [GenBank] ) (assay identification number Rn00667869_ml) were assay-on-demand gene expression products (Applied Biosystems). The gene-specific probes were labeled by using reporter dye FAM, and the β-actin internal control probe was labeled with a different reporter dye, VIC, at the 5' end. The primers for rat UGT1A7 (NM_130407 [GenBank] ) (Grams et al., 2000) and rat β-actin (Sugatani et al., 2006) were as reported previously. The thermal cycle conditions were as follows: incubate for 10 s at 95°C, followed by two-step polymerase chain reaction for 40 cycles of 95°C for 5 s followed by 60°C for 30 s. β-Actin was used to normalize gene expression in all the samples. -Fold induction values were calculated by subtracting the mean difference of gene and β-actin cycle threshold Ct numbers for each treatment group from the mean difference of gene and β-actin Ct numbers for the vehicle group and raising the difference to the power of 2 (2^-Ct).

Biochemical Analyses. Serum glucose concentration was determined by the hexokinase method using commercial reagents (R-Biopharm AG, Darmstadt, Germany). Serum insulin was measured using a double antibody radioimmunoassay kit specific for rat insulin (Eiken Chemical Co., Tokyo, Japan). Serum aspartate aminotransferase, alanine aminotransferase, and -glutamyl transpeptidase were measured using kits from Wako Pure Chemicals (Osaka, Japan). Serum levels of triacylglycerol and total cholesterol were measured enzymatically with kits from Shino Test (Tokyo, Japan). Frozen livers (about 0.5 g) were homogenized in 20 volumes (the SD group) or 100 volumes (the HF group) of 0.9% NaCl containing 0.1% Triton X-100, and the concentrations of triacylglycerol, total cholesterol, and nonesterified fatty acid were estimated with kits from Shino Test.

Study of Metabolism of APAP. APAP and AG in plasma were determined using a modification of a method previously published (Esteban et al., 1992). To 50 µl of plasma, 5 µl of 5-hydroxy-L-tryptophan (2.5 µg) as an internal standard and 10 µl of 35% w/v perchloric acid were added. After centrifugation for 10 min at 2200g, 10 µl of the supernatant was injected into a high-performance liquid chromatography system, which consisted of a CO-8020 injector, DP-8020 pump, Mightysil RP-18GP Guard Pak precolumn cartridge, Mightysil RP-18GP (4.6 x 250 mm) reverse-phase column, and a PD-8020 spectrophotometer (TOSOH, Tokyo, Japan). Detection was performed by measuring absorption at 254 nm. An elution gradient was applied, beginning with 15% acetonitrile in an aqueous solution of 20 mM phosphate buffer (pH 4.5) at a flow rate of 1.0 ml. After 6 min, acetonitrile was increased to 30% over 2 min. At 15 min, acetonitrile was increased to 70% over 2 min, and 5 min later was returned to 15%. Under these conditions, AG, internal standard, and APAP eluted at 2.8, 3.2, and 5.3 min, respectively. Standard curves for APAP and AG in plasma (2.5–800 µg/ml) were linear (r 0.997).

Statistics. Values are expressed as the mean ± S.E. All the data were analyzed using one-way analysis of variance. The difference between the means of the groups was tested for significance using Student's t test. The level of statistical significance was set at p < 0.05.

	Results

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Nutritional Status Modulates Hepatic Expression of UGT mRNA and Proteins in Male Rats. Consumption of the HF1 diet by male rats for 2 to 8 weeks and the HF2 diet for 8 weeks resulted in major increases in the absolute liver weight, relative liver weight, and liver lipids, including a marked accumulation of triacylglycerols, total cholesterol, and free fatty acids in the livers, with higher accumulation of lipids by the HF1 diet than the HF2 diet at 8 weeks (Fig. 1). Male rats fed the HF1 and HF2 diets for 8 weeks showed hepatic steatosis, which was not associated with an increase in serum aspartate aminotransferase, alanine aminotransferase, and -glutamyl transpeptidase levels (data not shown). Table 1 shows that mRNA levels of UGT1A1, UGT1A6, and UGT1A7 were increased, and the UGT2B1 mRNA level tended to increase in the liver but not the jejunum of male rats fed the HF1 diet for 8 weeks. The UGT1A1, UGT1A6, UGT1A7, and UGT2B1 mRNA levels did not increase in male rats fed the HF2 diet. To determine whether the observed changes in mRNA levels translated into altered hepatic protein levels, we determined the protein levels by Western blot analysis (Fig. 2). Consistent with the changes in the mRNA levels, the protein levels of UGT1A1, UGT1A6, UGT1A7, and UGT2B1 were significantly increased by consumption of the HF1 diet for 8 weeks (Fig. 2). A decrease in the protein level of CYP1A2, an increase in the UGT1A1 protein level, and a tendency toward an increase in the UGT1A6 protein level in the liver were found after 2 weeks of consumption of the HF1 diet. In contrast, the change in the protein levels of CYP3A and UGT1A7 in the liver was dependent on the intake period of the HF1 diet; the levels of these proteins in the HF1 diet group were decreased at 2 weeks and increased at 8 weeks, whereas in the HF2 diet group they were decreased at 8 weeks (Fig. 2). The hepatic microsomal contents of CYP2E1 and CYP4A, which is considered to function in the microsomal catalysis of lipid peroxides in nonalcoholic steatohepatitis (Leclercq et al., 2000), were significantly increased in the HF2 diet group but not in the HF1 diet group (Fig. 2).

View larger version (39K): [in this window] [in a new window]   FIG. 1. Effects of HF1 diet and HF2 diet on body weight, liver weight, and liver lipids in male rats. Rats (5 weeks of age) were fed SD, HF1, or HF2 diet for 2, 4, and 8 weeks and then sacrificed. Values are the mean ± S.E. of three to six determinations. *, p < 0.05, **, p < 0.01, ***, p < 0.001 for HF1 male rats versus SD male rats or HF2 male rats versus SD male rats; #, p < 0.05, ###, p < 0.001 for 4- or 8-week SD male rats versus 2-week SD male rats.

View larger version (50K): [in this window] [in a new window]   FIG. 2. Effects of HF1 diet and HF2 diet on expression of hepatic drug-metabolizing enzyme proteins in male rats. Rats (5 weeks of age) were fed SD, HF1, or HF2 diet for 2, 4, and 8 weeks and then sacrificed, and the microsomal proteins (20 µg/lane) were prepared and subjected to immunoblot analysis. Relative levels were expressed by taking the control values obtained from 2-week SD male rats as 100. Values are the mean ± S.E. of three or four determinations. *, p < 0.05, **, p < 0.01, ***, p < 0.001 for HF1 male rats versus SD male rats or HF2 male rats versus SD male rats; ##, p < 0.01, ###, p < 0.001 for 4- or 8-week SD male rats versus 2-week SD male rats.

Gender-Specific Difference in UGT Protein Levels in Livers of Rats Fed the SD or HF1 Diet. Consumption of the HF1 diet resulted in a major increase in liver lipids, including triacylglycerols, total cholesterol, and free fatty acids, after 4 weeks of the dietary regimen in both female and male rats, with the exception that serum glucose levels were significantly increased in male rats (1.53 ± 0.05 g/l in HF1 diet-fed male rats and 1.38 ± 0.03 g/l in SD diet-fed male rats, p < 0.01) but not in female rats (1.40 ± 0.02 and 1.43 ± 0.03 g/l in SD and HF1 diet-fed rats, respectively). Liver baseline levels of CYP1A2, UGT1A7, and UGT2B1 mRNA and proteins were higher in female rats than in male rats (Fig. 3 and Supplemental Fig. 1). Intake of the HF1 diet for 4 weeks decreased the CYP1A2 mRNA and protein levels in livers of male and female rats and increased UGT1A1 and UGT1A6 mRNA and protein levels in livers of male rats but not female rats (Fig. 3 and Supplemental Fig. 1). UGT1A7 and UGT2B1 mRNA and protein levels in female rats were decreased to similar levels as those in control male rats by intake of the HF1 diet for 4 weeks. In contrast, there was no significant change in protein levels of CYP2E1, CYP3A, or NADPH-P450 reductase in male and female rats after consumption of the HF1 diet (Fig. 3).

View larger version (28K): [in this window] [in a new window]   FIG. 3. Effects of HF1 diet on expression of hepatic drug-metabolizing enzyme proteins in male and female rats. Rats (7 weeks of age) were fed SD or HF1 diet for 4 weeks and then sacrificed, and the microsomal proteins (20 µg/lane) were prepared and subjected to immunoblot analysis. Relative levels were expressed by taking the control values obtained from SD male rats as 100. Values are the mean ± S.E. of five determinations. *, p < 0.05, **, p < 0.01, ***, p < 0.001 for HF1 male rats versus SD male rats or HF1 female rats versus SD female rats; #, p < 0.05, ###, p < 0.001 for SD female rats versus SD male rats.

Effect of HF1 Diet on the Hepatic Expression of Drug Transporters. Changes in the protein levels of several basolateral and canalicular hepatic transporters were assessed in male and female rats after consumption of the HF1 diet for 4 weeks (Fig. 4). In rats fed the SD diet, protein expression significantly differed between genders and was higher (Mrp3), comparable (Mrp2), or lower (Oatp1, Oatp2) in female rats than in male rats (Fig. 4). No significant changes of the Oatp1, Mrp2, or Mrp3 protein levels in male and female rats were detected after consumption of the HF1 diet, whereas the Oatp2 protein level tended to increase in the male rats and decrease in the female rats. Western blot immunostaining for -tubulin confirmed equivalent protein loading.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Effects of HF1 diet on expression of hepatic drug transporter proteins in male and female rats. Rats (7 weeks of age) were fed SD or HF1 diet for 4 weeks and then sacrificed, and the plasma membrane fraction (50 µg/lane) was prepared and subjected to immunoblot analysis. Relative levels were expressed by taking the control values obtained from SD male rats as 100. Values are the mean ± S.E. of four or five determinations. *, p < 0.05 for HF1 male rats versus SD male rats or HF1 female rats versus SD female rats; ##, p < 0.01, ###, p < 0.001 for SD female rats versus SD male rats; ++, p < 0.01, +++, p < 0.001 for HF1 female rats versus HF1 male rats.

View larger version (46K): [in this window] [in a new window]   FIG. 5. Plasma levels of APAP (A) and its AG (B) in male and female rats fed SD or HF1 diet for 4 weeks and then administered APAP (500 mg/kg). Rats (7 weeks of age) were fed the two diets for 4 weeks and then administered APAP (500 mg/kg), and then sacrificed. Values are the mean ± S.E. of five determinations. *, p < 0.05, **, p < 0.01 for HF1 male rats versus SD male rats or HF1 female rats versus SD female rats.

View larger version (44K): [in this window] [in a new window]   FIG. 6. Effects of HF1 diet on expression of transcription factor proteins in the liver cell nuclei of male and female rats fed SD or HF1 diet for 4 weeks. Rats (7 weeks of age) were fed the two diets for 4 weeks and then sacrificed, and the nuclear proteins (30 µg/lane) were prepared and subjected to immunoblot analysis. Relative levels were expressed by taking the control values obtained from SD male rats as 100. Values are the mean ± S.E. of four determinations. *, p < 0.05, **, p < 0.01 for HF1 male rats versus SD male rats or HF1 female rats versus SD female rats; ##, p < 0.01, ###, p < 0.001 for SD female rats versus SD male rats.

We further examined the mRNA levels of CAR and PPAR in livers of male and female rats fed the SD and HF1 diets. The baseline mRNA level of PPAR in female rats was half of the level in male rats, and the mRNA level of CAR and PPAR in male rats fed the HF1 diet significantly increased to 1.4- or 1.6-fold over the control level in male rats fed the SD diet, respectively, whereas there was no significant difference in the mRNA levels of CAR or PPAR in female rats (data not shown).

Effect of Treatment of Male Rats with AhR Ligand, CAR Activator, or PPAR Ligand on the Expression of P450 and UGT Proteins in the Liver. Rat CYP1A1 and UGT1A6 include the xenobiotic response element (XRE) sequences (Auyeung et al., 2003), but intake of the HF1 diet decreased the CYP1A1 protein level and increased the UGT1A6 protein level (Figs. 2, 3, and 7). To investigate whether the responses of CYP1A1 and UGT1A6 to AhR ligand PH were influenced by intake of the HF1 diet, we examined the effect of PH on the protein levels of CYP1A1, CYP1A2, UGT1A1, and UGT1A6 in livers of male rats fed the SD and HF1 diets. Treatment of the SD-fed male rats with PH increased the liver protein levels of CYP1A1, CYP1A2, and UGT1A6 but not UGT1A1. Whereas the extent of induction of CYP1A1 by PH was higher than that of CYP1A2, UGT1A6 was more markedly increased among these enzymes in male rats fed the SD diet. Moreover, treatment of the HF1-fed male rats with PH significantly elevated not only the protein levels of CYP1A1 and CYP1A2 that were decreased by the HF1 diet but also the UGT1A6 protein level to 12.6-, 4.1-, and 4.3-fold the vehicle-treated control levels, respectively (Fig. 7).

View larger version (38K): [in this window] [in a new window]   FIG. 7. Effects of PH treatment on expression of CYP1A1, CYP1A2, UGT1A1, and UGT1A6 proteins in male rats fed SD or HF1 diet for 8 weeks. Male rats (13 weeks of age) were administered PH (75 mg/kg, p.o.) or vehicle (C) once daily for 3 days and then sacrificed, and the microsomal proteins (20 µg/lane) were prepared and subjected to immunoblot analysis. Relative levels were expressed by taking the control values obtained from SD male rats as 100. Values are the mean ± S.E. of 3 determinants. *, p < 0.05, ***, p < 0.001 for PH-administered animals versus vehicle-administered animals.

Next, because the protein levels of CAR and PPAR were elevated in liver cell nuclei of male rats fed the HF1 diet, we examined the effect of the CAR activator PB on the protein levels of UGT1A1, UGT1A6, UGT1A7, and UGT2B1 compared with CYP2B (Fig. 8A) and the effect of the PPAR ligand CFB on the protein levels of CYP1A2, UGT1A1, and UGT1A6 compared with CYP4A (Fig. 8B). Treatment of SD-fed male rats with PB increased the liver protein levels of UGT1A1, UGT1A6, and UGT2B1 to 1.3-, 4.2-, and 2.5-fold over the vehicle-treated control levels; these extents of induction were similar to that of CYP2B (3.0-fold) (Fig. 8A). Furthermore, to investigate whether an increase in the PPAR protein level in liver cell nuclei was associated with decreased protein levels of CYP1A1 and CYP1A2 and increased UGT1A1 and UGT1A6 protein levels in male rats fed the HF1 diet, we treated male rats with the PPAR ligand CFB. As shown in Fig. 8B, administration of CFB to male rats reduced the protein level of CYP1A2 to 20% of the control level in the liver. In addition, the protein levels of UGT1A1 and UGT1A6 in the liver after treatment with CFB were increased to 1.8- and 1.9-fold over the vehicle-treated control levels, respectively; the extents of induction were similar to that of CYP4A, whose induction by CFB is mediated via PPAR (Zhou et al., 2002) (Fig. 8B).

View larger version (26K): [in this window] [in a new window]   FIG. 8. Effects of PB (A) or CFB (B) treatment on expression of hepatic CYP2B, UGT1A1, UGT1A6, UGT1A7, and UGT2B1 proteins (A) or hepatic CYP1A2, CYP4A, UGT1A1, and UGT1A6 proteins (B) in male rats fed the SD diet. Male rats (11 weeks of age) were administered PB (80 mg/kg, i.p.) or vehicle (0.9% sodium chloride) once daily for 4 days (A) or CFB (300 mg/kg, i.p.) or vehicle (corn oil) once daily for 5 days and then sacrificed, and the microsomal proteins (20 µg/lane) were prepared and subjected to immunoblot analysis. Relative levels are expressed by taking the control values obtained from SD animals as 100. Values are the mean ± S.E. of five determinations. *, p < 0.05, **, p < 0.01, ***, p < 0.001 for PB- or CFB-administered animals versus vehicle-administered animals.

	Discussion

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There have been many epidemiological studies on the inducibility of hepatic CYP1A enzymes by diet and its association with the metabolism of environmental and dietary carcinogens. Murray (2006) reported that dietary indolyl glucosinolates and flavonoids induce CYP1A expression either by direct ligand interaction with AhR or by augmenting the interaction of AhR with XRE in CYP1A1 and other target genes. Ciprofibrate, a PPAR ligand widely used in the treatment of hyperlipidemia, has been reported to decrease the ethoxyresorufin-O-deethylase activity of CYP1A1 in rats (Makowska et al., 1990). In addition, Shaban et al. (2004) showed that when Wistar rats were treated with the AhR ligand Sudan III and the PPAR ligand CFB for 3 days, AhR protein expression, CYP1A1/2 mRNA and protein expression, and the related metabolic activities of ethoxyresorufin-O-deethylase and methoxyresorufin-O-deethylase were down-regulated in a PPAR-dependent manner. Because the PPAR protein level in liver cell nuclei was elevated in male rats fed the HF1 diet, the suppression of CYP1A1 and CYP1A2 may be PPAR-dependent (Figs. 2, 3, 7, and 8B).

This study further showed that intake of the HF1 diet was associated with increases in hepatic UGT1A1 and UGT1A6 mRNA and protein levels (Table 1; Figs. 2, 3, and 7). UGT conjugate endogenous lipophilic substrates such as bilirubin, thyroxine, estradiol, and serotonin, as well as xenobiotics such as acetaminophen, morphine, nonsteroidal anti-inflammatory drugs, flavonoids, and carcinogens with glucuronic acid (Radominska-Pandya et al., 1999; Tukey and Strassburg, 2000). Environmental compounds such as certain fresh fruits and vegetables in addition to xenobiotics such as therapeutic drugs have been found to be UGT inducers; stimulation of estradiol glucuronidation in human liver microsomes by soy isoflavone daizein has been reported to be associated with a reduction in the incidence of hormonal cancer by soy food (Pfeiffer et al., 2005). In addition, marked differences in tissue- and gender-specific expression patterns of UGT have been shown to exist in mice, potentially influencing drug metabolism and pharmacokinetics (Buckley and Klaassen, 2007). Our study has shown a gender-related change in the diet-induced expression of UGT1A1 and UGT1A6 in rats (Fig. 3) for which the mechanism remains to be clarified.

The glucuronidation of APAP in humans has been reported to be caused mainly by UGT1A1, UGT1A6, UGT1A9, and UGT2B15 (Mutlib et al., 2006). Relatively high levels of UGT1A1 and UGT1A6 are expressed in rat liver. In rats, UGT1A9 is a pseudogene whose function is considered to be replaced by UGT1A7 (Emi et al., 1995). A recent study showed that UGT1A6 and UGT1A7 possess similar kinetic properties regarding APAP catalysis, and their relative contributions in vivo are predicted to depend on their levels of expression, which are determined by environmental exposure (Kessler et al., 2002). To investigate whether the increase in UGT1A1 and UGT1A6 protein levels by intake of the HF1 diet affects the in vivo metabolism of drugs in rats, in this study we focused on the in vivo metabolism of APAP, which is predominantly metabolized to glucuronide and sulfate. APAP AG, a monovalent organic anion formed in hepatocytes, is a substrate for Mrp2, which is responsible for the biliary excretion, and Mrp3, which exports products of hepatic metabolism back into the sinusoidal blood (Xiong et al., 2002). Approximately 50% of AG formed in hepatocytes is excreted into the bile, and the rest is exported into the blood via the basolateral membrane (Xiong et al., 2002). In this study, in male rats fed the SD and HF1 diets, no significant difference in the AUC of APAP was found, although the maximum concentration of APAP in the HF1 diet group was higher than that in the SD diet group (Fig. 5). However, the AUC of AG in the HF1 diet group of male rats was 2.1-fold higher than that in the SD diet group. In contrast, there were no significant differences in the AUC of APAP or AG between female rats fed the SD and HF1 diets, in accord with the lack of a significant increase in the expression of UGT1A1, UGT1A6, and UGT1A7. These observations indicate that the elevation in the AUC of AG may reflect the enhancement of the in vivo glucuronidation of APAP by increased UGT1A1 and UGT1A6 protein levels because no significant change in the expression of Mrp2 and Mrp3 proteins was found in HF1 diet-fed male rats (Fig. 4) and the protein levels of CYP1A1, CYP1A2, and CYP2E1, which oxidize APAP to produce NAPQI, were also not increased (Figs. 2, 3, and 7).

Transcriptional up-regulation of drug-metabolizing phase 1 enzymes by xenobiotics occurs via CAR (CYP2B induction), PXR (CYP3A induction), AhR (CYP1A induction), and PPAR (CYP4A induction). The drug-metabolizing phase 2 enzymes NAD(P)H:quinone oxidoreductase and UGT are also induced by the transcriptional factor Nrf2 (Shelby and Klaassen, 2006). Administration of the CAR activator PB to normal male rats increased UGT1A1 and UGT1A6 protein levels to a similar extent as CYP2B (Fig. 8A). Moreover, administration of the PPAR ligand CFB to normal male rats increased UGT1A1 and UGT1A6 protein levels to a similar extent as CYP4A (Fig. 8B). Intake of the HF1 diet decreased the protein levels of CYP1A1 and CYP1A2 and increased the protein levels of UGT1A1 and UGT1A6 in male rats, even though the CYP1A1 and UGT1A6 promoters include XRE sequences (Figs. 2, 3, and 7). In contrast, PH induced CYP1A1 and UGT1A6 in male rats fed not only the SD diet but also HF1 diet (Fig. 7). The present study has shown that gender-related changes in CAR and PPAR expression and in UGT1A1 and UGT1A6 expression occur in the liver cell nuclei of rats fed the HF1 diet (Figs. 3 and 6). These observations indicate that UGT1A1 and UGT1A6 may be induced via CAR and/or PPAR but not via AhR, although CAR and PPAR binding sites were identified in human UGT1A1 (Sugatani et al., 2001; Senekeo-Effenberger et al., 2007) and thought to occur in human UGT1A6 but not in rat UGT1A1 and UGT1A6. Consumption of a high-fat and high-sucrose diet caused increases in blood glucose and liver lipids (text and Supplemental Table 1). Dhe-Paganon et al. (2002) proposed that fatty acids or related molecules might be endogenous ligands for HNF4, and that HNF4 may function as a biosensor for fatty acids within the cell. Glucose itself has been shown to induce the expression of HNF4 mRNA in primary-cultured rat hepatocytes (Oyadomari et al., 2000). Additionally, Ding et al. (2006) showed that HNF4 directly regulates CAR gene expression through an HNF4-responsive element in its proximal promoter in mice. Thus, the enhanced expression of CAR may be mediated by HNF4 activation through elevated blood glucose and accumulated liver fatty acids. The reason for the gender-related response of PPAR in HF1 diet-fed rats is currently unclear (Fig. 6). However, it is possible that PPAR induced by intake of the HF1 diet may contribute to the increases in the protein levels of UGT1A1 and UGT1A6 and the decrease in the protein level of CYP1A2 in male rats, as these enzymes were similarly altered in CFB-treated male rats (Fig. 8B). CFB and the HF1 diet exerted differential effects on the induction of CYP4A, UGT1A1, and UGT1A6, suggesting that transcriptional cofactors and/or endogenous molecules other than PPAR may contribute to the transcriptional regulation of these genes. In conclusion, we speculate that the HF1 diet caused the elevation in the expression and activation of CAR and PPAR in liver cell nuclei, which may be also associated with the concurrent alteration of UGT1A1 and UGT1A6. We are now investigating the molecular mechanism of rat UGT1A1 and UGT1A6 induction via CAR and PPAR and cross-talk between CAR and PPAR.

	Acknowledgments

 
We thank Honami Hara, Ikuyo Yasuda, Norio Tezuka, Shoko Hashimoto, Nao Minowa, and Kasumi Yamakawa for excellent technical assistance.

	Footnotes

 
This work was in part supported by the COE21 Program, Global COE, Grant-in-Aid for Scientific Research (19590070, 19590151), and Cooperation of Innovative Technology and Advanced Research in Evolutional Area (CITY AREA) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.017731.

ABBREVIATIONS: HF1 diet, high-fat and high-sucrose diet; PB, phenobarbital; APAP, acetaminophen; NAPQI, N-acetyl-p-benzoquinoneimine; UGT, UDP-glucuronosyltransferase; AG, acetaminophen glucuronide; SD diet, standard diet; CAR, constitutive androstane receptor; gtPBREM, UGT1A1 phenobarbital responsive enhancer module; PXR, pregnane X receptor; AhR, aryl hydrocarbon receptor; PPAR, peroxisome proliferator-activated receptor; Nrf2, nuclear factor E2-related factor 2; HNF, hepatocyte nuclear factor; PGC, peroxisome proliferator-activated receptor-coactivator; HF2 diet, high-fat diet; PH, 1,7-phenanthroline; CFB, clofibrate; P450, cytochrome P450; Mrp, multidrug resistance-associated protein; RXR, retinoid X receptor; ARNT, AhR nuclear translocator; Oatp, organic anion transporter polypeptide; AUC, area under plasma concentration-time curve; XRE, xenobiotic response element.

The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

Address correspondence to: Junko Sugatani, Department of Pharmaco-Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. E-mail: sugatani{at}u-shizuoka-ken.ac.jp

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