Characterization of organic anion transporter regulation, glutathione metabolism and bile formation in the obese Zucker rat
Introduction
Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as the basis for inflammatory hepatitis and induction of liver cirrhosis [1]. Prevalence of NAFL in patients undergoing liver biopsy because of elevated aminotransferases varies between 15 and 40% [2], [3] and the disease is associated with obesity and type II diabetes mellitus [4], [5].
The Zucker rat represents an established animal model for insulin resistance and diabetes followed by fatty liver disease and steatohepatitis due to increased lipogenesis [6], [7]. The underlying genetic defect for the observed changes has been localized to a point mutation of codon 269 in the leptin receptor gene and leads to a lack of response to leptin [8].
We recently observed reduction of bile flow in homozygous Zucker rats suggesting alterations in the expression of transport proteins directly involved in bile formation. Hepatic transport proteins are crucial for the central metabolic function of the liver. Transporters in the canalicular membrane of hepatocytes mediate extrusion of endogenous and exogenous compounds, which have been detoxified in the liver. This eliminating function is served by Multidrug resistance-associated protein 2 (MRP2/ABCC2), Breast cancer resistance protein (BCRP/ABCG2) and Multidrug resistance transporter 1 (MDR1/ABCB1) [9], [10]. Additionally, canalicular membrane proteins mediate bile formation by secreting osmosensitive bile constituents into the canaliculus. This comprises secretion of bile salts by the bile salt export pump (BSEP/ABCB11)), glutathione (MRP2), phospholipids and cholesterol, which contribute to micellar formation mediated by MDR3/ABCB4 and ABCG5/G8 [9]. In the basolateral membrane, transport proteins serve metabolism by importing substrates (organic anion transporting polypeptides, OATPs, in rats Oatp1/Slco1a1, Oatp2/Slco1a4 and Oatp4/Slco1b2) or exporting products of hepatic metabolism back into the sinusoidal blood (MRP3 and 4/ABCC3 and 4) [9].
Alterations in the expression of these transport proteins may modulate the course of liver disease and help to understand changes in bile formation, drug elimination and the enormous vulnerability of fatty livers against various toxic insults. Therefore we studied for the first time expression and function of key hepatic organic anion transport proteins in Zucker rats.
Section snippets
Animal models
Eight-weeks-old male obese Zucker rats (fa/fa, 390±40 g) and their lean heterozygous littermates (Fa/fa, 280±50 g) (Charles River Wiga) were housed under a constant 12-h light/dark cycle and free access to water and rat chow (standard diet Altromin 1314). Animals received humane care in compliance with guidelines by the local animal welfare committee.
In vivo experiments
Rats were anaesthetized with pentobarbital (50 mg/kg intraperitoneally). After laparotomy and cannulation of the common bile duct with a PE-10 tube,
Zucker rats exhibit liver cell damage without histological inflammation
Clinicochemical characteristics of obese Zucker rats in comparison to their heterozygous littermates are given in Table 2. Livers of obese rats (fa/fa) showed microvesicular steatosis of approximately 50% of hepatocytes without inflammation, necrosis or fibrosis. In contrast, no histological abnormalities were found in livers of heterozygous animals (Fa/fa) (Fig. 1). Compared with lean animals, liver weight of obese Zucker rats increased by 20% (Table 2).
Bile flow and biliary secretion of transporter substrates
Total bile flow (BF) obtained in vivo
Discussion
There are five novel findings of our study. (i) We observed reduction of bile salt-dependent and bile salt-independent bile flow in homozygous Zucker rats. (ii) Reduction of bile salt secretion was paralleled by slightly reduced Nctp protein and significantly diminished Oatp2 expression while Bsep expression was unchanged. (iii) Impaired bile salt-independent bile flow was associated with a 50% reduction of biliary secretion of the Mrp2 substrates GSSG and DNPG. (iv) In line with this
Note added in proof
While this paper was under revision, a report by Pizarro et al. was recently published on the same topic (Gut 2004 Dec;53(12):1837–43).
Acknowledgements
The authors thank Sabine Beutelspacher, Ingrid Liβ, Aline Müller, Petra Schmitz, Gudrun Schwertfeger and Sonja Strauch for their excellent technical assistance as well as Dr D. Eaton (University of Washington, Seattle, WA) for kindly providing rat γ-GCS cDNA. Grant support: Supported by the Deutsche Forschungsgemeinschaft grant SFB542 TP C1 (to A.G., S.M., C.G.), DI 729/3-1 (to C.G.D.) and DFG FOR 440 (to A.L.G., M.B.)
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