Biochimica etBiophysicaActa, 1044(t990) 375-381 Elsevier

375

BB ~LIP 53422

U,ptake and metabolism of high-dens' ty iipoproteins by cultured rabbit hepatocytes Ross W i s h a r t a n d M a l c o l m Mack_innon Unit of Medicine. Fiinders Medical Centre, Bedford Park (Au.~tra!:a)

(Received 8 November 1989) (Revised manusc~pt received 9 Februar,,' !990)

Key words: Lipoprotein uptake; Lipoprotein metabolism; HDL; Cel! culture: (Rabbit hepatoc_yte)

T h e selective uptake and internalization of cove c o m p o n e n t s of high-density lipoproteins ( H D L ) were e x a m i n e d in primary m o n o l a y e r cultures of rabbit hepatocytes. U s i n g I t 4 C l m c r o s e as a surface marker covalentiy attached to apolipoprotein and [3H]cholesteryl linoleyl ether as a core marker, there was a 5--6-fold greater internalization o f cbolesteryl ether than sucrose-labeled apolilmprotein during 4 8 h o f culture. T h e rate o f uptake o f [~Hlcholesteryl linoleyl ether was 2 6 3 : 1 : 2 9 ng a p o H D L / m g cell protein per h during the initial 8 h of culture, but averaged 101 :t: 32 ng a p o H D L / m g cell protein per h over the 4 8 h culture p e d o ~ C o n c o m i t a n t with this apparent selective uptake o f cholesteryl ester core, there was a c h a n g e in the H D L s i z e distrilmtkm, w i | h the a p p e a r m ~ of a distinct population o f smaller 4.3 n m radius particles in addition to the originally predominant particles o f 4.9 run radius. This was a s s ~ . i a t e d with a significant reduction o f cholesteryl ester as a percentage o f lipoprotein m a s s from 15.5 + 1.2 to ! 1 . 0 5= 1.2 ( P < 0 . 0 6 1 ) and a reduction in cholesteryi e s t e r : p r o t e i n m a s s ratio f r o m 0 . 3 0 + 0 . 0 1 to 0.19-1- 0.01 ( P < 0.001). T h e r e w a s n o c h a n g e in the m a s s ratio o f H D L triacylglycerol to protein. T h u s rabbit h e p a t o c y t e s in culture exhibit the capacity to selectively extract cholesteryl ester f r o m H D I and produce smaller H D L particles.

Introduction T h e , e is accumulating evidence that a major function o f the high-density lipoproteins ( H D L ) is the delivery o f cholesterol to the liver. There are a n u m b e r of different yet equally plausible mechanisms for the delivery o f cholesterol to liver cells which m a y be surnmar=w.ed as follows. (a) T h e transfer o f unesterified cholesterol from H D L [1-3]. This in all p r o b a b i l i t y involves the bidirectional exchange o f free. c;~o!esterol b e t w e e n H D L a n d h e p a t o c y t e s a n d m a y not necessarily result in any change in H D L mass. Recer.t d a t a suggest that this process m a y not b e d e p e n d e n t on the binding o f H D L to the cell surface [41. (b) ~ h o i e particle or e n d o e y t o t i c u p t a k e o f H D L [3,5] with equivalent u p t a k e o f core and surface c o m p o n e n t s o f the lipoprotein. (c) The selective deliver 3, o f core ~.omponents of the H D L with a consistently

Abreviations: HDL, high-d©nsity lipoproteins; VLDL, very-low-density iipoprotein; EGTA, ethyleneglycol bis(~-aminoethyl ether)-N, Ntetraaeetic acid; PBS, phosphate-buffered saline; NBCS, new-born calf s e r u m , Correspondence: A,M. Mackinnon, Flinders Medical Centre, Bedford Park, 5042, Australia.

greater transfer o f cholesteryl esters than that of the surface a p o l i p o p r o t e i n s [6-9]. This preferential o r selective hcpatk, delivery of c h o l ~ t e r y l esters m a y have physiological significance in that ,a.~ have recently demo n s t r a t e d that cholesteryl ester-enriche~ H D L sttmulate the synthesis o f bile acids in cultured rat h e p a t o c y t e s [10]. (d) T h e transfer o f H D L cholesterol to other lipoproteins. After esterification b y the action o f lecithin :cholesterol acyltransferase on plasma H D L . the cholesterol m a y b e transferred to other lipoprotein classes with hepatic delivery resulting from interaction o f these lipoproteins with the liver [9,11]. In an earlier study of the m e t a b o l i s m o f H D L b y the perfused rabbit liver [9] we d e m o n s t r a t e d that the rabbit liver removed significantly more core =holestery! ~.gter than surface apoprotein from H D L . C o n c o m i t a n t l y , there was the a p p e a r a n c e in the perfusate of smaller H D L particles signif.;cantly depleted o f cholesteryl esters. However, since whole liver is c o m p o s e d o f a variety o f cell types, it is conceivable that cells other than h e p a t o c y t e s m a y have c o n t r i b u t e d significantly to this process [12]. Furthermore. in the ,,,hole liver perfusion studies there was an appreciable transfer of labeled cholesteryl esters from H D L to V L D L and it was possible that this transfer m a y have c o n t r i b u t e d to the

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376 differential uptake [9]. This report describes the interaction of labeled H D L with primary monolayer cultures of rabbit hepatocytes in which there was very little specific transfer of cholesteryl ester label to other lipoproteins. Materials and Methods

Materials Bovine insulin, soybean trypsin inhibitor, Hepes, E G T A and cyanuric chloride were obtained f r o m Sigma (St. Louis, MO, U.S.A.). New-born calf serum (NBCS) (heat-inactivated at 56 ° C for 30 rain), Dulbecco's modified Eagle's ( D M E ) m e d i u m , penicillin G (1650 units.mg) a n d streptomycin sulphate (731 u n i t s / m g ) were all obtained from G r a n d Island Biological (Santa Clara, CA, U.S.A.). Betore use, the m e d i u m was supplem e n t e d with 10 m M Hepes. Collagenase was obtained from B o e h r i n g e r - M a n n h e i m (F.R.G.). Tissue culture dishes, 90 m m in diameter, were obtained from Bunzl Industries ( H e n d e n , South Australia). High specific activity [U-14C]sucrose, [la,2a(n)- 3H]cholesteryl linoleyl ethe~, N C S tissue solubilizer and A C S II scintillant were supplied by A m e r s h a m International (U.K.). Rabbits Male N e w Zealand White rabbits weighing 2.5-3.5 kg were obtained from the Institute of Medical and Veterinary Science, Adelaide a n d were fed standard rabbit chow (Purina). Liver cell cultur~ Hepatocytes were isolated by a modification of the m e t h o d of Davis et ai. [13]. The animals were anaesthetized with N e m b u t a l and the liver flushed with non-recirculating Hanks' calcium-free buffered saline solution c o n t a i n i n g E G T A (6.25 mM). W h e n c o m p l e t e l y blanched, the liver was perfused for 1 5 - 2 0 rain with a recirculating solution c o n t a i n i n g collagenase (0.16 u n i t s / m l ) , trypsin inhibitor (50 # g / m l ) a n d CaCI 2 (0.3 raM) dissolved in Hanks" buffered saline. The liver was t , e n placed in H a n k s ' buffered saline and cells were c o m b e d away from the connective tissue. Dispersed cells were filtered t h r o u g h a 4 0 0 / t m mesh nylon gauze and the hepatocytes separated from n o n - p a r e n c h y m a l cells by e e n t n f u g a t i o n at 50 × g for 2 rain with at least 98% o f the final cell preparation being hepatocytes. T h e pe!let was c~trefully resuspended a n d recentrifuged twice at 5 0 . , g. ; real hepatocyte preparations were greater than 9")% w,~ole as assessed by T r y p a n blue dye exclusion. Cells were plated in 90 m m diameter plastic tissue culture dishes in 7 ml of Dulbt...oa's modified Eagle's m e d i u m ( 1 - 1 0 6 c e l l s / m l ) containii.g 10% NBCS, insulin ( 1 / t g / m l ) , streptomyei~l sulphate ~50 # g / m l ) and penicillin G (50 u n i t s / m l ) and placed :m ,: t, umidified incubator at 37 " C. After 4 h, the m e d i u m was ;,_pla,~ed

with 5 ml of fresh seri~m-tree Dulbecco's m o d i f i e d Eagles" medium.

Isolation of H D L Rabbit plasma H D L were isolated by centrifugation as the fraction of density 1.063-1.21 g / m l as described previously [9]. Labeling of H D L with [14C/sucrose Rabbit H D L was labeled in the protein m o i e t y with [U-14C]sucrose (spec. act. 20 G B q / m m o l ) by a modification of the m e t h o d of P i t t m a n et al. [14]. Labeled sucrose, activated by reaction with t.yanuric chloride (twice recrys*.aIized) was i n c u b a t e d with H D L for 3 h at r o o m temperature. After incubation, labeled H D L was separated from u n b o u n d label by exhaustive dialysis against 0.9% NaCl a n d then passed through a 0.22 /~m Millipore filter. In the final [14C]sucrose-labeled H D L preparation, less than 2% was soluble in 10% trichloroacetic acid a n d less t h a n 1% was lipid-extractable by the m e t h o d of F o l c h et ai. [15]. T h e specific activity o f the [~4C]sucrose-labeled H D L ranged from 16000 to 18000 d p m / / t g a p o l i p o p r o t e i n H D L . S D S - p o l y a c r y l a m i d e gel electrophoretic separation o f [14C]sucrose-labeled apolipoproteins d e m o n s t r a t e d that radioactivity was distributed in p r o p o r t i o n to the distribution o f a p o p r o t e i n s , but in some preparations partially purified a p o l i p o protein AI was covalently labeled with [14Clsucrose a n d reincc, rporated into H D L . In this situation the label was in apo AI alone. Labeling of H D L with [~H]cholesteryl iinoleyl ether R a b b i t H D L w a s l a b e l e d with [ l a , 2 a ( n ) 3H]cholesteryl hnoleyl ether (spec. act. 1.18 T B q / m m o l ) essentially by the m e t h o d described by Stein et al. [16]. In all preparations o f 3H-labeled H D L , greater than 97% of the label was lipid-extractable by the m e t h o d o f Folch et al. [15]. T h e final specific activity of 3H-labeled H D L ranged from 3000 to 5000 d p m / / L g apolipoprotein H D L . To minimise possible differences in labeled H D L pools, the 14C-sucrose-labeled H D L was also subjected to the same i n c u b a t i o n a n d reisolation as the 3H-etherlabeled H D L . With b o t h Z4C a n d 3H labelings, the effect o f the labeling p r o c e d u r e o n H D L zomposition a n d particle size distribution was d e t e r m i n e d with analysis of protein, p h o s p h o l i p i d , free a n d total cholesterol, esterified cholesterol a n d triacylglycerol by eolorimetric assay [9], a p o l i p o p r o t e i n c o m p o s i t i o n by SDS-polyacrylamide gel elcctrophoresis a n d H D L particle size distribution by gradi::nt gel electrophoresis [O]. N e i t h e r labeling p r o c e d u r e was associated with any c h a n g e in H D L composit-:on or particle size distribution. Cultu.-e experiments In previous rabbit liver perfusion studies [9], we had d e m o n s t r a t e d no evidence of sat~rability with H D L

377 c o n c e n t r a t i o n in t h e p e r f u s a t e r a n g i n g from 5% to 200% of n o r m a l p l a s m a H D L a n d p i l o t studies h a d d e m o n s t r a t e d n o s a t u r a b i l i t y in cell c u l t u r e studies with H D L c o n c e n t r a t i o n s o f 50 to 400 p g / m l o f media. W e therefore elected to u s e a c o n c e n t r a t i o n o f H D L in these e x p e r i m e n t s of 100 p g / m l . In the h e p a t o c y t e i n c u b a t i o n s with l a b e l e d H I ) L , c u l t u r e dishes w e r e h a r v e s t e d at 8 h intervals o v e r a 48 h i n c u b a t i o n p e r i o d b y the m e t h o d o f D a s h t i e t a l . i17]. I n the c u l t u r e e x p e r i m e n t s d e s i g n e d to e x a m i n e the e f f e c t o f h e p a t o c y t e s o n H D L size a n d c o m p o s i t i o n , H D L w a s i n c u b a t e d w i t h h e p a t o c y t e s as a b o v e at a c o n c e n t r a t i o n o f 100 ttg H D L p r o t e i n / m l m e d i a f o r 24 h. Tv, o e x p e r i m e n t a l c o n t r o l s w e r e c o n d u c t e d u n d e r the s a m e c o n d i t i o n s in the a b s e n c e o f h e p a t o c y t e s : in the first - a m e d i a c o n t r o l - H D L w a s i n c u b a t e d with fresh m e d i a at 4 o C; in t h e s e c o n d - a c u l t u r e c o n t r o l - H D L was incubated with media which had been preincubated w i t h cells f o r 24 h ( c o n d i t i o n e d media). At t h e c o m p l e tion o f i n c u b a t i o n , H D L p a r t i c l e size d i s t r i b u t i o n a n d c o m p o s i t i o n w e r e d e t e r m i n e d b y g r a d i e n t gel electrop h o r e s i s a n d e o l o r i m e t r i c a n a l y s i s as d e s c r i b e d previo u s l y [9]. T h e a p o l i p o p r o t e i n c o m p o s i t i o n o f the H D L p r i o r to i n c u b a t i o n w i t h cells w a s 7 5 - 8 0 % a p o AI, 1 0 - 1 2 % a p o A l l , 6 - 8 % a p o C a n d < 3% a p o E. T h e overall p a t t e r n o f a p o l i p o p r o t e i n c o m p o s i t i o n was n o t a l t e r e d b y i n c u b a t i o n . P r o t e i n c o n c e n t r a t i o n o f cell hom o g e n a t e s w a s d e t e r m i n e d b y L o w r y a s s a y [18].

Bile acid analysis Bile a c i d s in c u l t u r e m e d i a were a s s a y e d with a P e r k i n - E l m e r 8420 c a p i l l a r y gas-liquid c h r o m a t o g r a p h

[191.

to each p l a t e o f a series of h e p a t o c :to cultures set u p to d e t e r m i n e H D L u p t a k e as d e s c r i b e d above. Results

Hepatocyte uptake of H D L components H e p a t o c y t e u p t a k e of [14C]sucrose-labeled a n d [3H]cholester?¢l linoleyl e t h e r - l a b e l e d H D L is d e p i c t e d in Fig. 1. This u p t a k e r e p r e s e n t s n o n - t r y p s i n - r e l e a s a b l e label d e t e r m i n e d b y s u b t r a c t i n g t h e c e l i - a s s o c , a t e d t r y p s i n - r e l e a s a b l e label f r o m total c e l l - a s s o c i a t e d label at e a c h t i m e - p o i n t . I n c u b a t i o n o: ceil~ .a~-.;._ H D L at 4 ° C f o r 2 h a n d s u b s e q u e n t t r y p s i n t r e a t m e n t resulted in a release o f 93.0% o f the label a n d we p r e s u m e t h a t t h e m a j o r i t y o f n o n - t r y p s i n - r e l e a s a b l e label is internatised. A f t e r 16 h of c u l t u r e with l a b e l e d H D L , 15% o f total cell-associated label w a s released f r o m the cells b y t h e a d d i t i o n o f trypsin, while at 24 h, 4.9% a n d at 36 h 0.8% o f label w a s t r y p s i n - r e l e a s a b l e . Fig. 1 d e m o n s t r a t e s that at all time-points, there w a s a g r e a t e r internalization o f [~H]cholesteryl linoleyI e t h e r t h a n [14C]sucrose. T a b l e I, in w h i c h the rates o f u p t a k e of e a c h label are e x p r e s s e d in t e r m s o f the e q u i v a l e n t u p t a k e o f H D L protein, reinforces t h e d i s p a r a t e rates of internalization o f the c o r e a n d s u r f a c e labels o f H D L . T h u s for e a c h c u l t u r e p e r i o d , there w a s a b o u t 6-times g r e a t e r d e l i v e r y o f c o r e H D L c o m p o n e n t s t h a n of s u r f a c e [14C]sucrose-labeled a p o l i p o p r o t e i n . It is p r o b able that b o t h [14Clsucrose a n d [ 3 H ] e t h e r u p t a k e s b y h e p a t o c y t e s are in fact an u n d e r e s t i m a t e o f the a c t u a l u p t a k e . W h i l e t h e s e labels are to a large e x t e n t t r a p p e d within h e p a t o c y t e l y s o s o m e s a f t e r i n t e r n a l i z a t i o n [15,23], there will a l m o s t c e r t a i n l y b e s o m e l e a k a g e o f label f r o m the cells. T o d e t e r m i n e w h e t h e r leakage o f .

Addition of antibody to lipid transfer protein to cell cultures T o a d d r e s s the p o s s i b l e role o f t r a n s f e r of H D L c o r e c o m p o ~ e n t s b y lipid t r a n s f e r p r o t e i n to m o r e b u o y a n t l i p o p r o t e i n s a n d t h e s u b s e q u e n t h e p a t i c u p t a k e of these c o m p o n e n t s w i t h o u t direct H D L - h e p a t o c y t e interaction, w e e x a m i n e d the effect o f i n h i b i t i o n o f lipid transfer p r o t e i n activity o n selective H D L u p t a k e . A specific a n t i b o d y t o r a b b i t lipid t r a n s f e r p r o t e i n was g e n e r o u s l y p r o v i d e d b y Dr. M. A b b e y a n d the c h a r a c t e r i s t i c s a n d p r e p a r a t i o n o f this a n t i b o d y h a v e b e e n r e p o r t e d [20,21]. W e h a v e r e p o r t e d p r e v i o u s l y that lipid transfer p r o t e i n is s e c r e t e d b y r a b b i t liver, b u t activity w a s n o t high a n d liver p e r f u s a t e r e q u i r e d c o n s i d e r a b l e c o n c e n t r a t i o n f o r c o n s i s t e n t m e a s u r e m e n t o f activity [22]. Similarly, o u r e x p e r i m e n t s to d a t e i n d i c a t e d a l o w o u t p u t o f lipid transfer protein by cultured rabbit hepatocytes. Nevertheless, to e n s u r e c o m p l e t e inhibition o f t r a n s f e r p r o t e i n a c t i v i t y in cell c u l t u r e s the a m o u n t of a n t i b o d y r e q u i r e d for c o m p l e t e inhibition o f activity in 5 ml o f raL~it p l a s m a (the v o l u m e o f c u l t u r e m e d i u m o n e a c h plate) w a s d e t e r m i n e d a n d this a m o u n t o f a n t i b o d y w a s a d d e d

c

.t,,

-V

31 2

1

i

0

a

16

Ineub~

24

32

40

"~

Umn4,(11)

Fig. l. Uptake of [14C]sucros~- and [3H]cholesleryl linoleyl etherlabeled HDL by cultured rabbit hepatocytcs. 100 pg protein/m; of [14Clsucrose-labeled HI:)L or I~HJcholcsteryl linoleyl ether-labeled HDL was added to hepatocyte cultures at time zero. Cells were harvested after a brief incubation with trypsin (0.05q~) and cell radioacti~:y determined by hquid scintillation counting. Values represent mean :t: S.D. of at |east five experiments. Solid line represents incubation carried out over a continuous 48 h period. D o t t e d line represents uptake 24 h to 48 h when reedium at 24 h is replaced by fresh m e d i u m containing 100 u g / m l o f labeled H D L

378 TABLE 1 Rate of uptake of labeled HDL by cultured hepato~yte.~

Values are mean ± S.D.. seven animals each group, ng apo HDL/mg cell protein per h. Each incubation period is from zet'o lime Incubation period (h) 8 16 24 32 40 48

[ 3HJCholesteryl linoleyl ether 263 3:29 191 + 35 169± 32 160 ± 34 113-1-29 10l + 32

[ t*C/Sucrose

41.9 ± 4.2 30.0 ± 6.3 27.5 ± 5.3 25.4 ± 3.2 20.7± 5.6 19.5 + 4.1

[14C]sucrose c o u l d a c c o u n t f o r the o b s e r v e d d i s p a r i t y in t h e a m o u n t s o f i n t e r n a l i z e d labels, we assessed the e x t e n t o f [~4C]sucrose l e a ka ge f r o m t h e c u l t u r e d h e p a t o c y t e s . T h e [14C]sucrose-labeted H D L w a s inc u b a t e d with cells f o r 24 h, the m e d i a w e r e t h e n rep l a c e d with fresh m e d i a w i t h o u t a d d i t i o n a l H D L a n d t h e c u l t u r e s c o n t i n u e d f or a f u r t h e r 24 h, at w h i c h t i m e t he d i s t r i b u t i o n o f r a d i o a c t i v i t y b e t w e e n cells a n d m e d i a was d e t e r m i n e d . L e a k a g e o f [~4C]sucrose f r o m cells a f t e r 24 h was less t h a n 0.01% o f initial r a d i o a c t i v i t y / r a g cell p r o t e i n , a n d t h e r e f o r e c o u l d n o t a c c o u n t f o r t he o b s e r v e d 6 - f o l d d i s p a r i t y in t he u p t a k e o f the c o r e a n d s u r f a c e labels o f H D L d i n i n g t h e i n c u b a t i o n p e r i o d . A d d i t i o n a l l y , t he s e d a t a suggest t h a t w i t h t hese cells a n d w i t h th es e labels t h e r e is m i n i m a l r e t r o e n d o c y t o s i s of whole particle HDL taken up by hepatocytes. It is a p p a r e n t f r o m these d a t a ( T a b l e 1) t hat t h e rat e oF u p t a k ¢ o f b o t h labels d i m i n i s h e d w i t h c o n t i n u i n g t i m e o f c u l t u r e . T h e p o s s i b i l i t y t h a t this r e f l e c t e d a r e d u c t i o n in t h e vi a bi l i t y o f the h e p a t o c y t e s w a s exa m i n e d b y d e t e r m i m n g a n u m b e r o f p a r a m e t e r s o f cell viability. T h e r a t e o f o u t p u t o f V L D L t r i a c y l g l y c e r o l in f act i n c r e a s e d w i t h i n c r e e s i n g t i m e in c u l t = r e up t o 96 h. O v e r t h e first 24 h o f c u l t u r e , r a b b i t h e p a t o c y t e s s e c r e t e d 3.28 # g o f V L D L t r i a c y l g l y c e r o l / m g cell p r o t e i n p e r h. F r o m 24 to 48 h t he r a t e i n c r e a s e d t o 6.25 # g triacylg l y c e r o l / r a g cell p r o t e i n p e r h. A c o m p a r a b l e i n c r e a s e in cell f u n c t i o n was n o t e d w i t h bi l e acid s y n t h e s i s b y c u l t u r e d h e p a t o e y t e s : f o r t he initial 24 h, t h e rat.e o f bi!e acid s y n t h e s i s w as 90 p m o l / m g cell p r o t e i n p e r h w h i c h s u b s e q u e n t l y i n c r e a s e d t o 170 p m o I / m ~ , cell p r o t e i n p e r h f r o m 24 to 48 h. A., ''~"q:. "~= c x p i a n a t i o n f o r t h e d i m i n i s h i n g u p t a k e o f ~ . D L ~ o m t m n e n t s with t i m e m i g h t be d e p l e t i o n o f m e d i u m s u b s t r a t e , p a r t i c u l a r l y H D L c h o l e s t e r y l esters, as H D L is t a k e n u p a n d m c : a b o l i z e d b y t he cells. T h i s p o s s i b i l i t y w a s t e s t e d b y r e m o v i n g t he m e d i u m f r o m 24 h c u l t u r e s a n d r e p l z c i n g this ' ; , h t he original c o n c e n t r a t i o n o f f r e s h - l a b e l e d H D L . A s ~Ilustrated in Fig. 1, this d i d n o t result in a n y i n c r e a s e in tl r a t e o f u p t a k e o f H D L c h o l e s t e r y l e t h e r s a n d t he r a t e of u p t a k e

w i t h fresh H D L f r o m 24 h to 48 h c o u l d n o t b e s e p a r a t e d f r o m t hat d e m o n s t r a t e d in T a b l e I. A t h i r d explanation - the secretion of inhibitory factors with i n c r e a s i n g c u l t u r e t i m e - is a l s o e x c l u d e d b y t h e a b o v e medium change. It is u n l i k e l y , t h e r e f o r e , t h a t th e r e d u c t i o n w i t h t i m e in t he r a t e cJ: u p t a k e o f b o t h c o r e a n d s u r f a c e c o m p o n e n t s o f H D L is e x p l a i n e d b y impai,-ed cell f u n c t i o n , o r b y d e p l e t i o n o f a v a i l a b l e s u b s t r a t e a n d it is t h u s c o n c e i v a b l e that this r e p r e s e n t s d o w n r e g u l a t i o n o f a n uptake process. C h a n g e s in H D L

s i z e a n d c o m p o s i t i o n with c u l t u r e

C h a n g e s in H D L p a r t i c l e size r e s u l t i n g f r o m i n t e r a c tion o f t h e H D L with c u l t u r e d r a b b i t h e p a t o c y t e s a r e i l l u s t r a t e d in Fig. 2. T h e u p p e r p a n e l (a) r e p r e s e n t s t h e

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l)article radius (rim) Fig. 2. Particle size distribution 6f HDL after incubation with cultured hepatocytes assessed by gradient gel electroDhoresis. (a) HDL

incubated with fresh culture medium for 24 1~ at 37°C. lkis pattern was identical to that of unincubated HDL. (b) HDL incttbat~q for 22 h at 3 7 ° C with culture medium that had p-eviously been incubated with cultured hepatocytes for 24 h. (c) HDI incubated with cultured hepatocytes for 24 h at 37 o C.

379 gradient gel electrophoretic pattern or size distribution of H D L which have been in contact with cell culture m e d i u m alone, i.e., H D L having no interaction with hepatocytes or their products, it is a p p a r e n t that an H D L particle size o f 4.9 n m radius predominates. T h e middle panel (b) represents the particle size distribution of H D L incubated at 37~"C for 22 h with culture m e d i u m which had bezn p r e i n c u b a t e d with hepatocytes for 24 h ( ' c o n d i t i o n e d medium'). Essentially. incubation o f H D L with these c o n d i t i o n e d m e d i a (containing h e p a t o c y t e secretions such as V L D L a n d cholester:¢l ester transfer protein) had no effect on H D L particle size distribution. In contrast, the lower panel (c) illustrates the c h a n g e in H D L particle size distribution resulting from direct incubation of H D L with cultured hepatocytes for 24 h. This panel illustrates a m a r k e o shift in H D L particle size distribution with the app e a r a n c e of a distinct population of small particles o f radius 4.3 nm. N o t all incubations resulted in a shift as m a r k e d as illustrated in Fig. 2C. However, all incubations resulted in a clear increase in particles o f 4.3 n m radius. A similar shift in particle size distribution was observed when radic,-iabeled H D L was subjected to gradient gel electrophoresis. Examination of the distrbutton o f radioactivity within the gels revealed that in control incubations 12.1-14.2% o f radio-labeled H D L h a d a particle size radius o f less than 4.4 nm. After incubation with hepatocytes, 20.4-24.7% of radiolabeled H D L had a particle ~ e of less than 4.4 n m in radius. Thus, the changes in H D L particle size seen following incubation of cultured hepatocytes with H D L are very similar to those seen in short-term liver perfusions [9] and these cultures o f pure hepatocytes e m p h a size the role o1" the h e p a t o c y t e in this process. Importantly, cell culture for 24 h did not result in sufficient secretion of H D L to allow detection and sizing on gradient gels. The effect on H D L composition of interacting rabbit H D L w~th culture m e d i u m alone, culture m e d i u m rem o v e d f r o m h e p a t o c y t e culture (conditioned m e d i u m ) a n d cultured hepatocytes directly is illustrated in Table II. Only incubation o f H D L with cultured h e p a t o c y t e s for 24 h resulted in H D L particles with consistent compositional changes manifested by an increase in the protein and a reduction in the cholester2¢l esters as a percentage of H D L mass and a reduction in the cholesteryl e s t e r : p r o t e i n mass ratio. We have previously suggested [9] that the delivery o f labeled H D L c o m p o n e n t s to the h e p a t o c y t e m a y be the result o f transfer o f H D L core c o m p o n e n t s to other iipoproteins through the action o f cholesteryl ester transfer protein which we have d e m o n s t r a t e d is secreted b y rabbit liver [22]. This possibility was e x a m i n e d by d e t e r m i n i n g the transfer of [ 3H]cholesteryl linoleyl et:zer to other lipoproteins secreted by the cultured hepatocytes. C o n d i t i o n e d m e d i u m was o b t a i n e d from hepato-

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Fig. 3. Uptake of [a4C]suclose- and [)H]chole~teD, t linoleyl etherlabeled H D L by cultured rabbit hepatocytes after addition of antibody to lipid transfer protein. Experiment was conducted as described in Fig. I for control hepatocytes (solid line). In co-cultures, specific goat anti rabbit lipid transfer protein antibody was added and uptake assessed in presence of antibody (dotteo line).

cytes cultured for 24 h without a d d e d H D L . This m e d i u m (containing secreted V L D L and other proteins) was incubated with labeled 100 p t g / m l H D L for 24 h without any further ceil contact. The m e d i u m was then adjusted to d = 1.21 g / m l and centrifuged for 40 h as described u n d e r Materials and Methods. The supernatant was passed through a Superose 6B c o l u m n 19] and radioactivity in separated lipoprotein fractions was determined. In each experiment > 97% o f total radioactivity r e m a i n e d associated with H D L while 1.8-2.4% was recovered in the V L D L fraction, suggesting that very little lipid transfer protein is secreted during the 24 h culture. However, this question was further addressed by the addition of a n t i b o d y to lipid transfer protein to cultures in an a m o u n t that had been d e t e r m i n e d to inhibit rabbit plasma lipid transfer protein activity. The effect of the addition of a n t i b o d y of H D L core and surface c o m p o n e n t uptake is illustrated in Fig. 3. This d e m o n s t r a t e s that when a n t i b o d y is present t h r o u g h o u t the culture period there is an 8-12% reduction in cholesteryl ether uptake and ~ 15-29% reduction in labeled surface c o m p o n e n t uptake. These differences were not however, statistically significant, a n d at all times the uptake of the core c o m p o n e n t was m a r k e d l y greater than that of the surface c o m p o n e n t . Discussion

These studies of cultured rabbit h e p a t o c y t e interaction with labeled H D L c o n f i r m our previous observations [9] on the capacity o f the liver to generate small cholesteryi ester-depleted H D L . In the whole liver perfusion studies it was conceivable that cells o t h e r than hepatocytes were responsible for the observed changes in H D L [12]; the present hepatocyte culture study reinforces the role of the hepatocyte in this process.

380 E x a m i n a t i o n o f the c a p a c i t y of c u l t u r e d rabbit h e p a t o c y t e s to internalize H D L labeled with ' n o n - d e g r a d e a b l e ' c o r e and surface c o m p o n e n t s revealed a consistent disparity b e t w e e n cholesteryl ester and a p o l i p o p r o t e i n uptakes. W h e n the u p t a k e of each H D L label was expressed in terms o f a p p a r e n t H D L p r o t e i n uptake (i.e., w h o l e particle u p t a k e w o u l d be r e p r e s e n t e d b y equal u p t a k e o f b o t h tracers) there was a 6-fold greater u p t a k e of cholesteryl ether at a!l time-points. In this respect, r a b b i t h e p a t o c y t e s ~,ppear to h a v e a g r e a t e r c a p a c i t y for differential c h o l e s t e r y l ether u p t a k e than either rat [24] or pig h e p a t o c y t e s [25] in w h i c h a 2 - 3 - f o l d differential u p t a k e has b e e n r e p o r t e d . H o w e v e r , the highest rate o f u p t a k e of a p o l i p o p r o t e i n H D L in these p r e s e n t experiments, 41.9 + 4.2 ng a p o H D L / m g ceil p r o t e i n p e r h is l o w e r than that r e p o r t e d b y Soltys et al. [26] for r a b b i t h e p a t o c y t e s . T h e reason for this d i s p a r i t y is n o t i m m e d i a t e l y o b v i o u s , b u t d i f f e r e n c e s in cell prep a r a t i o n a n d c u l t u r e conditions, labeling p r o c e d u r e s a n d m e a s u r e m e n t o f d e g r a d a t i o n m a y all b e p a r t l y responsible. T h e differential u p t a k e o f l a b e l e d - H D L c o m p o n e n t s b y the r a b b i t h e p a t o c y t e s was c o n s i s t e n t w i t h the c h a n g e s o b s e r v e d in H D L c o m p o s i t i o n a n d particle size. A f t e r h e p a t o c y t e i n c u b a t i o n , there w a s a c h a n g e in H D L particle size with the a p p e a r a n c e o f a clearly d e f i n e d p o p u l a t i o n o f smaller particles o f r a d i u s 4.3 n m in a d d i t i o n to the originally p r e d o m i n a n t particles o f r a d i u s 4.9 nm. W h i l e the relatively triacylglycerol-rich r a b b i t H D L d o n o t s e p a r a t e into distinct H D L subclasses, this shift in particle size w o u l d c o m p a r e to a m o v e m e n t in h u m a n H D L f r o m H D L 2 to H D L 3 [27]. T h e a p p e a r a n c e o f a distinct s u b p o p u l a t i o n of smaller particles was clearly c o n f i r m e d b y an analysis o f the d i s t r i b u t i o n o f radioactive tracer within the total H D L p o p u l a t i o n . Thus, after i n c u b a t i o n o f l a b e l e d H D L with h e p a t o c y t e s there v,as a 2-fold increase in r a d i o a c t i v i t y in the H D L particles ,~.4 n m o r less in radius. This implies that the r a d i o a c t i v i t y in the smaller p o p u l a t i o n originated in the larger l a b e l e d H D L particles p r e s e n t in the m a j o r i t y o f particles at the initiation o f culture. A s p r e v i o u s l y r e p o r t e d with p e r f u s e d liver studies [9] this shift in particle size w a s a c c o m p a n i e d b y signific~'at c o m p o s i t i o n a l changes. W h i l e i n c u b a t i o n o f H D L with c u l t u r e m e d i u m a l o n e resulted in n o c h a n g e in H D L c o m p o s i t i o n , I-IDL i n c u b a t e d with h e p a t o c y t e s in culture for 24 h were significantly d e p l e t e d in m a s s per~ n t a g ~ ,., cJ,~flesteryl ester a n d a p p a r e n t l y e n r i c h e d in p r o t e i n a n d t,,acylglycerol. T h e m o s t significant c h a n g e w a s in the H D L eholesteryl es, er c o n t e n t , with a c o n c o m i t a n t r e d u c t i o n in the mass ratio o f cholesteryl e s t e r s : a p o l i p o p r o t e i n , suggesting tha, d e p l e t i o n o f this c o r e e o m p o n e n , o f H D L w&~ ;ntegral to ,r.e a p p e a r a n c e o f smaller particles. T h e m a s s ratio 4¢ triacyig l y c e r o l : p r o t e i n , w a s n o t a f f e c t e d b y incub~:~!~n o f H D L with h e p a t o c y t e s .

I: has b e e n s u £ g e s t e d that h e p a t i c lipase m a y generate smaller particles t h r o u g h the h y d r o l y s i s o f H D L triacylglycerols [28]. T h e triacylglycerol c o n t e n t o f H D L i n c u b a t e d with c u l t u r e d r a b b i t h e p a t o c y t e s was, h o w e,,er, n o t d e c r e a s e d in o u r s t u d i e s a n d , as n o t e d a b o v e , the ratio o f triacylglycerol to p r o t e i n r e m a i n e d c o n s t a n t . F u r t h e r m o r e , it has b e e n r e p o r t e d that h e p a t i c lipase activity m a y facilitate the d e l i v e r y o f H D L - f r e e c h o l e s t e r o l to the liver [291 , a l t h o u g h it is as yet u n c l e a r w h e t h e r h e p a t i c lipase is a l s o i n v o l v e d in the h e p a t i c delivery o f c h o l e s t e r y l esters [30]. O u r o b s e r v a t i o n that t h e preferential u p t a k e o f H D L c h o i e s t e r y l esters b y r a b b i t h e p a t o c y t e s is g r e a t e r t h a n that r e p o r t e d for t h e rat [24] is, h o w e v e r , i n c o n s i s t e n t w i t h an i n v o l v e m e n t o f h e p a t i c lipase, since t h e activity o f this e n z y m e in the r a b b i t is very m u c h l o w e r t h a n that in the rat [31]. O n e p o s s i b l e m e c h a n i s m for the p r e f e r e n t i a l u p t a k e o f H D L cholesteryl e s t e r b y h e p a t o c y t e s is the t r a n s f e r o f c h o l e s t e r y l esters f r o m H D L to o t h e r l i p o p r o t e i n s a n d the s e p a r a t e h e p a t i c d e l i v e r y o f t h e s e l i p o p r o t e i n s . In o u r earlier h e p a t i c p e r f u s i o n s t u d i e s [9] 9 . 4 - 1 1 . 0 % o f t h e H D L [ 3 H ] c h o l e s t e r y l e s t e r w a s r e c o v e r e d in the V L D L fraction after 90 rain p e r f u s i o n . In the p r e s e n t study, however, t h e l o w level o f t r a n s f e r of [3H]cholesteryl linoleyl e t h e r f r o m H D L to V L D L , 1 . 8 2.4% o v e r a 24 h culture, w o u l d s u g g e s t that a n y inv o l v e m e n t o f this m e c h a n i s m is o f m i n o r significance. S u c h a t r a n s f e r o f c h o l e s t e r y l esters t o V L D L h a s b e e n s h o w n to b e a s s o c i a t e d with a n e q u i m o l a r t r a n s f e r o f triacylglycerols into the H D L p a r t i c l e [32] w h i c h w o u l d result in an i n c r e a s e in size d u e to t r i a c y l g l y c e r o l ' s g r e a t e r m o l e c u l a r v o l u m e [331. H o w e v e r , given t h e o b s e r v e d r e d u c t i o n in p a r t i c l e size, it w o u l d a p p e a r that t h e r e m o v a l o f c o r e c h o l e s t e r o l is t h e p r e d o m i n a n t process. W h i l e t h e r e is a d e c r e a s e in the m a s s r a t i o o f c h o l e s t e r y l ester a n d p r o t e i n ( c o n s i s t e n t with a d e p l e tion o f c h o l e s t e r y l esters) t h e r e w a s n o c h a n g e in t h e m a s s r a t i o o f t r i a c y l g l y c e r o l a n d p r o t e i n , s u g g e s t i n g that there w a s n o real m a s s i n c r e a s e o f triacylglycerol in the H D L i n c u b a t e d with h e p a t o c y t e s . F u r t h e r m o r e , t h e inc u b a t i o n of c o n d i t i o n e d m e d i a c o l l e c t e d a f t e r 24 h o f culture (and containing VLDL and transfer proteins s e c r e t e d b y the cells) w i t h l a b e l e d H D L d e m o n s t r a t e d a t r a n s f e r to V L D L o f < 2.4% o f l a b e l e d " h o t e s t e r o l , i.e., v e r y m u c h less t h a n t h a t r e q u i r e d to e x p l a i n the o b served uptake data. in a r e c e n t s t o d y o f the u p t a k e o f H D L c h o l e s t e r y l esters b y c u l t u r e d h u m a n liver t u m o r cell ( H e p G 2 ) , G r a n o t et al. [34] d e m o n s t r a t e d t h a t the a d d i t i o n o f c h o l e s t e r y t ester t r a n s f e r p r o t e i n i n d u c e d the selective u p t a k e o f c h o l e s t e r y l esters. "/he role o f c h o l e s t e r y l e s t e r t r a n s f e r p r o t e i n s in e n h a n c i n g the s : l e c t i v e u p t a k e o f H D L c h o l e s t e r y l esters in the re, b b i t h a s no~. b e e n a d d r e s s e d in t h e p r e s e n t study. H o w e v e r , w e h a v e d e m o n s t r a t e d p r e v i o u s l y that cholester~j! e s t e r t r a n s f e r p r o tein is s e c r e t e d b y r a b b i t liver [22] a n d given the c o n d i -

381

tions o f culture, we would anticipate an increasing mass o f transfer protein accumulating in culture media with time. Thus, on a priori grounds, if transfer protein were an ib~portant mediator o f selective uptake o f H D L cholesteryl ester in the rabbit, we would anticipate that selective uptake would increase with time of culture, in fact, wc have demonstrated the converse, i.e., a decrease o f selective uptake with time. suggesting that the role o f cholesteryl ester transfer protein in this process in the rabbit may not be o f major importance. In addition, we have further tested thc po~bible role o f lipid transfer protein in these studies o f selective uptake by adding specific antibody to rabbit transfer protein in sufficient concentrations to completely inhibit transfer protein activity. With inhibition o f transfer protein activity there was essentially no change in the selective uptake process, indicating that this process in cultured rabbit hepatocytes is independent o f this protein. Rinninger and Pittman [24] have recently examined the regulation o f the process o f preferential H D L cholesteryl ester uptake in the rat and demonstrated that the uptake o f cholesteryl esters by rat hepatocytes was not responsive to changes in cellular cholesterol metabolism. While the issue o f the regulation o f this process was not addressed directly in the present study, there was a reproducible and significant reduction in uptake o f H D L cholesteryl esters with time in culture. The most obvious explanation for this would be a reduction in the metabolic viability o f the hepatocytes with increasing time in culture. This argument did not appear valid, however, when other parameters o f cell function were examined - bile acid synthesis and secretion increased [19], V L D L triacylglycerol secretion increased and It'C]acetate incorporation increased with time iu culture. Furthermore, replacement o f core-depleted H D L with fresh-labeled H D L did not result in an increase in uptake o f H D L components, suggesting that substrate depletion is not a satisfactory explanation for these observations. Thus, we believe that the data presented justify further examination of the regulation o f the selective hepatic uptake o f H D L cholesteryl esters by rabbit hepatocytes. Acknowledgements We would like to thank Dr. Malcolm Whiting for assaying bile acids and Irene Pryor for typing the manuscript. This study was supported by the National Health and Medical Research council and b~ a Grant in Aid from the ~4ational Heart Foundation o f Australia. References 1 Schwartz, C.C.. Halloran, L.G., Vlahcevic, Z.R., Gregory, D.I-I. and Swell, L. (1978~ Science 200. 62-64.

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