In vivo clearance of low-density lipoproteins and β-very-low-density lipoproteins in normal and hypercholesterolemic White Carneau pigeons

202

Btochim#ca et Btopl(~'sica A cttt. 1081 (iqgl) 202 21t} 199t ElsevierSciePce PuhlishersB,V, (Biomedical t)ivision) (.~005-2760/9| / ~;1)350 A DONIS 09052760910(1077F

BBALIP53539

In vivo clearance of low-density lipoproteins and B-very-low-density lipoproteins in normal

and hypercholesterolemic White Carneau pigeons J e r r y W . R e a g a n . Jr. * a n d R i c h a r d W . St. C l a i r The Arterif,.~clerosL~" Research Center. Department nf Pathology. Bowman Gray School of Medicine of Wake Forest Universio'. Winston-Salem, NC f U.S,A,)

(Received 20 April 1990) (Revised manuscriptreceived26 July 199(I)

Key words: Lipoproleinclearance: LDL: Chole.aeroF(Pigeon) Low-density lipoproteins (hLDL) and ~0-mi~ating-very-low-densi.ty lipoproteins (/I-VLDL) were isolated from the plasma of cholesterol-fed White Carneau (WC) pigeons and low-density lipoproteins (nLDL) were isolated from the plasma of grain-fed WC pigeons. The lipoproteins were radiolabeled with llSl or i311 and injected into normocholes. terolemic or hypercholesterolemic WC pigeons to determine their rate of clearance from the plasma. ~l-he fractional catabolic rate (FCR) of nLDL and hLDL in normocholesterolemic pigeons averaged 0.202 and 0.206 p o o l s / h , respectively./~-VLDL was cleared at a significantly slower rate of 0.155 p o o l s / h . The FCR of the same lipoproteins injected into hyperchoiesterolemic pigeons was reduced by 17% for nLDL, 50% for hLDL and 57% for 1O-VLDL, indicating that the effect of hypercholcsterolemia on clearance in vivo was different for the three lipoproteins. The FCR of reductively methylated pigeon LDL (MeLDL), which gives a measure of receptor-independent clearance of LDL, was shown previously ',o be 0.037 pools/h. These studies suggest therefore that LDL and ~0-VLDL are cleared from the plasma of norrnocholesterolemic and hypercholesterolemic pigeons at a rate substantially greater thar, that predicted for non-specific processes. Despite the reduction in the clearance rate of hLDL and/~-VLDL due to cholesterol feeding, the absolute amount of cholesterol tl:.at was cleared from the plasma by these lipoproleins was increased from approx. 200 m g / k g body weight per day in the normocholesterolemic pigeons to greater than 1000 m g / k g body weight per day in the hypercholesterolemic pigeons. This is due principally to the enrichment in cholesterol relative to protein of the lipoproteins isolated from cholesterol-fed pigeons and the failure of hypercholesterolemia to completely inhibit receptor-dependent clearance of LDL and /~-VLDL. The lower rate of clearance of 1O-VLDL relative ~o LDL is in marked contrast to mammalian/~-VLDL, which is cleared much faster than LDL, but is consistent with the lack of apo E on pigeon lipoproteins. Apo E is the apoprotein that is thought to be responsible for the rapid clearance o f / t . V L D L in normocholesterolemic mammals. The low rate of B-VLDL clearance in pigeons also suggests that pigeons lack an apolipoprotein that function like mammalian apo E.

Abbreviations: LDL. low-densitylipoprotein; /3-VLDI,, /3-very-lowdensity lipoprotein; MeLDL.methylaled LDL: WC, White Carneau pigeon: SR. Show Racer pigeon: FCR. fractional catabolic rate: ACR. absolute c:n:tbolic rate. * This work was performedas partial fulfillmentof the requirements for the degree of Doctor of Philosophy from the Bowman Gray School of Medicine of Wake Forest University. Present address: Department of Biochemistry,Dartmouth MedicalSchool. Hanover. NH, U.S.A, Correspondence: R.W. St. Clair, Department of Pathology, Bowman Gray School of Medicine, Wake Forest UniversityLWinston-Salem, N(" 27103, U.S,A.

Introduction Previous studies from this laboratory have shown that aortic smooth muscle cells, skin fibroblasts and embryo fibroblasts, grown in culture from explants from White Carneau (WC) and Show Racer (SR) pigeons do not express functional LDL receptors [1-3]. in contrast to these in vitro findings, subsequent studies revealed that more than 85% of the LDL cleared in vivo in pigeons consuming a cholesterol-free diet, occurred by a receptor-mediated process that closely resembled the LDL receptor pathway 14]. Diet-induced hypercholes-

203 terolemia, however, did not abolish receptor mediated clearance as it does in all mammalian species that have been studied to date. Even in pigeons with plasmz cholesterol concentrations grea:er than 1900 mg/dl. receptor-mediated clearance ,v~s Sttll re.~ponsible for 80% of the total L D L clearance [4]. These studies were conducted with LDL lhz~t w~:: isolated from the p~asma of nt, rmocholesterolernic grain-fed pigeons. Cholesterol feeding of piget;ns ,:an increase total plasma cholesterol concentrations to levels in excess of 2000 mg/dl which induces marked changes in the plasma lipoprotein profile [5,6]. Tius increase in plasma cholesterol concentration is accounted for by an increase in LDL concentration, a change in LDL composition and by the appearance of fl-migrating-verylow-density lipoproteins (fl-VLDL). /~-VLDL also appear in the plasma of other cholesterol-fed animals such as rabbits [71, rats [8], dogs [9], swine 110]. patas monkeys [11] and in the plasma of human beings with Type III hyperlipoproteinemia [121. Pigeon /3-VLDL has a number of similarities to mammalian fl-VLDL, These include a density of less than 1.006 g/ml, /3-mobility on agarose gel electrophoresis and a high concentration of cholesteryl esters. One notable difference, however, is the lack of apolipoprotein E ~apo E) on pigeon/~-VLDL (or any other pigeon lipoprotein) [5]. Apo E is a ..m.'~j,~r apoprotein of mammalian fl-VLDL [13] and has been shown to be the ligand primarily responsible for the high-affinity binding of /3-VLDL to LDL receptors [14,151. In addition to the data from this laboratory showing that pigeons express LDL receptors in vivo, we have also shown that pigeon peritoneal macrophages [16,17] and monocyte-derived macrophages [18] express receptors for pigeon/~-VLDL. Many of the properties of the pigeon/3-VLDL receptor are simila.r to the mammalian LDL receptor [19], with the exception that apo E containing lipoproteins such as rabbit ~R-VLDL are not effective competitors for binding to the pigeon/~-VLDL receptor. This suggests that, in contrast to the mammalian LDL receptor, apo E does not mediate the binding of pigeon/3-VLDL to the pigeon LDL receptor. In mammals, /3-VLDL is cleared from the plasma at a rapid rate with 50% of the injected dose being cleared from the plasma in 10 min or less [20-22[. This rapid rate of clearance is thought to be due to the high affinity of apo E for the LDL receptor [23]. In these cholesterol-fed mammals/3-VLDL clearance is reduced presumably due to saturation and down-regulation of the I.DL receptor [20-22]. The primary objective of the current study was to compare the rate of clearance by WC pigeons of pigeon /~-VLDL and LDL in order tt~ determine how the lack of apo E on pigeon /3-VLDL affects its clearance in vivo. The clearance of LDL isolated from the plasma of cholesterol-fed pigeons (hLDL) was determined as well,

since it has a size and compositkm intermedia,e between LDL from normocholesterolemic pigeons (nLDL) an~t ~q-VLDL. Results from these studies show that 3-VLDL is cleared from the plasma at a slower rate than 1 DL. which ',s consistent with the lack of apo E. Even though tb,.~ fractional catabolic rate (FCR) ~,f /3-VLDt. clearance :s less tFan LDL. the absolute rate of cholester~l clearance by/~-VLDL is similar to that of bLDL due to the enrichment in the amount of cholesterol per/~-VLDL particle. When this is coupled with the fai!ure ~f hypercholesterolemia ~o dc,wn-regulate/~-VI_ DL and LDL clearance, there is up to a 6-fold increase in the absolute amount of chol,~sterol turnover per day in hyper::holesterolemic pigeons. Methods

Lipoprotem isokltion and lahe!in:z To conduct these studies, ow-density lipoproteins (nLDL) were isolated by ultracentrifuga,ion of plasma from male WC pigeons fed a cho'esterol-free pelleted grain diet. LDL (hLDL) and ~-VLDL were isolated from the pk-sma of male WC pigeons fed a diet to which cholesterol and lard were added to a final concentration of 0.5% cholesterol (w/w) and 10,% lard (w/w). The pigeons were fed this diet for a minimum of 3 months before lipoproteins were isolated. Blood from animals that were fasted overnight was collected into tubes containing 1 mg/mi EDTA and 0.4% 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and kept at 4 ° C during the isolation procedure. ,8-VLDL was removed from the plasma by ultracentrifugation at 36000 rpm for 24 h at a density of 1.006 g/ml. To remove any residual LDL that might be present, the d < 1.006 g/ml fraction from the hypercholesterolemic pigeons was applied to a 4% agarose column (Bin-Gel A-15m) and eluted with a high-salt buffer containing 0.3 M NaCI, 0.1 M KHzPO4 and 0.01% EDTA (pH 7.4) [5]. This step was included because in cholesterol-fed birds the separation of LDL and /~-VI_.DL by ultracentrifugation is sometimes incomplete due to the larger size and lower density of some of the LDL particles. For plasma ~aken from normal pigeons the d < 1.006 g / m i fractitm was discarded since it contains only trace amounts of lipoproteins [5]. To obtain LDL the d > 1.006 g/ml fraction from both normal and hypercholesterolemie pigeons was adjusted to a density of 1.080 g / m l with solid KBr, ovedayered with a 1.063 g / m l KBr solution containing 0.01% EDTA and centrifuged at 36000 rpm for 24 h in a Beckman SW-40 rotor. The resulting 1.063 g/rot density fraction from hypercholesterolemic pigeons was also applied to the 4% agarose column to remove any contaminating/3-VLDL. LDL from normal pigeons was not further purified. The LDL isolated from both normal and hypercholesterolemic pigeons and the/~-VLDL from the hy-

204 percholesterolemic pigeons were exhaustively dialyzed against a solution containing 0.9% NaCI and 0.01% EDTA. and concentrated to about 3 mg p r o t e i n / m l by dialysis against Dextran (Dextran T 500. Pharmacia Biotechaology Products, Piscataway, N J). The L D L and fl-VLDL were sterilized by filtration d.rough a 0,45 btm M;!lipore filter and stored at 4°C. Lipoproteins were u.,,ed within 2 weeks. Lipoproteins were quantified on the basis of protein using the method of Lowry et al. [24] with bovine serum albumin as the standard. LDL and t%VLDL were labded with t2Sl or 1~I as indicated in the legends by the IodiE~ monochloride method [25]. Specific activities ranged from 156-550 c p m / n g of protein. More than 94% of the radioactivity of each lipoprotein was precipitable with 10% trichloroacetic acid (TC) and less than 15% was extractable in chlorofo, r m / m e t h a n o l (2 : 1, v/v).

agnost:cs High-Performance cholesterol reagent (No. 236691 ).

Stalls:its Statistical evalaation was carried out in order to test the effects of dielary induced hypercholesterolemia and lipoprolein type on kinetic parameters of lipoprotein turnover. For thi:. muitivariate ar,alysis of variance was used with diet a+ the grouping factor and lipoprotein particle type as the outcome variable [30]. Pairwise comparison.~ were performed using the Bonferroni method for mul.iple comparis.~,l [31]. Comparison between iipoprotein type injected into normocholesterolemic and hypercholesterolemic I:;geons was performed using a t-test [32]. Comparisons were considered statistically significant at P < (;.05. Results

Measurement of lipoprotein clearance in vivo Adult male WC pigeons were obtained from our breeding colony. The pigeons were fasted 12 h immediately prior to and dt~ring each experiment. Each bird was injected with the indicated labeled lipoprotcin or a combination of two of the three lipoproteins as shown in Table II. I mt blood samples were collected from the alar vein opposite to the one used for injection. The blood was placed into tubes containing EDTA at a final concentration of 1 m g / m l and the tubes were kept on ice. The first 1 ml blood sample was taken exactly 2 rain after injection of the radiolabeled lipoproteins. Other ] ml samples were collected at various time point:; from 15 rain to 24 h as indicated in the figures. At the final time point the birds were anesthetized with methoxyflurane (Metafane, Pittman-Moore, Washington Crossing, N J) and killed by decapitation, Plasma samples were precipitated in 10% trichloroacelic acid (TCA) and the TCA precipitable fraction counted for 12Sl- and I~]l-radioactivity using a Tracor 1185 Autogamma Spectrometer. Corrections were made for spill of I'qI-radioactivity into the 12SI-channel and all count:; were corrected for background and isotope decay. Plasma clearance of lipoproteins was determined from the T e A precipitable radioactivity. The slope and intercept of the plasma decay curves were calculated by nonlinear least-squares regression analysis using a two exponential formula by means of the R S / 1 fit function computer program [26]. The fractional catabolic rate (FCR) was calculated from these data by the method of Matthews [27], as described [4,28]. Cholo,;t~ol in the plasma of the birds and in the isolated lipoproteins was measured by an automated enzyma,,., procedure using the Teehnicon RA-1000 analyzer. The procedure is based on the method of Allain et al. [29] using the Boehringer-Mannheim Di-

fl-VLDL and h L D L isolated from the plasma of cholesterol-fed pigeons and n L D L isolated from the lasma of grain-fed pigeons, were labeled with ~2Sl or 1 . . . . I and rejected mto normocholesterolemtc or hyper-

~

'~'N.

0.

(9

3

I

I ....

I

10

0

5

tO

1.5

20

25

T~me (hr)

Fig. 1. Mean plasma decay curves for nLDL (O). hLDL 4o). and B-VLDL (m) in WC pigeons. Each bird was injected with the radiolabeled lipoprolein(s) as described in the legend to Table I[ and the plasma decay was determined from the TCA-precipitable plasma counts as described in the me|hods. The mean data are compiled from the decay curves for each of the individual pigeons shown in Table !!. Panel A is for normocholeslcrolcmic pigeons and panel B is for hypcrcholes|erolemicpigeons. Results are the mean _+S.E.

205 cholesterolemic male W C pigc,ms. T h e m e a n p l a s m a decay curves for each of these l i p o p m t e i n s are shown in Fig. 1. Like n L D L |4], h L D L a n d f l - V L D L were cleared from the p l a s m a in a m a n n e r that best fit the kinetics of a b i e x p o n e n t i a l die away. As shown in Fig. IA, h L D L was cleared from the p l a s m a of normocho|es~.erolemic pigeons at virtually the s a m e rate as n L D L . T h i s can be seen from the similar m e a n p l a s m a decay curves a n d also from the tt/2 of c l e a r a n c e as s h o w n in T a b l e I: 2.00 h for n L D L a n d 1,83 h for h L D L T h e fractional c a t a b o l i c rates ( F C R ) for l i p o p r o t e i n s .n i n d i v i d u a l pigeons are s h o w n in T a b l e II. C o n s i s t e n t with the o v e r l a p p i n g p l a s m a decay curves for n L D L a n d h L D L in n o r m o c h o l e s t e r o l e m i c p i g e o n s s h o w n in Fig. IA. the average F C R ' s also were n o t significantly d i f f e r e n t for n L D L or h L D L . A l t h o u g h the m e a n p l a s m a decay for ~ - V L D L (Fig, I A ) was s o m e w h a t slower t h a n for b o t h n L D L a n d h L D L , these differences did not reach statistical significance in n o r m o c h o l e s t e r o l e m i c p i g e o n s w h e n the 11/2 values of c l e a r a n c e were c o m p a r e d ( T a b l e i). T h e F C R of f l - V L D L 10.155 p o o l s / h ) however, was s i g n i f i c a n t l y slower t h a n that of n L D L a n d h L D L (Table 11). T h i s suggests t h a t the slow c o m p o n e n t of /3V L D L c l e a r a n c e was largely r e s p o n s i b l e for the signific a n t difference in F C R of B - V L D L as o p p o s e d to the n o n - s i g n i f i c a n t c h a n g e w h e n the tt, 2 values were c o m pared. Similar studies were c o n d u c t e d in cholesterol-fed pig e o n s that h a d a n a v e l a g e p l a s m a cholesterol c o n c e n t r a t i o n of 1655 m g / d l ( T a b l e IlL As s h o w n in Fig. IB, the rate of p l a s m a decay o f r a d i o l a b e l e d n L D L h L D L a n d

tABLE II Fra(-ttomd catuht,&" raft' of n L D L hLDI., and fl- l "I.DI. m m~nn,)t'hot•,"~.t'rolt't?ht (-%('1 attd i[vpcrchoiv.~wroiomc ¢iiC? H.'t."ptgomv

Normal LDL [nLDLL hLDL and [3-VLDL were irbected into the alar vein of normal and hypercholesterolemic WC piget,nx. These are the ~,a,ae birds for which the t~ z of lipoproteln clearance is shown in Table I. The F"R was calculated as des::ribed in Method.; from the slope and intercept for a r~vt',-exponentialfi; to the pktsma die away of TCA-preciplt,~ble radioactivity. Bird no.

N,ormtx:hol¢:.,lerolemic Expt. 1 1 207 2 251 Expt 2 1 210 2 217 3 221 4 223 5 229 6 235 "~ 236 3 238 9 275 Mean ± S,E.

Normal LDL (nLDL), hLDL, and ~I.VLDL "~ete ia]ected into the Mar vein of normal and hypercholesterolemic WC pigeons. Blood samples were collected as described in Methods. The data are the means of all values from two separate experiments. An average of ~5 tag of nLDL containing 19.62-lOs cpm ff t-'Sl, 30 ttg of hLDL containing 9.61.10 ~ cpm of IH1. and 43 /~g of B-VLDL conlainlng 7.76-10 ~ cpm of Psi or ~ 1 were injected into the birds. The f i , of clearance was derived from each bird's plasma decay curve. The data are from the same birds shown in Table I1 in which FCR's were calculated. q.,2 {h) (mean_+S.E.) nLDL hLDL #-VLDL

NC pigeons

tiC pigeons

2.00_+0.12 (n = 6) 1.83 +_0.14 (n = 8) 2.26_+0.52 (n = 5)

2.35__.0.18(n = 5) 3.68-+,).32(,)=8) 8.36-+0.96(n = 6)

Statistical significance ( P value.,,) Lipoproteins

NC

nLDL ws. hLDL nLDL vs. B-VLDL hI.DL vs. fl-VLDL

0.06 0.03 0.59

pigeons

t1C pigeons < O.IN)O1 < 0.tKKH < 0.0t}01

232 _6

F('R (p(×~ls/h ; nt.Dt, hLDL

0.170 0.195 0.215 0,197 0.2t8

11 12

B-VLDL

0.226 1}.1~2 0.218 0.2114

o.202 ± O.(X)8

0.164

tl.13~ (k h':,9 11.21)8 0.234

0.138

0 185

0.160 0.132

O.2t~ _ 0.(X17

0.155 + 0.009

0.215

i-lypcrc|'tole~,Icrolemi~: Expt. I 3 1820 4 2 620 F.xpt. 2 It) 643

TABLE 1 Tt/2 of clearance o] nLDL, hLDL and ~-VLDL ~n normocholesterolemi(" ( N (') and h.wercholt'ateroh'mir (11(') 14/(" pigeens

Plasmi chol. ! nag/dl')

0.093 0.118 0.I 12

0.085 0.08,2

8g~ 930

0,168 (}.211R

13 14 15 16"

!113 I O47 f656

0.155

17

2064 2856

0,13|

0.056 0.056

1655 +215

0.167 0.104 _+0.013 +(}.007

0.0.66 + 0.{816

18 Mean +S.E.

0.1,15 0.088

0.172

0.0~2 0.087

1%2

0.(}65 0.054

0.108

Statisucal signific:mcc( P values) Lipoprotcins

NC pigeons

ttC pigeons

nLI)l, vs. hLI)L nLDL w. B-\ LDL hLDL vs. fl-VLDL

0.97 0.1.~1 < 0.(Nllli

< 0~0001 < O.0001 < 0.001

f l - V L D L was r e d u c e d in the cholesterol-fed p i g e o n s c o m p a r e d to the rate of c l e a r a n c e of the same lipoproteins in n o r m a l pigeons. T h i s slower rate of c l e a r a n c e can also be seen in T a b l e I in which the tl/,. of c l e a r a n c e f r o m the p l a s m a was 1.2-times longer for n L D L , 2.1times longer for h L D L a n d 3.7-times longer for f l - V L D L in the cholesterol-fed pigeons, c o m p a r e d tt) the s a m e l i p o p r o t e i n s injected i n t o n o r m o c h o l e s t e r o l e m l c pigeons. All of these differences were highly statistically significant ( P < 0 . 0 0 0 1 ) . T h i s suggests that hyper-

206 cholesterolemia had a greater effect on the clearance of h L D L than on the clearance of nLDL and an even greater effecl or) the clezrance of /%V!_DL. Th~.s differential effect of hypercholesterolemia on ~he clearance of the three lipoproteins can also be seen in the calculation of FCR (Table II). Whereas there was no difference in the FCR of n L D L and h L D L in normocholesteroIemic pigeons, the mean FCR of h L D L was 38% less than the FCR of n L D L in the cholesterol-fed pigeons. and the FCR of ,8-VL,DL was 60% less than nLDL. In our previous study we showed that the FCR of methylated n L D L (MeLDL) averaged 0.037+0.003 pools/h (n = 17, x + S.E.) in normocholesterotemic pigeons and 0.040 + 0.002 p o o l s / h (n = 11) in hypercholestero[emic pigeons [4]. in normocholesterolemic pigeons the FCR for n L D L and h L D L was approx. 5.5-times greater than the FCR of MeLDL, and /3VLDL was more than 4-times greater. For these same lipoproteins injected into hypercholesterolemic pigeons the FCR was 4.5-, 2.8- and 1.8-times greater, respectively, than the FCR of MeLDL. As a result, nLDL, h L D L and ,8-VLDL were all cleared in vivo at a rate that exceeded that of nonspecific uptake. To better illustrate the differences in clearance rates of the three lipoproteins injected into normal and hypercholesterolemic pigeons, the mean FCR + S.E. for nLDL, h L D L and /3-VLDL in normocholesterolemic and hypercholesterotemic pigeons are shown in Fig. 2. Analysis of variance indicated a significant effect of both hyperchotesterolemia ( P < 0.01) and lipoprotein type ( P < 0 . 0 I ) on FCR. This is easily seen in Fig. 2 where there was a progressive decrease in F C R of hLDL and ~ - V L D L relative to n L D L in hypercholesterolemic animals, but in normocholesterolemic pigeons this was seen only for B - V L D L Cholesterol feeding reduced the FCR of n L D L by 17% compared to the F C R of n L D L in normal pigeons. This is somewhat less O.30 0.24 0.202

0,205

O.'t8 Q

0.155

0.12

01

0.0,5 0,00 nLDL

hLDL

/

0.24 [

0.1b i" 0.12 I 0.06 0,00 0.30

0.18

• 0q) 0 006 0.00 0.30 13-~_[3_

0.18

•

•

Q~

0

tB-VLDL

1

I

600

1200

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&

&

|

i

1800

2400

:3000

Total Plasma Cholesterol (mg/dl) Fig. 3. Relationshipof FCR to total plasma cholesterol concentration. For each of the birds shown in Table I for which the total plasma cholesterol concentration was known, the FCR for nLDL (ll), hLDL (e) and fl-VLDL (A) is plotted against the total plasma cholesterol concentration, Open symbols represent the mean+S,E, of the FCR for the same lipoproteins in normocholesterolemicpigeons (n = 6 for nLDL n=8 for hLDL and n=5 for /~'.VLDL). The FCR for MeLDL was 0.037 in normocholesterolemic pigeons and 0.040 in hypercholesterolemic pigeons [4]. These values were not significantly different from each other.

than the 27% reduction in L D L FCR that we saw previously with cholesterol feeding [4], probably due to the fact that the L D L FCR in the normocholesterolemic pigeons used in this study was lower than in the previous study, rather than any fundamental difference in the response of the hypercholesterolemic animals between the two studies. The FCR's for h L D L and flVLDL, however, were reduced by more than 50% in cholesterol-fed pigeons compared to the same lipoproteins injected into normal pigeons. The data in Fig. 2 include the average of all pigeons regardless of the degree of hypercholesterolemia. Since the plasma cholesterol concentrations ranged from 643 to 2856 m g / d l in these pigeons, the average reduction in FCR with cholesterol feeding could be misleading. Therefore, in Fig. 3, the F C R values have beer pie:ted against each pigeon's own total plasma cbc.!~s'. ~rol con-

iiiii!ii!ii

Fig. 2. Fractional catabolic rate (FCR) of N L D L , h L D L and fl-VLDL in normtxzholesterolemic (cross-hatched) and hypercholesterolemic (stippled) W C pigeons. Each bar represents the m e a n + S . E , of the F C R of the lipoproteins from the values for the individual birds shown in Table I1. n = 6, 8 and 5 for n L D L , h L D L and / ~ - V L D L respectively, injected into normocholesterolemic pigeons a n d n = 5, 8 and 6 for these same lipoproteins injected into hypercholesterolemic pigeons.

&

207 centration. For n L D L and h L D L there was not a significant correlation ( P = 0.164 and P = 0.288. respectively) between the lipoprotein F C R in hypercholesterolemic pigeons and the pigeon's total plasma cholesterol concentration. For fl-VLDL this correlation just reached significance at the 5% level ( P = 0.049). This suggests that there was not a strong relationship between the rate of clearance of L D L or B - V L D L with increasing plasma cholesterol concentrations. These correlation analyses were carried out using only the data from the hypercholesterolemic animals shown in Fig. 3 (closed symbols). The F C R of the same lipoproteins in normocholesterolemic pigeons is shown by the open symbols. The greatest change in FCR, particularly for h L D L and f l - V L D L took place over the plasma cholesterol range between that of the normochoiest: r,.)lemic pigeons (232 m g / d i ) and about 600 m g / d l . It is not possible to be more specific than this. however. since none of the hypercholesterolemic pigeons had plasma cholesterol concentrations less than 600 m g / d i . As a result, these data are most consistent with the conclusion that a relatively small increase in plasma cholesterol concentration produces nearly as great a decrease in F C R for all of the lipoproteins as do large increases in plasma cholestrol concentrations. The increase in L D L cholesterol and the appearance o f / ~ - V L D L with cholesterol feeding, results in significant increases in total plasma cholesterol concentrations in the pigeon [5], We have previously shm~n [61 that there is a highly significant correlation between total plasma cholesterol concentrations in cholesterol-fed pigeons and /)-VLDL cholesterol. Likewise there is a significant correlation between plasma cholesterol concentrations and L D L cholesterol at plasma ~.holesterol concentrations less than 1500 m g / d l [6]. Above 1500 m g / d l there is little further increase in L D L concentrations, Instead the additional increase in the plasma cholesterol concentrations is almost entirely the result of ~ - V L D L [61. Therefore, based on the plasma cholesterol concentration for each pigeon in the present study (Table I1) and the regression lines for the relationship between plasma LDI. and B - V L D L cholesterol concentrations [6], we estimaled the L D L and ,8-VLDL cholesterol concentrations of the plasma in the cholesterol-fed pigeons in the present study. With this information it was possible to estimate the absolute catabolic rates (ACR) ( m g / d l per h) for L D L and ,8-VLDL cholesterol. These data are shown in Fig. 4. Although there was a reduction in the F C R of h L D L and /)-VLDL in cholesterol-fed birds (Fig. 2), this was more than offset by the increase in cholesterol per lipoprotein particle, such that the absolute a m o u n t of cholesterol that was cleared from the plasma was increased substantially with increasing total plasma cholesterol concentrations. The absolute catabolic rate of the lipoprotein protein was also estimated and is

120 .

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f

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i

600

.

.

1200

.

.

. 1800

.

.

.

.

2400

3000

Total Plasma Chotesterol (rr~3/dl) Fin. 4. ~bsolute catabolic rate (ACR) ot nLDL. hLDL and /)+VLDL injected into cholesterot-f,.at pigetms with different degrees of hypercholestemlemia. The ACR of protein (,',o) anti cholesterol (Ae) of hLDL and B-VLDL are plotted against total plasma cholesterol concentrations. Cholesterol and protein ACR wa,;calculated by multiplying the lipoprotein cholesterol and lipoprolein protein concentratlons by the FCR. Lipoprloein cholesterol and protein concentrations ~ere estimated for the lipoprotcin particle using previously published data on the composition a n d concentration of lipoproteins from normc,cholesterolemie a n d hypercholesterolemic pigeons IFig. 2 of Ref. 6). W h e n the total plasma cholesterol ( T P C ) w a s le.~s than 1500 m g / d l there was a significant correlation between T P C a n d hLDL cholesterol and there was a significant correlation bet,.*,'oen T P C and the ratio of h L D L cholesterol to b L D L protein (chol/protein). W h e n the T P C exceeded 1500 m g / d L however, the h L D L cholesterol plateaued with a m e a n value of 948 m g / d l and the hLDL c h o l / p r o t e i n plateaued at 4.6. T h e increase in TPC beyond 1500 m g / d l was accounted for hy a continued increase in B-VLDL cholesterol. The following calculations were used to estimate 8.VLDL and hLDL cholesterol and c h o l / p r o l e i n ',-alues: lipoprotein cholesterol = (llotal plasma c h o l e s t e r o l s A ) - B ) where A = the slope of the regre~-ion

line :md B = the y-intercept of the regression line. Thus /)-VLDL cholesterol[ ~ ({total plasma cholesterol × 0.828)- 624.77)) and hLDL cholesterol Iv'hen TPC < 1500 mg/dl)= ({total plasma cholesterol x 0.705)-144.13). Lipoprotein protein values were calculated on the basis of the estimated ratio of lipoprotein cholesterol to lipoprotein protein. This value was dependent on the total plasma cholesterol concen*.ration. The regression lines describing this ratio were: /)-VLDL choL/protein ~ ((total plasma cholesterol × 0.0054) + 2.57q) and hLDL chol/protein (~hen TPC < 1500 mg/dl)= ((total plasma cholesterol x 0.0037) + 0.065).

shown in Fig. 4. Protein A C R did not increase at nearly the rate of cholesterol clearance. This is due to the fact that as the lip,~protein particle increases in size, it becomes enriched in cholesterol relative to protein [5].

208 Discussion

Our previous studies [4] have shown that nLDL cle:~.rance in normocholesterolemic White Carneau and Show Racer pigeons occurs at a biexponential rate with a FCR averaging 0.277 pools/h. At this rate of clearance only about 5~ of the labeled LDL remair,~s in the plasma after 24 h. In contrast, rcductively mcthylated LDL (MeLDL) is cleared at a much slower rate (FCR = 0.037 pools/h). Methylation is a modificanon that inhibits the binding of LDL to the LDL receptor [4,33], By comparing Ihe rates of clearance of [I2"~I]LDL and [t~I]MeLDL injected simultaneously into the same pigeons, it is possible to calculate the contribution of receptor-mediated mechanisms to LDL clearance Using these techniques we have shown that the FCR of nLDL in normocholesterolemic pigeons is, on average, 7-times faster than that of MeLDI. indicating that approx. 85% of the LDL clearance occurs by receptor-mediated mechanisms [4]. Cholesterol feeding reduces the FCR of nLDL by only 27% with nearly 80% of the LDL clearance still occurring by receptor-mediated mechanisms. In the study prese;,ted here we focused on the in vivo clearance of lipoproteins that are present in the plasma of cholesterol-fed White Carneau pigeons, The LDL from hypercholesterolemic pigeons is enriched in chnle~teryl e~!ers cempared to LDL from normal pigeons. Whereas the LDL isolated from normal pigeons contain about 23% cholesteryl ester by weight, the LDL isolated from hypercholesterotemic pigeons contain about 45% cholesteryl ester [5]. The LDL particles from cholesterol-fed pigeons are also. on average, about twice as Iarge( M~ = 6.36 • 10 r' for hLDL. 3.22 • 106 for nLDL). The fl-VLDL are even larger and more enriched in cholesteryl ester with molecular weights ranging up to 15.10 c', and containing as much as 55% cholesteryl ester by weight [5]. The presevt studies show that in normocholesterolemic pigeons there is not a significant difference in the clearance rate of nLDL and hLDL, and only a slight difference with ~-VLDL. if fl-VI,DL, like LDL, is cleared in pigeons primarily by an LDL receptor-like mechanism [4], one possible explanation for the reduced rate of clearance of fl-VLDL is that its greater size or altered composition changes its affinity or capacity for the LDL receptor. Indeed, previous studies from our laboratory [34] as well as those of others [35] have shown that the larger cholesteryl ester, rich LDL isolated from hypercholesterolemic rhesus monkeys has a higher affinity but a lower capacity for the LDL receptor. If a similar situation exists in vivo in pigeons, then the larger ~-VLDL might be cleared from the plasma at a slower rate owing to a reduced number of particles able to bind to a limited number of LDL receptors. This would result in a decreased FCR. The failure of the

hLDL FCR to be reduced even though they are also larger than nLDL. suggests either than there is a threshold of size before changes in capacity of binding to LDL receptors is expressed or that size and composition alone are not totally responsible for the difference in the clearance rates. The low rate of clearance of/3-VLDL compared to nLDL in the pigeon is in marked contrast to the situation in other species such as rabbits [20,22] and dogs [21], in which fl-VLDL is rapidly cleared from the plasma of normocholesterolemic animals. The fl.VLDL from these animals has a high content of apo E [13]. Lipoproteins that contain apo E have been shown to have a higher affinity for the LDL receptor than lipoproteins that contain only apo B [23]. Apo E may play an even more important role as the ligand for the binding of/3-VLDL to the LDL receptor [14,15]. Since pigeons do not have a protein of the size of apo E on any of their plasma lipoproteins [5], the low rate of clearance of pigeon/3-VLDL is consistent with the lack of an apoprotein that functions like apo E. It has been suggested [36] that apo AI may play the same role in avian species that apo E plays in mammals, particularly in regard to its ability to mobilize cholesterol and transport it to the liver for removal by LDL receptors. Although the results of this study do not rule out the possibility that pigeon apo A! may substitute for some of the functions of mammalian apo E, apo AI clearly does not substitute for apo E with respect to LDL clearance in vivo. If it did, it would be expected that pigeon fl-VLDL, which contains a significant amount of apo AI [5]. would be rapidly cleared from the circulation. Obviously this was not the case. This result is also consistent with previous studies from our laboratory showing that pigeon HDL which contains large amounts of apo AI does not bind to an LDL-like receptor on pigeon macrophages [16]. When LDL, hLDL or fl-VLDL were injected into hypercholesterolemic pigeons the FCR was reduced from 30-50% relative to the same lipoproteins injected into normal pigeons, in the present study, we did not determine receptor-dependent and receptor-independent clearance as we had done previously with nLDL [4]. Therefore it was not possible to determine the extent to which the reduction in lipoprotein clearance in the cholesterol-fed pigeons was due to a reduction in receptor-mediated clearance. Based on our previous study, however, in which receptor-mediated clearance of nLDL was reduced 32% with hypercholesterolemia while receptor-independent clearance was unchanged, it is reasonable to suggest that the reduction in the clearance of the lipoproteins used in this study also might be due to a slight, but incomplete, reductton in the receptormediated pathway. As shown in Fig. 3, this reduction in FCR had nearly plateaued at a plasma cholesterol concentration of about 600 mg/dl. Beyond this there was

209 little change in FCR with increasing plasma cholesterol concentration. One possible interpretation of these data is that LDL and fl-VLDL are being cleared by more than one mechanism. The decrease in FCR at plasma cholesterol concentrations of up t., about 600 m g / d l may reflect LDL receptor clearance that is down-regulated while the remainder of LDL and /3-VLDt. clearance occurs by some other mechanism that is not down-regulated by hypercholesterolemia. Alternatively it may be that all of the clearance is by the LDI. receptor pathway, but that this pathway is only partially down-regulated by hypcrcholesterolemia. Another possibility mentioned previously [4] is that LDL receptormediated clearance is not down-regulated in the pigeon by hypercholesterolemia, but that the slight reduction in FCR is the result of saturatian of receptors [37]. With the available data it is not possible to select among these possibilities, Regardless of the mechanism, the clearance of LDL and ,8-VLDL appears to be blocked by methylatmn as seen by the fact that the FCR of the lipoproteins never reached the level of non-specific clearance of MeLDL [4]. Pigeon n L D L h L D L and/3-VLDL all have measurable quantities of apo AI and several unidentified smaller apoproteins [5]. Since these non-apo B proteins, at least in nonavian models, are cleared from the plasma at a different rate than apo B [37], and since total TCA precipitable radioactivity, which would include all apoproteins, was used to measure lipoprotein clearance in these studies, we cannot discount t~e possibility that some of the reduced FCR of hLE; r. and /3-VLDL may be due to the exchange of these radiolabeled apopr. :*,eins to other lipoproteins. If this occurs it may be more prounced in cholesterol-fed pigeons than in normal pigeons, since there would be a larger pool of fl-VLDL and h L D L onto which the apoproteins could exchange, It seems unlikely, however, that the differences in lipoprotein clearance rates can be due entirely to differences in exchange of radiolabeled apoproteins, since the differences in clearance rates were readily apparent as early as 3 h after injection rather than becoming progressively greater at later time periods when the radiolabeled apo B had been largely cleared from the plasma. This question can only be resolved by future studies in which the clearance from the plasma of the different apoproteins is compared. Even though the h L D L and/~-VLDL were cleared at a lower rate than the n L D L in cholesterol-fed pigeons and thus exhibited a lower FCR, the ACR of cholesterol clearance is much higher in the hypercholesterolemic pigeons than in normal pigeons. The calculation of ACR for turnover of cholesterol assumes that the whole lipoprotein particle is cleared from the plasma as a unit. As a result, by knowing the amount of cholesterol per LDL or fl-VLDL particle it is possible to estimate the ACR of cholesterol from the clearance of the radio-

labeled lipopro~eins When ibis was done for normochotesterolemic pigeons the rate of cholesterol turnover was found to be approx. 209 m g / k g body weight per day. This compares to a value of about 250 m g / k g per day for the direct measurement of cho!esterol turnover in pigeons [39.40]. The high rate of cholesterol turnover may be a characteristic of birds as cholesterol turnover in the normocholesterolemic laying chicken is also extremely high (200 m g / k g per day) [41]. These values are approx. 10-fold greater than for ihe rate of cholesterol turnover in mammals such as African Green monkeys 121.1 m g / k g per day) [42]. Cholesterol feeding increases cholesterol turnover in pigeons to levels exceeding 1000 m g / k g per day. This value can be determined by taking the sum of the ACR of h L D L and fl-VLDL cholesterol. The large increase in daily cholesterol turnover in cholesterol-fed pigeons is the result of the increase in the amount of cholesterol per lipoprotein particle accompanied by only a small decrease in the FCR of these lipoproteins. The high rate of cholesterol turnover in the avian species may have evolved as a result of the large anmunts of plasma derived cholesterol needed for egg produclion [43|. This and our previous studies [4] have demonstrated j the presence of a highly efficient pathway in pigeons for the clearance of L D L and fl-VLDL in vivo. Clearance by this pathway is blocked by methylation of the lysine residues on the lipoproteins. This is similar to the LDL receptor pathway in mammals [33]. The presence of an LDL receptor-like pathway in vivo in pigeons is somewhat surprising, however, given our failure to demonstrate LDL receptors on smooth muscle cells, skin fibroblasts and embryo fibroblasts derived from explants in culture [1-3]. Preliminary data from our laboratory suggest that pigeon cells lose the expression of L D L receptors with passage in culture, but that early passage pigeon embryo cells and hepatocytes (unpublished data} and peritoneal macrophages [16.17] all display LDL receptor activity. As a result, the presence of an L D L receptor pathway in vivo is not inconsistent with our recent in vitro data. In summary, these studies have shown that iipoprorein clearance in pigeons is influenced by both lipoprorein type and hypercholesterolemia. Both LDL and /3-VLDL are cleared from the plasma of WC pigeons at a faster rate than MeLDL, suggesting that an LDL receptor-like pathway is responsible for their clearance. Unlike m a m m a l i a n 3 - V L D L /~-VLDL from cholesterol-fed pigeons is cleared from the plasma at a slower rate than LDL which is consistent with the lack of apo E on pigeon lipoproteins. The rate of clearance of nLDL, h L D L and fl-VLDL were reduced 30-50% in pigeons with diet-induceti hypercholesterolemia but when coupled with a large increase in the am~,unt of chc!,:steroi per lipoprotein particle resulted in a marked increase in the absolute amount of cholesterol cleared

210 per day. Thus, p i g e o n s a p p e a r to possess a m e c h a n i s m ( s ) for the c l e a r a n c e of !arge a m o u n t s of i i p o p r o t e i n cholesterol that is f u n c t i o n a l even in the face of gross hypercholesterolemia. Ac;,nowledgemenls W e a c k n o w l e d g e the excellent a s s i s t a n c e of Mrs, D o r i a n S e m a n a n d Ms. J a n e H o d g e Brooks in the prep'::,ation of this m a n u s c r i p t a n d Dr. T i m o t h y M o r g a n for the statistical a n a l y s i s of the data. T h i s work was s u p p o r t e d by S C O R G r a n t H L - 1 4 1 6 4 f r o m the N a tional Heart, L u n g a n d Blood I n s t i t u t e . Dr. R e a g a n was s u p p o r t e d by N a t i o n a l Research Service A w a r d I n s t i t u tional G r a n t HL-07115. References 1 RandtAph. R.K, and St, Clair, R.W. (1984) J. Lipid Res. 25, 888-902. 2 Randolph, R.K,, Smith, B.P. and St. Clair, R.W. (1984) J. Lipid Res. 25, 903-912. 3 St. Clair, R,W., Leighl, M.A. and Barakat. H.A. (1986) Arteriosclerosis 6. 170-177. 4 Reagan Jr., J.W., Miller, L.R. and St. Clair, R.W. (1990) J. Biol, Chem. 265. 9381-9391. 5 Barakal, H.A. and St. Clair, R.W. (1985) J. Lipid Res, 26, 12521268. 6 Sl. Clair, R.W., Randolph, R.K., Jokinen. J.P., Clarkson. T.B. and Barakat. H.A. (1986) Arteriosclerosis 6. 614-626. 7 Shore, V.G.. Shore. B. and Hart, R.G. (1974) Biochemistry 13. 1579-1585. 8 Mahley. R.W. and Holcombe, K.S. (1977) J. Lipid Res. 18, 314324. 9 Mahley, R.W., Weisgraber, K.H. and lnnerarity, T.L. (1974) Circ. Res. 35. 722-733. 10 Mahley, R.W.. Weisgraber, KH,. Innerarity, T.L., Brewer Jr.. H.B. and Assman, G. (1975) Biochemistry 14, 2817-2823. It Mahley. R,W, Weisgraber, K.H. and lnnerarity, T. (1976) Biochemistry 15, 2979-2985. 12 Mahley. R.W. and Rail Jr.. S.C. (1989} The Metabolic Basis of inherited Disease (Striver, C.R., Beaudet, A.L., Sly, W,S. and Valle. D.. eds.). Vol. i. pp. 1195-1213, McGraw-Hill, New York. 13 Mahley. R.W. (1978) Disturbances in Lipid and Lipoprotein Metabolism (Dietschy, J.M, Golto Jr., A.M. and Ontko. J.A., eds ), pp. t81-197, Am. Physiol. Soc., Belhesda. 14 Hui, D.Y.. Innerarity, T.L, and Mahley. R.W. (1984) J. Biol. Chem. 259, 860-869, 15 Harkes. L.. Van Duijne. A. and Van Berkel, T.J.C. (1989) Eur, J. Biochem. 180, 241-248. 16 Adelman, S.J. and St. Clair. R.W. (1988) J. Lipid Res. 29, 643-656. 17 Adelman, S.J. and SI. Clair. R.W. (1989) Arteriosclerosis 9, 673683.

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