Effects of bezafibrate, PPAR pan-agonist, and GW501516, PPARδ agonist, on development of steatohepatitis in mice fed a methionine- and choline-deficient diet
Introduction
Non-alcoholic steatohepatitis (NASH) is closely associated with metabolic syndrome, which is a lifestyle-related disease characterized by obesity, diabetes, hyperlipidaemia and hypertension. Although the pathogenesis of non-alcholic steatohepatitis is not well understood, a two-hit theory has been proposed (Day and James, 1998). According to this theory, hepatic steatosis is mainly caused by metabolic syndrome (the first hit). Then, the hepatic steatosis develops into non-alcholic steatohepatitis due to the effects of oxidative stress, reactive oxygen species, lipid peroxidation and/or any cytokine (the second hit) (Green, 2003). Since non-alcholic steatohepatitis can develop into cirrhosis via hepatic fibrosis (Matteoni et al., 1999) and finally into hepatocellular carcinoma (Jansen, 2004), the prognosis of non-alcholic steatohepatitis is very severe. For that reason, therapy for hepatic steatosis, metabolic syndrome and/or non-alcholic steatohepatitis itself is very important for patients with non-alcholic steatohepatitis. At present, ursodeoxycholic acid, vitamin E as an anti-oxidant agent or glycyrrhizin are used as allopathy for patients with non-alcholic steatohepatitis, in attempts to improve cholestasis and hepatic inflammation (Agrawal and Bonkovsky, 2002). Recently, troglitazone [a thiazolidinedione derivative that acts as a peroxisome proliferator-activated receptor (PPAR) γ agonist with insulin-sensitivity-enhancing properties] has been used to improve non-alcholic steatohepatitis via an improvement in the patient's hyperinsulinaemia (Caldwell et al., 2001). However, since successful therapeutic methods for non-alcholic steatohepatitis are not yet established, an effective drug for this condition still needs to be developed.
Bezafibrate (2-[4-[2-(4-chlorobenzamido)ethyl]phenoxy]-2-methylpropanoic acid) is already in clinical use as an anti-hyperlipidaemia drug and has been reported to be a PPAR pan-agonist (Willson et al., 2000, Inoue et al., 2002). Recently, it was demonstrated that bezafibrate prevented type 2 diabetes mellitus in patients with coronary artery disease and prevented the events of causing cardiac infarction (Tenenbaum et al., 2004, Tenenbaum et al., 2005). In addition, bezafibrate has an improvement effect on the non-alcholic steatohepatitis induced by tamoxifen, an anti-breast cancer drug (Saibara et al., 1999). However, reports clearly verifying the effect of bezafibrate against non-alcholic steatohepatitis are not found in the existing literature.
PPARs are closely associated with hepatic lipid metabolism and seem to play important roles in non-alcoholic fatty liver disease (Tanaka et al., 2005). An activation of PPARα may improve abnormal lipid metabolism in mice, while an activation of PPARγ may somewhat worsen it (Tanaka et al., 2005). On the other hand, it has been reported that a PPARδ agonist has profound anti-obese and anti-diabetic actions in animal models (Tanaka et al., 2003). However, the physiological role of PPARδ in non-alcholic steatohepatitis remains poorly understood.
In the present study, we evaluated the effects of bezafibrate and those of GW501516 (2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid; methyl-methyl-trifluoromethylphenyl-thiazolyl-methylsulfanyl-phenoxy-acetic acid, a PPARδ agonist) on the development of steatohepatitis in mice fed a methionine-choline deficient (MCD) diet, which is used widely for research on non-alcholic steatohepatitis (Ip et al., 2003, Weltman et al., 1996, Weltman et al., 1998). Using this model, we examined the efficacy of bezafibrate against non-alcholic steatohepatitis and also the relation between non-alcholic steatohepatitis and the functional role played by PPARδ.
Section snippets
Chemicals
Bezafibrate was obtained from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). GW501516 was synthesized by Kissei Pharmaceutical Co. Ltd. (Matsumoto, Japan).
Animal and experimental protocols
Male C57BL/6N mice, 7–8 weeks of age, were purchased from Charles River Japan Inc. (Kanagawa, Japan). They were housed individually in stainless-steel cages (260 × 230 × 180 mm) in an air-conditioned room at a temperature of 22–24 °C and a humidity of 37–64%, with a 12-h light/dark cycle. They were allowed a normal diet (CE-2, Clea Japan Inc.,
Effects of bezafibrate and GW501516 on biochemical parameters
Plasma triglyceride and HDL-cholesterol concentrations were lower in MCD-diet-fed control mice than in normal-diet-fed mice (Fig. 1A,B). In MCD-diet-fed mice, bezafibrate tended to inhibit the reduction in HDL-cholesterol concentration, while GW501516 significantly inhibited the reduction in HDL-cholesterol, but neither bezafibrate nor GW501516 had any effect on the plasma triglyceride concentration (Fig. 1A,B). The plasma adiponectin concentration was significantly higher in MCD-diet-fed
Discussion
PPARs are members of the superfamily of nuclear receptors that functions as fatty acid-activation transcription factors (Willson et al., 2000). PPARs are closely associated with hepatic lipid metabolism and seem to play important roles in non-alcoholic fatty liver disease (Tanaka et al., 2005). The isotypes of PPARs have been identified as PPARα, PPARγ and PPARδ. PPARα is expressed in liver and is involved in hepatic lipid metabolism (Rao and Reddy, 2001). It is activated by fibrate, which is
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