Excess Low Density Lipoprotein Research Articles

Physical-chemical interaction of heparin and human plasma low-density lipoproteins.

This study characterizes the physical-chemical interactions of heparin with human plasma low-density lipoproteins (LDL). A high reactive heparin (HRH) specific for the surface determinants of LDL was isolated by chromatography of commercial bovine lung heparin on LDL immobilized to AffiGel-10. HRH was derivatized with fluoresceinamine and repurified by affinity chromatography, and its interaction with LDL in solution was monitored by steady-state fluorescence polarization. Binding of LDL to fluoresceinamine-labeled HRH (FL . HRH) was saturable, reversible, and specific; HRH stoichiometrically displaced FL . HRH from the soluble complex, and acetylation of lysine residues on LDL blocked heparin binding. Titration of FL.HRH with excess LDL yielded soluble complexes with two LDL molecules per heparin chain (Mr 13,000) characterized by an apparent Kd of 1 microM. Titration of LDL with excess HRH resulted in two classes of heparin binding with two and five heparin molecules bound per LDL and apparent Kd values of 1 and 10 microM, respectively. At physiological pH and ionic strength, the soluble HRH-LDL complexes were maximally precipitated with 20-50 mM Ca2+. Insoluble complexes contained 2-10 HRH molecules per LDL with the final product stoichiometry dependent on the ratio of the reactants. The affinity of HRH for LDL in the insoluble complexes was estimated between 1 and 10 microM. Insoluble LDL-heparin complexes were readily dissociated with 1.0 M NaCl, and their formation was prevented by acetylation of the lysine residues on LDL.

Arterial uptake indices of low density lipoproteins after fatty streak formation in Cynomolgus monkeys.

To evaluate changes in arterial wall uptake of an atherogenic lipoprotein in early atherogenesis the uptake of labelled low density lipoprotein was measured in four segments of aorta in Cynomolgus monkeys with diet induced fatty streaks. The influx rate of low density lipoprotein was estimated from a simple kinetic model. The mean influx rate, expressed as plasma equivalents of tracer, was 69 nl.cm-2 per hour in fatty streak monkeys and 28 nl.cm-2 per hour in controls (p less than 0.001). Using additional assumptions, provisional estimates of the rates of apparent efflux were calculated. In fatty streak monkeys the mean apparent efflux rate, relative to the accumulation of tracer in the wall, did not differ significantly from the efflux rate in controls (8.7% per hour vs 7.9% per hour, p greater than 0.05). Thus intimal permeability to low density lipoprotein is increased at the fatty streak stage of lesion formation but apparent efflux may not be changed. The data fit the hypothesis that relative failure of efflux is a major cause of excess low density lipoprotein accumulation in the vessel wall in hypercholesterolaemia.

Specific high affinity binding and degradation of high density lipoproteins by isolated epithelial cells of human small intestine

The interaction of high density lipoproteins (HDL) with isolated epithelial cells of human small intestine (enterocytes) was studied. 125I-HDL 3 (density = 1,125 to 1,126 g/cm 3) exhibit a high-affinity (Kd = 8.3 × 10 −8. Bmax = 886 ng/mg cell protein), saturable and reversible binding to isolated enterocytes. In the presence of excess unlabeled HDL 3, the cell surface-bound 125I-HDL 3 are released into the medium nondegraded. Treatment of cells with pronase does not affect 125I-HDL 3 binding. The binding is accompanied with internalization and degradation of 125I-HDL 3. Chloroquine inhibits the degradation and increases 125I-HDL 3 uptake. A threefold excess of HDL 3 and HDL 2 inhibits the binding and degradation of 125I-HDL 3 by 60%, whereas a 20-fold excess of low density lipoproteins (LDL), only by 20%. HDL 3 (20 to 1,000 μg/mL) stimulates the synthesis of sterols and inhibits sterol ester synthesis in enterocytes. The obtained results make it possible to assume that epithelial cells of the small intestine may participate in the catabolism of HDL in human organism.

Uptake of low density lipoproteins by rat tissues. Special emphasis on the luteinized ovary.

The aim of this study was to determine how luteal cells of the hormone-primed (luteinized) ovary process low density lipoproteins (LDL). Ovary uptake of perfused 125I-LDL was assessed by tissue levels of radioactivity; the distribution of LDL protein in cells was assessed on autoradiograms of the fixed tissue; and the level of stimulation of steroidogenesis, as well as degradation of LDL protein, was assessed on effluent perfusion samples. Human LDL ligand used in these studies was rigorously defined biochemically and physiologically. Homologous (rat) LDL was used as a special ligand control. Other tissue controls included the use of perfused or in vivo-infused luteinized ovaries from animals pretreated to reduce circulating lipoprotein levels, perfused ovaries from a second hormone-primed model, perfused liver from estrogen-treated rats, and isolated and cultured cells from the same ovarian tissues used in the perfusion experiments. The results show that perfused LDL promptly stimulates steroidogenesis. However, the labeled protein moiety of the LDL is not interiorized by the luteal cells, nor is there evidence of LDL protein degradation in the effluent samples. In contrast, internalization of the ligand occurs when luteal cells are incubated with the ligand in vitro. We have observed also that uptake of the 125I-LDL by the ovary can be displaced equally well by excess unlabeled LDL or HDL3. Overall, these experiments suggest that in the intact luteinized ovary, LDL binds to the same sites on the cell surface where HDL "binds," and that LDL cholesterol must be obtained by these steroid hormone-producing cells by a mechanism that does not require internalization of the intact lipoprotein particle.

Nonenzymatic glycosylation products on collagen covalently trap low-density lipoprotein.

Advanced nonenzymatic glycosylation products capable of cross-linking proteins accumulate on collagen in vivo in proportion to time-averaged blood glucose concentration. In this report, we have evaluated the ability of advanced nonenzymatic glycosylation products formed on collagen in vitro to covalently bind low-density lipoprotein (LDL) in a manner similar to that which occurs in human atherosclerotic lesions. At constant LDL concentration, covalent trapping increased linearly with the extent of advanced glycosylation product formation, from 1.42 +/- 0.15 to 4.46 +/- 0.36 micrograms LDL protein/mg collagen. At a constant level of collagen advanced glycosylation product, LDL binding increased as a function of increasing LDL concentration. At an LDL-cholesterol level of 103 mg/dl, covalent trapping of LDL by nonenzymatic glycosylation products on collagen averaged 3.2 times as much as control (P less than 0.01). These data indicate that LDL is bound specifically by reactive products generated by nonenzymatic glycosylation of collagen, and suggest that excessive LDL trapping by hyperglycemia-induced advanced glycosylation endproducts may contribute to the accelerated development of atherosclerosis in patients with diabetes mellitus.

Characterization of high density lipoprotein binding to human adipocyte plasma membranes.

Freshly isolated human adipocytes showed specific uptake of 125I-labeled human high density lipoprotein (HDL2 and HDL3), a portion of which could be released by subsequent incubation with excess unlabeled ligand. To study the mechanism of HDL binding, sucrose gradient-purified adipocyte plasma membranes were incubated with radioiodinated lipoprotein particles under equilibrium conditions in the absence (total binding) or presence (nonspecific binding) of 100-fold excess unlabeled ligand. Specific binding of HDL2 and HDL3, calculated by subtracting nonspecific from total binding, was Ca++ independent, unaffected by EDTA, and not abolished by pronase treatment of the membranes. Modification of HDL3 by reductive methylation or cyclohexanedione treatment also failed to affect its binding to adipocyte plasma membranes. High salt concentration (200 mM NaCl) inhibited specific binding of HDL2 and HDL3 but had no effect on LDL binding. A significant portion of 125I-HDL2 or 125I-HDL3 binding was consistently inhibited by adding excess unlabeled LDL, but this inhibition was incomplete as compared with a similar molar excess of unlabeled HDL2 or HDL3. The role of apoproteins (apo) in HDL binding to adipocyte membranes was examined by comparing binding of HDL2 and HDL3 isolated from normal, abetalipoproteinemic (abeta) and apo E-deficient (apo E0) plasma. Specific binding was observed with all normal and mutant HDL particles. Furthermore, a significant portion (61-78%) of abeta-HDL2, apo E0-HDL2, and apo E0-HDL3 binding was inhibited by adding 100-fold excess of unlabeled low density lipoproteins (LDL). The cross-competition of LDL and HDL binding was confirmed by the ability of normal, abeta, and apo E0-HDL2 to completely inhibit 125I-LDL binding. These data suggest that HDL binding is independent of apo E and that the responsible apoprotein(s) of HDL complete with LDL-apo B for binding to the same or closely related site in the adipocyte plasma membrane. Normal and apo E0-HDL3 binding was also completely inhibited by normal HDL2, which suggested that HDL2 and HDL3 probably bind to the same site. Scatchard analysis of normal HDL2, normal HDL3, and apo E0-HDL3 binding data best fitted a one-component binding profile with similar equilibrium dissociation constants (40-96 nM). HDL3 binding was found to be effectively inhibited by anti-human apo AI or anti-human apo AII, but not by anti-human apo B antisera. This binding was also unaffected by monoclonal anti-human apo B or E antibodies known to inhibit binding of apo B or apo E containing lipoprotein to the LDL receptor of cultured fibroblasts. These findings, taken together, suggest that human fat cells possess HDL binding sites with apo AI and /or apo AII specificity. The significant but partial inhibition of HDL2 and HDL3 binding by LDL along with the complete inhibition of LDL binding by HDL2 and HDL3 tends to exclude a single binding site that interacts both lipoproteins and favors the interpretation that LDL and HDL particles bind to multiple recognition sites or to different conformation of the same lipoprotein binding domain on the human fat cell.

High-affinity association and degradation of 125I-labelled low density lipoproteins by human hepatocytes in primary culture

Catabolism of homologous low density lipoproteins (LDL) was studied in primary culture of human hepatocytes (HH). The cell association and degradation of 125I-labeled LDL ( 125I-LDL) were curvilinear functions of substrate concentration. Cell association and degradation of 125I-LDL were inhibited by excess unlabeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-LDL for cell association and degradation. Preincubation of HH with unlabeled LDL caused a 63% inhibition of the 125I-LDL degradation. It is concluded that the catabolism of LDL by HH proceeds in part through a receptor-mediated pathway similar to that demonstrated on extrahepatic cells. Human hepatocyte LDL Cell association Degradation High affinity association

Uptake of gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein by cultured rat granulosa cells: cellular mechanisms involved in lipoprotein metabolism and their importance to steroidogenesis.

We used electron microscopy, acid hydrolase cytochemistry, and biochemistry to analyze the uptake and metabolism of colloidal gold- and [3H]cholesteryl linoleate-labeled human low density lipoprotein (LDL) by cultured rat granulosa cells. The initial interaction of gold-LDL conjugates with granulosa cells occurred at binding sites diffusely distributed over the plasma membrane. After incubation with ligand in the cold, 99.9% of the conjugates were at the cell surface but less than 4% lay over coated pits. Uptake was specific since it was decreased 93-95% by excess unconjugated LDL and heparin, but only 34-38% by excess unconjugated human high density lipoprotein. LDL uptake was related to granulosa cell differentiation; well-luteinized cells bound 2-3 times as much gold-LDL as did poorly luteinized cells. Ligand internalization was initiated by warming and involved coated pits, coated vesicles, pale multivesicular bodies (MVBs), dense MVBs, and lysosomes. A key event in this process was the translocation of gold-LDL conjugates from the cell periphery to the Golgi zone. This step was carried out by the pale MVB, a prelysosomal compartment that behaves like an endosome. Granulosa cells exposed to LDL labeled with gold and [3H]cholesteryl linoleate converted [3H]sterol to [3H]progestin in a time-dependent manner. This conversion was paralleled by increased gold-labeling of lysosomes and blocked by chloroquine, an inhibitor of lysosomal activity. In brief, granulosa cells deliver LDL to lysosomes by a receptor-mediated mechanism for the hydrolysis of cholesteryl esters. The resulting cholesterol is, in turn, transferred to other cellular compartments, where conversion to steroid occurs. These events comprise the pathway used by steroid-secreting cells to obtain the LDL-cholesterol vital for steroidogenesis.

Regulation of low density lipoprotein receptor function in a human hepatoma cell line.

Low density lipoprotein (LDL) processing was investigated in a human hepatoma-derived cell line, Hep G2. Hep G2 cells bound, internalized and degraded LDL via a saturable, high affinity (Kd approximately 2 X 10(-8)M) pathway similar to that present in other mammalian cells. Although 80% of the uptake and degradation of 125I-LDL was inhibited by 40-fold excess native LDL, the same concentration of methylated LDL, which cannot bind to LDL receptors, had virtually no effect on processing. When added at low concentrations, the lysosomotropic agent, chloroquine, inhibited degradation (I50 approximately 15 microM) without affecting the rate of lipoprotein internalization. Receptor activity was decreased 60% by preincubation of the cells in medium containing a source of cholesterol (LDL or unesterified cholesterol) and increased 1.7-fold by preincubation with compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The Hep G2 cell line may prove a useful system both for the further study of hepatic lipoprotein metabolism and for the evaluation of new antihypercholesterolemic agents.

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4°C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (< 50 pg protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 μM) and colchicine (1 μM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.

Degradation of lipoproteins by human monocyte-derived macrophages. Evidence for two distinct processes for the degradation of abnormal very-low-density lipoprotein from subjects with type III hyperlipidaemia.

Human blood monocyte-derived macrophages that had been cultured in medium containing human serum for 7 days degraded the abnormal very-low-density lipoproteins (VLDL) from the plasma of subjects with type III hyperlipoproteinaemia by two distinct saturable processes. One process was stimulated when cells from normal subjects were preincubated with lipoprotein-free medium, was inhibited by excess unlabelled low-density lipoproteins (LDL) and was absent from cells from subjects with homozygous familial hypercholesterolaemia; on these criteria it was identified as an LDL-receptor-dependent process. Degradation by the second process was of equal magnitude in both cell types and was unaffected by excess unlabelled LDL or acetylated LDL. The activity of this process was reduced when the cells were preincubated in lipoprotein-free medium. The abnormal VLDL from the plasma of cholesterol-fed rabbits were also degraded by this process, which was similar to that in mouse peritoneal macrophages mediated by the receptor for VLDL of beta-electrophoretic mobility [Goldstein, Ho, Brown, Innerarity & Mahley (1980) J. Biol. Chem. 255, 1839-1848].

Interaction of serum lipoproteins with the intestine. Evidence for specific high density lipoprotein-binding sites on isolated rat intestinal mucosal cells

To determine if plasma lipoproteins interact and therefore possibly regulate intestinal lipoprotein metabolism, we investigated the binding, internalization, and degradation of 125I-labeled low density lipoprotein (LDL) and high density lipoprotein (HDL) by enzyme-dispersed rat intestinal mucosal cells. Both human and rat LDL and HDL were bound, internalized, and degraded in a concentration-dependent manner with calculated half-saturation occurring at approximately 30, 35, 35, and 15 micrograms/ml for human LDL, rat LDL, human HDL, and rat HDL, respectively. Isolated brush border membranes had no saturable or specific binding sites for 125I-labeled HDL or LDL, suggesting that lipoproteins may be bound to receptors on lateral or basal membranes of mucosal cells. Compared with HDL, LDL binding was characterized by a large non-specific component. LDL of human and the rat were not only displaced by excess LDL but at least as effectively by excess HDL of their own species. Labeled HDL was displaced by corresponding unlabeled lipoproteins, but human LDL could produce only minor displacement of human HDL3. ApoE-deficient rat HDL, separated by heparin-Sepharose affinity chromatography also showed highly specific saturable binding to intestinal cells. Thus, apparently two different lipoprotein binding sites exist in intestinal plasma membranes, one recognizing B and/or E apoproteins present in human and rat LDL and rat HDL while another binds human HDL3 and apoE-deficient rat HDL which contain A apoproteins as major components.

Binding properties of high-density lipoprotein subfractions and low-density lipoproteins to rabbit hepatocytes

Primary cultures of rabbit hepatocytes which were preincubated for 20 h in a medium containing lipoproteindeficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins 2 (HDL 2). The rate of degradation of HDL 2 was constant in incubations from 3 to 25 h. As the concentration of HDL 2 in the incubation medium was increased, binding reached saturation. At 37° C, half-maximal binding ( K m ) was achieved at a concentration of 7.3 μg of HDL 2 protein/ml (4.06·10 −8 M) and the maximum amount bound was 476 ng of HDL 2 protein/mg of cell protein. At 4°C, HDL 2 had a K m of 18.6 μg protein/ml (1.03·10 −7 M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL 2. Quantification of 125I-labeled HDL 2 binding to a specific receptor (based on incubation of cells at 4°C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45·10 − M. Excess HDL 2 inhibited the binding of both 125I-labeled HDL 2 and 125I-labeled HDL 3, but excess HDL 3 did not affect the binding of 125I-labeled HDL 3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL 2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL 2 and LDL from control and hypercholesterolemic rabbits inhibited the degrActation of 125I-labeled HDL 2, but HDL 3 did not. Treatment of HDL 2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL 2 degradation, suggesting that apolipoprotein E plays a critical role in triggering the degradative process. The effect of incubation with HDL on subsequent 125I-labeled LDL binding was time-dependent: a 20 h preincubation with HDL reduced the amount of 125I-labeled LDL binding by 40%; there was a similar effect on LDL bound in 6 h but not on LDL bound in 3 h. The binding of 125I-labeled LDL to isolated liver cellular membranes demonstrated saturation kinetics at 4°C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL 2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL 3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL 2 or HDL 3 to isolated liver membranes. Hepatocytes incubated with [2- 14C]acetate in the absence of lipoproteins incorporated more label into cellular cholesterol, nonsaponifiable lipids and total cellular lipid than hepatocytes incubated with [2- 14C]acetate in the presence of any lipoprotein fraction. However, the level of 14C-labeled lipids released into the medium was higher in the presence of medium lipoproteins, indicating that the effect of those lipoproteins was on the rate of release of cellular lipids rather than on the rate of synthesis.

Serum factors in experimental nephrosis prevent lymphocyte stimulation.

A factor found in the serum of rats made nephrotic by a single administration of puromycin aminonucleoside (PA) has been shown to inhibit the blastogenic response of normal rat spleen cells when stimulated with either phytohaemagglutinin (PHA) or concanavalin A (Con A). Spleen cells removed from the nephrotic animals also exhibited a reduced capacity to incorporate thymidine when cultured with normal serum. This potent inhibitor was not cytocidal, and inhibition was not due to a lack of components essential for normal growth. Inhibition was not due to the excess of low density lipoproteins found in the nephrotic serum. Increasing mitogen concentrations in the presence of the nephrotic serum had little effect on inducing thymidine uptake by the normal spleen cells, while washing of the cells removed from the nephrotic animals appeared to partially overcome suppression of blastogenesis. The inhibitory factor was found to be less effective if the spleen cells had already been triggered by mitogen to undergo transformation in normal serum before the addition of the nephrotic serum.

Difference in saturable binding of low density lipoprotein to liver membranes from normocholesterolemic subjects and patients with heterozygous familial hypercholesterolemia.

To investigate the possible role of the low density lipoprotein (LDL) receptor in the catabolism of LDL by the human liver, the binding of 125I-labeled LDL to membrane fractions prepared from human liver biopsies was determined. Biopsy samples taken for routine histology were obtained from seven patients with heterozygous familial hypercholesterolemia, one with non-familial hypercholesterolemia, and seven normocholesterolemic subjects. LDL was bound by the membranes from normal subjects in a saturable manner that was inhibited by 56% in the presence of excess LDL. Binding of LDL was also inhibited by modification of the lipoproteins with 1,2-cyclohexanedione. The amount of 125I-labeled LDL bound to membranes from familial hypercholesterolemic livers that could be displaced with excess LDL was significantly less than that bound by normocholesterolemic membranes. These observations suggest that LDL receptors are expressed in normal human liver and are defective in the livers of familial hypercholesterolemic patients.

The interaction of the high-density lipoprotein with cultured cells of bovine vascular endothelium.

Previous studies have shown that high‐density lipoprotein (HDL) is mitogenic for low‐density vascular endo thelial cell cultures. In contrast, low‐density lipoprotein(LDL) at physiological concentration is cytotoxic for this cell type [Tauber et al. (1980) J.Clin.Invest. 66, 696‐709]. In order to relate these observations to the physico‐chemical events occurring after the interaction of these lipoproteins with the cells, we have studied the binding, internalization, and degradation of HDL as a function of cell density and organization and have compared the results to those obtained with LDL.Cultured vascular endothelial cells bind HDL to an extent which, on a molar basis, is similar to that of LDL. Since an 80% loss of the LDL receptor sites in cells that were preexposed to LDL was not associated with a detectable decrease in HDL binding, it is likely that HDL and LDL binding sites exist on the cell surface as distinct entities A 5‐10 fold molar excess of LDL over 125 I‐HDL was almost as effective as a similar excess of unlabeled HDL in reducing the binding of 125, indicationg that LDL can comete with HDL for HDL binding sites. In contrast, 125 I‐LDL binding and uptake were reduced by less than 20% in the presence of a 50‐fold molar excess of unlabeled HDL. Both uptake and degradation of HDL were 10‐20 fold lower than those of LDL. HDL degradation was only slightly or not inhibited by chloroquine at a concentration which almost fully inhibited the degradation of LDL. Confluent and highly orgainized endothelial cell cultures bind HDL and LDL particles to an extent which is half of the amount bound to sparse or subconfluent culture However. The formation of a resting endothelial cell monlayer was associated with only a twofold decrease in the uptake of HDL, as compard to a 10‐20‐fold decrease in the uptake and degradation of LDL. In both sparse and confluent endothelial cultures, uptake via fluid pinocytosis non‐specific adscorptive endocytosis could account for most of the HDL uptake. The different pathways of internalization of HDL when added at high concentration to sparse and actively growing cultures.

Low density lipoprotein receptors and catabolism in primary cultures of rabbit hepatocytes

Rabbit 125I-labelled low density lipoproteins (LDL) were incubated with primary monolayer cultures of rabbit hepatocytes in studies designed to assess the role of liver in LDL catabolism at the cellular level. After hepatocytes were preincubated for 20 h in lipoprotein-free medium, they exhibited time- and concentration-dependent interaction with 125I-labelled LDL at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37° C) incubation with 50 μg LDL protein/ml, hepatocytes bound 400 ng (LDL protein)/mg (cell protein), internalized 280 ng/mg, and degraded 660 ng/mg. Internalization and degradation may be greater than indicated by these values since pulse studies suggested the presence of a deiodinase which attacks cell associated 125I-labelled LDL. The amounts of LDL bound to hepatocytes after 3 h (37° C) were similar to amounts for fibroblasts, but LDL internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled LDL showed the same amount of binding but 1.39 times more internalization and degradation than normolipidemic 125I-labelled LDL. Binding of both control and hyperlipidemic LDL was 3-fold greater at 24 and 42 h than at 0 or 3 h but addition of a 50-fold molar excess of high density lipoproteins (HDL) prevented increased LDL binding with time. Induction of specific high affinity receptors for binding LDL was shown to occur by preincubation of hepatocytes for increasing periods in lipoprotein-free medium and then measuring 125I-labelled LDL binding at 4° C in the presence and absence of excess unlabelled LDL. Finally, hepatocytes took up 40 times more LDL than sucrose or dextran over a 24-h period, an indication that the uptake of LDL occurs via some mechanism other than simple bulk fluid endocytosis.