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. 2004 Dec;53(12):1837-43.
doi: 10.1136/gut.2003.037689.

Bile secretory function in the obese Zucker rat: evidence of cholestasis and altered canalicular transport function

M Pizarro  1 , N Balasubramaniyan, N Solís, A Solar, I Duarte, J F Miquel, F J Suchy, M Trauner, L Accatino, M Ananthanarayanan, M Arrese
Affiliations

Bile secretory function in the obese Zucker rat: evidence of cholestasis and altered canalicular transport function

M Pizarro et al. Gut. 2004 Dec.

Abstract

Background: Obese Zucker rats (ZR) have been used as an experimental model for non-alcoholic fatty liver disease and are particularly susceptible to various types of liver injury. Bile secretory function has not been assessed in ZR.

Aim: To study bile secretion and expression of the main hepatobiliary transporters in ZR.

Methods: Bile flow and biliary secretion of lipids and glutathione were determined in eight and 14 week old obese ZR and their lean controls. Protein mass and mRNA of the Na(+)/taurocholate cotransporting polypeptide (Ntcp), the bile salt export pump (Bsep), and the multidrug resistant associated protein 2 (Mrp2) were assessed by western and northern blot, respectively. The effects of administration of a tumour necrosis factor alpha inactivator (etanercept) and an insulin sensitiser (rosiglitazone) were assessed in obese ZR while leptin was given to non-obese rats to study its effect on Mrp2 expression.

Results: ZR exhibited increased body weight and hyperlipidaemia. Only 14 week old obese ZR has fatty liver. Decreased bile flow and biliary lipid and glutathione secretion as well as reduced hepatic transport of both taurocholate and bromosulphthalein were found in obese ZR. Hepatic Mrp2 protein mass was markedly reduced (-70%) in obese rats while Ntcp and Bsep protein levels were similar to lean rats. Downregulation of Mrp2 seems to involve both transcriptional and post-transcriptional mechanisms probably related to insulin and leptin resistance.

Conclusions: Obese ZR exhibit an impaired bile secretory function with significant functional and molecular alterations consistent with mild cholestasis. A defective hepatobiliary transport capacity may be a contributory factor in rendering the obese ZR more susceptible to liver injury.

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Figures

Figure 6
Figure 6
Effect of hyperleptinaemia on multidrug resistant associated protein 2 (Mrp2) protein mass in non-obese Zucker rats (ZR). (A) Treated rats received leptin infusion (0.4 mg/kg/day) while control rats received vehicle (5 mmol/l sodium citrate, pH 7.4) infusion. Infusions were performed using osmotic minipumps implanted subcutaneously in the back for one week, as described in material and methods. Then, Mrp2 protein levels were measured in plasma membrane fractions of treated and untreated obese rat livers. Each band represents the results of a single animal. Representative experiments with three rats per group are shown. (B) Bar diagram showing western blot band volume as per cent of control animals. Leptin treated animals exhibited significantly higher levels of Mrp2. *p<0.05.
Figure 1
Figure 1
Liver histology in eight and 14 week old lean and obese Zucker rats. Micro- and macrovesicular steatosis was observed in a representative 14 week old obese rat while the other liver specimens displayed normal liver histology (final magnification 200×).
Figure 2
Figure 2
Protein expression of hepatic transporters in eight and 14 week old lean and obese Zucker rats (ZR). (A) Multidrug resistant associated protein 2 (Mrp2), bile salt export pump (Bsep), and multidrug resistant associated protein 3 (Mrp3) protein levels in plasma membrane fractions of lean and obese ZR livers. Approximately 50 μg of membrane proteins were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Immunoblotting was performed and bound antibodies were visualised as described in materials and methods. Each band represents the results of a single animal. Transport proteins are indicated on the left of each blot and animal groups at the top. Representative experiments with four rats per group are shown. β-Actin protein mass was used as a loading control. (B) Bar diagram showing western blot band volume as per cent of control for Mrp2, Bsep, and Mrp3. *p<0.05.
Figure 3
Figure 3
Decreased expression of hepatic canalicular transporter mRNA levels in eight and 14 week old Zucker rats (ZR). (A) Multidrug resistant associated protein 2 (mrp2), bile salt export pump (Bsep), and glyceraldehyde-3-phosphate dehydrogenase (gapdh) mRNA expression in lean and obese ZR. Total RNA was isolated from lean and obese animals and 5 µg polyA+RNA from each sample was fractionated on 1% formaldehyde-agarose probed with cDNAs for the various genes, as described in materials and methods. Each band represents the results of a single animal. Gene names are indicated on the left of each gel and animal groups at the top. (B) Bar diagram showing relative mRNA expression levels of mrp2, Bsep, and gapdh normalised to cyclophilin expression. *p<0.05 compared with controls.
Figure 4
Figure 4
Indirect immunofluorescent localisation of multidrug resistant associated protein 2 (Mrp2) in 14 week old control and obese Zucker rats (ZR). Frozen liver sections from control (A) and obese (B) ZR were used to assess qualitative distribution of Mrp2 by indirect immunofluorescence, as described in material and methods Decreased labelling of canalicular membranes was observed accounting for reduction in Mrp2 protein expression in obese ZR.
Figure 5
Figure 5
Effect of etanercept or rosiglitazone administration on multidrug resistant associated protein 2 (Mrp2) protein mass in obese Zucker rats (ZR). The anti-tumour necrosis factor α inhibitor etanercept (ETN) was given to 14 week old obese ZR in a single intraperitoneal injection of 8 mg/kg. Mrp2 protein mass was assessed 72 hours later. Rosiglitazone (RGZ) was given by gavage at a dose of 3 mg/kg/day over 10 days. Mrp2 protein levels in plasma membrane fractions of treated and untreated obese rat livers were measured as described in materials and methods. (A) Each band represents the results of a single animal. Representative experiments with three rats per group are shown. (B) Bar diagram showing western blot band volume as per cent of control animals. A significant increase in Mrp2 protein levels was seen after RGZ treatment while no significant changes were observed after etanercept administration. *p<0.05.
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