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Dietary Inulin Alleviates Hepatic Steatosis and Xenobiotics-Induced Liver Injury in Rats Fed a High-Fat and High-Sucrose Diet: Association with the Suppression of Hepatic Cytochrome P450 and Hepatocyte Nuclear Factor 4 Expression

Department of Pharmaco-Biochemistry and COE21, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka City, Shizuoka, Japan (J.S., M.O., K.Y., K.Y., M.M.); and Fuji Nihon Seito Corporation, Shizuoka City, Shizuoka, Japan (T.W.).

(Received April 15, 2006; accepted June 27, 2006)

	Abstract

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Inulin enzymatically synthesized from sucrose is a dietary component that completely escapes glucide digestion. Supplementing inulin to a high-fat and high-sucrose diet (HF) ameliorated hypertriglycemia and hepatic steatosis in 8-week-fed rats by suppressing elevated levels of serum triacylglycerols, fatty acids, and glucose, and the accumulation of hepatic triacylglycerols and fatty acids. Inulin intake prevented phenobarbital (PB)- and dexamethasone-induced liver injuries in the HF group. No significant alteration in the baseline expression of CYP2B, CYP2C11, CYP3A, and NADPH-cytochrome P450 (P450) reductase mRNAs and proteins was found. In contrast, baseline and PB-treated expressions of CYP2E1 mRNA were reduced in HF-fed rats. The induction of P450s in response to PB was affected by the nutritional status of the rats; mRNA levels of CYP2B1 and CYP3A1 after PB treatment, as assessed by quantitative real-time polymerase chain reaction analysis were reduced in the inulin-supplemented HF (HF+I) group, compared with those in the HF group. Western blot analysis detected the corresponding changes of CYP2B and CYP3A proteins. These alterations were correlated with changes in hepatic thiobarbituric acid-reactive substances. Furthermore, no significant difference in the expression of nuclear receptors constitutive androstane receptor, pregnane X receptor, and retinoid X receptor and coactivator peroxisome proliferator-activated receptor- coactivator 1 proteins was found in the hepatic nucleus between the HF and HF+I groups, but the expression of hepatocyte nuclear factor (HNF4) protein was significantly reduced in the HF+I group. Taken together, these results indicate that inulin intake ameliorates PB-induced liver injury, associated with a decline in lipid accumulation and PB-induced expression of CYP2B and CYP3A, which may be related by a reduction in the nuclear expression of HNF4.

Biochemical processes that generate oxidative stress can initiate hepatocellular injury, and CYP2B, CYP2C, and CYP3A, as well as CYP2E1 and CYP4A, are microsomal sources producing prooxidants in liver cells, which create oxidative stress under uncoupling with antioxidants. Here we found that the intake of a high-fat and high-sucrose diet (HF) for a long period (8 weeks) produced marked accumulation of hepatic and serum triacylglycerols in rats, leading to hypertriglycemia and hepatic steatosis but not hepatic necroinflammatory lesions. Also, there was no significant influence in the baseline levels of hepatic CYP2B, CYP2C, and CYP3A proteins, but levels of CYP2E1 and CYP4A proteins were reduced. In contrast, the administration of phenobarbital (PB) to HF-fed rats more promptly caused liver injury, leading to cell necrosis and inflammation, compared with the standard diet (SD)-fed rats and was associated with changes in PB-inducible P450 expression or via the constitutive androstane receptor (CAR)-mediated pathway associated with hepatic lipid accumulation.

Some dietary components that completely escape glucide digestion, such as resistant starch and oligofructose, have been demonstrated to exert systemic effects by modifying lipid metabolism (Younes et al., 1995; Daubioul et al., 2000). In contrast to starch, inulin is fermentable dietary fiber, resistant to hydrolysis by pancreatic amylase and saccharidases in the upper gastrointestinal tract. Previous studies by our laboratory have demonstrated that inulin was produced enzymatically from sucrose, and that supplementing inulin to the HF diet for 12 weeks reduced elevated body weight and serum and hepatic levels of triacylglycerols in rats (Wada et al., 2005). Interestingly, this study has demonstrated that PB-induced liver failure was suppressed in rats fed an inulin-supplemented HF diet. It is important to investigate whether the reduction in elevated levels of blood glucose and serum and hepatic lipids by supplementing inulin to the HF diet was associated with the down-regulation of P450 induction by PB, consequently leading to the suppression of liver injury. Hence, in this study, we characterized PB-induced P450 mRNA and protein profiles in connection with profiles of blood glucose and serum and hepatic lipids. Treatment of inulin-supplemented HF diet-fed rats with PB resulted in lower levels of CYP2B mRNA and protein than with only the SD or HF diet. PB induces CYP2B, CYP2C, and CYP3A via the activation of nuclear receptors, CAR and pregnane X receptor (PXR), which regulate the transactivation of those genes. Previous studies have demonstrated that hepatocyte nuclear receptor-4 (HNF4) is an important regulator of coordinate nuclear receptor (such as CAR and PXR)-mediated response to xenobiotics (Triona et al., 2003), and that peroxisome proliferator-activated receptor- coactivator-1 (PGC1) modulates the function of nuclear receptors such as CAR as a common coactivator (Puigserver and Spiegelman, 2003; Shiraki et al., 2003). Little is known about the effects of nutritional status on baseline and xenobiotic-mediated expression of nuclear receptors (CAR, PXR, RXR forming a heterodimer with CAR or PXR) and coactivator PGC1. Thus, we examined the effect of supplementing inulin to SD and HF diets on the hepatic expression of CAR, PXR, RXR, HNF4, and PGC1 mRNAs and nuclear expression of those proteins in rats.

	Materials and Methods

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Materials. Phenobarbital sodium (PB) was purchased from Wako Pure Chemicals (Osaka, Japan). Dexamethasone (DEX), pentoxyresorufin, and resorufin were obtained from Sigma-Aldrich (St. Louis, MO). Inulin, which consists of a linear polymer (average ratio of glucose/fructose, 1:17) having ß(2-1) linkages of D-fructose with one terminal glucose, was synthesized enzymatically from sucrose by inulin-producing enzyme as described previously (Wada et al., 2005). All other chemicals and solvents were of analytical grade and were obtained from commercial sources.

Experimental Animals. All studies followed protocols approved by the Institutional Animal Care and Life Committee, University of Shizuoka. Male Wistar rats were obtained from Japan SLC (Hamamatsu, Japan) at 4 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 animals were randomly assigned to SD, 5% inulin-supplemented standard diet (SD+I), HF, or 5% inulin-supplemented high-fat and high-sucrose diet (HF+I) for 4 and 8 weeks. The HF 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 [23.9% lipid, 56.8% carbohydrate, and 19.3% 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% humidity [12.9% lipid, 60.4% carbohydrate, and 26.7% protein (kJ)]. Three times per week, all rats were weighed, and food intake in grams was monitored. At 9 or 13 weeks of age, the rats in each group were administered PB (80 mg/kg) or vehicle (0.9% sodium chloride) by intraperitoneal injection once daily for 4 days, and dexamethasone (50 mg/kg) or vehicle (0.1 M citric acid in 0.5% methylcellulose) once daily for 3 days between 8:00 AM and 9:00 AM.

Blood and Tissue Sampling. The experiments were run 4 h after the last administration of PB. Rats were anesthetized with diethyl ether; 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 weighed. The liver median lobe was excised for the preparation of microsomes and nuclear extracts and RNA extraction, or was fixed in 4% paraformaldehyde in 0.12 M sodium phosphate (pH 7.3) for histological examination. Aliquots were snapfrozen and stored at –80°C until analysis. Sections were stained with hematoxylin-eosin (HE) or Oil Red O.

Preparation of Microsomes. Liver microsomes were prepared by differential centrifugation, first at 9,000g 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.

Preparation of Nuclear Extracts. 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 per ml, 1 µg of pepstatin per ml, 1 µg of aprotinin per ml, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄ with a Teflon-glass homogenizer with three strokes. 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 per ml, 1 µg of pepstatin per ml, 1 µg of aprotinin per ml, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM Na₃VO₄) with a Dounce homogenizer 10 times. 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 of the 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 dithiothreitol, 1 µg of leupeptin per ml, 1 µg of pepstatin per ml, 1 µg of aprotinin per 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 on a sodium dodecyl sulfate-12.5% polyacrylamide gel, electroblotted onto a polyvinylidene difluoride membrane (Millipore Corporation, Bedford MA). The immunoblots were incubated with the following primary antibodies: rat CYP2B1, rat CYP2C11, rat CYP2E1, rat CYP3A2, rat CYP4A1, and rat NADPH-P450 reductase (Daiichi Pure Chemicals Co., Tokyo, Japan), mouse CAR, mouse PXR, human RXR, human HNF4, human PGC1, and human histone H1 (Santa Cruz Biotechnology, Santa Cruz, CA). 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).

Determination of mRNA Levels. Total RNA was prepared from the liver using TRIzol reagent (Invitrogen, Carlsbad, CA). Samples were quantitated by spectrophotometry, and 1 µg of total RNA was used to generate cDNA by reverse transcription using SuperScript II (Invitrogen) 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., 2005), with an ABI PRISM 7000 Sequence Detector instrument (Applied Biosystems, Foster City, CA) using Premix Ex Taq reagent (TaKaRa Bio Inc., Otsu, Japan) for the TaqMan probe method or SYBR Premix Ex Taq reagent (TaKaRa Bio Inc.) for intercalation reaction with SYBR Green I according to the manufacturer's specifications. The TaqMan probes and primers for rat CAR and PXR were as reported previously (Hartley et al., 2004); and rat CYP2E1 (assay identification number Rn00580624 m1), rat HNF4 (assay identification number Rn00573309 m1), and rat ß-actin (assay identification number Rn00667869 m1) were assay-on-demand gene expression products (Applied Biosystems). The gene-specific probes were labeled by using reporter dye 5-carboxyfluorescein, and the ß-actin internal control probe was labeled with a different reporter dye, 6-carboxyrhodamine, at the 5' end. The primers for rat CYP2B1, rat CYP2B2, rat CYP3A1, and rat CYP3A2 (Yoshinari et al., 2004), rat CYP2C11 (Xu et al., 2001), rat CYP4A1, rat CYP4A2, rat CYP4A3 (Marji et al., 2002), rat RXR (Wang et al., 2002), and rat PGC1 (Zhang et al., 2005) were as reported previously; the primers for rat NADPH-cytochrome P450 reductase were 5'-CATGTCCGCAGACCCTGAA-3' (forward primer) and 5'-CGTATGTGGCCATGCAGAAG-3' (reverse primer), and the primers for rat ß-actin were 5'-TCCTAGCACCATGAAGATC-3' (forward primer) and 5'-AAACGCAGCTCAGTAACAG-3' (reverse primer). The thermal cycle conditions were as follows: hold 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. ß-Actin was used to normalize gene expression in all samples. Fold-induction values were calculated by subtracting the mean difference of gene and ß-actin cycle threshold Ct numbers for each treatment group from the mean difference of gene and ß-actin Ct numbers for the vehicle group and raising the difference to the power of 2 (2^–Ct).

Biochemical Analyses. Plasma concentrations of glucose and fructose were determined by the hexokinase method using commercial reagents (R-Biopharm AG, Darmstadt, Germany). Serum glucose concentration was determined by the glucose oxidase method with a commercially available kit from Shino Test (Tokyo, Japan). Plasma/serum insulin was measured using a double-antibody radioimmunoassay kit specific for rat insulin (Eiken Chemical Co., Tokyo, Japan). Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and -glutamyltranspeptidase (-GTP) were measured using kits from Wako Pure Chemicals. Serum levels of triacylglycerol, total cholesterol, and nonesterified fatty acid were measured enzymatically with kits from Shino Test. Serum concentrations of ß-hydroxybutyrate were measured using commercial reagents (Serotec Co., Sapporo, Japan). Frozen livers (about 0.5 g) were homogenized in 20 volumes (the SD and SD+I groups) and 100 volumes (the HF and HF+I groups) of 0.9% NaCl containing 0.1% Triton X-100, and the concentrations of triacylglycerol, total cholesterol, and nonesterified fatty acid were estimated with the above commercial kits. Frozen livers (about 0.5 g) were homogenized in 100 volumes of 0.9% NaCl, and thiobarbituric acid-reactive substances (TBARS) were measured in 20 µl of liver homogenate using a commercial assay kit (ZeptoMetrix Co., Buffalo, NY). Liver tumor necrosis factor (TNF) and interleukin 6 were measured in 12.5 µl of serum or liver homogenate using commercial enzyme-linked immunosorbent assay kits (GE Healthcare Bio-Sciences).

Resorufin Assay. The O-dealkylation of 7-pentoxyresorufin was measured fluorometrically by the production of resorufin using a Wallac 1420 ARVO plate reader (PerkinElmer Inc., Wellesley, MA) with excitation and emission wavelengths at 550 nm and 590 nm, respectively. The incubation mixtures contained Tris-HCl (100 mM, pH 7.4), MgCl₂ (3.3 mM), EDTA (1 mM), glucose 6-phosphate (3.3 mM), NADP (1.3 mM), glucose-6-phosphate dehydrogenase (0.4 U/ml), pentoxyresorufin (5 µM), and microsomal protein from control rats and PB-treated rats (25 and 2.5 µg/ml, respectively). NADPH was produced using the Gentest NADPH regenerating system (BD Biosciences, Woburn, MA). The mixtures were incubated for 10 min at 37°C. The formation rate of resorufin was determined by comparison to the fluorescence of known amounts of resorufin.

View larger version (80K): [in this window] [in a new window]   FIG. 1. Histologic analysis of liver from rats fed SD, SD+I, HF, or HF+I for 8 weeks and then administered PB (80 mg/kg, i.p.) or saline as vehicle once daily for 4 days. The livers were stained with HE (original magnification 200x) (A) and Oil Red O (original magnification 200x) (B). Blurred trabecular structure of the liver lobule with distinctly enlarged clear and vacuolated hepatocytes and numerous Oil Red O-positive hepatocytes can be seen throughout the hepatic lobule in rats fed HF. The lower intensity of Oil Red O staining can be seen in rats fed HF+I. Mixed inflammatory infiltrate with lymphocyte and polymorphonuclear neutrophil necroinflammation is dispersed in the liver lobule of rats administered PB, but rare necroinflammation can be seen in the liver lobule of rats administered vehicle. Scale bar indicates 100 µm.

View larger version (34K): [in this window] [in a new window]   FIG. 2. Inulin-supplemented diet normalizes serum AST and ALT activities in rats administered PB (80 mg/kg, i.p.) or DEX (50 mg/kg, p.o.). The rats were then sacrificed, and serum AST (A and D), ALT (B and E), and -GTP levels were measured. Values are the means ± S.E. for six to seven determinations in each group. **, p < 0.01, ***, p < 0.001 for HF- or HF+I-fed and PB-administered animals versus SD-fed and PB-administered animals; ###, p < 0.001 for HF+I-fed and PB-administered animals versus HF-fed and PB-administered animals.

	Results

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Dietary Inulin Ameliorates the Development of Fatty Livers in Rats Fed a High-Fat- and High-Sucrose-Supplemented Diet for 8 Weeks. We previously reported that supplementing inulin for 12 weeks reduced the elevation in body weight and serum and hepatic levels of triacylglycerols in rats fed high-fat (10% lard) and high-sucrose (60%) diet (HF), whereas inulin intake for 12 weeks in rats fed the SD diet containing naturally existing starch as carbohydrate did not affect the body weight and serum and hepatic levels of lipids (Wada et al., 2005). Consistent with these observations, supplementing inulin even for 8 weeks reduced the elevation of body weight, liver weight, serum levels of triacylglycerols, fatty acids and glucose, and hepatic levels of triacylglycerols in rats fed the HF diet, but did not significantly alter body weight, liver weight, and serum levels of lipids and glucose in rats fed the SD diet (Table 1). At this time, there were no significant differences among the four diet groups in the food intake and starting body weight (data not shown). No significant change in serum and hepatic levels of total cholesterol was found by inulin intake (Table 1). In contrast, the hepatic levels of triacylglycerol (72.6 ± 8.7 mg/g liver) in rats fed the HF diet for 8 weeks were significantly elevated compared with those in rats fed the HF diet for 4 weeks (43.8 ± 5.0 mg/g liver), and supplementing inulin to the HF diet suppressed the accumulation of hepatic triacylglycerols (44.8 ± 5.8 mg/g liver) in 8-week-fed rats. In addition, compared with hepatic free fatty acid levels in rats fed the SD diet for 8 weeks and in rats fed the HF diet for 4 weeks (18.5 ± 0.6 µEq free fatty acid/g liver), intake of the HF diet for 8 weeks resulted in significantly higher accumulation of hepatic free fatty acid, and supplementing inulin to the HF diet reduced the accumulation of hepatic free fatty acids in 8-week-fed rats (Table 1). These observations indicate that inulin intake suppressed the further development of hepatic steatosis. In addition, when we measured serum levels of insulin and ß-hydroxybutyrate in 8-week-fed rats, there was no significant change in either level in the four diet groups (data not shown). We interpret these data as indicating that feeding the HF diet for a long period (8 weeks) produced severe hepatic steatosis but did not induce diabetes.

Histological analysis of livers with HE and Oil Red O staining showed that steatosis was significantly improved after HF diet feeding for 8 weeks. As shown in Fig. 1, A and B, lipid droplets accumulated in the cytoplasm of hepatocytes in both periportal and pericentral areas in the HF group. In comparison, lipid droplet accumulation in hepatocytes was significantly reduced in the HF+I group.

Dietary Inulin Ameliorates Drug-Induced Liver Injury in Rats with Fatty Liver. PB administration has been reported to induce oxidative changes in rat liver, detected as elevated hydroxyl radical levels (Kinoshita et al., 2002). We compared drug-induced liver injury in rats fed the SD and HF diets. Treatment of 8-week-fed rats with PB at a dose of 80 mg/kg once daily for 4 days resulted in increased liver weights and liver-to-body weight ratios in the SD, SD+I, HF, and HF+I groups, and increased hepatic triacylglycerol levels in the HF and HF+I groups (Table 1). Staining of HF-fed rat liver with Oil Red O confirmed the lipid content of clear macrovacuoles (Fig. 1B), but there were few areas in the SD-fed rat liver stained with Oil Red O (data not shown). In addition to the further development of hepatic steatosis, there were areas of mixed inflammatory cell infiltration and hepatocyte necrosis dissecting the liver parenchyma after PB treatment (Fig. 1, A and B). Consistent with morphological changes, PB treatment of HF-fed rats resulted in significant increases in serum AST and ALT levels, but slight increases in serum -GTP levels, compared with those in the SD group (Fig. 2). At this time, the liver injury in the HF group was not accompanied by high levels of serum and hepatic TNF and interleukin 6, inasmuch as there was no significant difference of the TNF and interleukin 6 levels in the four diet groups (data not shown). Furthermore, treatment of rats with dexamethasone at a dose of 40 mg/kg once daily for 3 days (p.o.) resulted in significant increases in serum levels of AST and ALT in the SD, HF, and HF+I groups (Fig. 2). In contrast, inulin intake reduced the levels of serum AST and ALT in the HF group near to the levels in the SD group (Fig. 2). Correspondingly, hepatocyte necrosis was significantly attenuated in rats fed an inulin-supplemented HF diet after PB treatment (Fig. 1).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Effect of phenobarbital treatment on serum levels of AST and ALT in rats fed SD and HF for 4 and 8 weeks. Values are the means ± S.E. of six to seven determinations. ***, p < 0.001 for HF-fed and PB-administered animals versus SD-fed and PB-administered animals; +++, p < 0.001 for PB-administered animals versus vehicle-administered animals.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Inulin down-regulates TBARS levels in the livers of rats fed HF for 8 weeks and then administered PB once daily for 4 days. Values are the means ± 7 determinations. *, p < 0.05 for HF-fed and vehicle-administered animals versus SD-fed and vehicle-administered animals or HF-fed and PB-administered animals versus SD-fed and PB-administered animals; +, p < 0.05, ++, p < 0.01, +++, p < 0.001 for PB-administered animals versus vehicle-administered animals; #, p < 0.05, ##, p < 0.01 for HF+I-fed and vehicle-administered animals versus HF-fed and vehicle-administered animals or HF+I-fed and PB-administered animals versus HF-fed and PB-administered animals. A, TBARS (nmol/mg protein); B, TBARS (nmol/liver).

Hepatic Expression and Induction by PB of Enzyme Activities, mRNAs, and Proteins of P450s in Rats Fed the Four Diets. Since PB particularly induces CYP2B1/2B2 expression in rat liver, we first compared the enzyme activity of pentoxyresorufin O-dealkylation (PROD) activities in the four diet groups. As shown in Table 1, the HF and HF+I diets did not influence baseline or induced levels of microsomal protein. Although the HF diet did not influence the baseline and induced levels of PROD activity, lower PROD activity in the HF+I group than in the SD group after PB treatment was detected (Table 1).

To assess the effects of the SD, SD+I, HF, and HF+I diets on the hepatic expression of baseline and PB-treated P450 mRNAs, we measured hepatic mRNA levels in rats fed the four diets with and without PB treatment. Unexpectedly, fatty liver produced by feeding the HF diet down-regulated CYP2E1 mRNA expression to 69 ± 2% of the control (Table 2). The reduced levels were continued even after PB treatment for 4 days. Supplementing inulin to the HF diet did not reverse the mRNA levels of CYP2E1 (Table 2). In addition, there was no significant increase in the levels of CYP4A1, CYP4A2, and CYP4A3 in the vehicle- and PB-treated HF and HF+I groups.

As shown in Table 2, there was no significant change in baseline mRNA levels of CYP2B1/2B2, CYP2C11, CYP3A1/3A2, and NADPH-P450 reductase in the four diet groups. As expected, PB treatment markedly increased CYP2B1/2B2 mRNA levels in the four diet groups. PB-induced mRNA levels of CYP2B1 and CYP2B2 in the HF+I group were significantly lower than in the SD group. Whereas there was no significant change in PB-induced mRNA levels of CYP2B1 and CYP2B2 between the SD and SD+I groups, PB-induced expression of CYP2B1 mRNA in the HF+I group significantly declined compared with that in the HF group. PB-induced mRNA levels of CYP3A1 in the HF group were decreased compared with those in the SD group, and mRNA levels in the HF+I group were further decreased compared with those in the HF group. There was no significant difference in PB-induced mRNA levels of CYP3A2 in the four diet groups. The mRNA levels of NADPH-P450 reductase in the SD and SD+I groups were increased after PB treatment. Whereas there was no significant difference in PB-treated mRNA levels of NADPH-P450 reductase in the SD and HF groups, the levels in the HF+I group were lower than those in the SD group.

Next, we compared protein levels of P450s in the four diet groups. The baseline and treated protein levels of CYP4A and PB-treated levels of CYP2E1 were decreased in the HF and HF+I groups (Fig. 5). There was no significant change in the baseline protein levels of CYP2B, CYP2C, CYP3A, and NADPH-P450 reductase except for decreased levels of CYP2B protein in the HF+I group. The protein levels of CYP2B in the HF and HF+I groups after PB treatment were decreased to 76 ± 2% and 56 ± 1%, respectively, of those in the SD group (Fig. 5). In addition, CYP3A protein levels in the HF+I group after PB treatment were reduced to 79 ± 1% of those in the HF group. CYP2C11 protein levels in the SD and SD+I groups were elevated after PB treatment, but the levels in the HF and HF+I groups were not significantly influenced. In contrast, there was no significant increase in protein levels of CYP2E1, CYP4A, and NADPH-P450 reductase in the four diet groups after PB treatment.

View larger version (33K): [in this window] [in a new window]   FIG. 5. Effects of phenobarbital treatment on P450 protein levels in rats fed SD, SD+I, HF, and HF+I for 8 weeks. Rats were administered PB (80 mg/kg, i.p.) once daily for 4 days and then sacrificed, and the microsome proteins (20 µg/lane) were prepared and subjected to immunoblot analysis. The signal intensities were determined with a Fujix BAS-2000 bioimage analyzer (Fuji Photo Film, Tokyo, Japan). Relative levels are expressed by taking the control values obtained from SD-fed and vehicle-treated animals as 100. *, p < 0.05, **, p < 0.01, ***, p < 0.001 for SD+I-, HF-, or HF+I-fed and vehicle-administered animals versus SD-fed and vehicle-administered animals or SD+I-, HF-, or HF+I-fed and PB-administered animals versus SD-fed and PB-administered animals; ++, p < 0.01, +++, p < 0.001 for PB-administered animals versus vehicle-administered animals; ##, p < 0.01, ###, p < 0.001 for HF+I-fed and PB-administered animals versus HF-fed and PB-administered animals.

We further investigated the effect of the diets and PB treatment on the protein levels of nuclear receptors in the liver nucleus. As shown in Fig. 6, the protein levels of CAR in the nucleus were increased after PB treatment, but no difference was observed between the levels of the HF and HF+I groups. No significant difference was found in the baseline and PB-treated protein levels of PXR and RXR in the four diet groups. In contrast, the baseline and PB-treated protein levels of HNF4 in the HF group were decreased to 86 ± 5% and 77 ± 7%, respectively, of the ones in the SD group, and the baseline and PB-treated levels in the HF+I group were 78 ± 13% and 46 ± 5%, respectively, of those in the SD+I group. In addition, the treated protein levels in the HF+I group were significantly lower than in the HF group.

View larger version (34K): [in this window] [in a new window]   FIG. 6. Effects of phenobarbital treatment on nuclear receptor protein levels in rats fed SD, SD+I, HF, and HF+I for 8 weeks. Rats were fed the four diets for 8 weeks and then administered PB (80 mg/kg, i.p.) once daily for 4 days, and then sacrificed. The nuclear proteins (20 µg/lane) were prepared and subjected to immunoblot analysis. The signal intensities were determined with a Fujix BAS-2000 bioimage analyzer (Fuji Photo Film). Relative levels are expressed by taking the control values obtained from SD-fed and vehicle-treated animals as 100. *, p < 0.05, **, p < 0.01, ***, p < 0.001 for SD+I-, HF-, or HF+I-fed and vehicle-administered animals versus SD-fed and vehicle-administered animals or SD+I-, HF-, or HF+I-fed and PB-administered animals versus SD-fed and PB-administered animals; +, p < 0.05, +++, p < 0.001 for PB-administered animals versus vehicle-administered animals; ##, p < 0.01 for HF+I-fed and PB-administered animals versus HF-fed and PB-administered animals.

	Discussion

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This study demonstrates that feeding the high-lipid and high-sucrose diet for 8 weeks produces severe hepatic steatosis, which is associated with hepatic injury produced by the administration of xenobiotics such as PB and DEX (Table 1; Figs. 1 and 2). Although large amounts of lipid deposition were evident in the liver sections of the rats, there was no inflammation and fibrosis in the liver of control rats (Fig. 1). This model is not an experimental model of nonalcoholic steatohepatitis, but is beneficial for investigating the effects of lipid accumulation on xenobiotic-induced liver injury. Liver injury by PB treatment was found in the HF group of 8-week-fed rats but not 4-week-fed rats (Fig. 3), consistent with the accumulation of hepatic triacylglycerols in the HF group of 8-week-fed rats being markedly higher than that of 4-week-fed rats. As found in liver sections (Fig. 1) and serum levels of AST and ALT (Fig. 2), inulin intake attenuated lipid droplets and PB- and DEX-induced hepatotoxic injury. These results suggest that 1) marked lipid accumulation in hepatocytes may be associated with the induction of liver injury by xenobiotics, and 2) potential preventive effects of dietary inulin on blood glucose and lipid profiles, and hepatic lipid profiles may lead to the prevention of liver injury produced by xenobiotics. Steatosis produced by diets deficient in lipotropes such as choline and methionine (Weltman et al., 1996) and that induced by chemicals such as orotic acid (Su et al., 1999) are established as experimental models of nonalcoholic steatohepatitis and drug-mediated liver injury, respectively. Those models have been reported to be associated with liver injuries via the alteration of hepatic expression of drug-metabolizing enzymes such as CYP2E1, CYP4A, and CYP2C11. In addition, a pathophysiological status such as chemically induced and spontaneous diabetes has been demonstrated to influence the hepatic expression of CYP2B, CYP2C11, CYP3A, and CYP4A (Bellward et al., 1988; Dong et al., 1988; Favreau and Schenkman, 1988; Woodcroft et al., 1999). Nevertheless, the detailed effects of lipid accumulation on the expression of transcriptional factors, which regulate the gene expression of drug-metabolizing enzymes, remain to be clarified. This study establishes that dietary manipulation such as high lard and high sucrose produces hepatic steatosis associated with xenobiotic-induced liver injury (Figs. 1 and 2; Table 1). Thus, in this study, we examined the effects of lipid accumulation on baseline and xenobiotic-treated expression of CYP2E1, CYP2B, CYP2C11, CYP3A, and CYP4A mRNAs and proteins, and the association between alterations in the expression of P450s and transcriptin factors and xenobiotic-induced liver injury.

Leclercq et al. (2000) have demonstrated that CYP2E1 in wild-type mice but CYP4A in CYP2e1–/–mice is up-regulated in nonalcoholic steatohepatitis and plays a key role in the development of liver injury by initiating lipid peroxidation. In contrast, feeding a high-fat and high-sucrose diet down-regulated CYP2E1 and CYP4A mRNAs and proteins, and even after treatment with PB, the enzymes remained down-regulated (Table 2; Fig. 5). These results indicate that CYP2E1 and CYP4As seemed not to play a central role in this PB-induced liver injury.

Rats fed the HF+I diet exhibited reduced induction in response to PB treatment as evidenced by PROD activities and CYP2B mRNA and protein levels (Tables 1 and 2; Fig. 5). Zannikos et al. (1993) have demonstrated that rats fed the energy-dense diet for 32 weeks as compared with rats fed the control diet exhibit a lower increase in PROD activities after PB treatment (20-fold increase versus 45-fold increase), whereas similar amounts of protein and mRNA of CYP2B1/2B2 are found. Hepatic nutritional status in this experimental model appears to be rather different from the energy-dense diet model; lipid accumulation in the liver of rats fed the HF diet for 8 weeks was more than 2-fold higher than that of rats fed the energy-dense diet for 32 weeks. As pointed out by Zannikos et al. (1993), the change in the microsomal lipid environment based on diet and/or aging may influence enzyme activity.

PB activates CAR and PXR, which bind to and transactivate CYP2B and CYP3A promoters (Honkakoski et al., 1998; Lehmann et al., 1998). HNF4 synergistically enhanced CAR- and PXR-mediated xenobiotic induction of human CYP2C9 (Chen et al., 2005) and CYP3A (Tirona et al., 2003). Although there is no direct evidence that HNF4 synergistically enhances the CAR- and PXR-mediated xenobiotic induction of CYP2B, the CYP2B2 phenobarbital response unit contains an HNF4 binding site (Beaudet et al., 2005). In addition, single nucleotide polymorphism in a putative HNF4 binding site in the phenobarbital response element of CYP2B6 has been demonstrated to be correlated with lowered CYP2B6 expression (Lamba et al., 2003). By a study using HNF4 antisense RNA, it has been demonstrated that HNF4 is a general regulator of major drug-metabolizing P450s such as CYP3A4, CYP3A5, CYP2A6, CYP2B6, CYP2C9, and CYP2D6 (Jover et al., 2001). Thus, in this study, we assessed whether the reduced PB induction of CYP2B and CYP3A mRNAs and proteins in the HF+I group may be attributable to a decline in the expression of transcription factors. Although PGC1 has been demonstrated to mediate the ligand-independent activation of CAR (Shiraki et al., 2003), the levels of PGC1 mRNA and protein in the HF+I group were similar to those of the control of the SD+I and HF groups (Table 2; Fig. 6), suggesting that PGC1 may not play a central role in the preventive action of dietary inulin. There was no significant change based on HF and HF+I diets in the baseline and PB-treated expression of CAR, PXR, and RXR mRNAs and proteins except for increased levels of CAR mRNA in the vehicle-treated HF+I group (Table 2; Fig. 6). In contrast, HNF4 protein significantly declined in the liver nucleus of rats fed the HF and HF+I diets (Fig. 6). Thus, the reduced induction of hepatic CYP2B and CYP3A in the PB-treated HF+I group may be attributable to a decline in the expression of HNF4 protein in the liver nucleus. Serum glucose levels were increased in HF-fed and vehicle-treated rats, and inulin-supplemented diet suppressed the increase (Table 1). Furthermore, the levels in HF-fed rats after PB treatment exhibited a tendency to remain increased. Glucose itself has been demonstrated to induce the expression of HNF4 mRNA in primary-cultured rat hepatocytes (Oyadomari et al., 2000). Thus, the lower levels of HNF4 mRNA and protein in the PB-treated HF+I group than those in the HF group may be associated with suppression of increase in serum glucose by inulin. Dhe-Paganon et al. (2002) presume that fatty acids or related molecules might be the endogenous ligands for HNF4; that is, HNF4 may function as a biosensor for fatty acids within the cell. However, it remains to be addressed whether the decreased expression of HNF4 protein is influenced by the liver lipid environment; that is, marked accumulation of liver fatty acids as found in the HF and HF+I groups. Analysis of the HNF4 gene expression is currently progressing in our laboratory.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Proposed scheme of PB-induced liver injury associated with lipid accumulation and suppression by inulin. See text for explanations. GST, glutathione S transferase; FFA, free fatty acid; TG, triacylglycerol.

	Acknowledgments

 
We gratefully acknowledge Honami Hara, Ikuyo Yasuda, Norio Tezuka, Shoko Hashimoto, and Nao Minowa for excellent technical assistance.

	Footnotes

 
This work was in part supported by the COE21 Program, Grant-in-Aid for Scientific Research (15590063, 16590056), 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.106.010645.

ABBREVIATIONS: P450, cytochrome P450; CAR, constitutive androstane receptor; DEX, dexamethasone; HF, high-fat and high-sucrose diet; HF+I, inulin-supplemented HF; HNF, hepatocyte nuclear factor ; PB, phenobarbital; PGC1, peroxisome proliferator-activated receptor- coactivator 1; PROD, pentoxyresorufin O-dealkylase; PXR, pregnane X receptor; RXR, retinoid X receptor ; SD, standard diet; SD+I, inulin-supplemented SD; TBARS, thiobarbituric acid-reactive substances; TNF, tumor necrosis factor ; HE, hematoxylin-eosin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; -GTP, -glutamyltranspeptidase.

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

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

	References

Top Abstract Materials and Methods Results Discussion References

 


Beaudet MJ, Desrochers M, Lachaud AA, and Anderson A (2005) The CYP2B2 phenobarbital response unit contains binding sites for hepatocyte nuclear factor 4, PBX-PREP1, the thyroid hormone receptor beta and the liver X receptor. Biochem J 388: 407–418.[CrossRef][Medline]

Bellward CD, Chang T, Rodrigues B, McNeil JH, Maines S, Ryan DE, Levin W, and Thomas PE (1988) Hepatic cytochrome P450IV family proteins in streptozotocin-induced diabetes. Biochem J 268: 765–769.

Bogan AA, Dallas-Yang Q, Ruse MD Jr, Maeda Y, Jiang G, Nepomuceno L, Scanlan TS, Cohen FE, and Sladek FM (2000) Analysis of protein dimerization and ligand binding of orphan receptor HNF4. J Mol Biol 302: 831–851.[CrossRef][Medline]

Bugianesi E, Leone N, Vanni E, Marchesini G, Brunello F, Carucci P, Musso A, De Paolis P, Capussotti L, Salizzoni M, et al. (2002) Expanding the natural history of nonalcoholic steatohepatitis: from cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology 123: 134–140.[CrossRef]

Chalasani N, Gorski JC, Asghar MS, Asghar A, Foresman B, Hall SD, and Crabb DW (2003) Hepatic cytochrome P450 2E1 activity in nondiabetic patients with nonalcoholic steatohepatitis. Hepatology 37: 544–550.[CrossRef][Medline]

Chen Y, Kissling G, Negishi M, and Goldstein JA (2005) The nuclear receptors constitutive androstane receptor and pregnane X receptor cross-talk with hepatic nuclear factor 4 to synergistically activate the human CYP2C9 promoter. J Pharmacol Exp Ther 314: 1125–1133.[Abstract/Free Full Text]

Dai G, Chou N, He L, Gyamfi MA, Mendy AJ, Slit AL, Klaassen CD, and Wan YY (2005) Retinoid X receptor a regulates the expression of glutathione S-transferase genes and modulates acetaminophen-glutathione conjugation in mouse liver. Mol Pharmacol 68: 1590–1596.[Abstract/Free Full Text]

Daubioul CA, Taper HS, De Wispelaere LD, and Delzenne NM (2000) Dietary oligofructose lessens hepatic steatosis, but does not prevent hypertriglyceridemia in obese Zucker rats. J Nutr 130: 1314–1319.[Abstract/Free Full Text]

Dhe-Paganon S, Duda K, Iwamoto M, Chi Y-I, and Shoelson SE (2002) Crystal structure of the HNF4 ligand binding domain in complex with endogenous fatty acid ligand. J Biol Chem 277: 37973–37976.[Abstract/Free Full Text]

Dong Z, Hong J, Ma Q, Li D, Bullock J, Gonzalez FJ, Park SS, Gelboin HV, and Yang CS (1988) Mechanism of induction of cytochrome P450ac (P450j) in chemically induced and spontaneously diabetic rats. Arch Biochem Biophys 263: 29–35.[CrossRef][Medline]

Emery M, Fisher JM, Chien JY, Kharasch ED, Dellinger EP, Kowdley KV, and Thummel KE (2003) CYP2E1 activity before and after weight loss in morbidly obese subjects with nonalcoholic fatty liver disease. Hepatology 38: 428–435.[Medline]

Favreau LV and Schenkman JB (1988) Composition changes in hepatic microsomal cytochrome P450 during onset of streptozotocin-induced diabetes and during insulin treatment. Diabetes 37: 577–584.[Abstract]

Hartley DP, Dai X, He YD, Carlini EJ, Wang B, Huskey SW, Ulrich RG, Rushmore TH, Evers R, and Evans DC (2004) Activators of the rat pregnane X receptor differentially modulate hepatic and intestinal gene expression. Mol Pharmacol 65: 1159–1171.[Abstract/Free Full Text]

Hong J, Pan J, Gonzalez FJ, Gelboin HV, and Yang CS (1987) The induction of a specific form of cytochrome P450 (P450j) by fasting. Biochem Biophys Res Commun 142: 1077–1088.[CrossRef][Medline]

Honkakoski P, Zelko I, Sueyoshi T, and Negishi M (1998) The nuclear orphan receptor CAR-retinoid X receptor heterodimer activates the phenobarbital-responsive enhancer module of the CYP2B gene. Mol Cell Biol 18: 5652–5658.[Abstract/Free Full Text]

Johansson I, Ekstrom G, Scholte B, Puzycki D, Jornvall H, and Ingelman-Sundberg M (1988) Ethanol-, fasting-, and acetone-inducible cytochromes P450 in rat liver: regulation and characteristics of enzymes belonging to the IIB and IIE gene subfamilies. Biochemistry 27: 1925–1934.[CrossRef][Medline]

Jover R, Bort R, Gomez-Lechon MJ, and Castell JV (2001) Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes: a study using adenovirus-mediated antisense targeting. Hepatology 33: 668–675.[CrossRef][Medline]

Kinoshita A, Wanibuchi H, Imaoka S, Ogawa M, Masuda C, Morimura K, Funae Y, and Fukushima S (2002) Formation of 8-hydroxydeoxyguanosine and cell-cycle arrest in the rat liver via generation of oxidative stress by phenobarbital: association with expression profiles of p21^WAF1/Cip1, cycline D1 and Ogg1. Carcinogenesis 23: 341–349.[Abstract/Free Full Text]

Lamba V, Lamba J, Uda KY, Strom S, Davila J, Hancock ML, Fackenthal DJ, Rogan PK, Ring B, Wrighton SA, et al. (2003) Hepatic CYP2B6 expression: gender and ethnic differences and relationship to CYP2B6 genotype and CAR (constitutive androstane receptor) expression. J Pharmacol Exp Ther 307: 906–922.[Abstract/Free Full Text]

Leclercq IA, Farrell GC, Field J, Bell DR, Gonzalez FJ, and Robertson GR (2000) CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis. J Clin Investig 105: 1067–1075.[Medline]

Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, and Kliewer SA (1998) The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Investig 102: 1016–1023.[Medline]

Marji JS, Wang M-H, and Laniado-Schwartzman M (2002) Cytochrome P-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries. Am J Physiol 283: F60–F67.

Oyadomari S, Matsuno F, Chowdhury S, Kimura T, Iwase K, Araki E, Shichire M, Mori M, and Takiguchi M (2000) The gene for hepatocyte nuclear factor (HNF)-4 is activated by glucocorticoids and glucagon, and repressed by insulin in rat liver. FEBS Lett 478: 141–146.[CrossRef][Medline]

Puigserver P and Spiegelman BM (2003) Peroxisome proliferator-activated receptor- coactivator 1 (PGC-1): transcriptional coactivator and metabolic regulator. Endocr Rev 24: 78–90.[Abstract/Free Full Text]

Shiraki T, Sakai N, Kanaya E, and Jingami H (2003) Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor coactivator-1. J Biol Chem 278: 11344–11350.[Abstract/Free Full Text]

Su GM, Sefton RM, and Murray M (1999) Down-regulation of rat hepatic microsomal cytochromes P-450 in microvesicular steatosis induced by orotic acid. J Pharmacol Exp Ther 291: 953–959.[Abstract/Free Full Text]

Sugatani J, Nishitani S, Yamakawa K, Yoshinari K, Sueyoshi T, Nagishi M, and Miwa M (2005) Transcriptional regulation of human UGT1A1 gene expression: Activated glucocorticoid receptor enhances constitutive androstane receptor/pregnane X receptor-mediated UDP-glucuronosyltransferase 1A1 regulation with glucocorticoid receptor-interacting protein 1. Mol Pharmacol 67: 845–855.[Abstract/Free Full Text]

Tirona RG, Lee W, Leake BF, Lan L-B, Cline CB, Lamba V, Parviz F, Duncan SA, Inoue Y, Gonzalez FJ, et al. (2003) The orphan nuclear receptor HNF4a determines PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nat Med 9: 220–224.[CrossRef][Medline]

Wada T, Sugatani J, Terada E, Ohguchi M, and Miwa M (2005) Physicochemical characterization and biological effects of inulin enzymatically synthesized from sucrose. J Agric Food Chem 53: 1246–1253.[CrossRef][Medline]

Wang Q, Fujii H, and Knipp GT (2002) Expression of PPAR and RXR isoforms in the developing rat and human term placentas. Placenta 23: 661–671.[CrossRef][Medline]

Weltman MD, Farrell GC, Hall P, Ingelman-Sundberg M, and Liddle C (1998) Hepatic cytochrome P450 2E1 is increased in patients with nonalcoholic steatohepatitis. Hepatology 27: 128–133.[CrossRef][Medline]

Weltman MD, Farrell GC, and Liddle C (1996) Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation. Gastroenterology 111: 1645–1653.[CrossRef][Medline]

Wolf KK, Wood SG, Hunt JA, Walton-Strong BW, Yasuda K, Lan L, Duan SX, Hao Q, Wrighton SA, Jeffery EH, et al. (2005) Role of the nuclear receptor pregnane X receptor in acetaminophen hepatotoxicity. Drug Metab Dispos 33: 1827–1836.[Abstract/Free Full Text]

Woodcroft KJ and Novak RF (1999) Insulin differentially affects xenobiotic-enhanced cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther 289: 1121–1127.[Abstract/Free Full Text]

Xu Z, Kawai M, Bandiera SM, and Chang TKH (2001) Influence of dietary zinc deficiency during development on hepatic CYP2C11, CYP2C12, CYP3A2, CYP3A9, and CYP3A18 expression in postpubertal male rats. Biochem Pharmacol 62: 1283–1291.[CrossRef][Medline]

Yoshinari K, Sato T, Okino N, Sugatani J, and Miwa M (2004) Expression and induction of cytochromes P450 in rat white adipose tissue. J Pharmacol Exp Ther 311: 147–154.[Abstract/Free Full Text]

Younes H, Levrat MA, Demigne C, and Remesy C (1995) Resistant starch is more effective than cholestyramine as lipid-lowering agent in the rat. Lipids 30: 847–853.[Medline]

Zannikos PN, Bandyopadhyay AM, Robertson LW, and Blouin RA (1993) Effect of nutritional obesity on the induction of CYP2B enzymes following phenobarbital treatment. Drug Metab Dispos 21: 782–787.[Abstract]

Zhang J, Huang W, Chus SS, Wel P, and Moore DD (2002) Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science (Wash DC) 298: 422–424.[Abstract/Free Full Text]

Zhang P, Liu C, Zhang C, Zhang Y, Shen P, Zhang J, and Zhang C-Y (2005) Free fatty acids increase PGC-1 expression in isolated rat islets. FEBS Lett 579: 1446–1452.[CrossRef][Medline]

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