Receptor-independent low-density lipoprotein catabolism: Evaluation of 2-hydroxyacetaldehyde-treated lipoprotein as a probe for its measurement

This study examines the protein modification procedures available for inhibiting receptor recognition of low-density lipoprotein (LDL). Glycosylation with glucose, idose or ribose blocks the interaction of the lipoprotein with the high-affinity LDL receptor on cultured fibroblast membranes and delays its clearance from the plasma of rabbits. However, the prolonged incubation required in the process also changes the metabolic properties of the lipoprotein. An alternative approach using 2-hydroxyacetaldehyde-treated LDL completely blocks receptor recognition. This modified tracer has the same metabolic properties as the reductively methylated lipoprotein in rabbits and appears to be a suitable probe for the measurement of the receptor-independent LDL catabolic pathway in humans.     

Effect of adeno-associated virus-mediated transfer of low density lipoprotein receptor gene on treatment of hypercholesterolemia

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Comparative binding and degradation of lipoprotein(a) and low density lipoprotein by human monocyte-derived macrophages.

The binding and degradation of equimolar concentrations of lipoprotein(a) (Lp(a)) and low density lipoprotein (LDL) isolated from the same individual were studied in primary cultures of human monocyte-derived macrophages (HMDM). At 4 degrees C, LDL receptor-mediated binding of both Lp(a) and LDL was of low affinity, being 0.8 and 0.23 microM, respectively. Competitive binding studies indicated that the binding of Lp(a) to HMDM was competed 63% by excess LDL. In contrast to the 4 degrees C binding data, the degradation of Lp(a) at 37 degrees C was mainly nonspecific because the amount of Lp(a) processed by the LDL receptor pathway in 5 h was 17% that of LDL. According to pulse-chase experiments, this phenomenon may be accounted for by the facts that less Lp(a) is bound to HMDM at 37 degrees C and that Lp(a) has a lower intrinsic degradation rate and was not due to increased intracellular accumulation or retroendocytosis of the lipoprotein. Degradation of both lipoproteins was primarily lysosomal and only modestly affected by up- or down-regulation of the LDL receptor. The rate of retroendocytosis in HMDM was approximately equal to the degradation rate and appeared to be independent of the type of lipoprotein used, up- or down-regulation of the LDL receptor, or the presence of the lysosomotropic agent chloroquine. Overall, the results indicate that HMDM degrade Lp(a) mainly via a nonspecific pathway with only 25% of total Lp(a) degradation occurring through the LDL receptor pathway. As both 37 degrees C degradation and 4 degrees C binding of LDL are mainly LDL receptor specific, the different metabolic behavior observed at 37 degrees C suggests that Lp(a) undergoes temperature-induced conformational changes on cooling to 4 degrees C that allows better recognition of Lp(a) by the LDL receptor at a temperature lower than the physiological temperature of 37 degrees C. How apo(a) affects these structural changes remains to be established.     

Effects of cholestyramine on receptor-mediated plasma clearance and tissue uptake of human low density lipoproteins in the rabbit

This study examines the effects of cholestyramine (2 g/day) on the plasma clearance and tissue uptake of human low density lipoprotein (LDL) in rabbits. 1,2-Cyclohexanedione modification of human LDL abolishes its recognition by high affinity cell membrane receptors in vitro and delays its plasma clearance in comparison to native LDL. Consequently, the difference between the fractional rates of catabolism of simultaneously injected native and cyclohexanedione-treated LDL is an index of in vivo receptor-mediated clearance of the lipoprotein. When human 125I-LDL and 131I-cyclohexanedione-treated LDL were injected into rabbits, 44% of the lipoprotein was cleared from the plasma by the receptor mechanism. Various tissues were removed from the animals at the end of the turnover study and their relative uptakes of 125I native and 131I-cyclohexanedione-treated LDL were measured. All exhibited receptor activity to some extent, incorporating more native than cyclohexanedione-modified LDL. The greatest receptor activity per g of tissue was found in lymph nodes, spleen, and liver and, in terms of whole organ uptake, the liver played a major role in LDL catabolism. Treatment of the rabbits with cholestyramine lowered the circulating LDL cholesterol level by promoting its clearance (120%, p < 0.001) via the receptor pathway. This was associated with a virtual doubling of receptor-mediated incorporation of the lipoprotein into the liver. These results suggest that the drain which cholestyramine induces in the hepatic cholesterol pool promotes LDL receptor activity in this organ and thereby lowers the level of circulating LDL.     

Increase in hepatic low-density lipoprotein receptor activity during pregnancy in Watanabe heritable hyperlipidemic rabbits; an animal model for familial hypercholesterolemia

In homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, the serum cholesterol level and serum low-density lipoprotein (LDL) level decreased from 562 +/- 76 (mean +/- S.E.) to 144 +/- 34 mg/dl and 410 +/- 56 to 90 +/- 25 mg/dl, respectively, during pregnancy, although the LDL receptor in this rabbit is genetically deficient. When Tyroxapol, which inhibits the degradation of very-low-density lipoprotein (VLDL), as well as Triton WR-1339, was injected into WHHL rabbits, the rate of the increase in serum cholesterol level in pregnant rabbits was not statistically different from that in non-pregnant rabbits. This result implied that the secretion rate of VLDL-cholesterol, the precursor of LDL-cholesterol, did not decrease during pregnancy. The amount of 125I-labeled LDL bound to LDL receptor was increased 1.8-fold in normal rabbits (from 29.3 +/- 4.3 to 52.3 +/- 4.6 ng/mg protein) and 12-fold in WHHL rabbits (from 0.5 +/- 0.2 to 6.0 +/- 0.7 ng/mg protein) during pregnancy. These results suggest that the decrease in serum cholesterol level in WHHL rabbits during pregnancy was associated with an increase in hepatic LDL receptor activity, which plays an important role in the regulation of serum cholesterol level.     

Cellular signalling by lipoprotein receptors

Lipoprotein receptors are commonly thought merely to mediate the internalization of lipoprotein particles or the exchange of lipids at the cell surface. Recent findings have now implicated these multifunctional receptors in cellular signalling mechanisms that extend beyond simple ligand endocytosis. By mediating the cellular uptake of lipophilic vitamins and hormones, megalin, a member of the LDL receptor gene family, regulates critical hormonal and metabolic processes. Other members of the LDL receptor family interact with cytoplasmic adaptor and scaffold proteins, which allows them to transmit signals directly across the plasma membrane of the target cell. This sheds a new light on the emerging roles of lipoprotein receptors in pathologic disease processes such as Alzheimer's disease.     

Binding of low-density lipoprotein and chylomicron remnants to the hepatic low-density lipoprotein receptor of dogs, rats and rabbits demonstrated by ligand blotting. Failure to detect a distinct chylomicron-remnant-binding protein by ligand blotting

In this paper, human low-density lipoprotein (LDL), rat chylomicron remnants and very-low-density lipoproteins of beta-mobility from cholesterol-fed rabbits (beta VLDL) have been shown to bind strongly to a protein present in solubilised liver membranes of rats, rabbits and dogs by ligand blotting with biotin-modified lipoproteins. This binding protein was identified as the LDL-receptor on several criteria. First, binding of the lipoproteins to the receptor was saturable and Ca2+-dependent; secondly, the apparent relative molecular mass of the binding protein (ranging from 128,000 in the rabbit, 145,000 in the rat to 147,000 in the dog) was similar to that of the purified bovine LDL receptor. Finally, binding activity was greatly increased in the livers of rats treated with oestrogen in pharmacological doses and absent from the liver of Watanabe heritable hyperlipidaemic (WHHL) rabbits that have a genetic defect in the LDL receptor. Some binding was also observed to a high-molecular-mass protein present in solubilised liver membranes of rats and rabbits, which, in rabbits at least, shared antigenic determinants with rabbit apoB and was not likely to be related to the LDL receptor as it was present in equal amounts in normal and WHHL rabbits. No evidence was obtained for a specific chylomicron remnant binding protein, distinct from the LDL receptor, whose activity could be detected in solubilised liver membranes by ligand blotting although a variety of solubilisation and fractionation conditions were employed.     

Metabolic evidence for sequestration of low-density lipoprotein in abdominal aorta of normal rabbits

In rabbits, atherosclerosis develops preferentially at branch sites compared with the adjacent uniform aorta. This study investigated the hypothesis that low-density lipoprotein (LDL) is "sequestered" (present in a form that exchanges slowly with plasma LDL) in the aortas of normal rabbits and that more LDL is sequestered at branch sites. Thus 33 normal rabbits were injected with LDL labeled with (125)I-labeled tyramine cellobiose ((125)I-TC) to trace both undegraded LDL and aortic LDL degradation products. For 25 rabbits, LDL was also labeled with (131)I to trace undegraded LDL alone. The time-dependent aortic (125)I-TC and (131)I accumulation was determined from 0.6 to 120 h after injection. Compartmental modeling provided metabolic evidence for sequestration of LDL at the branch (P < 0.01) and uniform (P < 0.005) abdominal aorta. Concentrations of sequestered LDL were 109 +/- 28% higher (P < 0.0005) for branch sites. LDL mean residence time was 23.5 +/- 3.1 h for branch sites, 7.6 +/- 3.5 h longer (P < 0.05) than for the uniform abdominal aorta. Enhanced retention of higher concentrations of sequestered LDL at branch sites could account for the increased susceptibility of these aortic sites to atherosclerosis.     

Malondialdehyde modification of lipoprotein(a) produces avid uptake by human monocyte-macrophages.

Increased plasma levels of the apoB-100-containing lipoprotein(a) (Lp(a)) are associated with an increased risk for atherosclerosis and myocardial infarction, but the mechanisms by which lipoprotein(a) may accelerate these processes remain obscure. In this study we have investigated the impact of the association of apoprotein(a) with the low density lipoprotein (LDL)-like Lp(a) particle upon specificity of receptor recognition after lipoprotein modification by malondialdehyde or transition metal-induced oxidation. We have determined that radioiodination labels both apoprotein components of Lp(a), that malondialdehyde modification produces an anionic lipoprotein comparable to native Lp(a) in Stokes' radius, and that N,N'-disubstituted 1-amino-3-iminopropene derivatives preferentially cross-link apoprotein(a) to apoB-100 protein. Like LDL, native Lp(a) is recognized in human monocyte-macrophages by the LDL receptor. Like LDL, progressive modification of Lp(a) by malondialdehyde abolishes lipoprotein recognition by the LDL receptor and produces uptake and hydrolysis by the scavenger receptor of human monocyte-macrophages. We propose that intimal retention of Lp(a) by extracellular components of the atherosclerotic reaction places the lipoprotein in a microenvironment favoring subsequent peroxidative modification. The chronic production of lipid peroxide-modified Lp(a) together with unmitigated cellular clearance by scavenger receptors may contribute to the accumulation of lipoprotein-derived lipid in macrophage-derived foam cells of the atherosclerotic reaction.     

Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies.

The low density lipoprotein (LDL) receptor has been purified to homogeneity from rabbit liver by a combination of DEAE-Sephacel chromatography, LDL-Sepharose 4B chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The receptor protein had a pI of 4.45 and an Mr of 120 x 10(3)-125 x 10(3) in SDS gels under non-reducing conditions. Incubation of the LDL receptor with neuraminidase decreased its Mr to 105 x 10(3)-110 x 10(3) and increased its pI from 4.45 to 5.25. The purified receptor exhibited all the properties of the membrane-bound receptor including Ca2+-dependent binding of rabbit and human LDL but not of methylated LDL or high density lipoprotein. The amount of LDL receptor present in rabbit liver was measured by a quantitative blotting procedure employing a newly developed rat anti-receptor monoclonal antibody. The affinity and specificity of this monoclonal antibody allowed the quantification of the LDL receptor in detergent extracts of liver homogenate, thus eliminating the loss of receptor associated with the preparation of membrane fractions prior to receptor assay. Livers from adult female New Zealand White rabbits contained 149 +/- 13 ng of LDL receptor/mg of liver protein. Administration of pharmacological doses of 17 alpha-ethinyloestradiol raised the concentration of LDL receptor in liver to 312 +/- 25 ng/mg of liver protein.     

Modification of low density lipoprotein by lipoprotein lipase or hepatic lipase induces enhanced uptake and cholesterol accumulation in cells

Incubation of low density lipoprotein(s) (LDL) with either lipoprotein lipase or hepatic lipase led to modification of the core lipid composition of LDL. Both lipases modified LDL by substantially reducing core triglyceride content without producing marked differences in size, charge, or lipid peroxide content in comparison to native LDL. The triglyceride-depleted forms of LDL that result from treatment with these two enzymes were degraded at approximately twice the rate of native LDL by human monocyte-derived macrophages (HMDM). Lipase-modified LDL degradation was inhibited by chloroquine, suggesting lysosomal involvement in LDL cellular processing. The increased degradation by macrophages of the LDL modified by these lipases was accompanied by enhanced cholesterol esterification rates, as well as by an increase in cellular free and esterified cholesterol content. In a patient with hepatic triglyceride lipase deficiency, degradation of the triglyceride-rich LDL by HMDM was approximately half that of normal LDL. Following in vitro incubation of LDL from this patient with either lipoprotein or hepatic lipase, lipoprotein degradation increased to normal. Several lines of evidence indicate that LDL modified by both lipases were taken up by the LDL receptor and not by the scavenger receptor. 1) The degradation of lipase-modified LDL in nonphagocytic cells (human skin fibroblast and arterial smooth muscle cells) as well as in phagocytic cells (HMDM, J-774, HL-60, and U-937 cell lines) could be dissociated from that of acetylated LDL and was always higher than that of native LDL. A similar pattern was found for cellular cholesterol esterification and cholesterol mass. 2) LDL receptor-negative fibroblasts did not degrade lipase-modified LDL. 3) A monoclonal antibody to the LDL receptor inhibited macrophage degradation of the lipase-modified LDL. 4) Excess amounts of unlabeled LDL competed substantially with 125I-labeled lipase-modified LDL for degradation by both macrophages and fibroblasts. Thus, lipase-modified LDL can cause significant cholesterol accumulation in macrophages even though it is taken up by LDL and not by the scavenger receptor. This effect could possibly be related to the reduced triglyceride content in the core of LDL, which may alter presentation of the LDL receptor-binding domain of apolipoprotein B on the particle surface, thereby leading to increased recognition and cellular uptake via the LDL receptor pathway.     

A novel mechanism by which probucol lowers low density lipoprotein levels demonstrated in the LDL receptor-deficient rabbit

Treatment of low density lipoprotein (LDL) receptor-deficient rabbits (WHHL rabbits) with probucol (1% w/w in a chow diet) lowered their LDL-cholesterol levels by 36%, consonant with the reported effectiveness of the drug in patients deficient in the LDL receptor. Initial studies of LDL fractional catabolic rate (FCR) using 125I-labeled LDL prepared from the serum of untreated WHHL rabbits showed no difference between probucol-treated WHHL rabbits and untreated WHHL rabbits. When, however, 125I-labeled LDL was prepared from donor WHHL rabbits under treatment with probucol and injected back into them, the FCR was found to be increased by about 50% above that measured simultaneously using 131I-labeled LDL prepared from untreated WHHL donors. The labeled LDL from probucol-treated donors was also metabolized more rapidly than that from untreated donors when injected into untreated WHHL rabbits or into untreated wild-type New Zealand White rabbits. Finally, it was shown that rabbit skin fibroblasts in culture degraded labeled LDL prepared from probucol-treated WHHL rabbits more rapidly than that prepared from untreated WHHL donors. This was true both for normal rabbit fibroblasts and also for WHHL skin fibroblasts, although the absolute degradation rates in the latter were, of course, much lower for both forms of LDL. The data indicate that a major mechanism by which probucol lowers LDL levels relates not to changes in the cellular mechanisms for LDL uptake or to changes in LDL production but rather to intrinsic changes in the structure and metabolism of the plasma LDL of the probucol-treated animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of human serum low density lipoprotein by oxidation--characterization and pathophysiological implications

Plasma low density lipoprotein (LDL) can undergo free radical oxidation either catalyzed by divalent cations, such as Cu2+ or Fe2+ or promoted by incubation with cultured cells such as endothelial cells, smooth muscle cells and monocytes. The content of vitamin E, beta-carotene and unsaturated fatty acids is decreased in oxidized LDL. A breakdown of apolipoprotein-B (apoB), hydrolysis of the phospholipids, an increase of thiobarbituric acid reactive substances and the generation of aldehydes also occur. Changes in the ratio of lipid to protein, the electrophoretic mobility and the fluorescent properties have also been reported to accompany oxidation of this lipoprotein. The functional changes of oxidized LDL include its recognition by the scavenger receptor on macrophages, its cytotoxicity especially to proliferating cells, its chemotactic properties with respect to monocyte-macrophages and its regulation of platelet-derived growth factor-like protein (PDGFc) production by endothelial cells. In this article we summarize some of the contributions to this topic and present speculations relating oxidized LDL to pathological conditions such as atherosclerosis.     

Characterization of the estrogen-induced lipoprotein receptor of rat liver

The ethinyl estradiol-induced lipoprotein receptor of rat liver was purified and characterized. Liver membranes were prepared from ethinyl estradiol-treated rats, solubilized, and subjected to DEAE chromatography. A fraction with a high specific activity for low density lipoprotein (LDL) binding was isolated and used to immunize mice. Hybridomas were prepared from their spleen cells, and a clone that secreted an IgG antibody, which cross-reacted with an ethinyl estradiol-induced protein of the same molecular weight as the bovine adrenal LDL receptor, was expanded. This antibody, designated P1B3, immunoprecipitated the induced lipoprotein receptor. P1B3 was used to purify the receptor, and a polyclonal antibody was raised against the pure protein. This antibody recognized a protein of similar molecular weight in rat liver, adult dog liver, and human skin fibroblasts, thus demonstrating that the induced rat lipoprotein receptor was related to the LDL receptor of other species. This receptor is present in normal rat liver, and its content is reduced by feeding an atherogenic diet, but not by feeding a diet containing 0.5% cholesterol. Moreover, cholestyramine supplementation of the diet did not induce the receptor on liver membranes. The polyclonal antibody could prevent the binding of LDL to liver membranes from control or ethinyl estradiol-treated rats. It decreased chylomicron remnant binding to membranes from ethinyl estradiol-treated membranes, but did not affect chylomicron remnant binding to liver membranes of untreated rats, a result compatible with the existence of a distinct receptor for these latter particles. The amount of LDL receptor-independent, specific remnant binding was the same in both control and ethinyl estradiol-treated rats. This is consistent with the concept that the remnant receptor is not regulated by this treatment. Based on the above, we conclude that the ethinyl estradiol-induced lipoprotein receptor of rat liver is biochemically and immunologically similar to the LDL receptor of other species. It is present on the liver of normal adult rats and could account for LDL as well as beta VLDL and HDLc removal. Although it may contribute to chylomicron remnant removal, there appears to be a second unrelated receptor or process which recognizes this lipoprotein.     

Pigeon aortic smooth muscle cells lack a functional low density lipoprotein receptor pathway

The low density lipoprotein (LDL) receptor pathway was studied in aortic smooth muscle cells from atherosclerosis-susceptible White Carneau pigeons and compared with rhesus monkey cells whose LDL receptor pathway has been previously characterized. Pigeon LDL was bound with high affinity in a saturable manner to both pigeon and monkey aortic smooth muscle cells. The kinetics of binding were different, however. LDL binding to pigeon cells exhibited positive cooperativity at low LDL concentrations and at least two classes of binding sites. The same pigeon LDL bound to monkey cells in a manner consistent with a single class of binding sites. Thus, these differences were a property of the pigeon cells and not the result of differences in the LDL. On the average, pigeon cells bound less than 50% the amount of LDL as monkey cells. Despite the surface binding to pigeon cells, little of the LDL was internalized, whereas pigeon LDL was actively internalized by monkey cells. Consistent with this observation, chloroquine and leupeptin had no effect on accumulation of LDL or on LDL degradation by pigeon cells, and incubation of pigeon cells with LDL produced no increase in cellular cholesteryl ester content. Binding of LDL to pigeon cells also differed from that of monkey cells by being unaffected by pretreatment with the proteolytic enzyme pronase, and by not requiring calcium. Binding was not specific for LDL since acetyl-LDL, and to a lesser degree HDL, were able to compete for LDL binding. Incubation with lipoprotein-deficient serum decreased LDL binding in pigeon cells while 25-OH cholesterol caused an increase in binding; both effects are opposite of that seen with the same LDL in mammalian cells. Preincubation with LDL or cholesterol dissolved in ethanol were without effect on LDL binding in pigeon cells, even though they produced significant increases in cellular free cholesterol content. In spite of the failure to internalize LDL, there was considerable degradation of LDL. This apparently occurred on the cell surface rather than by internalization and degradation within the lysosomes as occurs in mammalian cells. The functional significance of LDL binding to pigeon smooth muscle cells is unclear. The characteristics of binding resemble that of a nonspecific lipoprotein receptor referred to by others as the "lipoprotein receptor" or the "EDTA-insensitive receptor." It is apparent, however, that White Carneau pigeon aortic smooth muscle cells lack a functional LDL receptor pathway and in this way resemble cells from human beings with homozygous familial hypercholesterolemia or from Watanabe rabbits.     

Low-density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very-low-density lipoprotein to low-density lipoproteins

Using thrombin and trypsin as probes, we determined: first, that low-density lipoprotein (LDL) receptor binding determinants switch from apolipoprotein (apo) E to apo-B within the very-low-density lipoprotein (VLDL) Sf 20-60 region of the metabolic cascade from VLDL1 (Sf 100-400) of hypertriglyceridemic (HTG) human subjects to LDL. Second, two different conformations of apo-E exist in HTG-VLDL Sf greater than 60, one accessible (greater than or equal to 1 mol/mol of particle) and one inaccessible (1-2 mol/mol) to both thrombin and the LDL receptor; normal VLDL (Sf greater than 60) have only the inaccessible conformation and therefore do not bind to the LDL receptor. Third, thrombin degrades apo-B into large fragments, three of which have electrophoretic mobilities similar to B-48, B-74, and B-26; this, however, has no effect on apo-B-mediated receptor binding. Fibroblast studies showed that thrombin could abolish receptor uptake of HTG-VLDL1 and HTG-VLDL2 (Sf 60-100), had little or no effect on HTG-VLDL3 (Sf 20-60), and no effect on uptake of intermediate-density lipoprotein (IDL) or LDL. Trypsin abolished the binding of HTG-VLDL1 and HTG-VLDL2, reduced that of HTG-VLDL3, but had little to no effect on IDL or LDL binding. Immunochemical techniques revealed that thrombin cleaved some apo-E into the E-22 and E-12 fragments; after trypsin treatment no apo-E was detected in any HTG-lipoprotein. Normal VLDL subclasses contained less apo-E than the corresponding HTG-VLDL subclasses and it was not cleaved by thrombin. Apo-B immunoreactivities of VLDL subclasses were not significantly changed after treatment with thrombin, although thrombin cleaved some of the B-100 of each VLDL subclass, and all apo-B in IDL and LDL, into 4-6 major large fragments. Trypsin converted all of the apo-B of each lipoprotein into smaller fragments (Mr less than 100,000). We conclude that apo-E of the thrombin-accessible conformation mediates uptake of HTG-VLDL1 and HTG-VLDL2 but that apo-B alone is sufficient to mediate receptor binding of IDL and LDL; the switch from apo-E to apo-B as the primary or sufficient binding determinant occurs within the VLDL3 (Sf 20-60) region of the metabolic cascade, where receptor binding first appears in VLDL subclasses from normal subjects.     

Role of a calf thymus preparation in the degradation of native and reductively methylated low density lipoprotein.

1. The clearance of low density lipoprotein (LDL) is mediated by a specific LDL receptor pathway and by an alternative metabolic pathway that is responsible for the receptor-independent LDL catabolism. 2. This alternative catabolism can be studied in vivo using a preparation of chemically modified LDL that are reductively methylated. 3. Recently we showed that a calf thymus protein extract affects the cholesterol metabolism via activation of LDL catabolism. 4. The aim of this study was to investigate whether in vivo the specific LDL receptor pathway and the independent LDL receptor pathway are affected by thymus treatment. 5. The results obtained injecting in rats native and chemically modified 125I-LDL to probe the receptor independent pathway, show that the thymus gland decreases serum cholesterol by activation of the specific LDL receptor pathway. 6. This effect is mainly evident in liver and kidney that represent organs in which the specific LDL receptors are widely present.     

Receptor-mediated catabolism and tissue uptake of human low density lipoprotein in the cholesterol-fed, atherosclerotic rabbit

The LDL receptor pathway, which was delineated in cultured cells, is now known to operate in vivo. In this study we have measured the plasma clearances and tissue uptakes of native and chemically modified (1,2-cyclohexanedione-treated or reductively methylated) LDL in rabbits in order to determine the response of the pathway to a high-cholesterol diet. 1 week on the diet increased circulating LDL and suppressed its receptor-mediated plasma clearance and uptake into all tissues. The fractional catabolic rate of the lipoprotein via the receptor-independent route also fell. Continuation of the feeding program for 12 weeks accentuated these changes and virtually eliminated receptor uptake into all tissues so that the plasma decay curves of native and cyclohexanedione-treated LDL were superimposable. Lipoprotein assimilation by the aorta, however, did not follow this general trend. This tissue, after 12 weeks, was variably infiltrated by atheromatous deposits and the appearance of these lesions was associated with a substantial increase in the relative uptakes of both native and chemically modified (cyclohexanedione-treated and reductively methylated) LDL. We concluded (a) that expansion of tissue cholesterol pools virtually abolishes LDL receptor activity in rabbits; and (b) that LDL assimilation (both apparently receptor-mediated and receptor-independent) paradoxically increases at sites where the aorta is affected by atheromatous lesions.     

Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts.

An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals.     

Phosphorylation of serine 833 in cytoplasmic domain of low density lipoprotein receptor by a high molecular weight enzyme resembling casein kinase II

A soluble protein kinase that phosphorylates the last serine residue (Ser-833) in the cytoplasmic domain of the low density lipoprotein (LDL) receptor was purified about 1300-fold from the cytosol of bovine adrenal cortex. The LDL receptor kinase shared several properties with casein kinase II: use of either GTP or ATP; phosphorylation of a typical casein kinase II recognition sequence in the LDL receptor (a serine followed by a cluster of three negatively charged amino acids); and inhibition by heparin. The LDL receptor kinase differed from classic casein kinase II in the following respects: its apparent molecular weight on gel filtration was approximately 500,000 as opposed to the usual molecular weight of 130,000 for casein kinase II; its affinity for the LDL receptor (apparent Km approximately 5 nM) was much greater than its affinity for casein (approximately 10 microM); and its activity was inhibited by polylysine, an agent that stimulates casein kinase II. The physiologic role of this unusual kinase, if any, is unknown.