Effect of AY-25,712 on fatty acid metabolism in rats

Effect of AY-25,712 on fatty acid metabolism in rats

Atherosclerosis, 45 ( 1982) 28 I-290 Elsevier Scientific Publishers Ireland, 281 Ltd. Effect of AY-25,712 on Fatty Acid Metabolism in Rats M.N. Caye...

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Atherosclerosis, 45 ( 1982) 28 I-290 Elsevier Scientific Publishers Ireland,

281 Ltd.

Effect of AY-25,712 on Fatty Acid Metabolism in Rats M.N. Cayen, M.A. Kallai-Sanfacon, J. Dubuc, D. Dvornik Department of Biochemistry, Ayerst Research Laboratories,

E. Greselin

and

Montreal H3C 351, Quebec (Canada)

(Received 18 March, 1982) (Revised, received 16 June, 1982) (Accepted 16 June, 1982)

suunnaly

The effect of AY-25,7 12 [2-methyl-2-phenyl-3(2H)-furanone-5-carboxylic acid] on various aspects of free fatty acid (FFA) and triglyceride metabolism was studied in male rats. Serum triglycerides were lowered by a single oral dose of AY-25,712 or nicotinic acid, but not of clofibrate. Unlike with clofibrate, when AY-25,712 or nicotinic acid was given in the diet, serum triglycerides were not affected. In vitro, both AY-25,712 and nicotinic acid suppressed the theophylline-induced FFA release by epididymal fat pads, but had no effect on lipolysis induced by norepinephrine. Both AY-25,712 and nicotinic acid enhanced the activity of adipose tissue lipoprotein lipase. The initial decrease in plasma FFA and triglycerides, and in liver triglycerides after a single oral dose of nicotinic acid was followed by a rebound to levels which, at later time intervals, were significantly higher than in controls. AY-25,712 was more potent than nicotinic acid in lowering plasma FFA and triglycerides as well as liver triglycerides, but produced no such rebound effect. The data show that, except for the absence of this rebound effect, the mode of action of AY-25,712 in rats resembles that of nicotinic acid and differs from that of clofibrate. Key words: AY-25,712 Lipoprotein

- Clofbrate - Free fatty acids - Lipid lipase - Nicotinic acid - Rat - Triglycerides

mobilization

-

Introduction

In the preceding report [ 11,studies were described on the lipid-lowering properties of AY-25,712 [2-methyl-2-phenyl-3(2H)-furanone-5-carboxylic acid] in normal and 0021-9150/82/0000-0000/$02.75

0 1982 Elsevier Scientific

Publishers

Ireland,

Ltd.

282

hyperlipidemic rats. Relatively low doses of AY-25,712 were found to decrease serum triglycerides and low density lipoprotein (LDL)-cholesterol concentrations. Unlike clofibrate and its congeners, AY-25,712 did not increase liver weight. In this paper, we report on the effects of AY-25,712 on various aspects of fatty acid metabolism, and on the dependence of its hypotriglyceridemic activity on the mode of administration. Comparisons were made with clofibrate and nicotinic acid, and it was found that, in general, the effects of AY-25,712 in rats resembled those produced by nicotinic acid.

Materials and Methods Animals and diets

Male albino Sprague-Dawley rats with unlimited access to water and Purina Laboratory Chow were kept under observation for 2-3 days before each study. Compounds were suspended in 2% Tween-80 and administered by gastric intubation. Analyses

Free fatty acid (FFA) concentrations in plasma or released from epididymal fat pads were determined by the semiautomated method of Kraml [2], based on previously reported techniques [3,4]. In view of the finding that the antilipolytic activity of nicotinic acid, used for comparison, is better expressed in older rats [5], epididymal fat pads were obtained from animals weighing 400-420 g. Fat pads were minced, a single pool was prepared, and aliquots of 100-130 mg were incubated for 30 min at 37’C in Krebs-Ringer bicarbonate buffer (pH 7.4) in the presence of FFA-free bovine serum albumin and test compounds. After acidification, the color was developed in the chloroform extract by the addition of a copper nitrate/triethanolamine and sodium diethyldithiocarbamide solution, and read at 440 nm. Plasma, serum and liver triglycerides were measured by a semiautomated technique [6]. Lipoprotein lipase activity in adipose tissue [7,8] was assessed by pre-incubating epididymal fat pads in a medium containing heparin, and subsequently incubating the released enzyme with [2-3H]glycerol trioleate (Amersham/Searle), and measuring the released [3H]glycerol. Radioactivity was determined in a Beckman LS-8000 liquid scintillation spectrometer. Statistical

analyses

One-way analysis of variance was used to determine subsequent testing for statistical significance. The two-tailed Student t-test was then used to determine significance of difference between group means. Experimental Lipid mobilization

by adipose tissue in vitro

Neither AY-25,712 nor nicotinic acid (at 1 X 10e3 M and 5 X 10V4M) altered the

283

TABLE

1

EFFECT OF AY-25,712 AND NICOTINIC ACID ON THEOPHYLLINE (3X lO-3 M)-INDUCED FREE FATTY ACID (FFA) RELEASE BY RAT EPIDIDYMAL FAT PADS IN VITRO Data are expressed theophylline-induced

as meanf increase

Concentration

SEM for 5 replicates. Values in parenthesis represent percent inhibition of in FFA release above basal values. Results from three studies are presented.

gmole

FFA released/g

tissue.30

min

(M)

1 x10-s 5x 10-4 1 x 10-4 5x 10-5 3 x 10-S 1 x10-5

aP


Theophylline

Theophylline +AY-25,712

Theophylline + nicotinic acid

8.73 kO.97 8.73 to.97 10.43 kO.69 10.43~0.69 6.57kO.78 6.57kO.78

3.6650.16 b (-85) 3.96~0.10= (-80) 4.77kO.24 b (-73) 5.93kO.25 b (-58) 7.43kO.42 7.47 t 1.03

3.3520.20 3.13*0.15 2.99-cO.08 2.76~0.15 3.10~0.10B 3.43 to.26

b b b b

(-90) (-94) (-96) (-98) (-88) a ( - 79)

b P co.001.

norepinephrine (1 X lop5 M)-induced FFA release from rat epididymal fat pads (data not shown). However, both compounds significantly inhibited the lipolysis induced by theophylline (3 X low3 M) (Table 1). Nicotinic acid was about 10 times more potent (based on the concentration which caused a 75% inhibition) than AY-25,712 in reducing the theophylline-induced FFA release; the minimal effective concentration of AY-25,712 was 5 X lo-’ M. Adipose tissue lipoprotein lipase Three h after food withdrawal, male rats (weighing 200-220 g) were given AY-25,712 or nicotinic acid as single oral doses of 125 or 250 mg/kg. Control animals received the vehicle (2% Tween-80) only. The rats were killed with ether 2 h after dosing, and lipoprotein lipase activity in the epididymal fat pads was measured. Neither drug affected lipoprotein lipase at a dose of 125 mg/kg; however, both TABLE

2

EFFECT OF A SINGLE LIPOPROTEIN LIPASE Data

are expressed

Dose

DOSE

OF AY-25,712

OR NICOTINIC

as mean c SEM for 8 rats/group. nmole

glycerol

released/g

Results

ACID

ON ADIPOSE

from two studies are presented.

tissue. h

(mg/kg)

125 250 B P
Control

AY-25,712

1.59kO.19 1.74~040

2.36kO.26 6.27-cO.94

Nicotinic

a

acid

1.7OkO.21 7.81 -co.74 a

TISSUE

284 TABLE

3

EFFECT OF A SINGLE DOSE OF AY-25,712, NICOTINIC TRIGLYCERIDE CONCENTRATIONS IN RATS Data expressed

as mean*

Group

Control AY-25,712 AY-25,712 AY-25,712 Nicotinic acid Clofibrate

ACID AND CLOFIBRATE

ON SERUM

SEM for 10 rats/group. Dose

Serum triglycerides

(mg/W

(mg/dl)

25 65 250 125 240

135 -c 10.6 80% 9.6= 7Ok 8.7 b 55* 5.2 b 58-c 5.4b 121~11.1

a PCO.01; b P
AY-25,712 and nicotinic acid at a dose of 250 mg/kg produced a three-fold increase in enzyme activity (Table2). Both compounds significantly lowered serum triglycerides at both dose levels (data not shown). Serum triglycerides

after a single dose

Groups of rats (weighing 200-22Og), with unlimited access to water and chow were given single oral doses of AY-25,712 (25, 65 or 250 mg/kg), nicotinic acid (125 mg/kg) or clofibrate (240 mg/kg). Animals were decapitated 3 h later and serum triglycerides were measured. The data in Table3 show that a single oral dose of AY-25,712 and nicotinic acid decreased serum triglyceride concentrations, while clofibrate had no effect. Serum triglycerides after administration

by garage or by admixture

to the diet

Groups of rats (weighing 140- 150 g), were fed for 1 week Purina chow supplemented with 0.065% 0.1% or 0.25% AY-25,712; 0.062% or 0.25% nicotinic acid, or 0.125% or 0.25% clofibrate. Other groups of rats were given the corresponding doses daily by gavage, i.e. AY-25,712 at 65, 100 or 250 mg/kg, nicotinic acid at 62 or 250 mg/kg, or clofibrate at 125 or 250 mg/kg. The animals receiving the compound by gavage were decapitated 3 h after the last dose, while those ingesting the drugs with the diet were killed at 11: 30 a.m. Serum triglyceride concentrations were measured. The results in Table4 show that, when given by gavage, all drugs at the doses tested significantly decreased serum triglycerides. However, when given with the diet, both AY-25,712 and nicotinic acid did not affect serum triglyceride concentrations even at the highest dose (250 mg/kg). Similar results were obtained when the drugs were given by gavage or with the diet for 3 weeks (data not shown). In all studies, gavage or dietary administration of AY-25,712 or nicotinic acid did not alter food intake or body weight gain.

285

TABLE 4 EFFECT OF ONE WEEK TREATMENT WITH AY-25,712, NICOTINIC ACID AND CLOFIBRATE GIVEN IN THE DIET OR DAILY BY GAVAGE ON SERUM TRIGLYCERIDE CONCENTRATIONS IN RATS Data expressed

as mean*

SEM for 10 rats/group.

Results

from three studies are presented. -

Drug

Dose

Serum triglycerides

(mg/dl) -

(mg/kg/day) Diet

Gavage -

AY-25,712

Nicotinic

acid

Clofibrate

Control

Treated

Control

Treated

65 100 250

134i: 11.0 107’ 9.1 134’11.0

133* 10.1 922 1.3 123% 14.6

1092 10.3 109s 10.3 _

53-4.9 a 5123.6” _

62 250

107% 1072

42’3.8 a 43 2 3.1 a

125 250

107* 9.1 134)ll.O

6.9 6.9

95% 95%

4.6 6.3

1022 1022

6.4 6.4

542 46*

5.9 a 5.5 a

109% 10.3 _

61*5.2a _

a P
Plasma free fatty acids

Rats (weighing 200-220 g) were fasted overnight and given by gavage AY-25,712 or nicotinic acid as single doses of 1, 2, 5, 10, 25, 65 and 250 mg/kg. The animals were killed with ether at 2 h after dosing, blood was collected, and plasma concentrations of FFA and triglycerides were measured. Pilot studies showed that aqueous solutions of AY-25,712, nicotinic acid and clofibric acid (the active metabolite of clofibrate) at concentrations up to 300 pg/ml (the anticipated maximal drug TABLE 5 EFFECT OF A SINGLE DOSE OF AY-25,712 TRIGLYCERIDES IN FASTED RATS Rats were killed 2 h after dosing. Dose

0

1 2 5 10 25 65 250 a P co.05;

Plasma

OR NICOTINIC

Data are expressed

Plasma

FFA (pequiv./l)

AY-25,712

Nicotinic

335k21.3 278’25.8 326143.0 268-40.3 168*35.0b 128k24.7’ 1002 15.0 c 67% 16.6’

335221.3 358-c 23.5 324 r 24.7 398* 38.0 369-c 19.7 202k52.1 a 21 6.6c 37% 7.4c

b P
= P-=0.001.

as mean%SEM

acid

ACID

ON PLASMA

FFA

for 8 rats/group. triglycerides

(mg/dl)

AY-25,712

Nicotinic

acid

66.3k5.70 49.629.15 36.2 * 5.79 b 39.7k6.54 b 28.9”2.15’ 23.2% 3.21’ 32.8 2 3.08 ’ 20.2 2 2.43 ’

66.3* 5.70 65.2 -c 9.06 73.42682 56.4 2 7.75 77.525.14 27.5 ‘4.86 = 17.21’2.24’ 24.6” 1.40c

AND

286

concentration in plasma) did not produce any “apparent FFA” with the procedure used. As shown in Table5, a single dose of AY-25,712 or nicotinic acid produced dose-dependent decreases in plasma FFA and triglyceride concentrations. The lowest doses which significantly lowered plasma FFA were 10 mg/kg for AY-25,712 and 25 mg/kg for nicotinic acid, while the minimal triglyceride-lowering doses were 2 mg/kg for AY-25,712 and 25 mg/kg for nicotinic acid. Rebound in lipid concentrations When rats are treated with nicotinic acid, the initial decrease in plasma FFA concentrations is followed by a rebound increase to levels significantly higher than those in matched controls [9]; the rebound in plasma FFA is followed by an increase in liver triglyceride levels [lo]. Studies were therefore conducted to determine whether AY-25,712 produces a rebound in plasma FFA, and in plasma and liver triglycerides. Rats (weighing 200-220 g) were fasted overnight, then given by gavage single doses of AY-25,712 or nicotinic acid at a level of 300 mg/kg. Groups of animals (including vehicle-treated controls) were killed with ether at 0.5, 1, 2, 3, and 6 h after dosing. In a subsequent study, using the same experimental design, groups of rats were killed at 8, 10, 12, 15, 18 and 24 h after dosing. Plasma FFA, and plasma and liver triglyceride concentrations were measured. The data presented in

+60-

+40

+60-

-

??

Lull 123

1

'

1

1

I

I

6

8

10

12

15

18

t 24

,I,, 123

Hours after Dosing

III, 8

8

I, 10

12

15

18

A 24

Hours after Dosing

Fig. 1. Effect of AY-25,712 and nicotinic acid at a dose of 300 mg/kg on plasma FFA concentrations in fasted rats. Each point represents the mean* SEM of 8 animals. Values significantly different (P ~0.05) from controls killed at the same time after dosing with vehicle are indicated with an asterisk. Fig. 2. Effect of AY-25,712 and nicotinic acid at a dose of 300 mg/kg tions in fasted rats (see legend to Fig. 1).

on plasma triglyceride concentra-

287

+150i5 b +125-

-50

; II,,

2

6

8

10

12

,

,

15

18

I 24

Hours after Dosing Fig. 3. Effect of AY-25,712 and nicotinic in fasted rats (see legend to Fig. 1).

acid at a dose of 300 mg/kg

on liver triglyceride

concentrations

Figs. l-3 comprise the results from both studies, and are expressed as percentage change from the appropriate vehicle-treated controls. Both AY-25,712 and nicotinic acid rapidly decreased plasma FFA concentrations; at OS-3 h after dosing, the FFA levels were 80-90% lower than in controls (Fig. 1). and were still significantly lower at 6 h after dosing. However, in rats given nicotinic acid, FFA levels were significantly higher than controls at 8- 12 h after dosing, and returning to control values at 15 h. No such rebound occurred in rats given AY-25,712: plasma FFA returned to basal levels at 10 h after dosing and remained within 20% of controls for the duration of the study. The plasma triglyceride lowering by AY-25,712 and nicotinic acid became statistically significant 2 h after dosing and, in rats given AY-25,712, remained significant for 24 h, i.e. for the duration of the study (Fig. 2). In contradistinction, the hypotriglyceridemic effect of nicotinic acid lasted about 10 h and was followed at 15-18 h by a rebound to concentrations much higher than in control, returning to control levels only at 24 h after dosing. Both AY-25,712 and nicotinic acid significantly decreased liver triglyceride concentrations in rats killed at 2 and 6 h after dosing (Fig. 3). With AY-25,712, liver triglycerides remained somewhat lower than in controls up to 24 h, though the differences were not statistically significant in all groups. On the other hand, in nicotinic acid-treated rats, liver triglyceride concentrations were markedly elevated from 10 to 24 h after dosing; at 15-18 h after dosing, liver triglycerides were more than 150% higher than in control rats.

Discussion

In vitro, both AY-25,712 and nicotinic acid inhibited theophylline-induced lipolysis (Table 1) but had no significant effect on FFA release induced by norepineph-

288

rine. The results suggest that the effect of AY-25,712 on lipid mobilization in vitro is similar to that of nicotinic acid. Other investigators have reported that lipolysis induced by methylxanthines is more sensitive to nicotinic acid than that induced by catecholamines [5,1 I]. Recently, it was suggested [ 121 that the antilipolytic effects of nicotinic acid, prostaglandin E, and cu-adrenergic drugs are influenced by adenosine released from the cells during incubation, and that maximal antilipolytic activity of these agents is detected only in the absence of adenosine, e.g. in the presence of theophylline which can release the adenosine deaminase. The effects of AY-25,712 on FFA and triglyceride metabolism are also similar to those of nicotinic acid. Thus, both AY-25,712 and, as reported previously [13], nicotinic acid enhanced the activity of adipose tissue lipoprotein lipase (Table2). Also, under our experimental conditions, a single dose of AY-25,712 (25 mg/kg) or of nicotinic acid (125 mg/kg) produced a significant decrease in serum triglycerides; in contrast, a single dose of clofibrate (240 mg/kg) had no effect (Table3). Furthermore, chronic treatment with AY-25,712 or nicotinic acid was hypotriglyceridemic when given by gavage, but not when administered with the diet at doses up to 250 mg/kg/day (Table4). Similar findings with &pyridylcarbinol [ 141 and nicotinic acid [ 151have been reported previously. Clofibrate, on the other hand, was active when given either by gavage or with the diet. The reason for the inactivity of AY-25,712 when mixed with the diet is not known. Unpublished studies from our laboratories have shown that the serum concentration of AY-25,712 in rats killed after administration of the drug in the diet at a dose equivalent to 250 mg/kg/day was higher than the peak serum drug concentration in rats given AY-25,712 by gavage at an (active) dose of 10 mg/kg/day. Thus, these as well as other studies have shown that the bioavailability of AY-25,712 when mixed with Purina chow is not impaired when given to rats. A single oral dose of AY-25,712 or nicotinic acid lowered plasma FFA and triglyceride levels (Table 5) as well as liver triglycerides in fasted rats. In spite of the finding that, in vitro, nicotinic acid was more potent in inhibiting the theophyllineinduced lipolysis (Table l), in vivo, the minimal effective doses of nicotinic acid were 2-10 times higher than those of AY-25,712, depending upon the parameter measured. The reason for this apparent discrepancy is unclear, although similar differences were reported between nicotinic acid and acipimox, an antilipolytic agent found to be less potent than nicotinic acid in vitro [ 161, but more active in vivo [ 171. The initial decrease in plasma FFA concentrations after nicotinic acid administration is followed by a rebound increase to levels higher than controls in both rats [9] and man [ 181. Increased plasma FFA in man have been reported to be associated with serious ventricular arrhythmias [ 19,201, possibly due to FFA-induced hypoxia [21]. In our studies, nicotinic acid, at a single dose of 300 mg/kg, produced initial decreases followed by significant increases of plasma FFA and triglycerides, and of liver triglycerides (Figs. l-3). No such rebound occurred with AY-25,712 at a dose of 300 mg/kg: plasma FFA were markedly decreased at 30 min after dosing, returning to control levels at 10 h and remaining there up to 24 h after dosing. Plasma triglycerides remained significantly lower than controls during the entire 24-h interval after AY-25,712 administration, and liver triglycerides up to 15 h. The

289

absence of rebound in plasma FFA and plasma and liver triglycerides in rats given AY-25,712 was, to date, the only qualitative difference in the lipid effects of AY-25,712 and nicotinic acid. The previously reported nicotinic acid-induced increase in liver triglycerides in rats [20-221 appears to be correlated with the rebound in plasma FFA [22]. The mechanism of the rebound in plasma FFA induced by nicotinic acid has not been elucidated. However, the triglyceride accumulation in the liver has been ascribed to the biotransformation of nicotinic acid [23] to N-methylnicotinamide, its major metabolite in rats and man [24]. At high doses, nicotinic acid can act as a “methyl trap”, i.e. by utilizing methyl groups which are normally used for choline and/or methionine synthesis, nicotinic acid can induce choline deficiency resulting in lipid accumulation in the liver [23]. Such a process cannot occur with the structurally different AY-25.712.

Acknowledgements

The authors thank Judy Zilber, Jane Wylie and Lison Dubreuil for their excellent technical assistance.

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