EXPERIMENTAL STUDIES Jpn Circ J 1999; 63: 471–477
Combined Effects of Probucol and Bezafibrate on Lipoprotein Metabolism and Liver Cholesteryl Ester Transfer Protein mRNA in Cholesterol-Fed Rabbits
Jiafu Ou, MD; Keijiro Saku, MD; Shiro Jimi, PhD*; Yuan-Lan Liao, MD; Takao Ohta, MD**; Bo Zhang, PhD; Kikuo Arakawa, MD
Probucol decreases and bezafibrate increases plasma high density lipoprotein-cholesterol (HDL-C) levels in humans. This study was performed to determine whether the HDL-C-lowering effects of probucol could be reversed by treatment with bezafibrate in hypercholesterolemic rabbits. Forty-nine normolipidemic Japanese White rabbits were divided into 5 groups [group 1: normal chow; group 2: 0.2% cholesterol (Ch) diet; group 3: 0.2% Ch and 1% probucol diet; group 4: 0.2% Ch and 1% bezafibrate diet; group 5: 0.2% Ch and 1% probucol plus 1% bezafibrate diet] and treated for 8 weeks. Plasma lipids, cholesteryl ester transfer protein (CETP) activ- ity in the lipoprotein-deficient plasma fraction, CETP mRNA in liver tissue and plasma drug concentrations were investigated. Serum total cholesterol (TC) increased after the rabbits in groups 2, 3, 4 and 5 were fed Ch, but overall, no significant differences were observed in serum TC and triglyceride (TG) among these groups. Serum HDL-C levels increased (p<0.01) in the bezafibrate-treated group, but a significant (p<0.05) reduction in HDL-C was observed in both the Ch + probucol (group 3) and Ch + probucol plus bezafibrate (group 5) groups; no significant difference was observed between groups 3 and 5. Significant correlation (p<0.01) was found between serum low density lipoprotein cholesterol (LDL-C) levels and plasma probucol concentrations in groups 3 and 5, but no correlation was found between plasma concentrations of probucol/bezafibrate and serum HDL-C levels. CETP activity in the lipoprotein-deficient plasma fraction increased in the Ch-, Ch + probucol-, and Ch + probu- col and bezafibrate-fed groups (groups 2, 3 and 5, respectively), whereas a significant reduction in this activity was observed in the Ch + bezafibrate-fed group (group 4). An analysis of covariance showed that the CETP activity responded more sensitively to drug treatment than did the serum HDL-C level. CETP mRNA in liver tissue was assessed by Northern blotting at 8 weeks, but no changes were observed among the 5 groups. Probucol decreased and bezafibrate increased serum HDL-C levels, through CETP activity without affecting liver CETP mRNA levels, and the decrease in HDL-C levels produced by probucol could not be reversed by bezafibrate. (Jpn Circ J 1999; 63: 471–477) Key Words: Bezafibrate; Cholesteryl ester transfer protein; Hypercholesterolemia; Lipids; Lipoproteins; mRNA; Probucol
robucol is a potent hypolipidemic agent that has been HDL-C levels in patients with various types of dyslipopro- reported to have antioxidant effects that may retard teinemia.9–11 The large-scale, 5-year Bezafibrate Coronary P the progression or accelerate the regression of Atherosclerosis Intervention Trial (BECAIT) demonstrated atherosclerosis.1,2 Probucol is known to lower high-density that bezafibrate slowed the progression of coronary athero- lipoprotein-cholesterol (HDL-C) levels and this reduction sclerosis, and reduced coronary events in young survivors of HDL-C has been shown to be associated with increased of myocardial infarction;12,13 bezafibrate effectively cholesteryl ester transfer protein (CETP) activity3 and lowered the serum very low density lipoprotein (VLDL)- mass.4 Yamamoto et al showed that a reduction in achilles TG, VLDL-C, and fibrinogen levels, but no change was tendon xanthoma was related to a decrease in HDL-C levels observed in low density lipoprotein-cholesterol (LDL-C). after probucol treatment.5 Recent studies have shown that it Therefore, some factor (s) other than LDL-C may play reduces the rate of restenosis after balloon coronary angio- important roles in the progression/regression of coronary plasty.6,7 However, a clinical study found that probucol had lesions.12,13 Generally, direct evidence of the antiatheroscle- no effect on the regression of femoral atherosclerosis.8 rotic effects of fibrates in animal studies is rare.14 An excep- Bezafibrate is a homologue of clofibrate that has been tion is fenofibrate,15 another homologue of clofibrate, which shown to reduce plasma triglyceride (TG) and increase has been shown to inhibit an early event in the atherogene- sis of rabbits fed a cholesterol diet; however, this action has (Received November 2, 1998; revised manuscript received December been also shown to be independent of the hypolipidemic 28, 1998; accepted February 5, 1999) effects of fenofibrate.15 Departments of Internal Medicine and *Pathology, Fukuoka Univer- Combination therapy with drugs that use different mech- sity School of Medicine, Fukuoka and **Department of Pediatrics, anisms has been evaluated in several studies of hyper- Faculty of Medicine, University of the Ryukyus, Okinawa, Japan lipoproteinemia;16–20 however, it is unclear whether probu- Mailing address: Keijiro Saku, MD, PhD, FACP, Department of Internal Medicine, Fukuoka University School of Medicine, 45-1-7 col-reduced HDL levels should be treated by drugs, and if Nanakuma Jonanku, Fukuoka 814-0180, Japan. E-mail: hh035399@ so, whether the addition of bezafibrate could possibly msat.fukuoka-u.ac.jp reverse probucol-reduced HDL levels. In addition, the
Japanese Circulation Journal Vol.63, June 1999 472 OU J et al. combination of probucol and fibrates quite often induces an ing [3H]-cholesterol oleate ([3H]-DBP) were prepared. To extreme reduction in HDL-C,16,20,21 and the mechanism of remove total lipoproteins from the rabbit plasma sample, this phenomenon is unclear. the plasma density was adjusted to d=1.21g/ml by KBr, In the present study, we investigated the effects of and then ultracentrifuged in a Beckman TL-100 Tabletop probucol, bezafibrate and their combination on lipoprotein Ultracentrifuge (Beckman Instruments, USA) for 3.5h at metabolism in hypercholesterolemic rabbits. We used 165,000 × g. The bottom fraction was collected and cholesterol-fed rabbits because in a previous HDL-apo A-I dialyzed against 10mmol/L Tris-HCl, 150mmol/L NaCl kinetic study we found that probucol had a greater effect in (pH7.4) for 24 h with 2 changes of dialysate buffer. The the hypercholesterolemic state, and that treatment with a total CET activity in lipoprotein-deficient (d>1.21 g/ml) similar dose of gemfibrozil, another homologue of clofi- plasma was determined in terms of the percentage of [3H]- brate, increased the rate of apolipoprotein (apo) A-I synthe- cholesterol oleate transferred from discoidal bilayer parti- sis in Watanabe heritable hyperlipidemic (WHHL) cles to human LDL.26 The reaction mixture containing 110 rabbits.14 The combined effects of probucol and bezafibrate μl of [3H]-DBP (1,587.3 Bq (42.9 nCi)) as a donor of on CETP gene expression in vivo were also examined, cholesterol ester and 130μg of LDL protein as an acceptor because there has been no previous report on the effects of was incubated with 10μl of lipoprotein-deficient (d>1.21 their interaction. The underlying mechanism of the HDL g/ml) plasma for 60 min at 37°C in the presence of 1.4 deficiency was also investigated. To the best of our knowl- mmol/L 2,2'-dithio-bis-(5'-nitropyridine), an inhibitor of edge, this is the first report to demonstrate the effect of lecithin-cholesterol acyltransferase (LCAT). After incuba- probucol/bezafibrate on plasma CETP activity and liver tion, 30μl of 0.1% dextran sulphate and 30μl of 60mmol/L CETP mRNA levels in hypercholesterolemic rabbits. magnesium chloride were added to the reaction mixture. The mixture was kept on ice for 20min and centrifuged at 10,500 × g for 10 min. The supernatant was collected and Methods the pellets (LDL) were dissolved in 0.1 mol/L NaOH. Rabbits Radioactivities in both the supernatant and pellets were Male Japanese White normolipidemic rabbits (ages counted using a Beckman liquid scintillation counter. The approximately 4 months and weighing 2.1–2.6kg) were result of the assay without a sample was used as nonspe- obtained from Kyudo Co, Ltd (Fukuoka, Japan). All of the cific CETP activity. CETP activity was expressed as total animals were housed individually under a 12-h light/dark CETP activity minus nonspecific CETP activity. cycle. The project was assessed and approved by the Ethics Committee of Fukuoka University. Northern Blot Analysis The rabbits were anesthetized with sodium phenobarbi- Study Groups tal. The liver tissue was excised, immediately frozen in Rabbits were divided into 5 groups: group 1 (N), normal liquid N2, and then stored at –80°C until use. Total cellular chow diet for 8 weeks (n=10); group 2 (Ch), 0.2% choles- RNA was extracted from the liver tissue with RNAzol B terol diet for 8 weeks (n=10); group 3 (Ch+P), 1% probucol (Biotecx Laboratories, Inc). Twenty micrograms of total with 0.2% cholesterol diet for 8 weeks (n=10); group 4 RNA was fractionated on 1% agarose formaldehyde gel (Ch+B), 1% bezafibrate with 0.2% cholesterol diet for 8 and blotted onto a positively charged nylon membrane weeks (n=10); and group 5 (Ch+P+B), 1% probucol plus (Amersham). A cDNA probe (479-base fragment, 1% bezafibrate with 0.2% cholesterol diet for 8 weeks nucleotides 437–915) for rabbit CETP27 was generated by (n=9). Each dietary treatment was started on the same day. reverse transcription-polymerase chain reaction (RT-PCR) using a GeneAmp RNA PCR Kit (Perkin Elmer), and then Preparation of Probucol and Bezafibrate Diets labeled with [32P]-deoxy-cytidinetriphosphate (dCTP) Pure probucol and bezafibrate powders (supplied by using a rediprime random labeling kit (Amersham). The Daiichi Pharmaceutical Co, Tokyo, and Kissei Co, Ltd, primers used for RT-PCR of rabbit CETP were (sense) 5'- Matsumoto, respectively) were added to the basal animal CTTTCCATAAACTGCTCCTG-3', and (antisense) 5'- chow (RC-4 or RC-4 with 0.2% cholesterol, Oriental Yeast, CTGGTTGGTGTCGAAACCCT-3'. The membrane was Tokyo) at the a concentration of 1%. No organic solvent hybridized with a labeled CETP probe, as described else- was used. Crude fat and cholesterol contents of RC-4 were where,28 and then washed with 0.1% sodium dodecyl 3.0g and 2mg per 100g of dry food, respectively. A choles- sulfate (SDS) / 0.1 × SSC at 65°C. The hybridized terol diet was prepared by adding 0.2% cholesterol to RC-4. membrane was exposed to Kodak BioMax film for 2 days at –80°C. The membrane was rehybridized with human Isolation of Lipoproteins glyceraldehyde-3-phosphate dehydrogenase (G3PDH) Blood samples were taken in the morning after an (Clontech) as described earlier, after washing with 1% SDS overnight fast. Serum was adjusted to a density (d) of 1.006 for 10min at 100°C. The proportions of CETP mRNA and g/ml for VLDL, 1.063 g/ml for low-density lipoproteins G3PDH mRNA on the autoradiograph were determined by (LDL), and 1.21g/ml for HDL, using KBr solution, before densitometric scanning (300 A Computing Densitometer being ultracentrifuged.22 Lipoprotein fractions were then and Image Quant Software v3.0 Fast Scan, Molecular isolated. Serum total cholesterol (TC) and TG, and lipids Dynamics). The CETP mRNA levels were normalized to from each of the lipoprotein fractions, were measured by G3PDH mRNA and expressed as a percentage of the enzymatic methods.23,24 G3PDH mRNA level.
Determination of Cholesteryl Ester Plasma Probucol and Bezafibrate Concentrations Transfer Protein (CETP) Activity To measure the concentrations of probucol, the lipid The activity of CETP was determined according to the fraction from 1ml of plasma was extracted. Forty micro- method of Kato et al.25 Discoidal bilayer particles contain- liters of the sample was analyzed by high-performance
Japanese Circulation Journal Vol.63, June 1999 Effects of Probucol and Bezafibrate on HDL Metabolism 473 ## ## ## ## ## ## 0.14** 48.60** 22.07 134.11** 11.72* 61.25** 52.27** 179.41** 11.72* 1.68** 1.15** 1.48** 11.67* ± ± ± ± ± ± ± ± ± ± ± ± ± 0.12 2.56 1.44 52.04 9.67 42.78 6.88 5.47 10.55 359.27 5.49 19.93 14.50 187.89 6.21 149.67 15.67 422.78 6.00 11.47 5.49 19.93 3.50 17.38 9.49 22.33 ± ± ± ± ± ± ± ± ± ± ± ± ± 9.40 23.20 53.93 ## ## ## ## ## ## 0.15** 2.32 11.64 38.67 43.73** 2.80 2.64** 18.07 6.57* 11.20 109.64** 16.67 51.51** 69.11 46.00** 10.93 148.24** 42.67 6.57** 11.20 6.61 11.96** 12.13* ± ± ± ± ± ± ± ± ± ± ± ± ± Fig1. Serum HDL-C levels in the 5 groups before and after treat- ment with probucol and/or bezafibrate. Group 1 (N), normal chow diet for 8 weeks (n=10); Group 2 (Ch), 0.2% cholesterol diet for 8 weeks (n=10); Group 3 (Ch+P), 1% probucol with 0.2% cholesterol diet for 8 weeks (n=10); Group 4 (Ch+B), 1% bezafibrate with 0.2% cholesterol diet for 8 weeks (n=10); Group 5 (Ch+P+B), 1% probucol 0.11 2.62 8.04 38.80 0.92 59.88 6.24 9.36 5.41 17.04 7.38 294.72 7.57 194.90 4.99 132.72 7.83 385.80 5.41 17.04 4.14 31.20 7.08 44.28 1.93 12.40 ± ± ± ± ± ± ± ± ± ± ± ± plus 1% bezafibrate with 0.2% cholesterol diet for 8 weeks (n=9). The ± values are expressed as means±SD. *p<0.01 vs before treatment. 21.18 49.96 p<0.01 vs after treatment of group 2 by ANOVA. ## # # liquid chromatography (HPLC)27,29 using a Waters Associates Instrument (Models 510 and 481, Milford, MA, 14.17 37.30 0.15** 2.34 2.48** 19.12 21.67** 1.82 6.64* 10.14 143.20** 11.40 6.64**52.70** 10.14 61.70 53.52** 7.92 158.85** 34.40 6.35 8.08 4.37** 4.80** (Ch+B): 0.2% cholesterol plus 1% bezafibrate diet; group 5 (Ch+P+B): ± ± ± ± ± ± ± ± ± ± ± ± USA) equipped with a 4.5mm id ×250mm Nucleosil 5C18 ± column (Chemco Osaka, Japan). The column was eluted with 3% acetic acid and acetonitrile (15:85, v/v) at a flow rate of 2.0ml/min. The effluent was monitored at 254nm. For the serum bezafibrate concentration, 20μl of the 9.93 31.80 0.10 2.53 3.82 8.58 1.84 34.24 5.31 13.86 6.88 25.26 4.37 12.00 4.93 311.76 5.319.04 13.86 160.80 3.30 125.16 8.11 358.00 sample was analyzed by HPLC equipped with a 4.6mm id 2.38 9.36 ± ± ± ± ± ± ± ± ± ± ± ± ×150mm TOSOH ODS 80 TM 5μm column. The column ± was eluted with 20mmol/L phosphate K (pH3.6):methanol (60:96, v/v) at a flow rate of 0.9ml/min. The effluent was monitored at 228nm. The plasma probucol and bezafibrate concentrations were linear between 0 and 10μg/ml, and the 7.19 33.50 1.52* 18.24 0.13** 2.29 3.99* 7.62 51.97* 1.60 4.17** 50.34 4.95* 21.84 3.99* 7.62 108.90** 9.06 2.98* 7.64 50.67** 60.70 42.24** 6.24 150.16** 32.50 lowest detectable concentration was 0.1μg/ml. p<0.05 vs after treatment of group 2 by ANOVA; ± ± ± ± ± ± ± ± ± ± ± ± ± # Statistical Analysis Data were analyzed using Statview-J 4.11 and Super- ANOVA for the Macintosh. The data are presented as means ± SD. Changes in serum lipids and lipoproteins 4.93 29.10 5.71 7.68 0.12 2.56 3.63 13.98 1.46 40.02 5.72 34.26 4.09 17.87 3.63 13.98 5.42 299.76 1.86 7.44 7.56 176.50 2.98 128.22 8.87 357.60 ± ± ± ± ± ± ± ± ± ± ± ± during the study period were analyzed by the paired t-test. ± The differences between the groups during the study were examined by an analysis of variance (ANOVA), followed by Fisher’s test, and an analysis of covariance (ANCOVA). The relationships between serum lipid parameters and plasma probucol and bezafibrate concentrations were 7.50 32.60 3.13 12.78 2.74* 2.22 0.11** 2.34 4.44 3.36** 51.58 9.42 2.57 21.87 28.01 10.44 2.98* 10.40 3.36**12.33* 9.42 12.59 61.40 6.36 30.48 34.50 ± ± ± ± ± ± ± ± ± ± ± ± examined by simple regression analysis. ±
Results Table1 shows the changes in serum TC, HDL-C, LDL- C, VLDL-C, TG, HDL-TG, LDL-TG, VLDL-TG, phos- Group 1 (N) Group 2 (Ch) Group 3 (Ch+P) Group 4 (Ch+B) Group 5 (Ch+P+B) 10.33 31.70 2.53 13.44 1.45 3.46 0.12 2.79 2.86 4.57 49.14 12.30 2.64 22.60 4.61 28.68 5.00 5.96 4.575.89 12.30 3.63 70.10 16.02 6.13 54.40 ± ± ± ± ± ± ± ± ± ± ± ± pholipid (PL), HDL-PL, LDL-PL, VLDL-PL, and body ± weight (BW) in all of the rabbits throughout the entire Before After Before After Before After Before After Before After study period. Serum TC increased after rabbits in groups 2–5 were fed cholesterol (Ch), but overall, no significant differences were observed in serum TC and TG in these groups. Treatment with probucol alone (group 3) and probucol plus bezafibrate (group 5) reduced serum HDL-C (p<0.01) (21.8±4.4mg/dl at 0 weeks vs 12.0±4.8mg/dl at 8 weeks, p<0.01 and 23.2±6.0mg/dl at 0 weeks vs 11.5±1.5 TG (mg/dl) 30.80 HDL-TG (mg/dl) 12.84 VLDL-C (mg/dl) 1.78 BW (kg) 2.35 HDL-PL (mg/dl)VLDL-PL (mg/dl) 52.62 9.06 HDL-C (mg/dl) 23.04 LDL-C (mg/dl) 12.48 VLDL-TG (mg/dl) 8.90 LDL-TG (mg/dl)PL (mg/dl) 9.06 LDL-PL (mg/dl) 63.30 7.92 TC (mg/dl) 37.30 Table 1 Changes in Serum Lipids, Lipoproteins Levels and Body Weight (BW) Group 1 (N): normal chow; group 2 (Ch): 0.2% cholesterol diet; 3 (Ch+P): plus 1% probucol 4 mg/dl at 8 weeks, p<0.01, respectively), while bezafibrate plus 1% probucol and bezafibrate diet. *p<0.05 vs before treatment by paired t-test; **p<0.01
Japanese Circulation Journal Vol.63, June 1999 474 OU J et al.
Fig 3. Cholesteryl ester transfer protein (CETP) activity was assessed in the lipoprotein-deficient plasma fraction in each group. Significant increases in CETP activity were observed in groups 2, 3 and 5, whereas a significant reduction in CETP activity was observed in group 4. *p<0.01 vs before treatment, #p<0.01 between groups.
Fig 4. The relationship between the change in cholesteryl ester transfer protein (CETP) activity and the change in high density lipoprotein-cholesterol (HDL-C) in groups 1 ( ), 2 ( ), 3 ( ), 4 ( ) Fig2. The relationships between (A) the plasma probucol concen- and 5 ( ). tration and the serum LDL-C level in group 3 ( ) and group 5 ( ), (B) the plasma probucol concentration and serum HDL-C level in group 3 ( ) and group 5 ( ), and (C) the plasma bezafibrate concen- tration and serum HDL-C level in group 4 ( ) and group 5 ( ). bezafibrate concentrations. Significant positive correlation was observed between serum LDL-C levels and plasma