Showa Univ. J. Med. Sci. 7(2), 173•`182, December 1995 Original Effects of Clofibrate Derivatives on Hyperlipidemia Induced by a Cholesterol-Free, High-Fructose Diet in Rats Hideyukl KURISHIMA,Sadao NAKAYAMA,Minoru FURUYA and Katsuji OGUCHI Abstract: The effects of the clofibrate derivatives fenofibrate (FF), bezafibrate (BF), and clinofibrate (CF), on hyperlipidemia induced by a cholesterol-free, high-fructose diet (HFD) in rats were investigated. Feeding of HFD for 2 weeks increased the high-density lipoprotein subfraction (HDL1) and decreased the low-density lipoprotein (LDL) fraction. The levels of total cholesterol (TC), free cholesterol, triglyceride (TG), and phospholipid in serum were increased by HFD feeding. Administration of CF inhibited the increase in HDL1 content. All three agents inhibited the decrease in LDL level. Both BF and CF decreased VLDL level. Administration of FF, BF, or CF inhibited the increases of serum lipids, especially that of TC and TG. The inhibitory effects of CF on HFD- induced increases in HDL1, TC, and TG were greater than those of FF and BF. These results demonstrate that FF, BF, and CF improve the intrinsic hyper- lipidemia induced by HFD feeding in rats. Key words: fenofibrate, bezafibrate, clinofibrate, fructose-induced hyperlipide- mia, lipoprotein. Introduction Clofibrate is one of the most effective antihypertriglycedemic agents currently available. However, because of its adverse effects, such as hepatomegaly1, several derivatives, such as clinofibrate (CF) and bezafibrate (BF) have been developed which are more effective and have fewer adverse effects. For example, it has been shown that the hypolipidemic effect of CF is greater than that of clofibrate while its tendency to produce hepatomegaly is less1. The inhibitory effects of BF on hepatic enzymes involved in cholesterol metabolism are greater than those of clofibrate2. Furthermore, another derivative, fenofibrate (FF) is now under clinical investigation in Japan as a possible antihyperlipidemic drug. The feeding of a cholesterol-free, high-fructose diet (HFD) induces hyperlipidemia in rats3-5. This HFD-induced hyperlipidemia is associated with enhancement of cholesterol and triglyceride synthesis in the liver; therefore, HFD feeding is a model of intrinsic hyper- lipidemia which would be very useful in evaluation of the effects of hypolipidemic agents on intrinsic hyperlipidemia. The present study was conducted to compare the effects of CF, BF, and FF on HFD- induced hyperlipidemia in rats. Department of Pharmacology, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. 174 Hideyuki KURISHIMA, et al. Materials and Methods Animals, diets and drugs Four-week-old male Sprague-Dawley rats (Sankyo Labo-Service Co., Tokyo, Japan) were individually housed in wire mesh cages and kept in a room controlled at a temperature of 24•}1•Ž and a relative humidity of 55•}10 % with a 12-hour light and dark cycle. The HFD (Oriental Yeast Co., Tokyo, Japan) contained 68.0% fructose, 23.7 % casein, 0.3 % DL- methionine, 0.01 % choline chloride, 1.0 % corn oil, 2.0% cellulose, 4.0% minerals, and 1.0% vitamins. Minerals and vitamines were provided according to Iwata's method4). Clofibrate derivatives-FF, BF, and CF-were kindly donated by Grelan Pharmaceutical Co. (Tokyo, Japan). HFD-induced hyperlipidemia After prefeeding with HFD for 1 day, rats were randomly divided into eight groups (five rats/group). Intrinsic hyperlipidemia was induced by feeding of HFD for 14 days. Control rats were fed a normal diet (ND: MF, Oriental Yeast Co., Tokyo, Japan). Test drugs (FF, BF, CF) were suspended in 1.0% Tween 80 solution and given orally at doses of 10 and 30 mg/kg/day for 14 days with HFD feeding. ND-fed and one group of HFD-fed rats were given 1.0% Tween 80 solution alone at 2.0 ml/kg. Blood was collected and the liver was removed 24 hours after final administration of test drugs and Tween 80 solution. After fasting for 18 hours, rats were anesthetized with sodium pentobarbital (30 mg/kg, i.p.). The abdomen was opened, and approximately 5 ml of blood was collected from the inferior vena cava with a disposable syringe. After perfusion with ice-cold saline, the liver was removed and weighted. Lipids in the liver were extracted according to the method of Bragdon6). Serum was separated by centrifugation at 1,280•~g for 15 minutes. Analysis of serum lipoproteins Serum lipoproteins were assayed by the prestaining disk polyacrylamide gel electrophoresis method7). Three layers of acrylamide were stacked; acrylamide concentrations were 1.875% for the stacking gel and 3 % and 5 % for the running gels. Serum (100ƒÊl) was stained with 30 pJ of saturated Sudan Black B in ethyleneglycol for 20 hours. Prestained serum was mixed with 100ƒÊl of 20% sucrose solution, and then 100ƒÊl of the mixture was added onto the stacking gel. Electrophoresis was carried out with Tris/glycine buffer (0.05 mM, pH 8.4) at 1.5 mA/disk. The densities of lipoprotein bands were measured with a densito- meter (F-808, Cosmo Co., Ltd., Tokyo, Japan). Lipoprotein levels were obtained from the peak heights (mm) in the densitograph of lipoprotein bands in the electrophoretic disk gel, and pre-ƒÀ, ƒÀ, and ƒ¿ (ƒ¿1, ƒ¿2, ƒ¿3) lipoprotein in the electrophoretic lipoprotein bands were expressed as very lowdensity lipoprotein (VLDL), lowdensity lipoprotein (LDL) and high- density lipoprotein (HDL3, HDL2, HDLI), respectively. Analysis o f lipid, protein, and enzyme Commercial kits (Wako Pure Chemical Industries Ltd.) were used for assay of lipid, protein, and enzyme in serum, HDL fraction and liver. The HDL fraction in serum was separated by a HDL-cholesterol precipitation reagent. Levels of total cholesterol (TC), free cholesterol (FC), triglyceride (TG), phospholipid (PL), and nonesterified fatty acid (NEFA) in serum, HDL fraction, and the liver were determined with the following assay kits: Cholesterol C-II, Free cholesterol C, Triglyceride G, Phospholipid B and NEFA. Levels of protein and transaminases (aspartate aminotransferase EAST] ; alanine aminotransferase Fibrates and Fructose-fed Hyperlipidemia 175 Body weight LW and LIB ratio Fig. 1. Effects of fenofibrate (FF), bezafibrate (BF), and clinofibrate (CF) on body weight and liver weight in rats fed a cholesterol-free, high-fructose diet (HFD) for 2 weeks. Each column represents the mean•}S.D. of five rats. BW-0: body weight on the 1st day of HFD feeding and test drug administration; BW-14: body weight on the final day of the experiment; LW: liver weight; L/B: liver weight/body weight atio; ND: normal diet; HFD: cholesterol-free, high-fructose diet; FF: fenofibrate; BF: bezafibrate; CF: clinofibrate; * p•ƒ0.05, ** p•ƒ0.01: compared with ND, •‹p•ƒ 0.01, 00 p•ƒ0.001: compared with HFD, *p•ƒ0.01, * p•ƒ0.001: compared with CF. [ALT]) in serum were determined with the A/G B and Transaminase C-II assay kits. Total protein {TP) level in the liver was determined by the method of Lowry et a1.8, and expressed as milligrams per gram of liver. Statistical analysis Statistical significance was evaluated using Student's t-test. A p-value of less than 0.05 was considered statistically significant. 176 Hideyuki KURISHIMA, et al. Fig. 2. Effects of FF, BF, and CF on protein and transaminases in serum of rats fed HFD for 2 weeks. TP: total protein; AST: aspartate aminotransferase; ALT: alanine aminotransferase; •‹p•ƒ0.05, •‹•‹p•ƒ0.01, •‹•‹•‹ p •ƒ0.001: compared with HFD . See explanation in Fig. 1. Results Body weight, liver weight, food intake, and TP content in liver The body weight of all rats increased daily, but the weight gain of rats fed HFD was lower than that of rats fed ND. Therefore, the body weight of HFD-fed rats were significantly lower than that of ND-fed rats (Fig. 1), most likely resulting from the lower daily intake of food by HFD-fed rats (10.8-14.8 g/day) than by ND-fed rats (16.0-16.4 g/day) . The liver weight of rats fed HFD alone and rats fed ND were not significantly different. However, the liver weight to body weight (L/B) ratio was significantly higher in rats fed Fibrates and Fructose-fed Hyperlipidemia 177 Fig. 3. Effects of FF, BF, and CF on serum high-density lipoprotein (HDL) in rats fed HFD for 2 weeks. Data of HDL level were obtained from the peak heights (mm) on the densitograph of lipoprotein bands in the electrophoretic disk gel. HDL1,HDL2, HDL3: subfractions of HDL, * p•ƒ0.001: compared with ND, * p•ƒ0.001: compared with CF. See explanations in Figs. 1 and 2. Fig. 4. Effects of FF, BF, and CF on serum lipoproteins in rats fed HFD for 2 weeks. VLDL: very low density lipoprotein, LDL: low-density lipoprotein, Al-NEFA: albumin-combined nonesterified fatty acid, *p•ƒ0.05, **p•ƒ0.001: compared with CF. See explanations in Figs. 1 and 3. HFD alone than that of rats fed ND. In rats that received FF (30 mg/kg, p.o.) and BF (10 or 30 mg/kg, p.o.), liver weight and L/B ratio were significantly higher than those of rats fed HFD alone (Fig. 1). The TP levels in the liver were slightly lower after feeding of HFD alone (p•ƒ0.1). However, FF and BF at 10 mg/kg inhibited the decrease in TP level in 178 Hideyuki KURISHIMA, et al. Fig. 5. Effects of FF, BF, and CF on serum lipids in rats fed HFD for 2 weeks. TC: total cholesterol, FC in serum HDL, * p•ƒ0.001: compared with ND , •‹p•ƒ0.05, 00 p•ƒ 0.01, 000 p•ƒ0.001: compared with HD, * p•ƒ0.05, ** p•ƒ0 .01, ***p•ƒ0.001: compared with CF.