297

Biochimica et Biophysica Acta, 1044 (1990) 297-304 Elsevier BBAI IP 53417

Cholesterol is required for the secretion of the very-low-density lipoprotein: in vivo studies Bobby V. Khan, Thomas V. Fungwe, Henry G. Wilcox and Murray Heimb,~rg Deportment o f Pharmacology, University o f Tennessee-Memphis, The Health Science Center. ?demphis, TN (U.S.A.) (Received 29 September 1989) (Revised manuscript received 27 February 1 9 ~ )

Key words: VLDL; Cholesterol; Lipoprotein secretio~ (Rat)

Rats were fed for I week with a standard chow diet, a diet suppiemented with lovastatin (0.1%), or a diet supplemented with both Iovastafin and d l o l ~ t e r o l ( 0 . 1 / 0 . 1 % ) , to study effects of depletion of a putative hepatic metabolic pool o~ cholesterol o n secretion of the very-low-density lipopmtein (VLDL) in the intact animal. Triton WR-1339 (50 mg/lO0 g body wt.) o r the 0.9% NaC! vehicle alone was given intravenously via a rmcrai vein. T r e a t m e n t with Iovastatin decreased the secretion of all plasma V L D L lipkls, the average decrease Mter 2 h for V L D L triacyiglyceroi, plmspholipld, cholesterol and cholesteryl e s t e r being 45%. W h e n both Iovastatin and d m t e s t e r o l were included in the diet. the secretion of V L D L triacylglyc~eroi and ~ i p i d was similar to that of control animals, while secretion of V L D L cholesterol and d m k ~ e r y l ester was increased. T r e a t m e n t with Iovastatin reduced the hepatic concentration of cholesteryl esters 42% without affecting free dmlesterol, in r.elmrate exl~riments, in vivo synthesis of cholesterol was determined ! h a f t e r intraperltoneal administration of ~ H 2 0 . Incoqmration into hepatic and plasma f r e e d m ; e s t e r o l and ¢holesteryl esters was greater in the rats fed Iovastafin than in control animals, c o n c u r r e n t with decreased V L D L secretion. T h e metabolism of V L D L was determined in vivo by intravenous administration of I ~ I - V L D L . T h e fractional d e a x a n ~ r a t e s of t ~ I-VLDL f r o m the plasma were simnar among the three experimental groups. Synthesis of hepatic triacyl~yeerol from ll-'4CIoleate in vivo was similar in all t r e a t m e n t g r o e l ~ incorimcatiort into plasma triaeylglycerol was reduced with Iovastafin t r e a l m e n t mKI reversed partially by inclusion of 0.1% d m l e ~ e r o l in the iovastatin diet. Plasma concentrations of ~iacylglycerol fo|lowed patterns of incorporation of ll-14Cloleate. Triacylgtycerol concentration in the liver increased when cholesterol was included in the diet. In companion experiments, i ~ t i o n of [l-MCloleate into p e d u s a t e triacylglycerol in vitro was reduced with perfttsed livers from Iovastatin-treated animal~ In these experiments, oxidation of fatty a d d into C O 2 and p e r d d o r i e ackl-soinble cotmts was not affected by Iovws¢~tin, added either to the diet o r to the perfusate in vitro. It appears, therefore, that Iovastatin does not affect trlacylglycerol synthesis o r fatty acid oxidation, which per se might reduce formation and secretion ol VLDL. T h e s e data, therefore, strengthen the hypothesis that reduced availability of cholesterol in a putative hepatic metabolic pool, required for secretion and transport of triacylglycerol in the v LDL, is a factor contributing to decreased secretion of the VLDL.

Introduction T h e liver secretes the very-low-densi!y lipoprotein (VLDL), the major function of which is to transport

Abbreviations: VLDL, very-low-density lipoprotein; H M G - C o A reductas¢. 3-hydroxymethyl-3-glutaryl coenzyme A reduetase; BSA. bovine serum albumin; E D T A ethylene diamine tetfaacA~lic acid; F C R , fractional cle.~rance rate; CPT-I, ca,rnitine palmitoy| acyltransferase; ACAT, acyl-CoA cholesterol acyl transferase. Correspondence: M. Heimberg. Department of Pharmacology, University of Tennessee-Memphis, The Health Science Center, Memphis, "IN 38163, U.S.A.

triacylglycerol of e n d o g e n o u s origin from the L:ver to other tissues. The p r i m a l substrates for the stimulation of synthesis and secretion of the V L D L are free fatty acids, which increase the secretion of all lipid and protein c o m p o n e n t s of the V L D L [1-5]. F a t t y acids also stimulate de novo biosynthesis of cholesterol and activate microsomal H M G - C o A reductase [5] a n d cytosolic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthase and H M G ~.~,A synthase [6]. W e postulated previously that the surface lipid and apoprotein c o m p o n e n t s o f the V L D L are essential to the secretion of triacylglycerol in the V L D L and that a reduction in synthesis or availability of any o f these c o m p o n e n t s can impair the secretion of the V L D L by

0005-2760/90/$03.50 © 1990 Elsevier Science Pubfishers B.V. (Biomedical Division)

298 the liver [5]. W e r e p o r t e d recently th=t cholesterol is required for secretion of V L D L b y the isol~.':.d p e r f u s e d rat liver [7,8]. By diminishing the p u t a t i v e m e t a b o l i c h e p a t i c pool o f cholesterol, the o u t p u t o f V L D L w a s r e d u c e d severely. F u r t h e r m o r e , by p r o v i s i o n o f cholesterol either b y d i e t a r y means, or b y a d d i t i o n to the m e d i u m in vitro, o u t p u t o f V L D L w a s o n c e again e n h a n c e d . T h e d e c r e a s e d net o u t p u t o f V L D L was clearly the result of r e d u c e d secretion, a n d not t h e result of increased c l e a r a n c e of V L D L b y the liver [8]. T o validate f u r t h e r these studies with the isolated p e r f u s e d liver, related e x p e r i m e n t s were p e r f o r m e d in vivo. T o s t u d y effects o f d e p l e t i o n o f a p u t a t i v e h e p a t i c m e t a b o l i c p o o l o f c h o l e s t e r o l on V L D L o u t p u t in vivo, T r i t o n W R - 1 3 3 9 w a s injected i n t r a v e n o u s l y t o block i n t r a v a s c u l a r c a t a b o l i s m o f V L D L and to m e a s u r e in vivo o u t p u t o f V L D L in the plasma. T h e fractional c l e a r a n c e rate of ~25I-VLDL was d e t e r m i n e d , f r o m which synthesis rates o f V L D L p r o t e i n w e r e calculated. Synthesis of cholesterol in these in vivo e x p c r i m e n t s was d e t e r m i n e d b y m e a s u r i n g i n c o r p o r a t i o n o f 3 H 2 0 into h e p a t i c a n d p l a s m a - f r e e cholesterol a n d cholesteryl esters. T h e net rate o f s y n t h e s i s o f triacylglycerol from i n c o r p o r a t i o n of [1-~4C]ol~'at_e w a s d e t e r m i n e d to c o m p a r e the effects o f lovastatin a n d cholesterol f e e d i n g in the isolated p e r f u s e d liver a n d in vivo. F a t t y acid oxidation rates were also e x a m i n e d in the p e r f u s e d liver. T h e results o f these e x p e r i m e n t s s u p p o r t the c o n c l u sions derived f r o m e x p e r i m e n t s w i t h the isolated perf u s e d liver that cholesterol is essential f o r the secretion o f the V L D L a n d thc l~ansport o f triacylglycerol in the p l a s m a . Preliminary r e p o r t s o f this w o r k h a v e a p p e a r e d [9]. Methods T r e a t m e n t o f animals N o r m a l m a l e S p r a g u e - D a w l e y rats o b t a i n e d from, H a r l a n ( I n d i a n a p o l i s , I N ) w e r e m a i n t a i n e d ad libitum on w a t e r a n d either a P u r i n a r o d e n t c h o w diet, a diet s u p p l e m e n t e d with 0.1% lovaslatin, o r a diet s u p p l e m e n t e d with b o t h 0.1% lovastatin a n d 0.1% cholesterol [8]. R a t s wer~ m a i n t a i n e d o n diets for 1 w e e k a n d were h o u s e d u n d e r a n o r m a l light-dark cycle, with lights on f r o m "~6L, ~o 1900. R a t s were a l l o w e d free access to f o o d and ater until the time o f the e x p e r i m e n t . N o d i f f e r e n c e s in f o o d i n t a k e o r b o d y weight were o b s e r v e d a m o n g a n y of the three experin,:~atal g r o u p s . Determina*,i~,n o f output o f V L D L in c.a " T o e v a l u a t e the effects o f d e p l e t i o n o t the p u t a t i v e h e p a t i c m e t a b o l i c p o o l o f cholesterol, a n d ~,.,h~,equent repletion, rats were injected w i t h T r i t o n W R - 1 3 3 ~ , a n o n i o n i c d e t e r g e n t w h i c h has b e e n u s e d w i d e l y as an i n d i c a t o r o f V L D L s e c r e t i o n in vivo [10,11]. A n i m a l s w e r e a n e s t h e t i z e d lightly with diethyl ether, a n d T r i t o n

W R - 1 3 3 9 (20 g / d l in 0.9% N a C I , 50 m g / 1 0 0 g b o d y wt.), or 0.9% N a C I a l o n e was given i n t r a v e n o u s l y v i a a sacral vein. A n i m a l s w e r e a l l o w e d to regain c o n s c i o u s ness. A f t e r 2 h, the a n i m a l s were a n e s t h e t i z e d w i t h p e n t o b a r b i t a l (60 m g / 1 0 0 g b u d 3 wt.) a n d w e r e killed b y e x s a n g u i n a t i o n from the a b d o m i n a l aorta. T h e b l o o d ( 8 - 1 0 ml), t r e a t e d with 0 . 2 - 0 . 5 ml E D T A (6%), w a s c e n t r i f u g e d to s e p a r a t e the p l a s m a f r o m the cells. P o r t i o n s o f t h e p l a s m a from individual a n i m a l s w e r e l a y e r e d u n d e r 6 ml of 0.9% N a C ! in p o l y c a r b o n a t e c e n t r i f u g e t u b e s a n d c e n t r i f u g e d in a B e c k m a n 4 0 r o t o r at 1 2 0 0 0 r p m for 30 m i n at 13 ° C a n d the s u p e r n a t a n t c h y l o m i c r o n fraction w a s r e m o v e d [12]. T h e i n f r a n a t a n t fraction w a s t h e n c e n t r i f u g e d for 18 h at 1 3 ° C a n d 3 9 0 0 0 r p m to isolate t h e V L D L [13]. T h e d i f f e r e n c e s in t h e c o n c e n t r a t i o n s o f V L D L triacyiglycerol b e t w e e n the a n i m a l s i n j e c t e d with T r i t o n W R - 1 3 3 9 a n d t h o s e inj e c t e d with 0.9% N a C I w e r e u s e d to c a l c u l a t e the o u t p u t o f V L D L lipids in vivo [12]. Synthesis o f cholesterol in vivo R a t s w e r e f e d either a c h o w diet, o r a d i e t s u p p l e m e n t e d with iovastatin, as d e s c r i b e d a b o v e . O n the d a y o f the e x p e r i m e n t , rats w e r e i n j e c t e d with 5 m C i 3 H 2 0 i n t r a p e r i t o n e a l l y , as d e s c r i b e d p r e v i o u s l y [14]. A f t e r 1 h, t h e rats w e r e a n e s t h e t i z e d with p e n t o b a r b i t a l a n d w e r e e x s a n g u i n a t e d . E D T A p l a s m a w a s i s o l a t e d b y centrifu g a t i o n ; livers w e r e p e r f u s e d w i t h 50 ml ice-cold 0.9% N a C ! to r e m o v e b l o o d a n d w e r e i s o l a t e d surgically [151. Incorporation of 3HzO into plasma and hepatic c h o l e s t e r o l was m e a s u r e d . M e t a b o l i s m o f I : 5 1 - V L D L in vivo R a t s w e r e fed, as d e s c r i b e d a b o v e , w i t h c h o w a l o n e , c h o w v,.~th lovastatin, o r c h o w w i t h b o t h l o v a s t a t i n a n d cholesterol. V L D L w a s p r e p a r e d u s i n g the i s o l a t e d p e r f u s e d liver, as d e s c r i b e d p r e v i o u s l y [13]. T h e i s o l a t e d V L D L w a s l a b e l e d with Na12Sl [16] a n d w a s u s e d as t h e s u b s t r a t e for m e a s u r e m e n t o f u p t a k e o f V L D L . O n the d a y o f the e x p e r i m e n t , rats w e r e i n j e c t e d w i t h r a d i o i o d i n a t e d V L D L (0.33 • 10 6 d p m / 1 0 0 g b o d y wt.) v i a a sacral vein. R a t s were killed b y e x s a n g u i n a t i o n , as des c r i b e d a b o v e , at v a r i o u s t i m e intervals, u p to 8 h. T h e V L D L w a s i s o l a t e d f r o m the E D T A p l a s m a . V L D L w a s p r e c i p i t a t e d in 10% T C A , u s i n g b o v i n e s e r u m a l b u m i n as a p r o t e i n - c a r r i e r . L i p i d w a s then e x t r a c t e d f r o m the T C A p r e c i p i t a t e with C H C I ~ / C H 3 O H (2:1, v/v) [13,17]. T h e radioactivit]t o f V L D L t o t a l p r o t e i n w a s d e t e r m i n e d b y c o u n t i n g the l i p i d - i n s o l u b l e p r o d u c t in a M i c r o m e d i c 4 / 6 0 0 0 A u t o m a t i c Gat~.ma C o u n t e r . T h e m a s s o f p r o t e i n "~n the V L D L w a s d e t e r m i n e d , as d e scribed p r e v i o u s l y [18]. Metabolis,,, o f [ l - l ~ C ] o l e a t e in vivo R a t s w e r e fed a c h o w diet, a diet s u p p l e m e n t e d w i t h IovaslatL-., or a diet s u p p l e m e n t e d w i t h b o t h l o v a s t a t i n

299 a r d cholesterol, as described above. E D T A plasma from normal-fed male rats was iqolated by centrifugation and used to prepare a complex with [1-~4C]oleate (6.5-107 d p n ) / m l plasma) [191. Rats were injected with 3.3 #Ci [1-~4C]oleate/100 g body weight via a sacral vein. After 1 h, the rats were killed by exsanguination from the abdominal aorta under pentobarbital anesthesia and livers were removed surgically after flushing with 0.9% NaCI. The E D T A plasma was separated from the erythrocytes and 1.5-ml aliquots of plasma and 1.0-g samples of liver were removed for analysis of mass and radioactivity in lipid fractio~:s.

Metabolism of [1- ~~C]oleate by the isolated perfused liver T o correlate the above in vivo experiments with hepatic metabolism of [1-~4C]oleate in vitro, the following experiments were carried o u t using the isolated perfused liver. Rats were fed either a chow diet, o r a diet s u p p l e m e n t e d with 0.1% lovastatin. Livers were r e m o v e d surgically and were perfused, a~ described previously [8], using [1-14C]oleate as a tracer. Bovine erythrocytes were not included in the perfus.-~e in these experiments [8]. T h e livers were perfuz,ed for 20 min with the basal m e d i u m for a period o f equilibration. A n oleate-albumin complex (6 g / d l purified BSA) was infused at a c o n s t a n t rate (11.7 m l / h and 166 /xmol ' e a t e / h a n d 2 btCi [1-~4C]oleate); the final concentration o f the oleate at the end o f the perfusion was 1.6 raM.

In s o m e

experiments,

lovaztatin

in e t h a n o l

w~-s

a d d e d to the perfusion m e d i u m and infusate to give a final concentratior, of ethanol of 0.5% a n d 100 ~g l o v a s t a t i n / m l . Perfusions were carried out for 4 h. Perfusate was collected at 2 and 4 h a n d liver samples were o b t a i n e d at the termination of the experiment [8]. ~*COa was collected hourly by trapping in 10~ K O H [201. As an estimate of ketogenesis from [1-~4C]oleate, 1.0 ml o f 15% HCIO 4 was a d d e d to 1.0 ml of perfusate: the precipitate was centrifuged at 3000 r p m for 5 rain and the acid-soluble radioactivity was d e t e r m i n e d by liquid scintillation spectrometry in Ecolume; samples were c o u n t e d for 2 rain, or 1% error, in a Beckman counter. For analysis of 14CO2, an aliquot of the ~4CO2 trapped in 10% K O H was distilled into 1 M h y a m i n e hydrochloride, after which the h y a m i n e was transferred to Ecolume a n d c o u n t e d in a Beckman c o u n t e r for 2 rain, or 1% e r r o : [201.

Analytical procedures T h e livers were homogenized a n d samples of liver, V L D L and plasma were extracted with chloio~'orm/ m e t h a n o l (2: 1, v / v ) [12,171. Aliquots of the washed c h l o r o f o r m extracts were separated into individual lipid classes by thin-layer c h r o m a t o g r a p h y on Silica-gel G plates with a solvent mixture of p e t r o l e u m e t h e r / d i e t h y l e t h e r / a c e t i c acid (84 : 15 : !, v / v ) [2I]. T h e radioactivity

in the lipid fractions was measured by liquid-scintillation spectrometry for 2 rain or 1% error, asing Ecolurne as a scintillation c o c k t a i l Mass determinations were m a d e on lipid fraction- f,~," triacylglycerol I2~], phospholipid [23] and for free a n d esterihed cnolesteroi i24I.

Materials Lovastatin was a generous gift ~),pp!i~,~ by A.~' Alberts o f Merck Sharp and D o h m e Research Laboratories. Oleic acid was o b t a i n e d from N u - C h e c k - P r e p (Elysian, MN). l 1 - 14C]Oleate a n d Na i 25t were purchased from N e w England N u c l e a r Corp. Bovine serum alb u m i n (fraction V) was o b t a i n e d f r o m Sigma and was delioidated and purified before use. as described previously [21]. Thin-layer plates of Silica-gel G were purchased from Analtech. Ecolume was pur~:l,ased from ICN Biochemicals. (lrvine. CA). Free chole~,ierol used for diets was of c o m m e r c i a l grade, with 98% purity. All other chemicals used in this study were of analytical grade. StatLvlics Values ar~ reported as m e a n s + standard error of m e a s u r e m e n t (S.E.). Statistical significance of the d a t a was calculatr,~d by using a two-tailed Student's t-test at P < 0.05. Results

Determination of output of I~ DL in t:i,.~ Hepatic V L D L o u t p u t after feeding lovastatin to rats was investigated in vivo. O u t p u t was calculated from differences between concentrations o f plasma V L D L 2 h following injectien o f either Triton WR-1339 or the 0.9% NaC! vehicle (Fig. 1). N o differences were observed in V L D L lipid c o n c e n t r a t i o n s between t r e a t m e n t groups when 0.9% NaCI was administered. With lovastatin treatment, the secretion of all V L D L Iipids was reduced, as indicated by lower plasma c o n c e n t r a tions after administration of Triton WR-1339 c o m p a r e d to the controls. The average decrease of secretion of V L D L triacylglycerol, phospholipid, choles:~rol and cholesteryl ester was 45%. "v~hen both I~vastatin and cholesterol were included in the diet, V L D L triacylglycerol and phospholipid secretion increased, approaching ihat of the control. Moreover, the increase in free cholesterol a n d cholesteryl ester o f the V L D L was larger than the increase in VLDL-triacylglycerol a n d VLDL-phospholipid in the group receiving dietary cholesterol. N o differences were observed in the molar ,'atios of p h o s p h o l i p i d / t r i a c y l g l y c e r o l , c h o l e s t e r o l / t r i acylglycerol, or cholesteryl e s t e r / t r i a c y l g l y c e r o t in the V L D L between controls and the g r o u p of rats fed lovastatin (Table 1), suggesting similarities in the lit~id composition of the secreted V L D L ; however, ratios lor

300 TABLE I Molar rattos o f lipids m ~,te p l a s m a V L D L 0.08

A

0.07 (n

I

1,,o

0.06

5

P-_.. _J

0.05

-.1 o

0.04

pt

Rats were i=-,jected i n t r a v e n o u s l y with T r i t o n W g - 1 3 3 9 . P l a s m a V L D L was isolated by u l t r a c e n t r i f a g a t l o n after 2 h, as described in the text. N u m b e r of o b s e r v a t i o n s is in parentheses, t~ll values are m e a n s ± S.E. Identification of groups: C O N , rats fed an a n s u p p ! e m e n t e d c h o w diet; L O V , rats fed a ,-how diet s u p p l e m e n t e d with 0.1% lo~,astatin, LOV + C, rats fed a c h o w diet s u p p l e m e n t e d with 0.1% Iovastatin and 0.1~ cho;~sterol. PL, p h o s p h o l i p i d ; C. cholesterol; CE, cholesteryl egtel'.

~L

0.03

CON(7) LOV(7) LOV+C(6)

0.02 0.01

PL/TG

C/TO

CE/TG

30.8±1.1 3~.9~1.3 32.8±3.3

13.6±0.4 13.4±0.4 1 6 . 0 ± 0 . 6 ~b

8.4±0.6 9.7±1.5 11.1±0.4 a

a Differs (P < 0.05) f r o m C O N , b Differs ( P < 0.05) f r o m LOV.

0.00 2 #

..

B

the group receiving both iovastatin a n d cholesterol suggested t h e . s e c r e t i o n of V L D L part;cles relatively enriched in cholesterol and cLolesteryl esters.

¢,,0

...t

o .a-

t2

:E

#

-1

CON

~.~)tt

Effects o f t r e a t m e n t with Iovastatin on synthesis a n d concentration o f p l a s m a a n d hepatic cholesterol

A n i m a l s were treated, as described in M e t h o d s . O n the day of the experiment, 5 m C i of -~H20 were injected intraperitoneally and after 1 h, the rats were e x s a n g u i n a t e d a n d the livers were removed. Cholesterol (C) a n d cholesteryl ester (CE) o f the liver and p l a s m a w e r e separated and i n c o r p o r a t i o n o f 3 H 2 0 was m e a s u r e d b y liquid scintilla~ioh spectrometry. I n c o r p o r a t i o n into cholesteryl ester and specific activity of cholesteryl ester ~,re based on the total molecule. N u m b e r of observations is indicated in parentheses. Identification of groups: C O N , rats fed an u n s u p p l e m e n t e d c h o w diet; L O V , rats fed a c h o w diet s u p p l e m e n t e d with 0.1% l o v a s t a t i n D a t a are means ± S.E. Liver

LOV+C

Iri;_ . • , c - o ~ at of ¢LDI~ I~jJids iO ~ivO following t r e a t m e n t with Triton ~ R-1 ,39_ Rats were i # ~ t ¢ ~ l x,i$ the sacral v e i n either with 0.9% N a C t ~r with F,-iton W l ~ l ' t 3 9 ~50 m g / k g ) , as described in the text. After 2 h, rats were e x s f t ~ i r i o t ~ t , p l a s m a s e p a r a t e d and the V L D L isolated by ultracentrifq~ti~rJ. , ;5. 1 A presents the plasma concentration of V L D L after '~)~:ti~ft dl ,~.~% N a C l ; Fig. 1B is the plasma c o n c e n t r a t i o n of VLI~I~ ~ft% t~lminis~, .~;on o f T r i t o n W R 1339, and Fig. I C is the c ~ \ ~ t ~ art!put ol V L D I . lipids (the difference between groups tt-~lA~! M/~tb saline at T r . : - , - WR-1339, assuming plasma v o l u m e to I ~ ~ ~)t Lady wt. [20]. ,,~2' tara are m e a n s ± S . E . Identification o f ~ t t i ~ s : ~ O N , rats fed a n urtoupplemented c h o w diet; LOV. rats t ~ ~ ¢hO~v diet s u p p l e m e n t e d with 0.1% iovastatin; L O V + C , rats feq ~ ~ i * ~ diet suDplemented with 0.1% Iovastatin and 0.1% cholest~f~j" j' i)iffers { P < 0 . 0 5 ) from C O N . # Differs ( f ~ 0.(l~) from L O V .

Plasma

C !. I n c o r p o r a t i o n C O N (5) LOV(5)

CE

3. Mass Con LOV

CE

d p m / g liver p e r h

dpm/ml plasma

6 9 4 ± 71 1"79:t:25 4887±378 • 692±76 •

i54± 19 1258±177a

2. Specific a c t i v i t y d p m / / ~ m o l CON LOV

C

199± 1 310+

dpm/t~mol

22 111 • 1

1 9 9 + 31 3 4 6 ± 54 b 1 3 5 ± 2 2 b 3 4 3 ± 173 • 2 8 4 0 ± 5 1 8 J.b ~ 7 ± 2 5 ~.b

F t m o l / g liver 3.51 + 0 . 0 5 3.73 ± 0.09

148±24 421±32"

/~ m o l / m i

0.90±0.04

plasma

~ 46 ~-0.03

1.10+0.04

0.52 ± 0.02 • 0.47 + 0.04

1 . 0 3 4- 0 . O l

• Differs from L:ON ( P < 0.05). b Differs from respective values for liver ( P < 0.05).

301 greater than controls, as were the specific activities ( d p m / t t m o l ) (Table II). It is also evident from this table that the specific activity of the cholesterol in the plasma is higher than that in the liver in either group. This o b s e l - a t i o n is similar to that previously reported in perfused liver studies and is further support for the existence o f and utilization o f a precursor pool of cholesterol for secreted cholesterol, e.g., VLDL, which is i n d e p e n d e n t from (i.e., not in rap~d equilibrium with) the total pool of hepatic cholesterol. In confirmation of previous experiments with perfused livers [8], feeding 0.1% lovastatin for 1 week reduced the hepatic concentration o f cholesteryl esters by 42% but did not change that of free cholesterol.

10

I.

A

U

uJ

g

8

o.

>,

G

4

Q. 0

" B - - OON

GON

Metabolism o f 125I-VLDL in vivo N o differences in rates of disappearance of tracer ~2SI-VLDL from the plasma were observed a m o n g the chow, iovastatin-fed and lovastatin plus cholesterol-fed groups d u r i n g the 8-h period (Fig. 2A). The concentrations of plasma V L D L protein in each group remained c o n s t a n t d u r i n g the experimental period. T h e m e a n c o n c e n t r a t i o n of plasma V L D L protein in the control group was 81.1 + 9.7 tLg/ml plasma ( m e a n s and stand a r d error were calculated for values at all time points, n = 22) a n d was r e d u c e d in the rats fed lovastatin (33.6 + 7 . 0 / L g / m l plasma, n ----24). W h e n the diet of the l o v a s t a t i n g r o u p was s u p p l e m e n t e d w i t h 0.1% cholesterol, the c o n c e n t r a t i o n of V L D L protein increased (60.3 + 7 . 7 / x g / m l plasma, n = 23), although it r e m a i n e d bt, low control levels. T h e specific activities of total V L D L protein ( d p m / m g protein) reflect these differences, being higher in the group of rats fed Iovastadn (Fig. 2B). T h e fractional clearance rate a n d the p r o d u c t i o n rate of V L D L protein were caIculated from these d a t a [25,26]. T h e fractional clearance rates of V L D L protein for the chow-fed, Iovastatin-fed a n d lovastafin plus cholesterol-fed groups were 0.29, 0.25 and 0 . ! 9 / h , respectively; the p r o d u c t i o n rates of V L D L protein for the three groups were 94.0. 33.6 and 47.9 / t g / 1 0 0 g b o d y w t . / h , respectively. Metabolism o f [1- t *C]oleate in vivo T h e in vivo metabolism of [l-t4CIoleate was investigated. In rats fed lovastatin for 1 week, the iecorporation of oleate into hepatic triacylglycerol was not different from cuntrols 1 h afte r administration (Table IIIA), nor was :here any difference in the mass of triacylglycerol or in the specific activity of hepatic triacylglycerol, A d d i t i o n of cholestt, rol to the diet of the lovastatin-fed group increased the mass of hepatic triacylglycerol without altering incorporation of [1=4C]oleate into the triacylglycerol of the liver; consequently, ;he specific activity ( d p m / / ~ m o l ) o f hepatic triacylglycerol was lower than either control or lovastatin-fed groups. T h e c o n c e n t r a t i o n o f plasma triacyl-

=

300000

LOV LOV+C

\

• ~o~oo

0

O0

t0

20

30

40

5.0

60

7Q

~C,

HOURS F i g , 2. F r a c t i o n a l c l e a r a n c e o f 12~I-VLDL f r o m t h e p l a s m a . R a t s w e r e fed, as d e s c r i b e d in the M e t h o d s soction. O n the d a y o f the expcrirncnt, rats were i n j e c t e d w i t h 0 . 3 3 . i 0 ~ d p m / 1 0 0 g b o d y w~. o f t2~I-VLDL. R a t s w e r e exs,,nguinatect at various intervals a n d the V L D L was isolated f r o m e a c h p l a s m a s a m p l e by u l t r a c e n t r i f u g a t i o n . I d e n t i f i c a t i o n o f g r o u p s : C O N , g r o u p o f rats fed an u n s u p p l e m e n t e d c h o w diet: LOV. g r o u p o f r a t s fed a c h o w diet s u p p l e m e n t e d with 0.1% lovastatin: L O V + C, g r o u p o f rats [ed a c h o w die*, s u p p l e m e n t e d with O . l ~ Iovastatin a n d 0.1c~ cholesterol. Fig. 2A rcprcsent~ the r a d i o a c t i v i t y in the t c t a | V L D L p r o t e i n ( d p m / m i p l a s m a ) at intervals d u r i n g the 8-h e x p e r i m e n t was m e a s u r e d . L e a s t me,a n - s q u a r e p l o t s o f the e l i m i n a t i o n p a r t o f the curves were c a l c u l a t e d . T h e fractional c l e a r a n c e r a t e was c a l c u l a t e d f r o m these plots. S.E.s arc < 209~ o f the m e a n s . T h e n u m b e r o f o b s e r v a t i o n s is that d e s c r i b e d in T a b l e i I l . Fig. 2B ~epresents the specific r a d i o a c t i v i t y o f total V L D L p r o t e i n after injoclion o f I25I-VLDL T h e r e are f o u r o b s e r v a t i o n s for e a c h time p o i n t , e x c e p t at t = 1 a n d 4 h for the C O N g r o u p a n d at t = l h for the L O V + C g r o u p ( n = 3 f o r e a c h group). D a t a are m e a n s ; S.E.s are not p r e s e n t e d . LOV d i f f e r s ( P < 0.05) f r o m C O N at all time p o i n t s . L O V + C d i f f e r s ( P < : 0 . 0 5 ) f r o m C O N at t = 0 . 0 S 3 , 0.25, ] at~A_ 4 h a n d differs f r o m LOb' at t ~ 1 a n d 8 h.

glycerol a n d incorporr.tion of [1-14C]oleate was less in the rats fed lovastatin than in controls, yet specific activity was not altered. A n increase in mass of plasma triacylglycerol a n d incorporation of oleate was observed in the lovastatin group treated with cholesterol; the specific activity of plasma triacylglycerol, however, was lower than that of either the control or lovastalin-f,~d groups. The specific activity of plasma triacylglycerols

302 synthesized f r o m [I-~4C]oleate e x c e e d e d that of h e p a t i c triacylglycerol, suggesting that in vivo, triacylglycerols secreted by the liver are n o t in r a p i d e q u i l i b r i u m with the total mass of hepatic tfiacy!g!y,'~rc,!¢ h u t are derived f r o m a smaller m e t a b o l i c pool.

Metabolism of [1-J+C]oteate by the isolated perfused liver The m e t z b o l i s m o f [1-14C]oleate was s t u d i e d also in t h e isolated p e r f u s e d liver. T h e d i s t r i b u t i o n o f r a d | o a c tivity from oleate a m o n g p r o d u c t s o f esterification a n d o x i d a t i o n is p r e s e n t e d in T a b l e IV. in all t r e a t m e n t groups, the m a j o r i t y o f o l e a t e was ester±fled to triacylglycerol. A p p r o x . 8 5 - 9 0 % o f total p e r f u s a t e triacylglycerol is f o u n d in t h e V L D L [27,28]. N o d i f f e r e n c e s w e r e o b s e r v e d in total i n c o r p o r a t i o n (liver plus perfusate) a m o n g a n y o f the t r e a t m e n t groups. A higher p e r c e n t a g e o f triacylglyceroi r a d i o a c t i v i t y w a s o b s e r v e d in livers from rats treated w i t h lovastatin in the diet, f o l l o w i n g p e r f u s i o n for 4 h. A d d i t i o n of l o v a s t a t i n to the m e d i u m d i d n o t h a v e such an action. In c o n t r a s t , t h e p e r c e n t a g e o f r a d i o a c t i v i t y in p e r f u s a t e triacylglycerol wan lower ( P < 0.05) with l o v a s t a t i n t r e a t m e n t in vivo or in vitro c o m p a r e d to the c o n t r o l g r o u p . T r e a t m e n t with lo',astatin d i d not a f f e c t the i n c o r p o r a tion of oleate into the other lipid fractions o f the total p e r f u s a t e o r liver ( V L D L w a s not isolated). T h e p r o d u c t s of o x i d a t i o n were also m e a s u r e d . Clearly, the addition o f Iovastatin, either to the diet a n d / o r in vitro, d i d not affect p r o d u c t i o n o f t4CO2. Similarly, i n c o r p o -

T A B L E !I!

T A B L E IV

Percent of infused [I- I'lC]oleate incorporated into metabolic products by the i¢olated perfused liver Rats w e r e t r e a t e d , as de.~cribed in the text. T r e a t m e n t g r o u p s a r e d e s i g n a t e d as follows: C O N , c o n t r o l s ; L(V). l o v a s t a t m a d d e d ;,~ v i t r o only; LOV, i c v a s t a t i n a d d e d to t h e diet o n l y ; L O V + L(V), l o v a s t a t i n a d d e d to the d i e t (0.1%) a n d p e r f u s a t e (100 ! ~ , / m l ) . The a c i d - s o l u b l e radioactivity w a s c o n s i d e r e d t o b e a n i n d e x o f ketogenesis. T G , triacylglycerol; PL, p h o s p h o l i p i d ; C, c h o l e s t e r o l ; C E , c h o l e s t e r y l ester: M G , m o n o a c y l g l y c e r o l ; D G . diacyiglyceroi. C / M G / D G are the lipid f r a c t i o n s t h a t are n o t s e p a r a t e d a f t e r t h i n - l a y e r c h r o m a t o g r a p h y with a solvent m i x t u r e o f p e t r o l e u m e t h e r / d i e t h y l e t h e r / a c e t i c a c i d (84 : 15 : 1, v / v ) . D a t a p r e s e n t e d a r e m e a n s ± S.E. N u m b e r o f o b s e r v a tions f o r each g r o u p , n = 4. CON

LgVI

Ester±fled p r o d u c t s Perfusate TG I 1.5 ± 1.5 C/MG/DG 0 . 1 ± 0.1 PL 0 . 2 ± 0.1 CE 0.1+ 0.l Liver TG C/MG/DG

L O V + L(V)

6 . 7 _ 1.5 a 0.2± 0.l 0.1 + 0 . | 0 . I ± 0.I

3.5 ± 0.7 a 0 . 1 ± 0.l 0 . l ± 0.1 0 . 1 ± 0.1

1.1 0.1 0.4 0.2

15.2___4.5 0.9-t-0.3 3.8±1.0 0.7±0.3

19.8_+ !.0± 4.6+ 0.7+_

1.7 a 2 t . 3 ± 0.2 0.6± 0.7 3.4± 0.1 0.6±

4.9 a 0,3 0.4 0.2

1.4 1.8

10.1±1.1 9.4±1.1

10.5± 10.0+

1.3 0.7

0.7 1.0

F F A n o t taken u p b y t h e liver 2 5 . 0 ± 5.5 ~,i.' 7o".6±12.5

31.7+4.2 7ha__00

2 6 . 3 + 5.6 81.4-1-14.9

PL

CE

15.2___ 0.8_+ 3.9± 07±

5.8 ± 1.8 a 0.1±0,1 0.1 + 0 . 1 0.1_+0.1

LOV

Oxidized products CO z 11.2+ A,~idsolubl¢ 9.6+ radioactivity

9.7+ 8.8±

3 0 , 7 + 8.5 76.9:t:11.6

a D i f f e r s ( P < 0.05) f r o m C O N .

Synthesis of triacylglycerol in vwo from [1 - t +C]oleate O n the d a y of the e x p e r i m e n t , rats received 3 . 3 / . t C i / 1 0 0 g b o d y wt. o f [ l J 4 C ] o l e a t e i n t r a v e n o u s l y as a c o m p l e x ,vitb rat s e r u m . A f t e r I h, h e p a t i c a n d p l a s m a lipids were isolated a n d radio c o u n t e d , as des c r i b e d in the text. N u m b e r o f o b s e r v a t i o n s is i n d i c a t e d in p a r e n t h e ,~es. D a t a are m e a n s ± S.E. I d e n t i f i c a t i o n of g r o u p s : C O N , rats fed an u n s u p p l e m e n t e d c h o w diet; LOV, rats fed a c h o w diet s u p p l e m e n t e d w i t h 0.17o Iovastatin; L O V + C , g r o u p s u p p l e m e n t e d with 0.1~. iovastatin a n d 0.1% cholesterol+ Liver

Plasma

A, I n c o r p o r a t i o n '-. ~7 " ,:~, LO" t s ) L(_'V + C (5)

dpm/g 246 469 + 28 544 2 7 0 7 6 2 ± 33 124 224057 ± 62 330

dpm/g 129 438 ± 12 363 74400-+ 12418 a 98 206 ± 11 149 ..h

B. Mass CON LOV LOV + C

/tmol T G , y liver 1.83 4- 0.07 1.88 4-0.07 2 6,1 ± 0.14 .,b

pmol/ml plasma 0+366 ± 0.039 0.235 ± 0.034 " 0.387 -I-0.020 h

C, Specifiz a¢,ivity CON L~V LOV+C

dp:a/pmot TG 135 204 + 10668 144 554 :t: 17 230 87308 :k 16284 tl, b

0v +)tmol T G 333 7.~+ £ 36 ~ 13 310 316 ± 30 527 2464375=33203 a

• Differs ( P < 0.05) f r o m C O N . b Differs ( P < 0,05) f r o m L O V .

ration of radioactivity from [1-14C]oleate i n t o a c i d - s o l u ble c o u n t s w a s not different than controls. Discussion T r e a t m e n t o f rats with 0.1% l o v a s t a t i n for t week, which is s u g g e s t e d to reduce a p u t a t i v e hepatic m e t a bolic p o o l o f cholesterol, d e c r e a s e d o u t p u t o f V L D L lipids in vivo, and r e d u c e d the hepatic c o n c e n t r a t i o n o f cholesteryi ester, s u p p o r t i n ~ data derived f r o m experim e n t s using the isolated p e r f u s e d liver in vitro [8]. Decreased output of the VLDL triacylglycerol, p h o s p h o l i p i d , cholesterol, a n d cholesteryl esters were observed. T h e m o l a r ratios o f V L D L lipids relative to triacylglycerol secretion s u g g e s t secretion o f fewer V L D L particles that were similar in c o m p o s i t i o n to controls, w h e n secretion o f V L D L w a s curtailed. W h e n b o t h c h o l e s t e r o l a n d l o v a s t a t i n were a d d e d to the diet, secretion o f V L D L lipids w a s increased; p l a s m a c o n centrations o f V L D L - c h o l e s t e r o l a n d V L D L - c h o l e s t e r y l ester, moreover, were higher than c o n t r o l levels, s u g g e s t i~lg secretion o f V L D L particles e n r i c h e d in cholesterol.

303 Higher ratios of cholesterol to triacylglycerol in the V L D L when 0.1% cholesterol was included in the diet, were o b s e r v e d in o u r previous study using the isolated perfused liver 18]. It is surprising that in vivo cholesteret synthesis from 3 H 2 0 was greater in lovastatin-fed ~-,nimals, although it is k n o w n that the drug is a competitive inhibitor of I t M G - C o A reductase. W e had shown that in the perfused liver, lovastatin a d d e d in vitro is indeed inhibitory to cholesterol synthesis; however, livers from lovastatinfed rats exhibited a m a r k e d increase in cholesterol synthesis w h e n p e r f u s e d in the absence o f a d d e d lovastatin [8]. Induction o f the enzyme during exposure to Iovastatin is a plausible explanation for this finding. Additionally, the c o n c e n t r a t i o n o f lovastatin in the liver when r e m o v e d from the animal for perfusion in the a b s e n c e of a d d e d d r u g m a y rapidly decrease due to s u b s e q u e n t m e t a b o l i s m a n d dilution in the system. T h e question remains, however, why, in the intact animal fed lovastatin for 1 week, increased incorporation o f 3HzO into cholesterol was observed. It is k n o w n that inhibition o f cholesterol synthesis is o b s e r v e d within 1 - 4 h after intraperitoneai injection o f c o m p a c t i n [14] and Iovastatin (Alberts, A.W., unpublished d a t a ) into rats, b u t that this effect s o o n disappears and is replaced b y increased rates o f synthesis. Presumably, in the animal rtu~eiving lovastatin in the diet, maximal inhibitory effects are evident primarily during the light-dark cycle coincident with maximal f o o d intake. A s the intake o f food diminishes, the effects of e n z y m e induction b e c o m e more prominent. M o r e investigations are needed to d e t e r m i n e the m e c h a n i s m s b y which lovastatin treatment o f the animal r e d u c e d the hepatic c o n c e n t r a t i o n o f cholesteryl esters. T h e u p t a k e o f V L D L in vivo was not observed to be altered by t r e a t m e n t o f the rat with lovastatin. Experiments using ~2SI-VLDL indicated no differences in the fractional clearance rate o f the lipoprotein a m o n g the experimental groups. Clearly, however, the concentration of plasma V L D L protein in vivo was reduced with lovastatin t r e a t m e n t and increased with cholesterol supplementation. These observations are consistent with those o b t a i n e d using the isolated perfused liver [8] a n d with a report by G i n s b e r g et al. [26] o f a patient with cholesteryl ester storage disease in w h o m clearance of p l a s m a V L D L was u n c h a n g e d with lovastatin feeding. These data suggest that the result of Iovastatin feeding is to decrease synthesis a n d secretion o f the V L D L a n d not to increase clearance o f V L D L from the plasma. T h e metabolism of []-laC]oleic acid after depletion o f the p u t a t i v e pool o f hepatic cholesterol with lovastatin w a s studied in the intact rat and in the isolated laerfused rat liver. In the perfused liver, no change in total esterification p r o d u c t s (mostly triacylglycerol) was observed, although i n c o r p o r a t i o n o f oleate into hepatic triacylglycelul increased and incorporation into the total

perfusate was r e d u c e d Thus, although lovastatin did not change the synthesis of triacylglycerol from oleate, triacylglycerol a c c u m u l a t e d in the liver and less was secreted, presumabl? as V L D L . This confirms previously reported d a t a in studies with the isolated perfused liver [8]. This limitation on secre'ion ,~f triacylglycerol by the liver was reversible by provision of cholesterol 18]. As noted previously [291. fee,];.~ , ' ~ , ~ t,~ rats increased activity of carnitine palinitoyl ac~cl:r._,.t.~ferase (CPT-I) in rnitochondria isolated from nonperfused liver; however, data from our l a b o r a t o r y (Cook, G., Khan. B.. Wilcox. H . G . and Heimberg, M., U n p u b lished data) suggested that the activity o f CPT-I was n o t different f r o m controls when the livers were perfused, p r e s u m a b l y due to the rapid m e t a b o l i s m of the drug b y the liver. U n d e r o u r conditions of perfusion, oxidation of oleate, m e a s u r e d as incorporation of [l-14C]oleate into C O 2 a n d into perchloric acid-soluble substances, was not different with livers from control or !o~,as:atinfed rats. or when lovastatin was a d d e d to the m e d i u m in vitro. U n d e r these experimental conditions, the o b served reduction o f V!~DL o u t p u t by the liver did not a p p e a r to result from increased fatty acid oxidation and s u b s e q u e n t l y reduced fatty acid available for esterification and secretion. The in vivo studies with ll-laC}oleate also indicate that administration of lovastatin to rats did not alter the rates o f esterification from oleate. Specifically, hepatic synthesis o f triacylglycerol was n o t inhibited; rather, the o u t w a r d transport ot ~ triacylglycerol into plasma was reduced. O u r studies intricate that lovastatin d o e s not affect the synthesis o f triacylglycerol or pho>pholipid and that the o u t p u t o f V L D L is altered by causing changes in the putative metabolic pool o f cholesterol. It is o f interest that, when cholesterol was a d d e d to the diet of luvastatin-fed rats. the mass o f hepatic triacylglycerol was increased. Davis et al. [301 observed previously that feeding of 2% cholesterol to rats for 8 weeks i~creased the c o n c e n t r a t i o n o f triacylglycerol in h e p a t o c y t e s isolated from such animals. O u r experiments with 0.1% cholesterol s u p p l e m e m a t i o n for only 1 week had similar, if less p r o n o u n c e d , effects, in our earlier study with perfused livers from lovastatin-fed rats [8], addition o f cholesterol to the m e d i u m (either free cholesterol or h u m a n L D L j increased hepatic triacylglycerol, as well as the secretion of triacylglycerol. It is conceivable that cholesterol stimulates synthesis o f triacylglycerol. Clearly, potential mechanisms for this effect remain u n k n o w n at this time. The study reported here confirms prior experiments with the isolated perfused liver and strengthens the hypothesis taat cholesterol is a requirement for the secretion o f the V L D L b y the liver. An i n a d e q u a t e metabolic pool o f cholesterol in the liver does not a p p e a r to inhibit synthesis o f triacylglycerol from free fatty acid. It does appear, however, that rt:dt, ;d l e v e l of this pool impair the capacity of the liver to transpor'.

304 triacylglycerol in the form o f V L D L by the isolgied p e r f u s e d liver in vitro and in the intact animal. K a m et al. [311 o b s e r v e d recently that inhibition with S a n d o z 58-035 o f the activity of a c y l - C o A - c h o l e s t e r o l acyltransferase ( A C A T ) in C a C O - 2 cells in tissue c u l t u r e increased the c o n c e n t r a t i o n o f free cholesterol, d e p l e t e d t h e p o o l of cholesteryl esters, increased the triacylglycerol c o n c e n t r a t i o n 2 - 5 - f o l d a n d d e c r e a s e d the secretion o f triacyiglycerol-rich lipoproteins. -lhese o b servations s u p p o r t the c o n c e p t that a n a d e q u a t e h e p a t i c p o o l o f C E is required, directly or indirectly, for secretion o f the V L D L . A n interesting h y p o t h e s i s d e r i v e d from these experiments, a n d f r o m those using the isolated p e r f u s e d liver i81, relates to the o b s e r v a t i o n s that t r e a t m e n t o f the a n i m a l with H M G - C o A reductage i n h i b i t o r s increases the n u m b e r o f h e p a t i c L D L r e c e p t o r s [32]. T h e inc r e a s e d d e n s i t y o f L D L receptors, which w o u l d stimulate the u p t a k e o f p l a s m a I D L a n d L D L b y the liver, m a y be the r e s p o n s e o f the liver, after t r e a t m e n t o f the a n i m a l with iovastatin, to its physiological r e q u i r e m e n t o f an a p p r o p r i a t e s o u r c e of c h o l e s t e r o l for the h e p a t i c synthesis and secretion o f the V L D L and t r a n s p o r t o f triacylglyce~ ol.

Acknowledgements This research w a s s u p p o r t e d b y g r a n t H L - 2 7 8 5 0 f r o m the N a t i o n a l Institutes of H e a l t h , U.S. P u b l i c H e a t h Service. B.V.K. is e P r e d o c t o r a l F e l l o w o f t h e A m e r i c a n D i a b e t e s Association, T e n n e s s e e Affiliate. T.V.K. is a p o s t d o c t o r a l trainee s u p p o r t e d b y g r a n t H L - 0 7 6 4 1 f r o m the N . I . H . W e wish to thank B e n n e t t Bickneil, L y n d a D i s n e y a n d M i c h e l e W a l k e r for their e x p e r t technical assistance.

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