Hepatic expression of cytochrome P450 in type 2 diabetic Goto–Kakizaki rats

Chemico-Biological Interactions 195 (2012) 173–179

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Hepatic expression of cytochrome P450 in type 2 diabetic Goto–Kakizaki rats Soo Jin Oh a,b,1, Jong Min Choi a,1, Kang Uk Yun a, Jung Min Oh a, Hui Chan Kwak a, Jin-Gyo Oh c, Kye Sook Lee a, Bong-Hee Kim a, Tae-Hwe Heo c,⇑, Sang Kyum Kim a,⇑ a

College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea Bio-Evaluation Center, KRIBB, 685-1 Yangcheong-ri, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea c College of Pharmacy, The Catholic University of Korea, Bucheon, Kyunggi 420-743, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 11 November 2011 Received in revised form 28 December 2011 Accepted 28 December 2011 Available online 5 January 2012 Keywords: Diabetes GK rat Cytochrome P450 Hepatic metabolism Ketone body

a b s t r a c t Although hepatic expression of cytochrome P450 (CYP) changes markedly in diabetes, the role of ketone bodies in the regulation of CYP in diabetes is controversial. The present study was performed to determine the expression and activity of CYP in non-obese type II diabetic Goto–Kakizaki (GK) rats with normal levels of ketone bodies. In the present study, basal serum glucose levels increased 1.95-fold in GK rats, but acetoacetate and b-hydroxybutyrate levels were not significantly different. Hepatic expression of CYP reductase and CYP3A2 was up-regulated in the GK rats, and consequently, activities of CYP reductase and midazolam 4-hydroxylase, mainly catalyzed by CYP3A2, increased. In contrast, hepatic expression of CYP1A2 and CYP3A1 was down-regulated and the activities of 7-ethoxyresorufin-O-deethylase and 7-methoxyresorufin-O-demethylase, mainly catalyzed by CYP1A, also decreased in GK rats. Hepatic levels of microsomal protein and total CYP and hepatic expression of cytochrome b5, CYP1B1, CYP2B1 and CYP2C11 were not significantly different between the GK rats and normal Wistar rats. Moreover, the expression and activity of CYP2E1, reported to be up-regulated in diabetes with hyperketonemia, were not significantly different between GK rats and control rats, suggesting that elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. Our results showed that the expression of hepatic CYP is regulated in an isoform-specific manner. The present results also show that the GK rat is a useful animal model for the pathophysiological study of non-obese type II diabetes with normal ketone body levels. Ó 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diabetes has been reported to result in an altered expression and activity of hepatic cytochrome P450 (CYP)1A, CYP2B, CYP2E1, CYP3A, CYP4A and other drug-metabolizing enzymes [1]. Because insulin secretion, sensitivity (i.e. resistance) and/or levels are altered in diabetes, insulin may mediate changes in the expression of drugmetabolizing enzymes observed in the disease. In fact, insulin administration to chemically induced or spontaneously diabetic rats has been reported to restore drug-metabolizing enzyme activity and expression [2–7]. We have demonstrated that insulin regulates the expression of CYP2E1, alpha-class glutathione S-transferase (GST), microsomal epoxide hydrolase and glutathione synthesis enzyme in primary cultured rat hepatocytes [7–10]. Furthermore, previous

⇑ Corresponding authors. Address: College of Pharmacy, Chungnam National University, 220 Gung-dong, Yuseong-gu, Daejeon 305-764, Republic of Korea. Tel.: +82 42 821 5930; fax: +82 42 823 6566 (S.K. Kim), tel.: +82 2 2164 4053; fax: +82 2 2164 4059 (T.-H. Heo). E-mail addresses: [email protected] (T.-H. Heo), [email protected] (S.K. Kim). 1 These authors contributed equally to this work. 0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.12.010

studies have demonstrated that the expression of CYP2E1 is down-regulated by insulin in primary cultured rat hepatocytes and that insulin signaling pathways involving phosphatidylinositol 3-kinase/ribosomal p70 S6 kinase are active in the insulin-mediated regulation of CYP2E1 expression [7]. These results suggest that changes in drug-metabolizing enzyme expression and activity levels in diabetes may be attributable to alterations in insulin level and/or sensitivity. Some studies have researched the effect of ketone bodies on the regulation of CYP expression and activity [2,7,11]. Hyperketonemia is a very common feature in diabetes. Acetoacetate, one of the major ketone bodies, reduced CYP2E1 mRNA levels but increased CYP2E1 protein levels in primary cultured rat hepatocytes [11]. These results suggest that hepatic expression of CYP isoforms in diabetes may be regulated by nutrients as well as hormones. The objective of the present study was to determine hepatic expression and activity of CYP isoforms in GK rats, a non-obese diabetic Wistar substrain that develops type 2 diabetes early in life [12,13]. This rat model exhibits mild hyperglycemia, hyperinsulinemia, nephropathy, hepatic glucose overproduction and insulin resistance [14–16]. However, both serum acetoacetate and serum b-hydroxybutyrate, two major ketone bodies, are similar to those

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in control rats [16]. Thus, the GK rat model can identify changes in hepatic expression of CYP isoforms in diabetes without hyperketonemia.

measured by NADPH-dependent reduction of cytochrome c, as described by Strobel and Dignam [20]. 2.6. Immunoblot analysis

2. Materials and methods 2.1. Chemicals NADH, NADPH, glycerol, sodium dithionite (sodium hydrosulfite), cytochrome c, resorufin, ethoxyresorufin, methoxyresorufin, chlorzoxazone (CZX) and 6-OH-CZX were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Midazolam (MDZ) was obtained from Bukwang Pharmaceutical Co. (Seoul, Korea), and 10 -hydroxymidazolam was acquired from BD Biosciences, Inc. (San Diego, CA, USA). All other chemicals and solvents were of reagent grade or better. Cytochrome b5, P450 reductase, CYP1A1, CYP1A2, CYP1B1, CYP2B1, CYP2C11, CYP2E1, CYP3A1 and CYP3A2 antibodies were purchased from Detroit R&D (Detroit, MI, USA), and antiactin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, CA, USA). 2.2. Animals Normal male Wistar rats and male diabetic GK rats were obtained from Charles River Japan, Inc. (Central Lab Animal, Inc., Seoul, Korea) and were housed in humidity- (55 ± 5%) and temperature-controlled (23 ± 1 °C) rooms with a 12 h light/dark cycle for at least 1 week before experimentation. Laboratory chow and tap water were allowed ad libitum. All procedures were approved by the Chungnam National University Committee for the Use and Care of Animals. The experiments were performed using 9-week-old GK rats and age-matched Wistar rats. 2.3. Biochemical analysis of serum Blood was sampled from the abdominal aorta in rats under light ether anesthesia. Serum glucose levels in all the rats were measured using the MediSense Optium Kit (Abbott Laboratories, Bradford, MA, USA). Serum acetoacetate and b-hydroxybutyrate concentrations were measured by enzymatic methods using a JCA-BM8 autoanalyzer (JEOL Ltd., Tokyo, Japan). 2.4. Preparation of hepatic microsomes The liver was homogenized in a 3-fold volume of ice-cold buffer consisting of 0.154 M KCl, 50 mM Tris–HCl and 1 mM EDTA (pH 7.4). All subsequent steps were performed at 0–4 °C. The homogenate was centrifuged at 10,000g for 20 min, and the supernatant fraction was further centrifuged at 104,000g for 65 min. The microsomal pellet was suspended and recentrifuged at 104,000g for 65 min. The microsomes were diluted to an equivalent of 1.0 g of liver/1 ml of buffer. Protein was determined by the Lowry method, with bovine serum albumin as the standard [17]. 2.5. Determination of total CYP content, cytochrome b5 content and P450 reductase activity The hepatic microsomal CYP contents were estimated from the CO difference spectrum, as described by Omura and Sato [18], using a spectrophotometer (V-630; Jasco, Tokyo, Japan). Sodium dithionite was used as the reducing agent. The hepatic microsomal cytochrome b5 content was determined by measuring the difference in the spectra between oxidized and reduced microsomes using NADH as the reductant [19]. Reductase activity was

For the immunoblot analysis of cytochrome b5, P450 reductase and CYP isoforms, microsomes were diluted to 1.5 mg/ml with loading buffer that contained reducing agent. The microsomal lysate (10 ll) was resolved by 10% SDS–PAGE, transferred to nitrocellulose (Bio-Rad Laboratories, Hercules, CA, USA), blocked in 5% milk powder in PBST (0.1% Tween 20 in phosphate-buffered saline; PBS) and incubated with anti-CYP1A1 (1:3000 in 5% milk in PBST), anti-CYP1A2 (1:50,000 in 5% milk in PBST), anti-CYP1B1 (1:3000 in 5% milk in PBST), anti-CYP2B1 (1:3000 in 5% milk in PBST), antiCYP2C11 (1:3000 in 5% milk in PBST), anti-CYP2E1 (1:150,000 in 5% milk in PBST), anti-CYP3A1 (1:3000 in 5% milk in PBST), antiCYP3A2 (1:3000 in 5% milk in PBST), anti-cytochrome b5 (1:5000 in 5% milk in PBST), anti-P450 reductase (1:1000 in 5% milk in PBST) and anti-actin (1:3000 in 5% milk in PBST) overnight at 4 °C. Blots were washed six times with PBST, and then, incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:10,000 in 5% skim milk in PBST) or horseradish peroxidase-conjugated goat anti-mouse antibody (1:10,000 in 5% skim milk in PBST) for 2 h at room temperature. Proteins were detected by enhanced chemiluminescence on the Chemidoc (Bio-Rad Laboratories) or LAS 4000 Mini (Fuji Photo Film Co., Tokyo, Japan). Immunoquantification of the immunoblots was accomplished by the Quantity One analysis program (Bio-Rad Laboratories) or Multi Gauge 3.0 software (Fuji Photo Film Co.). 2.7. Microsomal enzyme assays Microsomal 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) activities were measured using a modification of the method described by Kobayashi et al. [21]. Reaction mixtures contained microsomal protein (0.25 mg/ml for EROD and MROD), the substrate (1 lM 7-ethoxyresorufin or 0.5 lM 7-methoxyresorufin), 5 mM MgCl2, 1 mM NADPH and 50 mM potassium phosphate (pH 7.4) in a total volume of 0.4 ml. After a 3 min preincubation period, the reaction was initiated by adding NADPH. After the incubation was performed at 37 °C for 6 min, the reaction was terminated by adding 0.8 ml of ice-cold methanol, and the mixture was centrifuged at 6,000g for 20 min. The supernatant was analyzed using a HPLC column (SCL-10A; Shimadzu, Kyoto, Japan) equipped with a fluorescence detector (RF-10AXL, Ex 560 nm and Em 585 nm; Shimadzu). Chromatographic separation was achieved using a Luna C18 column (4.6 mm  150 mm, 5 lm; Phenomenex, Torrance, CA, USA). The mobile phase was 20 mM phosphate buffer (pH 6.8)–methanol–acetonitrile (52:45:3, v/v/v), and the flow rate was 0.8 ml/min. The rate of MDZ hydroxylation by rat liver microsomes was measured by the formation of 4-hydroxy MDZ and 10 -hydroxy MDZ [22]. Reaction mixtures consisted of 0.5 mg/ml microsomal protein, 50 lM MDZ, 1 mM NADPH and 50 mM phosphate buffer (pH 7.4) in a total volume of 0.4 ml. After a 5 min preincubation period, the reaction was initiated by adding NADPH. The incubation was performed at 37 °C for 10 min. The reaction was terminated with ice-cold acetonitrile containing 10 lM phenacetin used as an internal standard, and the mixture was centrifuged at 14,000g for 20 min. The supernatant was analyzed using a HPLC column (SCL-10A; Shimadzu) equipped with a UV detector (SPD10Avp, 220 nm; Shimadzu). The mobile phase was 10 mM KH2PO4 (pH 7.4)–methanol–acetonitrile (40:37.5:22.5, v/v/v), and the flow rate was 1 ml/min. The rate of CZX hydroxylation by rat liver microsomes was measured by the formation of 6-hydroxy CZX [23]. Reaction mixtures

S.J. Oh et al. / Chemico-Biological Interactions 195 (2012) 173–179

consisted of 0.1 mg/ml microsomal protein, 40 mM CZX, 1 mM NADPH and 50 mM phosphate buffer (pH 7.4) in a total volume of 0.4 ml. After a 5 min preincubation period, the reaction was initiated by adding NADPH. The incubation was performed at 37 °C for 10 min. The reaction was terminated with ice-cold acetonitrile containing 100 nM diclofenac as an internal standard, and the mixture was centrifuged at 3000g for 10 min. The supernatant was analyzed by LC–ESI/MS/MS system consisting of an Agilent 1200 series HPLC system (Agilent Technologies, Wilmington, DE, USA) and an API 4000 LC–MS/MS system equipped with a Turbo V IonSpray source (Applied Biosystems, Foster City, CA, USA) operated in the negative ion mode. The sample injection volume was 10 ll, and the separation was performed on an Atlantis dC18 column (150  4.6 mm i.d., 5 lm; Waters, Milford, MA, USA) with a SecurityGuard C18 guard column (2.0  4.0 mm i.d., Phenomenex) maintained at 30 °C. The column was pre-equilibrated in 100% v/v solvent A (deionized water containing 0.1% v/v formic acid)/0% v/v solvent B (acetonitrile containing 0.1% v/v formic acid) at a flow rate of 0.7 ml/min. A linear gradient of the two solvents was used; it started at 100% A and held for 0.1 min, ramped to 50% A to 4 min, and held until 0.1 min. The TurboIonSpray interface was operated in the negative ion mode at 4500 V. The operating conditions were determined as follows: ion source temperature, 600 °C; nebulizing gas flow, 50 L/min; auxiliary gas flow, 4.0 L/min; curtain gas flow, 20 L/min; and collision energy, 91 eV. Multiple-reactionmonitoring mode using specific precursor/product ion transition was used for quantification. Detection of the ions was performed by monitoring the transitions of m/z 184 ? 120. Peak areas for metabolites were automatically integrated using Analyst software (version 1.5; Applied Biosystems).

2.8. Statistical analysis All data are presented as the mean ± SD for seven rats. Significant differences between groups were determined using an unpaired Student’s t-test. The acceptable level of significance was established at P < 0.05, except where indicated otherwise.

3. Results 3.1. Levels of serum glucose and serum ketone bodies in GK rats Body weights, but not liver weights, decreased significantly in GK rats compared to the same-aged Wistar rats, and consequently, liver weight per body weight in GK rats increased from 3.54 ± 0.20% to 4.51 ± 0.13%. Serum basal glucose levels of GK rats were 1.95-fold higher than those of Wistar rats (Table 1). No significant differences in serum acetoacetate and b-hydroxybutyrate concentrations were observed between the GK and control rats. These results support the idea that the GK rat is a model of diabetes mellitus without hyperketonemia [16]. In addition, the GK rats did not show liver injury as evaluated by serum enzyme activities of alanine aminotransferase (40 ± 8 IU/L in Wistar rats; 35 ± 5 IU/L in GK rats) and aspartate aminotransferase (76 ± 5 IU/L in Wistar

Table 1 Levels of serum glucose and serum ketone bodies in GK rats.

Glucose (mg/dl) Acetoacetate (mmol/L) b-Hydroxybutyrate (mmol/L)

Control

GK

171 ± 7 0.10 ± 0.02 0.22 ± 0.07

333 ± 65*** 0.10 ± 0.03 0.21 ± 0.06

Each value represents the mean ± SD for seven rats. Significantly different from the control at P < 0.001.

***

175

rats; 77 ± 8 IU/L in GK rats) and serum levels of albumin (3.1 ± 0.1 mg/mg in Wistar rats; 3.2 ± 0.20 mg/mg in GK rats). 3.2. Hepatic microsomal protein, total CYP, cytochrome b5 and CYP reductase in GK rats Levels of hepatic microsomal protein, total CYP, cytochrome b5 and CYP reductase were determined in hepatic microsomes isolated from the GK rats or Wistar rats (Fig. 1). Neither hepatic microsomal protein level nor hepatic total CYP content determined from CO difference spectra was significantly different between the GK rats and control rats (Fig. 1A and B). Hepatic cytochrome b5 levels determined by spectrophotometry and immunoblot analysis were not also different between the GK rats and control rats (Fig. 1C and D). Both hepatic CYP reductase activity and protein level, however, increased in GK rats (Fig. 1E and F). In addition, the level of hepatic microsomal actin, determined as the loading control, was not different between the two groups (Fig. 1G). 3.3. Expression and activity of CYP isoforms in liver microsomes of GK rats Hepatic expression of CYP isoforms, including CYP1A1, CYP1B1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A1 and CYP3A2 was measured in the GK rats (Fig. 2). Hepatic CYP1A1 was not detected in both GK rats and Wistar rats and CYP1B1 protein level was not significantly different between GK rats and control rats (data not shown). CYP1A2 and CYP3A1 protein levels decreased significantly in the GK rats compared to control rats (Fig. 2A and E). However, CYP3A2 protein level, a male-specific CYP isoform, was significantly increased 1.6-fold in the GK rats relative to control rats (Fig. 2F). CYP2B1, CYP2C11 and CYP2E1 protein levels were not significantly different between GK rats and Wistar control rats (Fig. 2B–D). To compare hepatic microsomal CYP-dependent activities between GK rats and control Wistar rats, EROD, MROD, CZX 6hydroxylase, MDZ 10 -hydroxylase and MDZ 4-hydroxylase activities were measured (Fig. 3). Ethoxyresorufin is a substrate sensitive to CYP1A1; CYP1A2, CYP2C6, CYP2C11 and CYP2E1 are also involved in ethoxyresorufin metabolism, while methoxyresorufin metabolism is primarily catalyzed by CYP1A1 and CYP1A2 [24,25]. CZX 6-hydroxylation is extensively mediated by the CYP2E1 isoform [23]. MDZ is predominantly metabolized to 10 -hydroxy MDZ and 4-hydroxy MDZ by CYP3A1 and CYP3A2 in rats, and it has thus been used as a selective CYP3A substrate [21,26]. In this study, hepatic microsomal activities of EROD and MROD significantly decreased in the GK rats (Fig. 3A and B). MDZ 10 -hydroxylase activity was slightly increased 1.2-fold (Fig. 3D) and MDZ 4-hydroxylase activity was significantly increased 1.36-fold in the GK rats relative to control rats (Fig. 3E). However, CZX 6-hydroxylase activity was not significantly different between the GK rats and control rats (Fig. 3C). 4. Discussion The GK rat is a non-obese type II diabetes animal model characterized by impaired glucose-stimulated insulin secretion in the pancreatic b-cells. In the present study, serum levels of glucose, but not acetoacetate or b-hydroxybutyrate, increased in GK rats, indicating that the GK rat is a model of diabetes mellitus without hyperketonemia. These results are consistent with previous results reported by Minehiro et al. [16]. Hepatic expression of CYP reductase and CYP3A2 was up-regulated in the GK rats. CYP reductase activity determined using cytochrome c as a substrate and midazolam hydroxylase activity, catalyzed by CYP3A, also increased. In contrast, hepatic expression of CYP1A2 and CYP3A1 was down-regulated. The

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Total CYP contents (nmol/mg protein)

A

20 15 10 5 0

CYP reductase activity (nmol/min/mg protein)

0.6 0.4 0.2

Control

GK

Control

GK

Control

GK

Control

GK

D

120

0.3 0.2

(% of control)

0.4

0.1

100 80 60 40 20

0.0

0

Control

E

GK

350

*

300

**

200

100

*

F

300 250 200 150 100 50

0

140 120

Actin (% of control)

0.8

140

C

0.5

400

1.0

GK

Cytochrome b5

(nmol/mg protein)

Cytochrome b5 contents

0.6

B

1.2

0.0

Control

CYP reductase (% of control)

Microsomal protein levels (mg protein/g liver)

25

0

Control

GK

Control

GK

Control

GK

Control

GK

G

100 80 60 40 20 0

Fig. 1. Hepatic microsomal protein levels (A), total CYP contents (B), cytochrome b5 levels determined by spectrophotometry (C) and immunoblot analysis (D), CYP reductase activity (E) and protein levels (F), and actin levels (G) in GK rats. Each value represents the mean ± SD for seven rats. ⁄Significantly different from the control at P < 0.05.

EROD and MROD activities, mainly catalyzed by CYP1A, also decreased. Hepatic levels of microsomal protein and total CYP, and hepatic expression of cytochrome b5, CYP1B1, CYP2B1, CYP2C11 and CYP2E1 were not significantly different between the GK rats and control rats. Hepatic microsomal activity of CZX 6-hydroxylase, mainly catalyzed by CYP2E1, was not different.

Hepatic CYP1A1 in rats is generally known to be constitutively expressed very early in development, but not at appreciable levels in the adult liver [27]. In the present study, hepatic CYP1A1 protein was not detected in either GK rats or Wistar controls, which may be attributable to using adult (9-week-old) GK rats and the agematched Wistar rats as the experimental model. Hepatic expression

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Control

150

GK

160

120 90

**

60 30

CYP2B1 (% of control)

CYP1A2 (% of control)

A

0

GK

GK

Control

GK

Control

GK

Control

GK

80

40

120

C CYP2E1 (% of control)

CYP2C11 (% of control)

Control

90 60 30 0

D

100 80 60 40 20 0

Control

GK

Control

GK

200

E

100 80

*** **

60 40 20 0

CYP3A2 (% of control)

CYP3A1 (% of control)

Control

120

GK

120

120

GK

B

0

Control

150

Control

***

F

150

100

50

0

Control

GK

Control

GK

Fig. 2. Hepatic microsomal expression of CYP1A2 (A), CYP2B1 (B), CYP2C11 (C), CYP2E1 (D), CYP3A1 (E) and CYP3A2 (F) in GK rats. Each value represents the mean ± SD for seven rats. ⁄⁄, ⁄⁄⁄Significantly different from the control at P < 0.01 or P < 0.001, respectively.

and activity of CYP1A2 have been reported to be up-regulated in chemically induced diabetic animals [28]. The present study showed that hepatic microsomal expression of CYP1A2 decreased in GK rats, which was correlated with decreased activities of EROD and MROD, mainly catalyzed by CYP1A. These results suggest that insulin resistance is not a major factor involved in CYP1A2 induction observed in diabetes. In the present study, MDZ 10 -hydroxylase activity increased slightly by 1.2-fold and MDZ 4-hydroxylase activity was significantly increased by 1.36-fold in the GK rats, although the protein levels of CYP3A1 and CYP3A2 were differently expressed in these diabetic rats. These results raise the possibility that CYP3A2 plays a more important role in MDZ metabolism than CYP3A1. In fact, the contribution of CYP3A isoforms in MDZ metabolism to 10 hydroxylation and 4-hydroxylation is different. 4-Hydroxylation

of MDZ is mainly metabolized by CYP3A2, but 10 -hydroxylation of MDZ is metabolized by approximately equal contributions of CYP3A1 and CYP3A2 [21,24]. CYP3A2, a male-specific CYP, was dominantly expressed in the liver of adult male rats relative to other members of the CYP3A subfamily [29]. These results suggest that the up-regulation of CYP3A2 protein in the GK rats may be responsible for the elevation of MDZ hydroxylation activities. In fact, our previous results showed that hepatic microsomal MDZ 10 -hydroxylase and MDZ 4-hydroxylase in male rats may be more sensitive to changes in CYP3A2 levels compared to CYP3A1 [27]. Our findings were also consistent with previous results that showed slight increases in CYP3A activity using testosterone as a substrate in GK rats [30]. Hepatic CYP2E1 expression has been reported to be up-regulated in diabetes, fasting, obesity and long-term alcohol consumption.

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S.J. Oh et al. / Chemico-Biological Interactions 195 (2012) 173–179 180

A

80

*

60

40

20

MROD (nmol/min/mg protein)

EROD (nmol/min/mg protein)

100

0

1'-OH-MDZ (nmol/min/mg protein)

6-OH-CZX (nmol/min/mg protein)

90 60 30

14

C

2

1

0

Control

GK

Control

GK

D

12 10 8 6 4 2 0

Control

4-OH-MDZ (nmol/min/mg protein)

**

GK

3

35

120

0

Control 4

B

150

E

GK

*

30 25 20 15 10 5 0

Control

GK

Fig. 3. Hepatic microsomal activities of EROD (A), MROD (B), CZX 6-hydroxylase (C), MDZ 10 -hydroxylase (D) and MDZ 4-hydroxylase (E) in GK rats. Each value represents the mean ± SD for seven rats. ⁄, ⁄⁄Significantly different from the control at P < 0.05 or P < 0.01, respectively.

These pathophysiologic states result in both altered nutritional status and hormonal regulation. Previous studies have demonstrated that the expression of CYP2E1 is down-regulated by insulin in primary cultured rat hepatocytes and that insulin signaling pathways involving phosphatidylinositol 3-kinase/ribosomal p70 S6 kinase are active in the insulin-mediated regulation of CYP2E1 expression [7]. The up-regulation of CYP2E1 in diabetic and fasted rats, however, has been attributed to elevated ketone body levels [31,32]. Abdelmegeed et al. [11] showed that acetoacetate, one of the major ketone bodies that increase in diabetes, reduced CYP2E1 mRNA levels, but increased CYP2E1 protein levels in primary cultured rat hepatocytes. Moreover, these studies also revealed that phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin/ribosomal p70 S6 kinase and protein kinase C are involved in the acetoacetate-mediated inhibition of CYP2E1 mRNA levels and gene transcription. In contrast, acetoacetate increased CYP2E1 protein levels through the concerted action of increased CYP2E1 translation and inhibition of protein degradation [11]. In the present study, hepatic expression and activity of CYP2E1 were not signifi-

cantly different between 9-week-old GK rats and age-matched Wistar rats. Our results raise the possibility that elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. In summary, our results show that the expression and activity of hepatic CYP isoforms are differentially regulated in the GK rat. CYP3A2, a male-specific CYP isoform, was up-regulated, and CYP1A2 and CYP3A1 were down-regulated in GK rats. In addition, CYP2E1 activity and expression were not affected by diabetic conditions with normal ketone body levels, suggesting that the elevation of ketone bodies plays a critical role in the up-regulation of hepatic CYP2E1 in diabetic rats. The present results also show that the GK rat is a useful animal model for pathophysiological study of non-obese type II diabetes with normal ketone body levels.

Conflict of interest statement The authors declare that they have no conflict of interest.

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