Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.     

UDPgalactose:glucosylceramide beta 1----4-galactosyltransferase activity in human proximal tubular cells from normal and familial hypercholesterolemic homozygotes

The activity of a galactosyltransferase (GalT-2) that catalyzes the transfer of galactose from uridinediphosphogalactose to glucosylceramide in cultured normal human proximal tubular (PT) cells was characterized with respect to substrate saturation and metal ion requirements. Using a membrane-bound enzyme source, optimum activity was obtained in the presence of 1.0 mM Mn2+/Mg2+ (1:1) and a detergent mixture, Triton X-100/Cutscum (1:2, v/v), 0.1 mg/ml. The apparent Km values for glucosylceramide and UDP[14C]galactose were 3 microM and 0.5 microM, respectively. The Vmax values for glucosylceramide and UDP[U-14C]galactose were 0.12 nmol/mg protein per 2 h and 173 nmol/mg protein per 2 h, respectively. The purified 14C-labelled product comigrated with authentic lactosylceramide (LacCer) on TLC and HPLC analysis. The presence of a terminal beta-[14C]galactosyl group in the enzymatic product was proved by its cleavage (79%) by beta-galactosidase. Following the development of optimal assay conditions in normal PT cells, GalT-2 activity was next measured in urinary PT cells from homozygous familial hypercholesterolemic (FH) patients previously shown to accumulate large amounts of lactosylceramide. Urinary PT cells from familial hypercholesterolemic homozygous patients contained 35% higher GalT-2 activity as compared to control cells. We speculate that elevated GalT-2 activity may contribute to the storage of LacCer in FH-PT cells.     

Degradation by cultured monocyte-derived macrophages from normal and familial hypercholesterolaemic subjects of modified and unmodified low-density lipoproteins.

Human blood monocyte-derived macrophages that had been cultured for 7 days in the presence of 20% whole human serum exhibited saturable degradation of low-density lipoprotein (LDL). This degradation could be abolished by pre-incubating the cells with a high concentration of LDL in the medium and increased by pre-incubating the cells in medium containing lipoprotein-deficient serum. Cells obtained from the blood of homozygous familial-hypercholesterolaemic (FH) patients only exhibited a low rate of non-saturable degradation of LDL, even when pre-incubated without lipoproteins. Thus the saturable degradation of LDL by normal cells was mediated by the LDL receptors that are defective in FH patients and little LDL was taken up and degraded through any of the other endocytotic processes present in macrophages. Degradation by normal cells pre-incubated with lipoprotein-deficient serum had a higher apparent affinity for LDL than that of cells maintained in whole serum, which suggests that incubation with lipoprotein-deficient serum may not only induce the formation of LDL receptors but may also have a direct effect on the receptors themselves. Monocyte-derived macrophages from normal and FH subjects showed similar saturable degradation of acetylated LDL and also of LDL complexed with dextran sulphate. Maximal degradation of each was in the same range as the degradation of unmodified LDL by normal cells, and was not increased if the cells were pre-incubated with lipoprotein-deficient serum.     

Low density lipoprotein receptor activity in homozygous familial hypercholesterolemia fibroblasts

We have identified specific low affinity low density lipoprotein (LDL) receptors in skin fibroblasts from two patients previously classified as having LDL receptor-negative homozygous familial hypercholesterolemia (FHC). Km and maximum capacity for cell-associated and degraded 125I-LDL were determined by two independent methods, a traditional technique in which increasing amounts of 125I-LDL were added until receptor saturation was achieved and a new technique in which the displacement of a small amount of 125I-LDL tracer was observed during the addition of variable amounts of unlabeled LDL. The Km for specific cell-associated 125I-LDL in FHC cells was 3.5-7.3 times that of normal cells and the maximum specific capacity was reduced to 11% of normal. Thus, some FHC cells have reduced affinity as well as reduced capacity for LDL. The FHC cell receptors share many but not all properties of the normal skin fibroblast LDL receptor. Specific degradation of bound 125I-LDL occurred concomitantly with LDL binding and was greatly reduced by the addition of chloroquine, an inhibitor of lysosomal function. Preincubation of FHC cells with cholesterol or LDL resulted in significant suppression of receptor function. Modification of lysine residues of LDL abolished receptor activity in both normal and FHC cells. Treatment of FHC cells with compactin, a cholesterol synthesis inhibitor, resulted in significant increases in specific 125I-LDL binding and degradation compared to FHC cells without compactin treatment. Normal cells also showed increases in 125I-LDL binding and degradation with compactin treatment, but the mean percentage increase in specific 125I-LDL degradation was significantly greater in FHC cells (strain GM 2000, 160 +/- 18%) than in normal cells (29 +/- 8%).     

Regulation of glycosphingolipid glycosyltransferase by low density lipoprotein receptors in cultured human proximal tubular cells

We have shown previously that low density lipoproteins (LDL) suppressed the synthesis of lactosylceramide in normal human proximal tubular cells, but stimulated such synthesis in proximal tubular cells from LDL receptor negative subjects (Chatterjee, S., Clarke, K., and Kwiterovich, P.O., Jr. (1986) J. Biol. Chem. 261, 13474-13479). To understand the mechanism(s) of this effect of LDL, we have studied here the effects of LDL on the activity of UDP-GalCer:beta-galactosyltransferase (GalT-2). Maximum suppression (70-80%) of the activity of GalT-2 in normal proximal tubular cells at 37 degrees C occurred at a LDL concentration of 25 micrograms/ml medium. Such suppression was not observed either when the cells were incubated with LDL at 4 degrees C, or when the cells were preincubated with leupeptin, followed by incubation with LDL at 37 degrees C. High density lipoproteins and fetuin did not suppress the activity of GalT-2 in normal proximal tubular cells. In contrast LDL modified by reductive methylation (M-LDL, 100 micrograms/ml) stimulated the activity of GalT-2, approximately 3-fold. The effects of LDL and M-LDL were not related to their glycosphingolipid content. Much less suppression and stimulation of the activity of GalT-2 in proximal tubular cells by LDL and M-LDL, respectively, was found in normal human skin fibroblasts, Chinese hamster ovary cells, and bovine smooth muscle cells, suggesting that the LDL-mediated effect may be tissue-specific. In cells grown to very high density, the activity of the LDL receptor is decreased, and there was less suppression of GalT-2 activity by LDL. In normal proximal tubular cells, LDL stimulated the activity of UDP-Gal:LacCer, alpha-galactosyltransferase activity, UDP-Gal:LcOse3Cer, beta-galactosyltransferase, and CMP-NeuAc:LacCer,alpha-sialyltransferase activity but did not alter the activity of sulfotransferase. In conclusion, LDL that entered the normal proximal tubular cells via the LDL receptor-mediated pathway decreased GalT-2 activity, an effect that was dependent upon the binding, internalization, and degradation of receptor-bound LDL. In contrast LDL that entered normal or LDL receptor-negative proximal tubular cells via an LDL receptor-independent pathway failed to suppress GalT-2 activity, and led to a stimulation of LacCer synthesis.     

Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver.

Newly absorbed retinol is transported in association with chylomicrons and their remnants. In addition, after intake of high doses of retinol, significant amounts are also found in low-density lipoprotein (LDL). As both chylomicron remnants and LDL may be taken up by cells via the LDL receptor, and retinoids inhibit proliferation of some leukaemic cells, we have studied the uptake of retinol in leukaemic cells via the LDL-receptor pathway. HL-60 cells contain saturable binding sites for LDL. The binding of LDL to its receptor has a dissociation constant of about 3.2 x 10(-9) M, and the number of receptors per cell was calculated to be about 2700. Uptake of 125I-LDL by HL-60 cells was increased 2-fold by preincubating the cells with mevinolin. The presence of specific receptors for LDL on HL-60 cells was further confirmed by the finding that exogenous LDL cholesterol was able to up-regulate the ACAT (acyl-CoA: cholesterol acyltransferase) activity of HL-60 cells. We then tested the uptake of retinyl ester in leukaemic cells via the LDL-receptor pathway. HL-60 cells were incubated with LDL or chylomicron remnants labelled with [3H]retinyl palmitate. Uptake of retinyl ester associated with both LDL and chylomicron remnants was observed. Furthermore, the presence of excess LDL decreased the uptake by 75-100%, supporting the hypothesis that the uptake of retinyl ester occurred via the LDL receptor in HL-60 cells.     

Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.     

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the "apparent" Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column. Uptake of 125I-LDL by confluent monolayers of human skin fibroblasts was not changed by incubation with FM or by incubation with Hep-G2 conditioned medium. Taken together, these data demonstrate that LDL receptor activity in Hep-G2 cells is stimulated by a serum component. Furthermore, this serum factor shows some specificity for the LDL receptor pathway in liver-derived Hep-G2 cells.     

Separation of human urinary proximal tubular cells from familial hypercholesterolemic homozygotes by Ficoll gradient centrifugation. Morphological and biochemical characteristics

The isolation of renal proximal tubular (PT) cells from the fresh urinary sediment ( UrS ) of familial hypercholesterolemic (FH) homozygotes using discontinuous Ficoll gradient centrifugation was studied. For comparative purposes, cultured cells derived from normal human UrS or kidney were also studied. Unfractionated PT cells were nucleated and 90-99% of the cells excluded trypan blue. Both the unfractionated and fractionated cultured normal PT cells contained numerous empty cytoplasmic vesicles. In contrast, similar preparations from the UrS of FH homozygotes contained membrane-enclosed cytoplasmic vesicles that stained with the Papanicolaou (Pap) reagent and were strongly positive with a fluorescein-labeled antibody against lactosylceramide. The PT cells of FH homozygotes contained 2.0 to 2.5 fold higher activity of gamma-glutamyltransferase and alkaline phosphatase, respectively, than the unfractionated UrS cells. We conclude that human PT cells can be separated from other UrS cells by ficoll gradient centrifugation, and that most, if not all of the LacCer present in the UrS of FH homozygotes is associated with the PT cells. Purified PT cells should provide a useful model to test the biochemical mechanisms of LacCer accumulation in FH.     

A simple binding assay for the determination of low-density lipoprotein receptors in cell homogenates

A convenient binding assay has been developed for the determination of low-density lipoprotein (LDL) receptors in homogenates of cultured and freshly-isolated normal and malignant human cells. Cell homogenates were incubated with 125I-labeled LDL and the ligand bound to the homogenate particulates was separated from the unbound ligand by filtration. When the particulates of the homogenates were subsequently incubated with heparin, a fraction of the bound 125I-LDL was released. Previous studies on intact cells have shown that heparin exclusively releases LDL bound to its cell surface receptor. The heparin-sensitive binding of 125I-LDL to cell homogenate particulates represents LDL bound to its cell surface receptor as judged from the following criteria: (a) it was quantitatively similar to the heparin-sensitive binding of 125I-LDL to intact cells, (b) it showed a direct correlation to the receptor-mediated degradation of 125I-LDL by intact cells, (c) no heparin-sensitive binding could be detected in homogenates prepared from normal erythrocytes or from cultured fibroblasts from a patient with homozygous familial hypercholesterolemia (two types of cell lacking LDL receptors), (d) it was dependent on calcium and inhibited by EDTA, (e) it was susceptible to treatment with pronase, and (f) it was heat-labile. The assay developed should be of value in determining the number of LDL receptors in tissues, since it is far less time-consuming and requires less material than currently available methods.     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

The effect of concanavalin A-stimulated mononuclear cells on low density lipoprotein receptor activity of cultured fibroblasts

Secretory products of freshly isolated human circulating blood cells such as platelets, monocytes, and B lymphocytes, but not T lymphocytes, have previously been shown to enhance low density lipoprotein (LDL) metabolism by arterial wall cells. This study was undertaken to evaluate how secretory factor(s) from mononuclear cells that had been stimulated by concanavalin A (Con A) alters LDL receptor activity by cultured human skin fibroblasts. Conditioned medium from Con A-stimulated mononuclear cells produced an increase of 125I-LDL degradation accompanied by increased thymidine incorporation into DNA. The effect of conditioned medium from the Con A-stimulated mononuclear cells was mediated by the LDL receptor pathway. Degradation of HDL and methylated LDL, neither of which is taken up by the classical LDL receptor pathway, was not affected. The conditioned medium from these Con A-stimulated cells also failed to stimulate fluid pinocytosis, as measured by the uptake of [14C]sucrose. Some strains of fibroblasts, deficient in LDL receptors, responded to the conditioned medium from the Con A-stimulated mononuclear cells by increasing the very small amounts of LDL degraded by these cells. Fibroblasts from other homozygous familial hypercholesterolemic cell strains were unresponsive, however. The effect on LDL receptors was characterized by an increase in LDL receptor number without a change in the affinity of LDL for its receptor. Thus stimulated mononuclear cells secrete mitogens that also stimulate LDL receptor activity in human skin fibroblasts.     

Binding, internalization, and degradation of high density lipoprotein by cultured normal human fibroblasts.

Comparative studies were made of the metabolism of plasma high density lipoprotein (HDL) and low density lipoprotein (LDL) by cultured normal human fibroblasts. On a molar basis, the surface binding of (125)I-HDL was only slightly less than that of (125)I-LDL, whereas the rates of internalization and degradation of (125)I-HDL were very low relative to those of (125)I-LDL. The relationships of internalization and degradation to binding suggested the presence of a saturable uptake mechanism for LDL functionally related to high-affinity binding. This was confirmed by the finding that the total uptake of (125)I-LDL (internalized plus degraded) at 5 micro g LDL protein/ml was 100-fold greater than that attributable to fluid or bulk pinocytosis, quantified with [(14)C]sucrose, and 10-fold greater than that attributable to the sum of fluid endocytosis and adsorptive endocytosis. In contrast, (125)I-HDL uptake could be almost completely accounted for by the uptake of medium during pinocytosis and by invagination of surface membrane (bearing bound lipoprotein) during pinocytosis. These findings imply that, at most, only a small fraction of bound HDL binds to the high-affinity LDL receptor and/or that HDL binding there is internalized very slowly. The rate of (125)I-HDL degradation by cultured fibroblasts (per unit cell mass) exceeded an estimate of the turnover rate of HDL in vivo, suggesting that peripheral tissues may contribute to HDL catabolism. In accordance with their differing rates of uptake and cholesterol content, LDL increased the cholesterol content of fibroblasts and selectively inhibited sterol biosynthesis, whereas HDL had neither effect.     

Radiolabeled sucrose covalently linked to protein. A device for quantifying degradation of plasma proteins catabolized by lysosomal mechanisms.

A general method is described for assessing the degradation of proteins metabolized by lysosomal mechanisms. The method depends on the lysosomal trapping of sucrose which is covalently bound to the protein of interest and thus caried into the lysosome with it. The validity of the method was demonstrated in vitro in studies of the catabolism of low density lipoprotein (LDL) by cultured fibroblasts. Sucrose-derivatized LDL was not distinguished from 125I-LDL by fibroblasts, either in terms of surface binding or rate of uptake. 14C from [14C]sucrose-LDL accumulated in the cells as predicted; very little appeared in the trichloroacetic acid-soluble fraction of the medium (2% of total uptake). 14C-labeled metabolites in the cells (modal apparent Mr = 1000-2000) were separated from undegraded LDL by gel filtration. LDL degradation calculated from the 14C metabolites accumulating intracellularly was in excellent agreement with that calculated from paired studies using 125I-LDL. Finally, the validity of the method was demonstrated in vivo using asialofetuin, a protein previously shown to be selectively taken up and degraded by the liver. In principle, the method described should be applicable to the study of the sites of degradation of any of the plasma proteins.     

Analysis of low-density lipoprotein catabolism by primary cultures of hepatic cells from normal and low-density lipoprotein receptor knockout mice

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that (125)I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K(d)) (31.2 and 22.9 microg LDL-protein/ml, respectively). The maximal binding capacity (B(max)) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of (125)I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. (125)I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of (125)I-LDL and (3)H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.     

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.     

Processing and characterization of the low density lipoprotein receptor in the human colonic carcinoma cell subclone HT29-18: A potential pathway for delivering therapeutic drugs and genes

Low density lipoprotein (LDL) processing has been investigated in the subcloned human colonic carcinoma cell line HT29-18. LDL binding at 4°C was a saturable process in relation to time and LDL concentration. The Kd for LDL binding was 11 g/ml. ApoE-free HDL₃ or acetylated LDL did not significantly compete with¹²⁵I-LDL binding, up to 500 g/ml.¹²⁵I-LDL binding was decreased by 70% in HT29-18 cells preincubated for 24 hours in culture medium containing 100 g/ml unlabelled LDL. Ligand blotting studies performed on HT29-18 homogenates using colloidal gold labelled LDL indicated the presence of one autoradiographic band corresponding to an apparent molecular weight of 130 kDa, which is consistent with the previously reported molecular weight of the LDL receptor in human fibroblasts. At 37°C,¹²⁵I-LDL was actively internalized by HT29-18 cells and lysosomal degradation occurred as demonstrated by the inhibitory effect of chloroquine. LDL uptake and degradation by HT29-18 cells also resulted in a marked decrease in endogenous sterol synthesis. These data demonstrate that the HT29-18 human cancerous intestinal cells are able to specifically bind and internalize LDL, and that LDL processing results in down-regulation of sterol biosynthesis. Thus, intestinal epithelial cells possess specific LDL receptors that can be exploited to accomplish drug delivery and gene transfer via the receptor-mediated endocytosis pathway.Abbreviations HDL, HCL₃ high density lipoprotein - LDL low density lipoprotein     

Reconstitution of LDL with Lipophilic Fluorescein Derivatives: Quantitative Analysis of the Receptor Activity of Human Lymphocytes

Abstract

Low density lipoprotein (LDL), the major cholesterol transport protein in human plasma, consists of an apolar core of cholesteryl esters surrounded by a polar shell containing phospho-lipids, unesterified cholesterol and protein. In the current paper we report the absorption and fluorescence spectra of members of a new class of lipophilic fluorescein derivatives which were designed to be reconstituted into the core of LDL in place of the native cholesteryl esters. One of these derivates, cholesteryl 12–0-[methyl 3–0-methyl-5′(6′)-carboxyfluorescein]ricinoleyl carbonate (MMC) was reconstituted into the core of LDL. The resultant fluorescent reconstituted LDL was used in conjunction with flow cytometry to quantify the LDL receptor activity of fresh blood lymphocytes derived from normal individuals and from patients with the heterozygous and homozygous forms of familial hypercholesterolemia (FH). The LDL receptor activities of the heterozygous and homozygous FH lymphocytes were approximately 37% and 1% of normal, respectively. LDL reconstituted with these lipophilic fluorescein derivatives will be valuable in studying LDL metabolism and may be useful for the diagnosis of FH.     

Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived macrophages.

Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.     

Cellular localization of the receptor-dependent and receptor-independent uptake of human LDL in the liver of normal and 17 alpha-ethinyl estradiol-treated rats

The cellular localization in the liver of the receptor-dependent and -independent uptake of human low density lipoprotein (LDL) in normal and 17 alpha-ethinyl estradiol-treated rats was investigated by the simultaneous in vivo injection of human 131I-LDL and human reductive methylated 125I-LDL. The cells were subsequently isolated by a low temperature method. In untreated rats, after 30 min of in vivo circulation of human LDL, 57% of the receptor-dependent liver-association of human LDL occurs in non-parenchymal cells and 43% in parenchymal cells. Estradiol treatment of rats for 3 days selectively increases the receptor-dependent cell-association of human LDL with hepatocytes (17-fold), while the receptor-dependent cell-association with non-parenchymal cells is not affected.