Lipid metabolism in cultured cells. XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic and tumor virus-transformed human fibroblasts.

Serum lipoproteins control cell cholesterol content by regulating its uptake, biosynthesis, and excretion. Monolayers of cultured fibroblasts were used to study interactions with human high density (HDL) and low density (LDL) lipoproteins doubly labeled with [(3)H]cholesterol and (125)I in the apoprotein moiety. In the binding assay for LDL, the absence of specific LDL receptors in type II hypercholesterolemic fibroblasts was confirmed, whereas monolayers of virus-transformed human lung fibroblasts (VA-4) exhibited LDL binding characteristics essentially the same as normal lung fibroblasts. In the studies of HDL binding, specific HDL binding sites were demonstrated in normal and virus-transformed fibroblasts. In addition, type II hypercholesterolemic cells, despite the loss of LDL receptors, retained normal HDL binding sites. No significant competition was displayed between the two lipoprotein classes for their respective binding sites over a 5-fold concentration range. In VA-4 cells, the amount of lipoprotein required to saturate half the receptor sites was 3.5 micro g/ml (9 x 10(-9) M) for LDL and 9.1 micro g/ml (9 x 10(-8) M) for HDL. Pronase treatment reduced LDL binding by more than half but had no effect on HDL binding. Chloroquine, a lysomal enzyme inhibitor, stimulated net LDL uptake 3.5-fold by increasing internalized LDL but had essentially no effect on HDL uptake. Further experiments were conducted using doubly labeled lipoproteins to characterize the interaction of LDL and HDL with cells. While the cholesterol and protein moieties of LDL were incorporated into cells at similar rates, the uptake of the cholesterol moiety of HDL was 5 to 10 times more rapid than that of the protein component. Furthermore, the apoprotein component of LDL is extensively degraded following exposure, whereas the apoprotein moiety of HDL retains its macromolecular chromatographic characteristics. These results indicate that HDL and LDL bind to cultured cells at separate sites and that further processing of the two lipoprotein classes appears to take place by fundamentally different mechanisms.-Wu, J-D., J. Butler, and J. M. Bailey. Lipid metabolism in cultured cells XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic, and tumor virus-transformed human fibroblasts.     

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

Uptake of lipoprotein-associated alpha-tocopherol by primary porcine brain capillary endothelial cells

From the severe neurological syndromes resulting from vitamin E deficiency, it is evident that an adequate supply of the brain with alpha-tocopherol (alphaTocH), the biologically most active member of the vitamin E family, is of utmost importance. However, uptake mechanisms of alphaTocH in cells constituting the blood-brain barrier are obscure. Therefore, we studied the interaction of low (LDL) and high (HDL) density lipoproteins (the major carriers of alphaTocH in the circulation) with monolayers of primary porcine brain capillary endothelial cells (pBCECs) and compared the ability of these two lipoprotein classes to transfer lipoprotein-associated alphaTocH to pBCECs. With regard to potential binding proteins, we could identify the presence of the LDL receptor and a putative HDL3 binding protein with an apparent molecular mass of 100 kDa. At 4 degrees C, pBCECs bound LDL with high affinity (K(D) = 6 nM) and apolipoprotein E-free HDL3 with low affinity (98 nM). The binding capacity was 20,000 (LDL) and 200,000 (HDL3) lipoprotein particles per cell. alphaTocH uptake was approximately threefold higher from HDL3 than from LDL when [14C]alphaTocH-labeled lipoprotein preparations were used. The majority of HDL3-associated alphaTocH was taken up in a lipoprotein particle-independent manner, exceeding HDL3 holoparticle uptake 8- to 20-fold. This uptake route is less important for LDL-associated alphaTocH (alphaTocH uptake approximately 1.5-fold higher than holoparticle uptake). In line with tracer experiments, mass transfer studies with unlabeled lipoproteins revealed that alphaTocH uptake from HDL3 was almost fivefold more efficient than from LDL. Biodiscrimination studies indicated that uptake efficacy for the eight different stereoisomers of synthetic alphaTocH is nearly identical. Our findings indicate that HDL could play a major role in supplying the central nervous system with alphaTocH in vivo.     

Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture.

Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18-24 h incubation in the presence of LDL at 100 microgram LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 microgram LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 microgram LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 microgram/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

Uptake and degradation of high density lipoprotein: comparison of fibroblasts from normal subjects and from homozygous familial hypercholesterolemic subjects.

Fibroblasts cultured from the skin of subjects with homozygous familial hyperlipoproteinemia (HFH) internalize and degrade low density lipoproteins at a much lower rate than do fibroblasts from normal subjects. Evidence has been presented that this reflects the absence from such mutant cells of specialized binding sites with high affinity for low density lipoproteins. The specificity of this membrane defect in familial hypercholesterolemia is further supported by the present studies comparing the metabolism of low density lipoproteins (LDL) and high density lipoproteins (HDL) in normal fibroblasts and in fibroblasts from HFH patients. The surface binding (trypsin-releasable (125)I) of (125)I-labeled LDL by HFH cells was approximately 30% of that by normal cells at a concentration of 5 micro g LDL protein per ml. At the same concentration the internalization (cell-associated (125)I after trypsinization) and degradation (trichloroacetic acid-soluble non-iodide (125)I) of (125)I-labeled LDL were less than 10% of the values obtained with normal cells. In contrast, the binding of (125)I-labeled HDL to HFH cells was actually somewhat greater than that to normal cells. Despite this, the internalization and degradation of (125)I-labeled HDL by HFH cells averaged only 70% of that by normal cells. [(3)H]- or [(14)C]Sucrose uptake, a measure of fluid uptake by pinocytosis, was similar in normal and HFH fibroblasts. These findings are consistent with the proposal that fibroblasts from subjects with HFH lack high-affinity receptors for LDL. These receptors do not play a significant role in HDL binding and uptake. Instead, as previously proposed, HDL appears to bind randomly on the cell surface and its internalization is not facilitated by the specific mechanism that internalizes LDL. The small but significant abnormalities in HDL binding and internalization, however, suggest that there may be additional primary or secondary abnormalities of membrane structure and function in HFH cells. Finally, the observed overall rate of uptake of LDL (that internalized plus that degraded) by HFH fibroblasts was considerably greater than that expected from fluid endocytosis alone. This implies that adsorptive endocytosis, associated with binding to low-affinity sites on the cell surface, may play a significant role in LDL degradation by HFH cells, even though it does not regulate endogenous cholesterol synthesis in these cells.     

Low and high density lipoprotein metabolism in primary cultures of hepatic cells from normal and apolipoprotein E knockout mice.

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE-containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE-free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein-3 (HDL(3)) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL(3) metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (Bmax) of LDL, but not of HDL(3), is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL(3) holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE-HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4-fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB-100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL(3) by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL-CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL(3)-CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL(3) by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL(3) lipoproteins.     

Very low density lipoprotein binding to cultured aortic endothelium

Because of very low density lipoprotein's (VLDL) potential atherogenicity and the demonstration that VLDL can bind to other cells, we examined the interaction of human VLDL with cultured porcine aortic endothelium. The lipoprotein-cell interaction had many properties similar to those seen with the binding of a ligand to a cell surface receptor. It was time and temperature dependent, saturable, and reversible. Scatchard analysis of competition data suggested that there may be more than one class of binding site. The affinity of the low affinity site was similar to that for low density lipoprotein (LDL). Also, the capacity of endothelial cells to bind VLDL was similar to that for LDL, when related to apo B (i.e., particle) concentration. Not only was unlabelled VLDL able to compete for VLDL binding sites, but so was LDL and high density lipoprotein (HDL). The maximal competition either by LDL or by HDL was less than that by VLDL. The maximal competition by HDL was more than by LDL. The VLDL binding was dependent on Ca2+. It was not changed by the content of lipoprotein in the medium in which cells were grown prior to the binding studies. These observations suggest that VLDL binding to endothelial cells is similar in some respects, but not in all, to the binding of LDL. Comparison of the data with endothelial cells to previous data with adipocytes also indicated differences between the interaction of these two cell types with VLDL. It is possible that this binding process may be involved in the formation of atherogenic remnants of triglyceride-rich lipoproteins on the endothelial surface of large blood vessels.     

Androgen and FSH synergistically stimulate lipoprotein degradation and utilization by ovary granulosa cells

Androgen can directly modulate the induction of steroidogenic enzymes by FSH (follicle stimulating hormone) in ovary granulosa cells. In studies of its mechanism of action, we examined the androgen effect on granulosa cell interaction with lipoproteins, the physiologic source of cholesterol. After granulosa cells were cultured for 48 hours with and without androgen and/or FSH, the cells were incubated for 24 hours with ¹²⁵I-lipoproteins [human high density lipoprotein (HDL), rat HDL, or human low density lipoprotein (LDL)]. The media were then analyzed for lipoprotein protein coat degradation products (mainly ¹²⁵I-monoiodotyrosine) and progestin [mainly 20α-dihydroprogesterone (20α-DHP)]. In the absence of FSH and androgen, 2 × 10⁵ granulosa cells degraded basal levels of all three lipoproteins, but produced no measurable 20α-DHP. The addition of 10^?7 M androstenedione (A), testosterone (T), or 5α-dihydrotestosterone (DHT) had no effect on lipoprotein protein degradation or 20α-DHP production. FSH alone stimulated lipoprotein protein degradation by 50 to 300% while the addition of androgen synergistically augmented the FSH-stimulated 20α-DHP production as well as protein coat degradation of all three lipoproteins. DHT and T were both effective, indicating that androgens themselves, and not estrogen products, were responsible for the effect on lipoprotein protein degradation and 20α-DHP production. The addition of a 10-fold excess cyproterone acetate (an anti-androgen) inhibited the effect of T, suggesting that the action of T was mediated by the granulosa cell androgen receptor. Androgen and FSH also synergistically stimulated the production of ³H-progestin when the granulosa cells were incubated with either ³H-cholesterol ester core labeled human HDL or similarly labeled human LDL. This report demonstrates that androgen, in combination with FSH, augments the steroidogenic pathway of the granulosa cell from the degradation of lipoprotein and utilization of the cholesterol ester core, to the production of progestin product.     

Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.     

Low density lipoprotein binding, internalization, and degradation in human adipose cells.

Human adipose tissue derives its cholesterol primarily from circulating lipoproteins. To study fat cell-lipoprotein interactions, low density lipoprotein (LDL) uptake and metabolism were examined using isolated human adipocytes. The 125I-labelled LDL (d = 1.025-1.045) was bound and incorporated by human fat cells in a dose-dependent manner with an apparent Km of 6.9 + 0.9 microgram LDL protein/mL and a Vmax of 15-80 microgram LDL protein/mg lipid per 2 h. In time-course studies, LDL uptake was characterized by rapid initial binding followed by a linear accumulation for at least 4 h. The 125I-labelled LDL degradation products (trichloroacetic acid soluble iodopeptides) accumulated in the incubation medium in a progressive manner with time. Azide and F- inhibited LDL internalization and degradation, suggesting that these processes are energy dependent. Binding and cellular internalization of 125I-labelled LDL lacked lipoprotein class specificity in that excess (25-fold) unlabelled very low density lipoprotein (VLDL) (d less than 1.006) and high density lipoprotein (HDL) (d = 1.075-1.21) inhibited binding and internalization of 125I-labelled LDL. On an equivalent protein basis HDL was the most potent. The 125I-labelled LDL binding to an adipocyte plasma membrane preparation was a saturable process and almost completely abolished by a three- to four-fold greater concentration of HDL. The binding, internalization, and degradation of LDL by human adipocytes resembled that reported by other mesenchymal cells and could account for a significant proportion of in vivo LDL catabolism. It is further suggested that adipose tissue is an important site of LDL and HDL interactions.     

A comparative study of surface binding of human low density and high density lipoproteins to human fibroblasts: regulation by sterols and susceptibility to proteolytic digestion.

Binding of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein (HDL) was determined in cultured human fibroblasts from a normal subject and two subjects with homozygous familial hypercholesterolemia (HFH). Binding was assayed at 0 degree C to minimize the internalization of labeled lipoproteins. The binding of LDL and of HDL were compared following interventions reported to affect LDL binding in normal fibroblast. LDL binding to normal cells increased two to three fold 24 hours after transfer from medium containing whole fetal calf serum to medium containing lipoprotein-deficient fetal calf serum. This increase was completely blocked in the presence of cycloheximide (200 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). This increased capacity of normal fibroblasts to bind LDL could be reduced 70-80% by a subsequent 18-hour incubation with cholesterol (50 microgram/ml) or 7-ketocholesterol (2.5 microgram/ml). In contrast, no significant change in HDL binding to normal fibroblasts was observed after any of these interventions. HFH cells to show any significant change in either LDL binding or HDL binding following these interventions. These results suggest that HDL binding sites on normal fibroblasts are for the most part distinct from LDL binding sites. They also support the conclusion that LDL binding sites on HFH cells are for the most part qualitatively different from those on normal cells.     

Receptors for homologous plasma lipoproteins on a rat hepatoma cell line

Hepatocytes express on their surfaces more than one class of receptors capable of mediating the internalization of lipoproteins. However, relatively little is known about the binding characteristics of hepatic receptors for various lipoproteins, about the regulation of the receptors, and about the consequences for intracellular lipid metabolism of uptake of lipoproteins via different classes of receptors. The aim of the present studies was to characterize the binding and degradation of various lipoproteins and their mutual competition for cellular processing. Since these kinds of studies may be more easily carried out in continuous established hepatoma cell lines than in nondividing primary hepatocyte cultures, we examined the lipoprotein receptor functions of a well differentiated rat hepatoma (H-35). Cells were grown to confluence in Eagle's minimal essential medium in 15% newborn calf serum. Medium then was changed to 15% lipoprotein-deficient serum for 44 hr before experiments. External binding of 125I-labeled rat plasma and intestinal lymph lipoproteins was assessed at 4 degrees C. Cellular uptake and degradation were assessed at 37 degrees C. Lipoproteins were isolated by fixed angle or zonal ultracentrifugation or by heparin affinity column chromatography and characterized as to their lipid and apoprotein compositions. Labeled low density (LDL), high density (HDL2), non-apoE-HDL, very low density lipoproteins (VLDL), and chylomicron remnants (CM-R) each manifested specific and saturable binding and degradation by the hepatoma cells. Competition experiments indicated that separate receptors were present for LDL, HDL2, and CM-R. Most of HDL2 appeared to be bound to the non-apoE-HDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transport of lipids from high and low density lipoproteins via scavenger receptor-BI.

The scavenger receptor-BI (SR-BI) delivers sterols from circulating lipoproteins to tissues, but the relative potency of individual lipoproteins and the transported cholesterol has not been studied in detail. In this study, we used Chinese hamster ovary cells that express recombinant mouse SR-BI but have no functional low density lipoprotein (LDL) receptors (ldlA7-SRBI cells) to compare the fate of lipids transferred from high or low density lipoproteins to cells by SR-BI. HDL and LDL were equally effective in mediating the transfer of [(3)H]cholesterol to cells. Only 5% of the free cholesterol transferred to cells was esterified, in direct contrast to the findings in the cells that express LDL receptors in which 50% of the transported cholesterol was esterified. Almost all the free cholesterol transferred from lipoproteins to cells was rapidly excreted when the ldlA7-SRBI cells were switched to media containing unlabeled lipoproteins. SR-BI expression was associated with an increase in selective cholesteryl ester uptake from both lipoproteins, but HDL was a more effective donor. HDL and LDL were equally effective in delivering cholesterol to the intracellular regulatory pool via SR-BI. These data indicate that SR-BI is able to exchange cholesterol rapidly between lipoproteins and cell membranes and can mediate the uptake of cholesteryl esters from both classes of lipoproteins.     

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.     

Analysis of the binding and association of human intermediate density lipoproteins to HepG2 cells.

The binding of human intermediate density lipoproteins (IDL) to HepG2 cells was studied. We found that human 125I-IDL interact with a binding site of high-affinity (Kd 0.74 micrograms/ml, Bmax 0.049 micrograms/mg cell protein) and a binding site of lower affinity (Kd 86.8 micrograms/ml; Bmax 0.53 micrograms/mg cell protein). The high-affinity binding sites show characteristics of LDL-receptors since they interact with IDL and low-density lipoproteins (LDL) and are calcium dependent. The low-affinity binding sites are calcium-independent and interact with IDL, LDL, high density lipoproteins-3 (HDL3), apolipoprotein (apo) E-liposomes, apoCs-liposomes, apoA-I-liposomes but not with liposomes containing albumin or erythrocyte membrane proteins. Therefore, HepG2 cells have on their surface a binding site that resembles or is identical to the lipoprotein binding site (LBS) that we found on rat liver membranes (Brissette and No?l (1986) J. Biol. Chem. 261, 6847-6852). Internalization, degradation and cholesterol ester selective uptake were determined in the presence or in the absence of a sufficient amount of human HDL3 to abolish the interaction of IDL to the LBS in order to obtain information on the function of this site. Our results suggest that the LBS participates in the internalization of IDL but not in their degradation and that it is responsible for the selective uptake of cholesterol esters of IDL.     

The estradiol-stimulated lipoprotein receptor of rat liver. A binding site that membrane mediates the uptake of rat lipoproteins containing apoproteins B and E

Hepatic catabolism of lipoproteins containing apolipoproteins B or E is enhanced in rats treated with pharmacologic doses of 17 alpha-ethinyl estradiol. Liver membranes prepared from these rats exhibit an increased number of receptor sites that bind 125I-labeled human low density lipoproteins (LDL) in vitro. In the present studies, this estradiol-stimulated hepatic receptor was shown to recognize the following rat lipoproteins: LDL, very low density lipoproteins obtained from liver perfusates (hepatic VLDL), and VLDL-remnants prepared by intravenous injection of hepatic VLDL into functionally eviscerated rats. The receptor also recognized synthetic lamellar complexes of lecithin and rat apoprotein E as well as canine high density lipoproteins containing apoprotein E (apo E-HDLc). It did not recognize human HDL or rat HDL deficient in apoprotein E. Much smaller amounts of this high affinity binding site were also found on liver membranes from untreated rats, the number of such sites increasing more than 10-fold after the animals were treated with estradiol. Each of the rat lipoproteins recognized by this receptor was taken up more rapidly by perfused livers from estrogen-treated rats. In addition, enrichment of hepatic VLDL with C-apoproteins lowered the ability of these lipoproteins to bind to the estradiol-stimulated receptor and diminished their rate of uptake by the perfused liver of estrogen-treated rats, just as it did in normal rats. The current data indicate that under the influence of pharmacologic doses of estradiol the liver of the rat contains increased amounts of a functional lipoprotein receptor that binds lipoproteins containing apoproteins B and E. This hepatic lipoprotein receptor appears to mediate the uptake and degradation of lipoproteins by the normal liver as well as the liver of estradiol-treated rats. The hepatic receptor bears a close functional resemblance to the LDL receptor previously characterized on extrahepatic cells.     

Insulin-secreting beta-cell dysfunction induced by human lipoproteins

Diabetes is associated with significant changes in plasma concentrations of lipoproteins. We tested the hypothesis that lipoproteins modulate the function and survival of insulin-secreting cells. We first detected the presence of several receptors that participate in the binding and processing of plasma lipoproteins and confirmed the internalization of fluorescent low density lipoprotein (LDL) and high density lipoprotein (HDL) particles in insulin-secreting beta-cells. Purified human very low density lipoprotein (VLDL) and LDL particles reduced insulin mRNA levels and beta-cell proliferation and induced a dose-dependent increase in the rate of apoptosis. In mice lacking the LDL receptor, islets showed a dramatic decrease in LDL uptake and were partially resistant to apoptosis caused by LDL. VLDL-induced apoptosis of beta-cells involved caspase-3 cleavage and reduction in the levels of the c-Jun N-terminal kinase-interacting protein-1. In contrast, the proapoptotic signaling of lipoproteins was antagonized by HDL particles or by a small peptide inhibitor of c-Jun N-terminal kinase. The protective effects of HDL were mediated, in part, by inhibition of caspase-3 cleavage and activation of Akt/protein kinase B. In conclusion, human lipoproteins are critical regulators of beta-cell survival and may therefore contribute to the beta-cell dysfunction observed during the development of type 2 diabetes.     

Effect of apoE on triglyceride emulsion interaction with hepatocyte and hepatoma G2 cells

The uptake and internalization of a triglyceride emulsion by rat hepatocytes in culture less than 24 hr was either inhibited or uninfluenced by apoE. ApoE significantly increased the uptake of these emulsions in later cultures. Specific low density lipoprotein (LDL) binding was similar for hepatocyte monolayers prior to and after 24 hr. Rat hepatocytes in culture for 2 days, which were treated with collagenase, detached and then replated within 1 hr and were apoE-responsive in 2 hr. Heparin inhibited the apoE stimulation in both hepatocytes and hepatoma monolayers. Heparin wash of hepatocytes or hepatoma cells incubated with apoE-[14C]triolein emulsions at 4 degrees C resulted in a considerable loss in radiolabeled cell lipid. A similar wash after 37 degrees C incubations produced little loss suggesting internalization. Hepatocytes had lower affinity but similar apoE-emulsion binding capacity compared to hepatoma cells. Triolein emulsions with apoE were significantly more rapidly metabolized by the hepatocyte than unsupplemented emulsions. The apoE-mediated hepatocyte lipid uptake was inhibited by apoC proteins. High molar ratios of free fatty acid/albumin also suppressed hepatocyte apoE-mediated lipid uptake. Both rat high density lipoprotein (HDL) and LDL inhibited with a potency directly related to their content of apoE. Human LDL and HDL without apoE also inhibited the interaction with less potency than the rat lipoproteins. Human HDL inhibition was diminished after removal of apoC proteins.     

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.