The genetically obese Zucker rat arose from cross-breeding between Sherman and Merck stock M rats and obesity is transmitted as a Mendelian recessive trait due to a mutation on a single gene, called “fa”, for “fatty” (Zucker and Zucker, 1961). The symptoms of this genetic obesity share many similarities with those in obese patients with insulin-resistance or Type II diabetes. Therefore, the obese Zucker rat has been widely used as a genetic model of obesity and insulin resistance (Bray, 1977; Kurtz et al., 1989; Krief and Bazin, 1991).

Synthetic agonists for the peroxisome proliferator-activated receptors (PPAR) α (fibrates) and PPAR γ (thiazolidinediones) are widely used in the treatment of dyslipidemia and diabetes, respectively (Wahli et al., 1995; Schoonjans et al., 1996; Kersten et al., 2000; Berger and Moller, 2002). MRL-I, (2R)-7-[3-[2-chloro-4-(4-fluorophenoxy)phenoxy]propoxy]-2-ethyl-3,4-dihydro-2H-benzopyran-2-carboxylic acid (Fig. 1), is a PPAR α/γ dual agonist that is structurally distinct from marketed PPAR agonists. Its plasma glucose-lowering efficacy in db/db mice is similar to that of rosiglitazone, whereas its lipid-lowering efficacy is better than that of fibrates (Koyama et al., 2004). MRL-II, (2R)-7-[3-[2-chloro-4-(2,2,2-trifluoroethoxy)phenoxy]propoxy]-3,4-dihydro-2-methyl-2H-benzopyran-2-carboxylic acid (Fig. 1), is chemically similar to MRL-I but has PPAR α-selective agonist properties. The cholesterol- and triglyceride-lowering potency of MRL-II was shown to be superior to that of fenofibrate in various in vivo models (H. Koyama, J. K. Boueres, D. J. Miller, J. P. Berger, K. L. MacNaul, L. J. Kelly, T. W. Doebber, P. R. Wang, M. C. Ippolito, Y.-S. Chao, et al., manuscript in preparation).

Previous work has shown altered pharmacokinetic characteristics of some drugs in obese subjects (Abernethy and Greenblatt, 1982); however, the relevance of clinical obesity to drug metabolism, disposition, and excretion is poorly characterized. Pharmacokinetic differences between obese Zucker and lean Sprague-Dawley rats also have been reported with, for example, phenobarbital (Brouwer et al., 1984), but no explanation for this observation was provided.

In the present study, we compared the pharmacokinetics of MRL-I and MRL-II in obese Zucker and Sprague-Dawley rats. The former strain of rats is used as an animal model for obesity and diabetes and the latter strain is used commonly in drug metabolism and toxicology studies. To begin to address the possible underlying mechanisms behind differences in the pharmacokinetics of xenobiotics between these two strains, the in vitro kinetics of glucuronidation and plasma protein binding of MRL-I and MRL-II in obese Zucker and Sprague-Dawley rats were evaluated. In addition, the mRNA levels of xenobiotic transporters and hepatic drug-metabolizing enzymes in these strains were determined.

Materials and Methods

Chemicals. MRL-I, [ethyl-¹⁴C]MRL-I, MRL-II, [¹³CD₃]MRL-II, and [methyl-¹⁴C]MRL-II (Fig. 1) were synthesized at Merck Research Laboratories and were provided as their arginine salts. Alamethicin, saccharolactone, and UDPGA were purchased from Sigma-Aldrich (St. Louis, MO). Acetonitrile and methanol (HPLC grade) were obtained from Fisher Scientific Co. (Pittsburgh, PA). All other reagents were of analytical or HPLC grade.

Animals. Male Sprague-Dawley and obese Zucker rats were obtained from Charles River Laboratories Inc. (Wilmington, MA). They were housed in cages and maintained on a 12-h light/dark cycle. Access to food and water was allowed ad libitum. Rats used in this study were 14 to 16 weeks old; the Sprague-Dawley rats weighed ∼300 g and the obese Zucker rats weighed ∼600 g. All procedures for animal experiments were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee.

Studies in Bile Duct-Cannulated Rats. [¹⁴C]MRL-I (1 mg/kg) or [¹⁴C]MRL-II (0.5 mg/kg) was dosed intravenously by a bolus injection into the femoral vein of male Sprague-Dawley rats whose bile ducts had been surgically cannulated (n = 3 or 4). Each rat was given ∼25 to 30 μCi of radioactivity. Bile and urine were collected for up to 72 h postdose at specific time intervals into bottles containing 0.5 M formate buffer, pH 3.0, to stabilize labile acyl glucuronide metabolites. Bile was collected at the following time intervals: predose, 0 to 1, 1 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 24, 24 to 48, and 48 to 72 h. Urine was collected at 24-h intervals up to 72 h postdose. Levels of radioactivity in the bile and urine were determined by liquid scintillation counting (Beckman LS 6500; Beckman Coulter, Fullerton, CA).

Metabolite Identification. Bile samples from the rats dosed with 1 mg/kg [¹⁴C]MRL-I were treated with an equal volume of acetonitrile, spun in a centrifuge at 16,000g for 10 min, and the supernatants were analyzed using an HPLC system consisting of a Shimadzu (Kyoto, Japan) series 200 pump, a Shimadzu series 200 autosampler, and a β-RAM scintillation detector (IN/US Systems, Inc., Tampa, FL). The samples were chromatographed on a 3.5-μm Zorbax RX-C18 column (150 × 4.6 mm; Agilent Technologies, Palo Alto, CA). The mobile phase consisted of 10 mM ammonium acetate in water (A) and 7.8 mM ammonium acetate in 92:8 acetonitrile/methanol, v/v (B), and the flow rate was 1 ml/min. The column was eluted with a linear gradient from 25 to 60% B over 30 min, followed by a linear gradient to 90% B in 1 min, and isocratic elution at 90% B for 5 min. Identification of [¹⁴C]MRL-I metabolites was achieved by LC-MS/MS using a PerkinElmerSciex Instruments (Boston, MA) API 3000 triple quadrupole mass spectrometer. The Turbo Ionspray source was operated in the negative ion mode at 400°C with an auxiliary nitrogen gas flow of 7.0 l/min. The HPLC effluent was split 1 to 4, with 0.2 ml/min directed into the mass spectrometer and the remaining 0.8 ml/min directed to a radioactivity flow-through detector (β-RAM; IN/US Systems, Inc.).

Identification of metabolites in bile samples from studies with [¹⁴C]MRL-II was achieved by HPLC analysis of acetonitrile extracts and comparison of retention times with chemically synthesized standards. Radiochromatograms were obtained using an on-line PerkinElmer Flow Scintillation Analyzer (PerkinElmer Life and Analytical Sciences, Boston, MA) at room temperature. Separation of metabolites was achieved using a PerkinElmer 200 series HPLC system equipped with a 5-mm Betasil Phenyl column (250 × 4.6 mm; Thermo Hypersil, Keystone Scientific Operations, Bellefonte, PA). The mobile phase consisted of 1 mM ammonium acetate in water/acetonitrile/acetic acid (95:5: 0.1, by volume) (A) and 1 mM ammonium acetate in acetonitrile/water/acetic acid (95:5:0.1, by volume) (B). Separation was achieved under isocratic conditions from 0 to 5 min at 10% B, followed by a 5-min linear gradient to 45% B, an isocratic hold for 10 min at 45% B, and then a linear gradient to 55% B in 10 min and to 95% B in 4 min, followed by a 7-min wash with 95% B. The flow rate was 1 ml/min.

In Vivo Pharmacokinetic Studies. Rats were anesthetized by intramuscular administration of a mixture consisting of 4 ml of ketamine (100 mg/ml), 2 ml of xylazine (20 mg/ml) and 1 ml of atropine (1 mg/ml), at 100 μl/100 g of body weight, and their femoral arteries and veins were cannulated surgically. After a full day of recovery from surgery, MRL-I or MRL-II in 0.9% saline was dosed intravenously via the femoral vein, at 1 or 2 mg/kg, respectively. Blood was drawn from the femoral artery into heparinized containers at regular intervals up to 48 h postdose. Plasma was obtained by centrifugation at 1600g for 10 min, acidified with 0.5 M formate buffer, pH 3.0 (300 μl buffer/ml plasma), and stored at -70°C until analysis by LC-MS/MS.

Quantification of MRL-I in Rat Plasma. Concentrations of MRL-I in rat plasma were determined by LC-MS/MS following acetonitrile precipitation of plasma proteins. Briefly, 65-μl aliquots of acidified plasma (corresponding to 50 μl of nonacidified plasma) were mixed with internal standard, (2R)-7-[3-[2-chloro-4-(4-phenoxy)phenoxy]propoxy]-2-ethyl-3,4-dihydro-2H-benzopyran-2-carboxylic acid, followed by the addition of 400 μl of acetonitrile. Samples were then spun in a centrifuge at 1600g for 10 min, and supernatants were transferred by a Tomtec unit (Qudra 96, model 320; Tomtec, Hamden, CT) and subjected to LC-MS/MS analysis. The LC-MS/MS system consisted of a PerkinElmer HPLC system and a PerkinElmerSciex API 3000 tandem mass spectrometer operated in the negative ionization mode using a Turbo Ionspray. Multiple reaction monitoring of the following precursor→product ion combinations was used for detection of analytes: MRL-I, m/z 499.5→237 and internal standard, m/z 481.4→219. The HPLC column (BetaBasic 18, 50 × 2 mm; Thermo Hypersil, Keystone Scientific Operations) was eluted with 70% A (1 mM ammonium acetate) and 30% B (acetonitrile) for 0.4 min, followed by a linear gradient to 90% B in 1 min, and a 1.5-min hold at 90% B with a flow rate of 0.2 ml/min. The analytical detection limit was 5 ng/ml.

Quantification of MRL-II in Rat Plasma. Concentrations of MRL-II in rat plasma were determined by LC-MS/MS as described above for MRL-I except that [¹³CD₃]MRL-II was used as the internal standard. Multiple reaction monitoring was used to detect the analytes, using the following precursor→product ion combinations: MRL-II, m/z 473.4→225.1 and [¹³CD₃]MRL-II, m/z 477.3→142.2. Samples were injected onto an ACE 5 phenyl column (100 × 2.1 mm, 5 μm; MAC-MOD Analytical, Inc., Chadds Ford, PA) and eluted with a mobile phase consisting of 1 mM ammonium acetate and 0.002% formic acid in water (A) and acetonitrile (B). Separation was achieved at 0.2 ml/min with a linear gradient of 30 to 95% B over 4 min and a 1-min hold at 95% B. The analytical detection limit was 5 ng/ml.

Determination of Pharmacokinetic Parameters. Concentrations of the analytes were computed from the peak area ratios (relative to internal standard) using external calibration curves. The pharmacokinetic parameters were calculated using Watson software (version 6; Watson Software Systems) or Microsoft Excel v 97 SR-1 (Microsoft, Redmond, WA) for noncompartmental models.

Determination of Reversible Plasma Protein Binding. Fresh EDTA-treated blood was collected from three male Sprague-Dawley and five male obese Zucker rats. Plasma was obtained by centrifugation at 1600g for 10 min. Aliquots of rat plasma (0.5 ml) were mixed with aqueous solutions of [¹⁴C]MRL-I or [¹⁴C]MRL-II to a final concentration of 1 μM. The resulting plasma samples were transferred into one side of dialysis cells (Bel-Art Products, Pequannock, NJ) which were separated by a dialysis membrane (Sigma-Aldrich) with a molecular weight cutoff of 12,400, and were dialyzed against isotonic phosphate buffer for 24 h at 37°C. The unbound fraction was determined by comparing the amount of radioactivity in 100-μl aliquots of buffer and plasma, determined by liquid scintillation counting.

Evaluation of the Expression of Xenobiotic Transporters and Hepatic Drug-Metabolizing Enzymes. The mRNA levels for several drug-metabolizing enzymes, including rat P450s, UGTs, and hepatic transporters for which specific oligonucleotide probes were available at Merck Research Laboratories, were evaluated in Sprague-Dawley and obese Zucker rats. Specific primers and probes for quantitative reverse transcription-polymerase chain reaction were developed as described by Wang et al. (2003). Total RNA was isolated from Sprague-Dawley and Zucker rat livers using the SV Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer's instructions. Samples were quantitated by spectrophotometry and diluted to a concentration of 15 μg/ml. RNA integrity was checked by agarose/formaldehyde gel electrophoresis. Samples were then assayed in triplicate 25-μl reactions using 25 ng of RNA per reaction. Gene-specific primers were used at 7.5 pmol per reaction, and the gene-specific probes were used at 5 pmol per reaction. Glyceraldehyde-3-phosphate dehydrogenase was used to normalize gene expression in all samples.

Preparation of Microsomes. Liver microsomes from Sprague-Dawley and obese Zucker rats were prepared as described by Raucy and Lasker (1991). Protein concentration was determined using the bicinchoninic acid method (Smith et al., 1985).

In Vitro Glucuronidation Metabolism. [¹⁴C]MRL-I or [¹⁴C]MRL-II was incubated with UDPGA-fortified Zucker and Sprague-Dawley rat liver microsomes. Final mixtures (200 μl) contained 100 mM Tris buffer, pH 7.5, 0.025 mg/ml alamethicin, 5 mM MgCl₂, 5 mM saccharolactone, and 5 mM UDPGA. The microsomal incubations were carried out at protein concentrations of 0.5 mg/ml. The microsomes were first incubated with alamethicin at 4°C for 15 min, followed by the addition of Tris buffer, saccharolactone, MgCl₂ and [¹⁴C]MRL-I, or [¹⁴C]MRL-II [0.5, 1, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, and 200 μM ([¹⁴C]MRL-II only)]. The reactions were started by the addition of UDPGA and allowed to proceed at 37°C for 20 and 30 min for [¹⁴C]MRL-I and [¹⁴C]MRL-II, respectively. The reactions were terminated by adding 200 μl of acetonitrile containing 2% formic acid. Samples were spun in a centrifuge at 16,000g for 10 min. The supernatants were analyzed by HPLC (vide infra), and the rate of metabolism was determined based on the formation of the acyl glucuronide. Kinetic parameters were calculated by fitting the experimental data from triplicate incubations to the Michaelis-Menten equations using SigmaPlot software (Enzyme Kinetics Module 1.1; SPSS Inc., Chicago, IL).

HPLC Analysis. The HPLC system consisted of a Shimadzu series 200 pump, a Shimadzu series 200 autosampler, and a β-RAM scintillation detector (IN/US Systems, Inc.). The samples from the liver microsomal incubations of [¹⁴C]MRL-I were injected onto a Zorbax SB-C8 column (150 × 4.6 mm; Agilent Technologies) and eluted at a flow rate of 1.5 ml/min with 10 mM ammonium acetate in water containing 0.1% acetic acid (A) and 7.2 mM ammonium acetate in a mixture of acetonitrile and methanol (92.8:7.2, v/v) containing 0.1% acetic acid in (B). A 15-min linear gradient from 40 to 70% B was used. The samples from the incubations with MRL-II were analyzed using a 5-μm BetaBasic C18 column (100 × 4.6 mm; Thermo Hypersil, Keystone Scientific Operations). The mobile phase consisted of 1 mM ammonium acetate in water/acetonitrile/acetic acid (95:5:0.1, by volume) (A) and 1 mM ammonium acetate in acetonitrile/water/acetic acid (95:5:0.1, by volume) (B). Separation was achieved under isocratic conditions from 0 to 5 min at 50% B, followed by a 6-min linear gradient to 90% B, and an isocratic hold for 3 min at 90% B. The flow rate was 1.5 ml/min. In both cases, radiochromatographic peaks were monitored using a β-RAM scintillation detector.

Results

Studies in Bile Duct-Cannulated Rats. The in vivo disposition of [¹⁴C]MRL-I and [¹⁴C]MRL-II was evaluated in Sprague-Dawley rats after a bolus intravenous administration. The extent of biliary and urinary excretion of radioactive dose is summarized in Table 1. As shown, most of the radioactivity was excreted into bile, with only ∼8 and 29% for [¹⁴C]MRL-I and [¹⁴C]MRL-II doses recovered in urine, respectively. The acyl glucuronide conjugate was the major radioactive component detected in bile from rats dosed with [¹⁴C]MRL-I and [¹⁴C]MRL-II, whereas bile samples from rats dosed with [¹⁴C]MRLII also contained the taurine conjugate of parent compound (Fig. 2).

Pharmacokinetics of MRL-I and MRL-II.Table 2 summarizes the pharmacokinetic parameters of MRL-I and MRL-II in Sprague-Dawley and obese Zucker rats following a single intravenous dose. The plasma clearance was lower (4-fold and 2-fold, respectively, for MRL-I and MRL-II) and the half-life was longer (7- and 3-fold, respectively) in obese Zucker rats compared with Sprague-Dawley rats, without any significant differences in the volume of distribution.

Reversible Plasma Protein Binding. The protein binding of [¹⁴C]MRL-I and [¹⁴C]MRL-II was investigated in plasma from Sprague-Dawley and obese Zucker rats. The free fraction of [¹⁴C]MRL-I and [¹⁴C]MRL-II was ∼3.9 and 4.2%, respectively, in both obese Zucker and Sprague-Dawley rats at a concentration of 1 μM. Plasma protein binding in Sprague-Dawley rats was not concentration-dependent between 1 and 50 μM (data not shown). From the results of in vivo studies, there was no indication of instability of [¹⁴C]MRL-I and [¹⁴C]MRL-II in plasma at 37°C.

Expression of Hepatic Drug-Metabolizing Enzymes and Xenobiotic Transporters. The mRNA levels (normalized relative to GADPH) of several drug-metabolizing enzymes and transporters in the liver from Sprague-Dawley and obese Zucker rats are shown in Table 3. In general, relatively lower expression levels were observed in obese Zucker rats compared with Sprague-Dawley rats. The mRNA levels of CYP2C11 and 3A1 and UGT1A1, 1A6, and 2B1 in Zucker rat liver were ∼30 to 60% of the levels in the livers of Sprague-Dawley rats. Similar decreases in Oatp2 (80%) and multidrug resistance-associated protein 2 (Mrp2) (50%) mRNA were observed in Zucker rats, whereas the expression of Mrp3 was similar in both strains.

In Vitro Glucuronidation. The UGT-mediated metabolism of MRL-I and MRL-II was evaluated in liver microsomes prepared from Sprague-Dawley and obese Zucker rats. The kinetic parameters V_max and K_m are summarized in Table 4. The CL_int values, calculated from V_max/K_m, were ∼2-fold higher in Sprague-Dawley rat liver microsomes, whereas the K_m values were comparable for both compounds.

Discussion

Genetically obese Zucker rats are characterized by the cosymptoms of obesity, hyperglycemia, hyperinsulinemia, and hyperlipidemia (Stevenson and Kaysen, 1999). Since these symptoms are similar to the clinical observations in obese patients with Type II diabetes, the Zucker rat has been used as a genetic model to study obesity, as well as a pharmacological model for the discovery of new drugs for the treatment of Type II diabetes and hyperlipidemia (Bray, 1977; Kurtz et al., 1989; Krief and Bazin, 1991).

In the studies reported herein with the PPAR agonists MRL-I and MRL-II, we have observed significant differences in their pharmacokinetics in genetically obese Zucker and lean Sprague-Dawley rats (Table 1). Previously, Brouwer et al. (1984) reported different pharmacokinetics of phenobarbital in the Zucker and Sprague-Dawley rat. After a single intravenous bolus administration, the plasma clearance of phenobarbital in the obese Zucker and lean Sprague-Dawley rats was 20 and 56 ml/h/kg, respectively, whereas the volume of distribution was comparable. However, no explanation was provided for these observations. Since the data from the present study were in agreement with those of Brouwer et al. (1984), we have proceeded to investigate the possible underlying mechanisms that caused these differences in the pharmacokinetics of MRL-I and MRL-II.

Altered serum protein binding, an important determinant of drug disposition, has been reported for propranolol in obese humans (Benedek et al., 1984) and for propranolol, diazepam, and phenytoin in obese Zucker rats (Benedek et al., 1985). They reported that concentrations of α₁-acid glycoprotein in the obese human were double those of normal-weight controls, resulting in increased serum protein binding of propranolol. Similarly, the plasma protein binding of propranolol and diazepam was higher in obese than in lean Zucker rat. In contrast, the binding of phenytoin, which is primarily bound to serum albumin, was lower in obese Zucker rats (Porter and Layzer, 1975). Based on the report on the serum chemistry by Benedek et al. (1985), albumin concentrations were comparable among obese Zucker, lean Zucker, and Sprague-Dawley rats. However, the concentrations of free fatty acid, and cholesterol and triglyceride levels were higher in obese Zucker rats compared with the lean Zucker and Sprague-Dawley rats, which may affect drug-serum binding. Serum free fatty acids have been reported to displace phenytoin from rat albumin binding sites (Colburn and Gibaldi, 1978). Therefore, phenytoin displacement by elevated serum free fatty acids is most likely the reason for the decreased binding of phenytoin to serum albumin. Also, the presence of increased lipoprotein and other serum protein concentrations appeared to be responsible for the significant increase of protein binding of diazepam in obese compared with lean Zucker rats (Benedek et al., 1985).

In light of this information, the reversible plasma protein binding of [¹⁴C]MRL-I and [¹⁴C]MRL-II was evaluated in Sprague-Dawley and obese Zucker rats. The present data indicated that altered protein binding was not one of the contributing factors to the different pharmacokinetics with MRL-I and MRL-II.

In Sprague-Dawley rats, MRL-I and MRL-II were shown to be eliminated primarily by glucuronidation of the carboxylic acid moiety, followed by excretion of the resulting acyl glucuronide predominantly into the bile (Table 1; Fig. 2). The rat UGTs catalyzing MRL-I and MRL-II have not been characterized; however, in vitro studies using baculovirus-expressed human UGT Supersomes indicated that MRL-I was a good substrate of UGT1A3 and, to a lesser extent, UGT1A6, 1A9, and 2B7, whereas UGTs1A3, 1A9, and 2B7 exhibited similar activity toward MRL-II, with UGT1A1 and 1A8 showing a much lower extent of glucuronidation (Kim et al., 2004). In light of this information, the expression of rat hepatic UGTs, 1A1, 1A6, and 2B1, in obese Zucker and Sprague-Dawley rats was evaluated. The results indicated that the mRNA expression of the UGTs was lower in Zucker than in Sprague-Dawley rats. Similarly, lower mRNA levels were observed for CYP3A1, CYP2C11, glutathione S-transferase, and quinone reductase. The activities of UGTs in both strains were further characterized in the in vitro glucuronidation kinetic studies. The CL_int, calculated from V_max/K_m, in liver microsomes of Sprague-Dawley rats, was ∼ 2-fold higher than that in obese Zucker rat liver microsomes, consistent with the observed differences in plasma clearance between the two strains (Table 3). The intrinsic clearance of [¹⁴C]MRL-I was higher than that of [¹⁴C]MRL-II, especially in Sprague-Dawley rats. In contrast, the plasma clearance of MRL-I was the same as that of MRL-II in Sprague-Dawley rats. These results are consistent with the observation that MRL-II is eliminated not only by acyl glucuronidation but also by taurine conjugation after intravenous administration (Table 1; Fig. 2).

Altered metabolism in hepatic microsomes and cytosol from genetically obese Zucker rats has been reported previously. The specific activities of P450 and glutathione S-transferase in obese Zucker rats were 50 to 70% lower than those in lean Zucker rats and Sprague-Dawley rats (Litterst, 1980). Additionally, altered expression of hepatic CYP2E1 and CYP4A in obese, diabetic ob/ob, and fa/fa Zucker rats has been reported (Enriquez et al., 1999; Roe et al., 1999). The data showing lower hepatic xenobiotic enzyme activities in genetically obese rats can be contrasted with the data reported for acetaminophen by Wong et al. (1986) in diet-induced obese rats, and by Chaudhary et al. (1993) in genetically obese Zucker rats. Wong et al. (1986) have shown enhanced glucuronidation and sulfate conjugation of acetaminophen in diet-induced obese rats. Chaudhary et al. (1993) reported a higher rate of acetaminophen glucuronidation in liver microsomes from obese Zucker rats than from lean control rats, but the rates of glucuronidation of 1-naphthol, estrone, and morphine were comparable between lean and obese Zucker rats. The reason(s) for these apparent discrepancies remains to be characterized; however, based on the report by Chaudhary et al. (1993), the effect of genetic obesity on glucuronidation appears to be substrate-specific. It is also possible that physiological differences, such as age, and environmental factors may be responsible for these differences. Galinsky et al. (1986) reported that the fraction excreted as the glucuronide and the partial clearance of acetaminophen glucuronidation increased with age. The animals used in our study were 14 to 16 weeks old and were relatively younger than the 5- to 6-month-old animals used by Chaudhary et al. (1993) in their study. Both multidrug resistance (mdr)1a and mdr1b knockout mice housed in Amsterdam had increased levels of CYP3A protein, whereas the hepatic P450 expression was unaffected or decreased in the genetically identical mdr1a and mdr1a/1b^-/- mice housed in the United States (Schuetz et al., 2000), suggesting that the expression of hepatic drug-metabolizing enzymes depends not only on the nature of the genetics but also on the environmental factors. Interestingly, enhanced glucuronidation of acetaminophen, as well as lorazepam and oxazepam, has been reported in obese humans (Abernethy and Greenblatt, 1982). Again, the reasons for this apparent discrepancy in glucuronidation between genetically obese Zucker rats and obese humans remains to be determined; however, different genetics leading to differences in physiology may be a contributing factor. The obesity, hyperglycemia, and hyperinsulinemia observed in obese Zucker rats are the result of a mutation on the leptin receptor (Campfield et al., 1996). However, human obesity is not believed to be related to leptin deficiency (Considine, 1996).

Rat Oatp2, a member of the multispecific organic anion-transporting polypeptide family (Noe et al., 1997; Abe et al., 1998), is expressed in the hepatic sinusoidal membrane and it transports anions, cations, and neutral compounds (Abe et al., 1998; Eckhardt et al., 1999). Mrp2 is a transporter that was originally recognized in rat hepatocyte canalicular membranes and has been shown to mediate the ATP-dependent transport of glutathione, glucuronide, and sulfate conjugates of lipophilic compounds, and several other endogenous and xenobiotic compounds (Jansen et al., 1985; Oude Elferink and Jansen 1994; Gerk and Vore, 2002). Another organic anion transporter, Mrp3, is expressed almost exclusively in the intestine, with much lower levels found in other tissues in Sprague-Dawley rats (Cherrington et al., 2002). This transporter is localized to the basolateral membrane where it is involved in the excretion of various organic anions from cells into the sinusoidal blood (Hirohashi et al., 1999; Renes et al., 2000).

In the present studies, it was determined that the expression of Oatp2 and, to a lesser extent, Mrp2 was lower in obese Zucker rats than in Sprague-Dawley rats. Both transporters are believed to play major roles in the disposition of organic anions, catalyzing, respectively, their initial uptake from blood into hepatocytes (Noe et al., 1997; Abe et al., 1998) and subsequent secretion into bile across the canalicular membrane (Gerk and Vore 2002; Kim et al., 2003). Thus, the lower mRNA levels of oatp2 in obese Zucker rats may have resulted in decreased sinusoidal hepatic uptake of MRL-I and MRL-II, as compared with Sprague-Dawley rats, and may, in part, be responsible for their lower plasma clearance. Similarly, since hepatic biliary excretion as acyl glucuronides is the major elimination pathway for both MRL-I and MRL-II, it is conceivable that the decrease in the expression of Mrp2 could have affected the biliary clearance of MRL-I and MRL-II. Additional studies are needed to determine definitely whether the decreased expression of oatp2 and Mrp2 in Zucker rats may have contributed to the decreased clearance of MRL-I and MRL-II compared with Sprague-Dawley rats.

In summary, we have demonstrated that the pharmacokinetics of MRL-I and MRL-II, compounds cleared principally by phase II metabolism, were significantly altered in genetically obese Zucker rats compared with Sprague-Dawley rats. Furthermore, differences in the mRNA levels of phase I and phase II hepatic drug-metabolizing enzymes and transporters were documented and may have contributed to the observed differences in pharmacokinetics between the two strains of rats.