Comparative Study on the in Vivo and in Vitro Antilipolytic Effects of Etofibrate, Nicotinic Acid and Clofibrate in the Rat
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ELSEVIER Environmental Toxicology and Pharmacology 2 (1996) 351-357 Comparative study on the in vivo and in vitro antilipolytic effects of etofibrate, nicotinic acid and clofibrate in the rat Carlos Bocos, Emilio Herrera * Facultad de Ciencias Experimentales y Tecnicas, Uniuersidad San Pablo-CEU, P.O. Box 67, 28660 Boadilla de! Monte, Madrid, Spain Received 22 April 1996; revised 12 August 1996; accepted 12 August 1996 Abstract The release of both glycerol and free fatty acids (FFA) into a medium by epididymal fat pad pieces from fed rats incubated in Krebs Ringer bicarbonate-albumin buffer supplemented or not with epinephrine decreased more in the presence of etofibrate than in the presence of equimolecular doses of nicotinic acid or clofibrate. The first drug was the only one to stimulate the rate of fatty acid re-esterification when incubations were done under basal conditions. By 3 h after their acute oral administration all three drugs decreased plasma FF A levels, although the effect from etofibrate was largest, the drugs enhanced or decreased plasma glycerol levels depending on both the dose and the time after treatment. Plasma triglycerides also decreased at 3 h after oral drug administration, and this effect was similar with etofibrate and nicotinic acid but less with clofibrate. With the exception of a decrease at 7 h after the highest dose (1.2 mmoljkg) of either etofibrate or nicotinic acid (but not clofibrate), plasma cholesterol levels remained stable at 7 h after the respective treatments. Thus, the hypocholesterolemic effect of these drugs seems secondary to their hypotriglyceridemic effect, which would be a consequence of their respective antilipolytic actions, and follows an efficiency sequence of etofibrate, nicotinic acid and clofibrate. Keywords: Nicotinic acid; Etofibrate; Clofibrate; Lipolysis; Triglyceride; Cholesterol; Hypolipidemic drug; (Rat) 1. Introduction acids (Fig. I), was developed after it showed the synergis tic effects of clofibrate and nicotinic acid in decreasing The 3-hydroxy-3-methyl glutaryl CoA reductase in elevated levels of plasma lipids (Rosenhamer and Carlson, hibitors are generally recognized as the most efficient 1980; Cayen et al., 1979). Etofibrate has been shown to be drugs for the reduction of elevated total and LDL a potent hypolipidemic agent in animals (Fabiani et al., cholesterol (Adult Treatment Panel II, 1993). However, the 1989; Herrera et al., 1988; Bocos et al., 1993; Bocos et al., substantial cost of these drugs and their limited effect in 1995) and humans (Series et al., 1988; Hendricks and raising HDL-cholesterol and decreasing elevated triglyc Jensen, 1986). The primary mode of action of nicotinic eride levels (Tikkanen et al., 1989; Gaw and Shepherd, acid is to inhibit lipolysis in adipose tissue by inhibiting of 1991) justifies the use of other hypolipidemic drugs that remedy these limitations. Both nicotinic acid and fibric CH3 acid derivatives still occupy a key place in our lipid-regu I lating formulary since both of them have been proven rAY O~~; COOCH,CH,OOC'[Q useful in the clinical management of primary hypertriglyc eridemia and primary hypercholesterolemia, although their C~ N use is commonly restricted to a second-line position for the Etofibrate latter condition (Probstfield and Hunninghake, 1994; Fat CH3 tore and Sirtori, 1991; Gaw and Shepherd, 1991). Etofi I brate (Lipo-Merz retard, Merz, Frankfurt/Main, Germany), 0~ - COOCH2CH3 HOOC,a the ethanediol-1,2 diester of the nicotinic and clofibric CH3 Cl N Clofibrate Nicotinic acid ' Corresponding author. Tel.: +34-1-351.05.50; Fax: +34-1- 351.04.96. Fig. I. Chemical formulas of the three compounds investigated. 1382-6689/96/$15.00 Copyright© 1996 Elsevier Science B.V. All rights reserved. PI/ S 1382-6689(96)00069-5 352 C. Bocos, E. Herrera/ Environmental Toxicology and Pharmacology 2 ( 1996) 351-357 adenylate cyclase activity (Fears, 1987; Fattore and Sirtori, weight) of either clofibrate, nicotinic acid or etofibrate 1991). This prevents the utilization of free fatty acids freshly suspended in 2% Tween 80 or the medium (con (FFA) in the hepatic synthesis of very low density lipopro trols) were administered to the rats by stomach tube with teins (VLDL) (Olsson et al., 1986), consequently reducing out anesthesia. At different times thereafter (3, 5 and 7 h) low-density lipoproteins (LDL) levels. The hypolipidemic blood was collected from the tip of the tail into ice chilled effects of clofibrate, its fibric acid derivatives and etofi heparinized receptacles. After centrifugation at 3000 X g brate are, however, very wide-spread and while they affect at 4°C for 15 min plasma was stored at - 30°C until several lipoprotein metabolism pathways, their primary processed. Aliquots of total plasma were used for FFA, mode of action still remains an enigma. Both clofibrate triglycerides and cholesterol analysis with commercial kits and etofibrate have also been shown to inhibit adipose (Wako Chemicals, Germany; Boehringer Mannheim, Ger tissue lipolytic activity (D'Costa et al., 1979; Bocos et al., many, and Menarini, Italy, respectively). Other plasma 1993). So, the present study compares the antilipolytic aliquots were deproteinized (Somogyi, 1945) for glycerol effects of nicotinic acid, clofibrate and etofibrate both in measurement (Garland and Randle, 1962). vivo and in vitro and relates these to their respective 2.3. Expression of the results effects on plasma triglycerides and cholesterol in normo lipidemic rats. Values correspond to means± S.E., and the statistical comparison between the groups was performed by the Student's t test. 2. Materials and methods 2.1. Jn vitro experiments 3. Results Male Sprague-Dawley rats weighing 180-220 g fed ad Adipose tissue lipolytic activity was measured in vitro libitum purina chow diet (Panlab, Barcelona, Spain) and using epididymal fat pad pieces from fed rats. The fat pads subjected to a 12 h on-off light cycle (7.00 to 19.00 h) were incubated either under basal conditions or under were decapitated and their epididymal fat pads pieces used lipolytic stimulating conditions created by the presence of to determine lipolytic activity following the method al epinephrine in the medium, which was supplemented or ready described by us (Dominguez and Herrera, 1976), not with two different doses of each drug (10-5 and 10- 3 with a few modifications. After weighing, fresh epididy M). As shown in Fig. 2A, nicotinic acid decreased the mal fat pads were cut into small pieces and 18-20 mg production of both glycerol and FFA whether tissues were were placed in vials containing 2 ml of Krebs Ringer incubated under basal conditions or in the presence of bicarbonate buffer (pH 7.4) supplemented with 20 mg/ml epinephrine, and although the effect of the 10-5 M dose of fatty acid-free bovine albumin. Half of the vials were was not significant when considering the glycerol produc also supplemented with 0.1 µ,g/ml of epinephrine bitar tion of tissue pieces incubated under basal conditions, it trate (Sigma Chemical Co., USA). At zero time 20 µ,l of was significant when expressed as a percentage of the dimethyl sulfoxide (from Merck, Darmstadt, Germany) respective control values (72.1 %, P < 0.05). As shown in containing a corresponding amount of either clofibrate, nicotinic acid or etofibrate kindly supplied by Merz Table I (Frankfurt, Germany) to give a final concentration of 0, Free fatty acid re-esterification index (FFA/glycerol) 5 3 10- or 10- M was added to the corresponding vial. Treatment Dose Medium of incubation Vials were capped, gassed for 5 min with O2/CO2 and Basal p + Epinephrine incubated for 120 min as previously described (Dominguez Controls 2.49±0.14 n.s. 2.67±0.09 and Herrera, 1976) and incubation stopped by placing the vials in an ice bath. In each experiment a set of vials Etofibrate 10-5 M 2.11±0.31 n.s. 2.38±0.22 3 corresponding to each of the conditions used was placed 10- M 1.87 ±0.30 ' <0.01 2.91 ±0.08 directly in an ice bath without incubation to determine the Clofibrate 10-5 M 2.39±0.17 n.s. 2.51 ±0.18 zero time values. An aliquot of 750 µ,l of each medium 10-3 M 2.39±0.16 n.s. 2.42±0.17 5 was treated with 10% HCIO4 for protein precipitation and Nicotinic ac. 10- M 2.67 ±0.14 n.s. 2.90±0.10 the neutralized supematants were used for glycerol deter 10-3 M 2.32±0.24 n.s. 2.66±0.15 mination (Garland and Randle, 1962). Another aliquot of Values are means± S.E. The free fatty acid re-esterification index was each medium was used for FF A determination with a calculated using the (nmol FFA/100 mg tissue):(nmol glycerol/JOO mg commercial kit (Wako Chemicals, Germany). tissue) ratio. Therefore, a value near 3 means low re-esterification of free fatty acids and a value near O means high reesterification. The total 2.2. Jn viva studies number of vials was eight in all the cases, from four separate experi ments. The asterisks correspond to the statistical comparisons versus the Male Sprague-Dawley rats weighing 210-240 g, main respective control value (incubations without the drug) (' P < 0.05), tained under the same conditions as above were used. At whereas P values correspond to the comparisons between incubation 10.00 h different doses (0.2, 0.5, 0.8 or 1.2 mmoljkg body done in the presence and the absence of epinephrine. n.s., not significant. C. Bocos, E. Herrera/ Environmental Toxicology and Pharmacology 2 ( 1996) 351-357 353 A Nicotinic acid 1000 1000 2250- BASAL + EPINEPHRINE 2250 BASAL + EPINEPHRINE 1 CD ::i I Ill CD ::i .!!! Ill .