Deficiency of low density lipoprotein receptors in liver and adrenal gland of the WHHL rabbit, an animal model of

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp , April 1981 Biochemistry Deficiency of low density lipoprotein receptors in liver and adrenal gland of the WHHL rabbit, an animal model of familial hypercholesterolemia (cholesterol metabolism/lipoproteins/(3-migrating very low density lipoprotein/cell surface) TORU KITA*, MICHAEL S. BROWN*, YOSHIO WATANABEt, AND JOSEPH L. GOLDSTEIN* *Deparments of Molecular Genetics and Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75235; and Experimental Animal Laboratory, School of Medicine, University of Ko, Ko 65, Japan Contributed by Michael S. Brown, January 16, 1981 ABSTRACT The WHHL (Watana heritable hyperlipidemic) rabbit has en proposed as an animal model for human familial hypercholesterolemia. Homozygous WHHL rabbits have marked increases in the plasma level of low density lipoprotein (LDL), removal of LDL from their plasma is delayed, and LDL receptors are absent from their cultured fibroblasts [Tanzawa, K., Shimada, Y., Kuroda, M., Tsujita, Y., Arai, M. & Watana, Y. (198) FEBS Lett. 118, 81-84]. We here report that membranes from the liver and adrenal gland of WHHL rabbits lack high-affinity LDL receptors. In normal rabbit membranes, binding of LDL to this receptor requires calcium and is inhibited by EDTA. The LDL receptor binds rabbit "I-laled f8-migrating very low density lipoprotein (fl-vldl), which contains apoproteins B and E, as well as rabbit "SI-laled LDL, which contains only apoprotein B. It does not bind high density lipoprotein or methyl- LDL. All of these properties are identical with those of the LDL receptor of cultured fibroblasts. We conclude that a deficiency of hepatic and adrenal LDL receptors contributes to the hypercholesterolemia of the WHHL rabbits. Familial hypercholesterolemia (FH) is one of the most common genetic diseases affecting humans. The disorder is caused by a series ofallelic mutations in the gene specifying the cell surface receptor for the plasma cholesterol-transport protein, low density lipoprotein (LDL) (1). Originally demonstrated in cultured skin fibroblasts, the LDL receptor binds LDL and facilitates its internalization and delivery to lysosomes, where the lipoprotein is degraded and its cholesterol is released for cellular use (2). Individuals homozygous for the most frequent mutant allele at the receptor locus (i.e., the receptor-negative allele) produce no functional LDL receptors. As a result, LDL is not degraded at a normal rate, and the lipoprotein accumulates in plasma, eventually depositing its cholesterol in arterial walls to produce atherosclerosis (1). Individuals heterozygous for the receptor-negative allele produce one-half the normal numr of LDL receptors and exhibit a less severe clinical syndrome (1). Attention has now turned to the distribution of LDL receptors in various body tissues and determination of the sites most severely affected by the receptor mutation. Freshly isolated blood mononuclear cells from normal humans express LDL receptors (3). The numr of these receptors is reduced by 5% in FH heterozygotes and by more than 95% in FH homozygotes (4). While these studies indicated that the LDL receptor defect is present in at least one cell type in vivo, they could not approach the question of the distribution of receptors in solid tissues. The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact Recent studies in rats (5-7), mice (7), young dogs (8), and rabbits (9) have shown that membranes from the liver and adrenal gland exhibit a specific binding site that resembles the LDL receptor of cultured fibroblasts and blood mononuclear cells. These binding sites share the following properties: (i) requirement for calcium and inhibition by EDTA; (ii) affinity for lipoproteins that contain apoprotein E greater than the affinity for LDL, which contains apoprotein B; (iii) no binding of methylated LDL (methyl-ldl) or high density lipoprotein (HDL); and (iv) sensitivity to destruction by Pronase. Whether the receptors in the liver or adrenal gland are genetically the same as the LDL receptor of fibroblasts and whether these liver and adrenal receptors are deficient in FH patients is unknown. A way to explore this question has come available as a result of the important discovery by Watana of the WHHL (Watana heritable hyperlipidemic) rabbit, an animal model for FH (1-12). Rabbits homozygous for this single-gene mutation resemble their human counterparts in having plasma cholesterol levels of 5-95 mg/dl (9% of which is contained in LDL) (12), extensive tissue cholesterol deposits (xanthomas), and severe atherosclerosis (11). Tanzawa et al. (12) recently reported that cultured fibroblasts from the homozygous WHHL rabbit lacked LDL receptor activity as determined by studies of binding and degradation of 125I-laled LDL ('25I-LDL) by intact cells. As a result of the receptor defect, LDL did not suppress 3-hydroxy-3-methylglutarvl-coenzyme-A reductase normally, nor did it stimulate cholesteryl ester synthesis normally in these cells (12). In all ofthese respects, the WHHL fibroblasts resembled fibroblasts from patients with receptor-negative homozygous FH (1). In vivo the fractional catabolic rate for intravenously administered 1251-LDL was lowered in the WHHL rabbit (12), as it is in FH patients (1). In the current studies, we sought to determine whether the LDL receptors of adrenal and liver membranes are absent in the WHHL rabbit. METHODS Rabbits. Male New Zealand White rabbits were purchased from Sunny Acres Rabbitry (Tyler, TX). Male Japanese White rabbits were obtained from Chubu Kagaku Shizai (Tokamatsu, Japan). Male homozygous WHHL rabbits were raised in Dallas from a mating pair ofhomozygous WHHL rabbits obtained from Watana (1-12). Animals consumed Purina Rabbit Laboratory Chow and were killed at 3 months of age unless otherwise indicated. Abbreviations: FH, familial hypercholesterolemia; HDL, high density lipoprotein; LDL, low density lipoprotein; /3-VLDL, /-migrating very low density lipoprotein; WHHL rabbit, Watana heritable hyperlipidemic rabbit.

2 Biochemistry: Kita et al. Lipoproteins. Blood was obtained from normal fasting 6- month-old male New Zealand White rabbits. LDL (p, g/ml) and HDL (p, g/ml) were isolated by ultracentrifugation from plasma prepared with the anticoagulant EDTA (9). The LDL was washed by recentrifugation at p 1.5 g/ml. Rabbit LDL isolated at p g/ml or p g/ml gave identical results in the membrane binding assay. a-migrating very low density lipoprotein (3-VLDL) was isolated from male New Zealand White rabbits fed a 2% cholesterol/1% corn oil diet (9). Canine l"i-laled apo-e- HDLC was kindly provided by R. W. Mahley (13). Methyl-LDL was prepared by treatment of human LDL (p, g/ ml) with formaldehyde plus sodium borohydride (14). The concentration of each lipoprotein is given in terms of its protein content. Rabbit LDL and J3-VLDL were radioiodinated with iodine monochloride (15) to specific activities ofabout 7 and 25 cpm/ ng, respectively. For LDL and /-VLDL, averages of 3% and 13% of the radioactivity, respectively, were extractable into chloroform/methanol. NaDodSO4/polyacrylamide gel electrophoresis (16) of rabbit 125I-LDL showed that 97% and 1% ofthe P2I radioactivity was in apoproteins B and E, respectively. 125I-,8-VLDL contained laled bands that corresponded to apoproteins B, E, A-I, A-IV, and C in the ratio of 15:6:2.5:1.5:1. Preparation of Membranes. Rabbit adrenal glands and liver were homogenized, and membranes (fraction sedimenting tween 8 and 1, X g) were prepared as descrid (9). Rabbit fibroblast membranes (fraction sedimenting tween 8 and 1, x g) were prepared as descrid for human fibroblasts (17). The fibroblasts were incubated for 48 hr in lipoprotein-deficient serum prior to harvest (17). Membrane pellets were frozen in liquid nitrogen and stored at - 19 C. On the day of an experiment, the pellets were resuspended in buffer A (5 mm NaCl/1 mm CaClJ2 mm Tris HCl, ph 8) by flushing through a 25-gauge needle. The suspensions were sonicated for 45 sec (5) and diluted with buffer A to a protein concentration of 1 mg/ml. Membranes could refrozen in suspension and resonicated (15 sec) at least once without loss of binding activity. Binding of 125I-Lipoproteins to Membranes. The standard binding assay (5-9) was conducted at ph 8 in 8,ul of buffer B (2 mm NaCl/.63 mm CaCl2/5 mm Tris-HCl/2 mg of bovine serum albumin per ml) containing 1 ug of membrane protein and the indicated amount of l25i-lipoprotein in the absence or presence of EDTA and in the absence or presence of excess unlaled lipoprotein. Tus were incubated for 6 min in an ice/water bath at C, and membrane-bound l25i-lipoprotein was measured by centrifugation at 1, X g in a Beckman Airfuge (5-9). Analysis of Binding Data. EDTA-sensitive binding was calculated by subtracting the amount of 12'I-lipoprotein bound in the presence of EDTA (EDTA-resistant binding) from that bound in the absence of EDTA. The apparent equilibrium dissociation constant (Kd) was determined from plots of the ratio of membrane-bound to free lipoprotein vs. membrane-bound lipoprotein according to the Scatchard method (18). Other Assays. Protein was determined by the method of Lowry et al. (19). Cholesterol content in liver specimens was determined by gas/liquid chromatography (2). RESULTS In confirmation of the data oftanzawa et al. (12), we found that intact fibroblasts from homozygous WHHL rabbits had <5% of normal LDL receptor activity as determined by measurement of binding, uptake, and degradation of 125I-LDL at 37 C (data not shown). WHHL fibroblasts also showed an absence Proc. Natl. Acad. Sci. USA 78 (1981) 2269 of high-affinity binding of l"i-laled canine apo-e-hdlc, a lipoprotein that is obtained from the plasma of cholesterol-fed dogs and that binds to the LDL receptor of normal human and rabbit fibroblasts with higher affinity than does LDL (13). To determine the effects of the WHHL mutation on LDL receptor activity in vivo, we studied the binding of rabbit 125I- LDL to membranes prepared from the liver and adrenal glands of normal and WHHL rabbits. Membranes from normal rabbit livers, when incubated with 125I-LDL at 3 Ag/ml in the presence of calcium, bound 52 ng of lmi-ldl per mg of membrane protein (Fig. 1A). Addition of a 1-fold excess of unlaled LDL reduced the binding to 8 ng/mg. On the other hand, the same amount of unlaled methyl-ldl reduced the binding of 125I-LDL only to 46 ng/mg. EDTA at concentrations of 1 mm or above reduced the binding of lmi-ldl by a maximum of about 6% to the range of 2 ng/mg. EDTA had no effect on the nonspecific binding that was observed in the presence of unlaled LDL. Binding that was observed in the presence of excess EDTA was saturable in that it was reduced further in the presence of unlaled LDL. However, this binding was reduced to an equal degree by unlaled methyl-ldl. The experiment of Fig. 1A indicates that normal rabbit liver membranes bind l"i-ldl at two sites, both of which are susceptible to competition by unlaled LDL. Binding at one site is inhibited by EDTA. This site accounted for 3 ng/mg out of the total of 52 ng/mg bound. Binding to this site was inhibited competitively by native LDL, but not by methyl-ldl. Hereafter, this site is designated as the "EDTA-sensitive site." This EDTA-sensitive site was destroyed by Pronase (15 pug/ml for 1 hr at 37 C). Optimal binding of lmi-ldl occurred over a broad ph range of 7-9 (data not shown). The second site, which accounted for about 14 ng/mg, was susceptible to competition by methyl-ldl as well as by native LDL (Fig. 1A). This site is designated as the "EDTA-resistant site." In addition to these two saturable sites, about 8 ng/mg of the 125I-LDL was bound to a site from which it could not displaced by native LDL. This nonsaturable binding presumably represents nonspecific sticking or trapping of the LDL in the membrane pellet and is not considered further. The significance of the distinction tween the EDTA-sensitive and EDTA-resistant sites came apparent when binding of 125I-LDL to liver membranes from WHHL rabbits was studied. In the absence of EDTA, these membranes bound about 18 ng/mg of protein (Fig. 1B). EDTA at concentrations up to 1 mm had no effect on this binding, indicating that the WHHL membranes were devoid ofdetectable EDTA-sensitive binding "O _ :5 W Q S. _{4 m N~ bl 6Q-A 4hl..B 2 i -, 1f EDTA, mm FIG. 1. Effect of EDTA on binding ofrabbit 125I-LDL to liver membranes from normal (A) and WHHL (B) rabbits. Each tu contained 1,ug ofmembrane protein,.63 mm CaCl2, 125I-LDL at 3,ug/ml, and the indicated concentration of EDTA in the presence of one of the following unlaled lipoproteins: e, none; o, rabbit LDL at 3 jig/ml; or A, human methyl-ldl at 3,ug/ml.

3 227 Biochemistry: Kita et al. sites. However, they possessed about the same numr of EDTA-resistant sites as did membranes from normal rabbits. As in normal rabbits, the EDTA-resistant binding site in the WHHL rabbits was susceptible to competition by methyl-ldl. Fig. 2 shows saturation curves for binding of '25I-LDL to liver membranes from normal and WHHL rabbits. In normal rabbits (Fig. 2A), total binding was inhibited partially by EDTA, as expected from Fig. 1. Unlaled LDL further reduced binding. In WHHL liver membranes, total binding of "I-LDL was lower than normal (Fig. 2B) and was not reduced by EDTA. Excess unlaled LDL reduced this EDTA-resistant binding, as it did in normal membranes. In Fig. 2C, the EDTA-sensitive component of 125I-LDL binding is presented. These data were calculated by subtracting 125I-LDL binding in the presence of EDTA from that in its absence. Normal rabbit liver membranes showed saturable binding of 125I-LDL to the EDTA-sensitive site; no such binding was detectable in WHHL membranes. In contrast, binding to the EDTA-resistant site was the same in normal and WHHL liver membranes (Fig. 2D). Scatchard analysis of 125I-LDL binding to the EDTA-sensitive site in normal liver membranes gave a single line whose slope and intercepts indicated an apparent Kd of 1.4 jig/ml and maximal binding of 39 ng/mg. The amount of EDTA-sensitive binding in WHHL membranes was essentially zero and could not analyzed. The EDTA-resistant site in both normal and WHHL liver membranes gave a single line whose slope and intercepts indicated a Kd of 18 ug/ml and maximal binding of 91 ng/mg. In experiments not shown, we found that human 125I- LDL bound to the EDTA-sensitive site of liver membranes from normal rabbits in a manner similar to rabbit 125I-LDL, with an apparent kd of 1.5 jkg/ml and maximal binding of 49 ng/mg protein. The LDL binding site of rabbit liver membranes also recognizes /3-VLDL, a cholesterol-rich remnant lipoprotein that accumulates in the plasma ofcholesterol-fed rabbits (9). In contrast to rabbit LDL, which is composed almost entirely of apoprotein B, /B-VLDL contains apoprotein E as well as apoproteins B and C (21, 22). When administered intravenously to normal rabbits, 125I-laled f3-vldl is rapidly cleared from the.a a1 a-8 -=,d 6 8,r 2 -O Proc. Natl. Acad. Sci. USA 78 (1981) if EDTA, mm FIG. 3. Effect of EDTA on binding of rabbit 125I-3VLDL to liver membranes from normal (A) and WHHL (B) rabbits. Each tu contained 1,g of membrane protein,.63 mm CaCl2, 125I-,3VLDL at.5 ug/ml, and the indicated concentration of EDTA in the presence of one of the following unlaled lipoproteins: e, none; *, unlaled rabbit 3-VLDL at 61,ug/ml; o, unlaled rabbit LDL at 3,g/ml; or A, unlaled human methyl-ldl at 3 gg/ml. circulation by a high-affinity receptor operating in the liver (9). Binding of 1 I-,-VLDL to normal rabbit liver membranes (Fig. 3A) showed an EDTA-sensitive and an EDTA-resistant component, like that for 125I-LDL. The EDTA-sensitive binding site was inhibited competitively by LDL and f3-vldl, but not by methyl-ldl. WHHL rabbit liver membranes lacked the EDTA-sensitive '25I-3-VLDL binding (Fig. 3B). Binding at the EDTA-resistant site in both normal and WHHL liver membranes was inhibited competitively by LDL as well as by methyl-ldl and f3-vldl (Fig. 3). As with 125I-LDL, saturation curves showed that total binding of '25I-P-VLDL to normal liver membranes was inhibited partially by EDTA (Fig. 4A). In contrast to their total lack of EDTA-sensitive 125I-LDL binding (Fig. 2C), WHHL liver membranes consistently showed detectable but markedly reduced EDTA-sensitive binding of '"I-/3-VLDL (Fig. 4C). The B r.., 4 2 W e -6 NO a8 a., -o 9:1 o Rabbit 1251-LDL,,g protein/ml FIG. 2. Saturation curves for binding of rabbit 1251-LDL to liver membranes from normal and WHHL rabbits. (A and B) Each tu contained 1,g of membrane protein from either normal (A) or WHHL (B) rabbits,.63 mm CaCl2, and the indicated concentration of 125i- LDL in the absence (e) or presence of either 2 mm EDTA (o) or 2 mm EDTA plus unlaled rabbit LDL at 1 mg/ml (A). (C andd) Values for the EDTA-sensitive and EDTA-resistant binding sites, respectively, for normal (o) and WHHL (i) rabbits Rabbit 125I-,8-VLDL, pg protein/ml FIG. 4. Saturation curves for binding of rabbit "'514-VLDL to liver membranes from normal and WHHL rabbits. (A andb) Each tu contained 1 ug of membrane protein from either normal (A) or WHHL (B) rabbits,.63 mm CaC12, and the indicated concentration of '"I-3-VLDL in the absence (.) or presence of either 5 mm EDTA (o) or 5 mm EDTA plus unlaled rabbit f3-vldl at 3,g/ml (A). (C and D) Values for the EDTA-sensitive and EDTA-resistant binding sites, respectively, for normal (n) and WHHL (i) rabbits.

4 EDTA-resistant binding of '25I-P-VLDL was similar in the membranes from the normal and WHHL rabbits (Fig. 4D). Scatchard plots ofthe data in Fig. 4 showed that in the normal liver membranes the EDTA-sensitive site for,3-vldl exhibited an apparent Kd of.67 ug/ml and maximal binding of 147 ng/mg. The amount of EDTA-sensitive binding in the WHHL rabbits was too small for evaluation. In normal rabbits the EDTA-resistant site showed a Kd of 5.6 jig/ml and maximal binding of 18 ng/mg. In WHHL rabbits the Kd was similar (5.3 pug/ml).and the maximal binding was reduced slightly to 116 ng/mg protein. In five separate binding experiments, the average Kd values for 125I-LDL and 251I-f-VLDL binding to the EDTA-sensitive site of normal rabbit liver membranes were 1. and.5 jag/ml, respectively. The average maximal amount of P-VLDL bound at saturation (14 ng/mg) was 4-fold greater than the amount of LDL bound (35 ng/mg). Similar binding parameters were obtained for normal New Zealand White rabbits and normal Japanese White rabbits at 3 and 6 months of age. Binding of 125I-LDL to normal liver membranes was inhibited competitively by unlaled -VLDL and LDL, /-VLDL ing about 8-fold more potent than LDL (Fig. 5A). HDL caused only a slight inhibition of binding at very high concentrations. Similar findings were obtained with regard to competition for 125I-f3-VLDL binding (Fig. SB). When the competition experiments were performed in the presence of EDTA so as to study the EDTA-resistant binding site, unlaled LDL, /3VLDL, and HDL were all effective in inhibiting the binding of 125I-LDL to normal liver membranes (data not shown). In the current experiments, as in prior studies (5, 6, 8, 9), we used the liver membrane fraction that sedimented tween 8 and 1, X g. This fraction exhibits the highest amount of binding per mg of membrane protein, but it contains only 3% of the total binding activity of the whole homogenate (5). In experiments not shown, we measured 125I-LDL binding to crude homogenates and membrane pellets prepared at 5 X g, 8 X g, and 1, X g. No EDTA-sensitive binding was detected in any of these fractions from the WHHL rabbit. In contrast, the EDTA-resistant binding of the various fractions was similar in normal and WHHL rabbits. Adrenal membranes from normal rabbits bound 125I-LDL with high affinity (Fig. 6A). The binding was reduced by EDTA. The combination of EDTA and an excess of unlaled LDL reduced the binding even further, a finding that was similar to that obtained with normal liver membranes. The binding curves in the adrenal membranes were similar to those with membranes. a -6 ~ Biochemistry: Kita et al. r._ o i. _ 1 Ei _ 8! _ 6-6 _ 4 -Io.O- 2 9 I O 1 3 C Competing unlaled lipoprotein,,ug proteirn/ml e FIG. 5. Ability of unlaled rabbit lipoproteins to compete with rabbit 125I-LDL (A) and 125I-f3VLDL (B) for binding to liver membranes from normal rabbits. Each tu contained 1,ug of membrane protein,.63 mm CaC12, and either 125I-LDL at 1.5,ug/ml (A) or 125k.,3VLDL at.5,ug/ml (B) in the presence ofthe indicated concentration of one of the following unlaled lipoproteins: *, none; e, (-VLDL; o, LDL; or A, HDL. Proc. Natl. Acad. Sci. USA 78 (1981) A _-~B 6o.~~~~~~~~~~~~~~~~5 ~15 ~ ~~~~~~~~~~~ "'5I-LDL, ug protein/ml FIG. 6. Saturation curves for binding ofrabbit 125ILDL to adrenal membranes (A) and cultured fibroblasts (B) from normal rabbits. (A) Each tu contained 1.ug ofmembrane CaCl2, protein,.63 mm and the indicated concentration(e) of 125I-LDL in the absence or presence of either 2 mm EDTA or 2 mm EDTA plus unlaled rabbit LDL at 3 ug/ml (A). (B) Fibroblasts were grown in lipoprotein-deficient serum for 48 hr prior to harvest (17). Each tu contained 1 pg of membrane protein,.63 mm CaC12, and the indicated concentration of 125I-LDL in the absence (e) or presence of either 5 mm E:DTA (o) or 5 mm EDTA plus unlaled rabbit LDL at 3 ug/ml (A). isolated from normal rabbit fibroblasts that had en incubated with lipoprotein-deficient serum so as to induce high receptor activity (Fig. 6B). The apparent Kd values for the EDTA-sensitive site of the adrenal and fibroblast membranes were 1 and 2,ug/ml, respectively. For comparative purposes, adrenal membranes were prepared from normal and WHHL rabbits and incubated with 2I- LDL at a concentration of 1.5 ug/ml (Table 1). Adrenal membranes from WHHL rabbits showed a complete absence of EDTA-sensitive LDL binding. In contrast, the small amount of EDTA-resistant binding was identical in the normal and WHHL membranes. DISCUSSION The current studies show that membranes from normal rabbit livers and adrenal glands contain two saturable binding sites, each ofwhich binds both LDL and f3-vldl. Both ofthese sites had a higher affinity for f3-vldl than for LDL. One site resembled the fibroblast LDL receptor in its requirement for calcium and its inhibition by EDTA. This site had an apparent K3 of about 1 tig/ml for rabbit 125I-LDL and.5,ug/ml for rabbit 125[-f3VLDL. It did not bind HDL or methyl-ldl. Thus, the specfficity of the EDTA-sensitive site was similar to that of the fibroblast LDL receptor (2, 14). The EDTA-sensitive site was severely deficient or absent in the livers and adrenals of the WHHL rabbits. The second site in liver membranes also bound both LDL and,b-vldl in a saturable manner. However, its affinity was Table 1. Binding of rabbit 125I-LDL to adrenal membranes from normal and WHHL rabbits '25I-LDL bound to membranes, Addition ng/mg protein to Normal WHHL assay rabbit rabbit None EDTA, 2 mm LDL, 3,ug/ml 7 5 EDTA +LDL 9 6 Each tu contained 6 ug of membrane protein,.63 mm CaCl2, 125I-LDL at 1.5,g/ml, and the indicated addition. 2

5 2272 Biochemistry:, Kita et al. 1/1th to 1/2th for both lipoproteins (kd = 18 and 5.6,Ag/ ml for rabbit '25I-LDL and 1 I-/3-VLDL, respectively) as compared with the first binding site. Binding at the second site was not inhibited by EDTA. In addition to binding LDL and' 3, VLDL, the EDTA-resistant site bound HDL and methyl-ldl. Thus, the specificity of the EDTA-resistant site differed from that of the fibroblast LDL receptor (2, 14). The EDTA-resistant site was present in normal amounts on liver membranes from WHHL rabbits. The EDTA-sensitive receptor must responsible for the removal of large amounts of LDL from plasma, cause when this site is absent in the WHHL rabbit the degradation of LDL in vivo is delayed and LDL accumulates in plasma (12). On the other hand, the functional significance of the EDTA-resistant binding site is not yet clear. This latter binding reaction resembles the EDTA-resistant, saturable binding of 125I-LDL to inert substances such as glass (23). Moreover, the EDTA-resistant binding lacks specificity in that LDL and HDL, which share no apoproteins, are both recognized. The absence of EDTA-sensitive binding sites for f3-vldl, as well as LDL, in the WHHL livers is ofpotential significance.,(3vldl particles are thought to represent remnants of chylomicrons (24). Like chylomicron remnants, f3-vldl contains large amounts of apoprotein E and is taken up with great efficiency by the rabbit liver in vivo (9, 24). More than 8% of intravenously administered 125I-8-VLDL is cleared by the liver within 5 min (9). If the EDTA-sensitive binding sites are responsible for this rapid uptake, then the hepatic uptake of 1"I-,-VLDL should retarded in the WHHL rabbits. In contrast, if rabbits take up chylomicron remnants by a mechanism that differs from the LDL receptor, then the uptake of'25i--vldl should not severely retarded in the WHHL rabbit. Because the LDL receptor in the liver is subject to regulation (8, 9), it is possible that the absence ofthis receptor in the liver of the WHHL rabbit is due to a metabolic suppression rather than to a genetic loss. If the genetic defect involved only extrahepatic LDL receptors and if this led to a high plasma LDL level owing to defective extrahepatic degradation, the high plasma LDL level might in turn suppress hepatic LDL receptors. Indeed, recent studies have shown that the EDTA-sensitive binding sites for 3VLDL and LDL in normal rabbits are reduced by about 6% after 8 days ofcholesterol feeding (9) and. by as much as 8% after one month of cholesterol feeding (unpublished observations). One observation argues against metabolic suppression ing responsible for the reduction in hepatic receptors in the liver of WHHL rabbits. In the cholesterolfed rabbit the suppression ofhepatic receptors occurs only after the hepatic content of cholesterol. has risen to extremely high levels [i.e., to >4 pg/mg of protein (9)]. In two homozygous WHHL rabbits studied to date, the hepatic cholesterol content was 15 and 23 pug/mg of-protein at 3 and 6'months of age, respectively. This is similar to the corresponding values in age- Proc. Natl. Acad. Sci. USA 78 (1981) and sex-matched normal rabbits (11 and 17 rag/mg, respectively). Further studies will necessary to determine definitively whether the LDL receptors in rabbit liver are the products of the same gene as the extrahepatic LDL receptors and thus genetically absent in the WHHL rabbits, or whether these receptors are the product ofa different gene and have en suppressed owing to the high plasma LDL level that results from a defect in receptor-mediated degradation of LDL in extrahepatic tissues. Richard Gibson, John Jaramillo, and Michael Funk provided excellent technical assistance. This research was supported by grants from the National Institutes of Health (HL-2948) and the Moss Heart Foundation. 1. Brown, M. S. & Goldstein, J. L. (1979) Harvey Lect. 73, Goldstein, J. L. & Brown, M. S. (1977) Annu. Rev. Biochem. 46, Ho, Y. K., Brown, M. S., Bilheimer, D. W. & Goldstein, J. L. (1976) J. Clin. Invest. 58, Bilheimer, D. W., Ho, Y. K., Brown, M. S., Anderson, R. G. W. & Goldstein, J. L. (1978) J. Clin. Invest. 61, Kovanen, P. T., Brown, M. S. & Goldstein, J. L. (1979)J. Biol. Chem. 254, Windler, E. E. T., Kovanen, P. T., Chao, Y.-S., Brown, M. S., Havel, R. J. & Goldstein, J. L. (198) J. Biol. Chem. 255, Kovanen, P. T., Goldstein, J. L., Chappell, D. A. & Brown, M. S. (198)J. Biol. Chem. 255, Kovanen, P. T., Bilheimer, D. W., Goldstein, J. L., Jaramillo, J. J. & Brown, M. S. (1981) Proc. Natl. Acad. Sci. USA 78, Kovanen, P. T., Brown, M. S., Basu, S. K., Bilheimer, D. W. & Goldstein, J. L. (1981) Proc. Natl. Acad. Sci. USA 78, Kondo, T. & Watana, Y. (1976) Exp. Anim. 24, Watana, Y. (198) Atherosclerosis 36, Tanzawa, K., Shimada, Y., Kuroda, M., Tsujita, Y., Arai, M. & Watana, Y. (198) FEBS Lett. 118, Innerarity, T. L., Pitas, R. E. & Mahley, R. W. (198) Biochemistry 19, Weisgrar, K. H., Innerarity, T. L. & Mahley, R. W. (1978)1. Biol. Chem. 253, Bilheimer, D. W., Eisenrg, S. & Levy, R. I. (1972) Biochim. Biophys. Acta 26, Kovanen, P. T., Schneider, W. J., Hillman, G. M., Goldstein, J. L. & Brown, M. S. (1979) J. Biol. Chem. 254, Basu, S. K., Goldstein, J. L., & Brown, M. S. (1978) J. Biol. Chem. 253, Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, Lowry,. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, Brown, M. S., Faust, J. R. & Goldstein, J. L. (1975)J. Clin. Invest. 55, Malley, R. W., Weisgrar, K. H. & Innerarity, T. (1974) Circ. Res. 35, Goldstein, J. L., Ho, Y. K., Brown, M. S., Innerarity, T. L. & Mahley, R. W. (198)J. Biol. Chem. 255, Dana, S. E., Brown, M. S. & Goldstein, J. L. (1977) Biochem. Biophys. Res. Commun. 74, Ross, A. C. & Zilversmit, D. B. (1977) J. Lipid Res..18,