Bioehimiea et Biophysica Acta. 1085( 1991) 131- 135

13 i

© 1991 ElsevierScience PublishersB.V. 0005-2760/91/$03.50

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Rapid Report

The effects of probucol on lipoprotein metabolism in the rat B a r t S t a e l s i, A r i e v a n T o l 2, H a n s J a n s e n 2 a n d J o h a n A u w e r x t i The Laboratory of Experimental Medicb)e and Endocrinology. Campus Gasthuisberg. Katholieke Unirersiteit. Lem'en (Belgium) and 2 Department of Biochemistry I. Medical Faculty. Erasmus Unit'ersity. Rotterdam (The Netherlands)

(Received II June 1991)

Key words: Gene expression;Hyperlipidemia;Atherosclerosis;ApolipoproteimLDL-receptor;Hypolipidemicdrag; Probucol The effects of probucol on liver and intestinal apolipoprotein, LDL-receptor and hepatic lipase gene expression, as well as plasma lipid and apolipoprotein levels and liver lipase activity were evaluated in male rats. AdmiIAstration of probueol decreased plasma triacylglycerols, without affecting plasma cholesterol. Plasma apo E and apo B concentrations increased after probucol. Since liver and intestinal apo B and apo E mRNA levels remained unchanged, this increase could be attributed to a delayed clearance by the LDL.t~'eptor, whose mRNA levels dropped by 50% in the liver. For the HDL-apolipoproteins, only liver aim A-IV mRNA levels decreased after probucol, which was reflected by a fall of plasma apo A-IV. Neither hepatic lipase activity nor mRNA levels were significantly influenced by probucol.

The hypolipidemic drug probucol is able to retard the progression of the atherosclerotic lesion in humans and rabbits [1-3]. On the one hand, prohucol is a potent antioxidant, which inhibits the oxidative modification of LDL [4] and consequently the uptake of oxidized LDL by macrophages in the atheroslerotic lesion [2,3,5,6]. O n the other hand, probucol effectively reduces plasma lipid concentrations. It is the only drug which decreases plasma LDL-cholesterol in patients suffering from homozygous familial hypercholesterolemia as well as in the LDL-receptor-deficient W H H L rabbit [7-9]. In the W H H L rabbit, prohucol changes the structure of the LDL-particl¢, thereby enhancing its removal via an LDL-receptor independent pathway [7]. However, probucol also lowers plasma HDL-cholesterol levels, which is the reason for the controversy on the use of probucol in the treatment of atherosclerosis [10]. At present little is known on the mechanism of action of probucol on plasma lipoprotein metabolism. One possible mechanism may be by influencing the expression of different genes involved in

Abbreviations: aim, aimliimprotein; LDL, Iow-densil3,lipoprotein; HDL, high-densitylipoprotein; VLDL. very.low-densityliimprotein; HL, hepatic lipase; WHHL, Watanabe heritable hyperlipidemic. Correspondence: J. Auwerx, Laboratoriumvoor ExperimenteleGeneeskunde en Endocrinologie(LEGENDO), Gasthuisberg (Onderwijs ¢n Navorsing),B-3000 Leuven, Belgium.

lipoprotein metabolism. Therefore we investigated the effects of probucol on the expression of different apolipoprotein genes, as well as on thc expression of genes coding for proteins in,/olvcd in lipoprotein conversion, such as hepatic lipase (a-HL), or clearance, such as the LDL-receptor. Consequently, it was investigated whether the changes in g e n t expression could explain the observed alterations in plasma apolipoprotein levels. Male Wistar rats (n = 4) were fed standard rat chow supplemented or not with 1% (w/w) probucol (Merrell Dow Pharmaceuticals, Cincinnati, U.S.A.) for 14 days. At the end of the experiment animals were fasted overnight, killed and blood was collected in EDTAcontaining tubes. Plasma total cholesterol, triacylglycerol, apo A-i, apo A-IV, apo B and apo E concentrations were measured as described [11-13]. Total plasma lipoproteins were flotated through a KBr-gradient, and apolipoproteins were separated by SDS-~lyacrylamide gel electrophoresis as described [12]. R N A was extracted from livers and intestines of individual animals, and northern and dot blots of total cellular RNA, labeling of probes, hybridizations and washing of filters were pcrfon'ned exactly as described [11-13]. The following probes were used: rat apo A-I, apo A-II, apo E and apo B eDNA clones; human LDL-receptor (pLDLR-3) and hepatic lipase clones; and a 42-mer oligonucleotide probe complementary to rat apo A-IV [11-15]. A chicken/~-actin eDNA clone was used as a control probe [16]. Autoradiograms of filters were ana-

132 lyzed by quantitative s c a n n i n g d e n s i t o m e t r y in the line a r r a n g e of film sensitivity exactly as d e s c r i b e d [11] a n d values are expressed in a b s o r b a n c e units relativc to the level o f the control animals. T r e a t m e n t with p r o b u c o l did not affect the b o d y weight, liver w e i g h t o r f o o d i n t a k e o f t h e a n i m a l s (Table I). P l a s m a triacylglycerols d e c r e a s e d a f t e r administration o f p r o b u c o l , w h e r e a s p l a s m a cholesterol r e m a i n e d u n c h a n g e d . T r e a t m e n t with p r o b u c o l did n o t influence liver o r intestinal a p o A - I o r a p o A-II m R N A levels (Table I, Fig. 1). In a d d i t i o n , p l a s m a a p o A - I c o n c e n t r a t i o n s r e m a i n e d c o n s t a n t ( T a b l e I, Fig. 2). In contrast, liver a p o A - I V m R N A levels d r o p p e d to nearly 5 0 % o f the controls, w h e r e a s intestinal a p o A - I V m R N A levels did n o t c h a n g e significantly ( T a b l e

I). T h e a l t e r a t i o n s in liver a p o A - I V m R N A as m e a s u r e d by dot blot hybridization a r e c o n f i r m e d by n o r t h e r n blot analysis (Fig. 1). T h e effects of this hypolipid e m i c d r u g o n h e p a t i c a n d intestinal a p o A - I V g e n e expression a r e t h e r e f o r e r e m i n i s c e n t o f t h e tissueselective r e g u l a t i o n o f a p o A - I V g e n e expression a f t e r t r e a t m e n t with several h o r m o n e s [12]. I n d e e d , h o r m o n e s also a l m o s t exclusively a f f e c t h e p a t i c a p o A - I V m R N A levels, w h e r e a s intestinal a p o A - I V m R N A levels r e m a i n u n a l t e r e d . A c c o m p a n y i n g the d e c r e a s e in h e p a t i c a p o A - I V g e n e expression, p l a s m a a p o A - I V levels d e c r e a s e a f t e r p r o b u c o l ( T a b l e I). T h i s d e c r e a s e as m e a s u r e d by elect r o - i m m u n o a s s a y , is c o n f i r m e d a f t e r flotation o f p l a s m a l i p o p r o t e i n s a n d s e p a r a t i o n o f a p o l i p o p r o t e i n s by

TABLE I Influence of probucol on body and liver weight, food intake, and lipid parameters

Adult male rats (n = 4) were treated with probucol (1%, wt./wt., in rat chow) for 14 days. At the end of the treatment period body and liver (expressed in g/100 g body weight) weights were recorded. The food intake was measured every 2 days throughout the period of treatment. Plasma lipid and aDoliDoprotein concentrations, liver lipase activity and liver and intestinal mRNA levels were measured and expressed as described [11-14]. Values represent the mean4-S.D. Statistically (t-test) significant differences from controls are indicated by an asterisk (P < 0.05).

Control

Probucol

(g) (g/100gbw) (g/day) (mg/100ml) (mg/100ml)

336 4- 3 2.914- 0.15 23 + 2 71 4- 7 96 4-29

329 42.75422 478 + 53 4-

(mg/100ml) (R.A.U.) (R.A.U.)

32.2 4- 1.6 100 +22 100 4- 9

30.4 + 2.0 117 :1:24 101 :l:tl

Apo A-It Liver mRNA

(R.A.U.)

100

Apo A-IV Plasma Liver mRNA Intestinal mRNA

(mg/100ml) (R.A.U,) (R.A.U.)

12.5 4- 1.6 100 ±34 I00 4-35

ADOB Plasma Liver mRNA IntestinalmRNA

(A.U.) (R.A.U.) (R.A.U.)

71 100 100

ADO E Plasma Liver mRNA

(mg/100ml) (R.A.U.)

8.6 4. t.5 100 4-18

11.7 4. 0.8 * 93 4- 8

LDL-REC. Liver mRNA

(R.A.U.)

100

44

HL Activity LivermRNA

(mU/gww) (R,A,U.)

1.75+ 0.20 100 +25

2.10+ 0.25 75 4- 9

fl-Actin Liver mRNA Intestinal mRNA

(R.A.U.) (R,A.U.)

100 100

98 81

Body weight Liv~rweight Food intake Cholesterol Triacylglycerols

5 0.19 t

5 7 *

ADo A-I

Plasma Liver mRNA Intestinal mRNA

4-15

4-14 4. 24 4-44

4-13

± 21 4. 25

85

4-13

9.5 4- 0.2 * 52 ± 1 3 " 79 4-30 105 100 116

5:15 * 4-14 4-28

+ 12 *

+ 7 4-30

133 content remain unchanged after probucol (Table 1 and Fig. 1). The results from this study, which are summarized in Fig. 3, show that probucol lowers plasma triacylglycerol levels in rats. The lipid lowering effect of probucol is most likely unrelated to its antioxidant action [2,3]. The lowering of plasma triacylglycerols is probably attributable to a reduction of VLDL production, since probucol has no stimulating activity on hepatic lipase (this study) or lipoprotein lipase [10,17,18]. In this as well as in other studies [19] plasma cholesterol concentrations remained unchanged, which contrasts with several studies showing a lowering of plasma cholesterol by probucoi [17,20,21]. The reason for this discrepancy is unknown, but may reflect differences in strain, diet, dose or duration of the treatment. Indeed, in one study probucol merely inhibited an increase in 7.1asma cholesterol after feeding a safflower oil-containing diet to the rats [17]. In addition, it is demonstrated that probucol may modulate plasma lipoprotein metabolism by changing the expression of genes involved in apolipoprotein synthesis and clearance. On the one hand, apo A-IV synthesis appears to decrease, since apo A-IV m R N A

SDS-PAGE (Fig. 2). In addition it can be seen that plasma apo A-I concentrations remain unchanged (Fig. 2). In contrast to the limited variation of apo B and apo E m R N A levels in liver and intestine (Table 1), plasma apo B and apo E concentrations increase markedly after probucol (Table I, Fig. 2). Since changes in apo B and apo E gene expression can not be invoked to explain the observed alterations in plasma apo B and apo E levels and since the majority of apo E a n d / o r apo B containing iipoproteins are cleared from plasma by the liver through interaction with the LDL-receptor, it was investigated next whether probucol influences liver LDL-receptor gene expression. Our results show that probucol reduces hepatic LDL-receptor m R N A levels by approx. 50% (Table I). Northern blot analysis confirms the alterations in liver LDL-receptor m R N A levels (Fig. 1), measured by dot-blot hybridization (Table I). Therefore, it appears that the catabolism of aoo B and apo E-containing lipoproteins is delayed after treatment with probucol due to a decreased expression of the LDL-receptor gene in the liver. Probucol did not influence significantly hepatic lipase m R N A levels and activity (Table I). Liver and intestinal B-aetin m R N A

A

B

Apo A-I

Apo A-II

C

O

Apo A-IV

E

LDL-Rec

Acfin

28S-

-

18S--

I I

--

"4-

--

"4-

--

+

--

+

--

28S

--18S

+

Fig. l. Northern blot analysisof the influenceof probuc'~lon liver mRNA l~v¢lsof apo A-I, apo A-If, apo A-IV, the LDL*roceptorand ~-actin. RNA was prepared from liversof control ( - ) or probucol ( + ) treated rats and subjectedto electrophoresisin a 1% agarose gel, transferred to a nylonmembrane(Hybond-N. Amersham)and hybridizedas described[11-13]. The locationof the 18 S and 28 S rRNA bands are indicated.

134

A p o A-IV-Ap o E , -

Apo A-I

Apo C +A-II

-,

"~IIllllb

~

~

t

,

:-~:~'~

/

~

~

t

,,,

PRO

CON

Fig. 2. Influenceof probncol(PRO) on plasmaapolipoproteinlevels• Adult male Wistar rats received probucol(1%, wt./wt., mixed in rat chowfor 14 days)or not (CON). Plasmalipoproteins(d < 1.21 g/ml) were isolated, delipidated and apolipoproteinswere separated by SDS-PAGelectroph0resisas described [12].

levels decrease in liver, which results in a lowering of plasma apo A-IV concentrations. In contrast, the expression of both apo A-I and apo A-II, the main HDL-constituents, is not affected by probucol. This sel~etive action of probueol on apo A-IV mRNA may therefore influence the composition of HDL. In humans, however, the decrease in plasma HDLcholesterol has been suggested to result from decreased synthetic rates of apo A-I and apo A-II [22,23]. On the other hand, plasma lipoprotein clearance is influenced by probucol. Indeed, LDL-receptor mRNA levels are lowered in probucol treated rats. In view of the unaltered liver and intestinal mRNA levels of both apo B and apo E, the increased plasma apo B and apo E concentrations indicate that the decrease in hepatic LDL-receptor gene expression results in a lower LDLreceptor activity, which may explain the reduction in the absolute rate of catabolism of apoLDL observed previously in rats treated with probucol [20]. These observations in rats contrast with the observations in humans that probueol increases both LDL and apo B fractional catabolic rate [10,22], but these effects in humans have been attributed mainly to an increase in LDL-receptor independent increase in the fractional catabolic rate of LDL [1,8]. In addition, other investigators found no consistent change in fractional catabolic rate of LDL [23,24]. Since the majority of plasma cholesterol is transported by an apo A-l-containing HDL-fraction in the rat, it is not surprising that plasma total cholesterol concentrations remain un-

PROBUCOL LIVER

PLASMA

RNA ~00A-~

-

/_~

C HOL ~ TG 1|

/#

ApoA-~--

Apo A-IV I ~ ApoA-IV { Apo B -- / C ApoB ~ Apo E - - / Apo E

/

HI_

INTESTINE RNA

/

~

o~

I~

&

A00A-

Apo A-IV -,'"LAoo B -,-

\

~ ~ CLEARANCE

Fig. 3. Influenceof probucol on plasmalipid and apolipoprotemconcentrationsand mRNA levelsfor apolipoproteins(apo), hepatic lipase (HL) and the LDL-receptor (LDL-R) in rat liver and intestine. Changesare semi-quantitativelyexpressedrelative to the levels in control rats (1 arrow = lessthan 2-fold differencefrom controls,2 arrows= 2-fold difference,3 arrows= 3-fold difference,etc...).

135 changed. In addition, previous studies showed a decrease in hepatic H M G - C o A reductase activity and sterol synthesis both in vivo and in vitro in isolated rat hepatocytes t r e a t e d with probucol [20,21,25,26], which could contribute to the unaltered plasma cholesterol levels. In conclusion, in addition to its antioxidant action, which inhibits the oxidative modification of L D L and thus decreases the rate at which L D L is taken up by m a c r o p h a g e f o a m cells in the fatty streak, probucol exerts a m a r k e d influence on p l a s m a lipoprotein and apolipoprotein concentrations in the rat by selectively influencing hepatic apo A - I V and L D L - r e e e p t o r gene expression.

Acknowledgements This work was s u p p o r t e d by an ILSI a w a r d and by a F G W O a w a r d (No. 3.0027.90) to J . A . J . A . is a research associate a n d B.S. is a r e s e a r c h assistant of the Belgian F o u n d a t i o n for Scientific R e s e a r c h ( N F W O / F N R S ) . W e t h a n k J. Rosseels, H.A. van Rijssel, A . N . R . D . D o r s m a n a n d M.IVi. G e e l h o e d - M i e r a s for their excellent technical assistance. W e acknowledge Merrell Dow P h a r m a c e u t i c a l s Inc. for the g e n e r o u s gift of probucol,

References 1 Yamamoto, A., Matsuzawa, Y., Yokoyama. S., Funahashi, T., Yamamura, T. and Kishino, B.-1. (1986) Am. J. Card~ol. 57, 29H-35H. 2 Kita. T., Nagano, Y., Yokode, M., lshii, K., Kume. N., Ooshima, A., Yoshida, H. and Kawai, C. (1987) Proc. Natl. Aead. Sci. U.S.A. 84, 5928-5931. 3 Carew, 'I.E., Schwenke, D.C. and Steinberg, D. (1987) Proe. Natl. Acad. Sci. U.S.A. 84. 7725-7729. 4 Parthasarathy, S., Young, S.G., Witztum, J.L.. Pittman, R.C. and Steinberg. D. (1986) J. Clin. Invest. 77, 641-644.

5 Yamamoto, A., Takaichi, S., Hara, H.. Nishikawa,O., Yokoyama, S., Yamamura, T. and Yamaguchi. T. (1986) Atheroselerosis 62, 21)9-217. 6 Yamamoto. A.. Hara, H.. Takaiehi, S., Wakasugi, J.-l. and Tomikawa. M. (1988) Am. J. Cardiol. 62, 31B-36n. 7 Naruszewic'z,M.. Carew, T.E., Pittman, R.C., Witztum. J.L. and Steinberg, D. (1984)J. Lipid Res. 25, 1206-1213. 8 Baker, S.G,. Joffe, B.I., Mendelsohn, D. and 5eftel, H.C. (1982) S. Aft. Med. J. 62, 7-11. 9 Yamamoto. A., Matsuzawa, Y., Kishino, B., Hayashi, R., Hirobe, K. and Kikkawa,T. (1983)Atherosclerosis 48,157-166. to Kesaniemi. Y.A. and Grundy. S.M. (1984) J. Lipid Res. L~;. 780-790. I 1 Staels, B.. Auwerx. J., Chan, L, Van Tol, A., Rosseneu, M. and Verhoeven. G. (1989)J. Lipid Res. 30, 1137-1145. 12 Staels. B., Van Tot, A.. Verhoeven, G. and Auwerx, J. (1990) Endocrinology 126, 2153-2163. 13 Staels, B., Jansen, H., Van Tol. A., Stahnke, G., Will, H,, Verhoeven. G. and Auwerx, J. (1990)J. Lipid Res. 31,1211-1218. 14 Staels, B., Van Tol, A., Chan, L., Will, H., Verhoeven, G. and Auwerx, J. (1990) Endocrinology 127, 1144-1152. 15 Chin. D.J.. Gil, G.. Russell, D,W., Liseum, L., Luskey, K.L., Basu, S.K., Okayama, H., Berg, P., Goldstein, J.L. and Brown, M.S. (1984) Nature 308, 613-617, 16 Cleveland, D.W., Lopata, M.A., McDonald, R.J., Cowan, M.J., Rutter, W,J. and Kirsehner, M.W. (1980) Cell 20, 95-105. 17 Strandberg, T., Kuusi, T,, Tilvis, R. and Mieuinen, T.A. (1981) Atheroselerosis 40, 193-201. 18 Helve, E. and Tikkanen, M.J. (1988) Atherosclerosis 72,189-197. 19 Shankar, R.. Sanis, J.D., Stanton, H. and Thomson, R. (1989) Atherosclerosis 78, 91-97. 20 Balasubraraaniam, S., Beins, D.M. and Simons, L.A. (1981)Clin. Sei. 61,615-619. 21 Li, J.R., Holets, R.J. and Konke, B.A. (1980) Atherosclerosis 36, 559-565. 22 Nestel, P.J, and Billington, T. (1981) Atherosclerosis 38, 203-209. 23 Attach. R.F., Stewart, ,LM., Boag. D.E., Packard, C.J., Lorimer, A.R. and Shepherd. J. (1983) J. Lipid Res. 24, 588-595. 24 Cortese, C., l~arenah, C.B., Miller, N.E and Lewis B, (1982) Atheroselerosis 44, 319-325. 25 Barnhart, J.W., Li. D.L. and Cheng, W.D. (1988) Am. J. Cardiol. 62, 52B-56B. 26 Miettinen, T.A. (1972) Atheroselesosis 15, 163-176.