Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

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.     

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatocytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptor-defective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.     

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.     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

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 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms 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 microM) and colchicine (1 microM) 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.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

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.     

Interaction of canine and swine lipoproteins with the low density lipoprotein receptor of fibroblasts as correlated with heparin/manganese precipitability.

Canine HDL1 and canine and swine HDLc were fractionated into several lipoprotein subpopulations by heparin/manganese precipitation. The ability of the various subfractions of HDL1 or HDLc to compete with 125I-labeled low density lipoproteins (LDL) for binding and degradation by human fibroblasts was compared. The HDL1 or HDLc which precipitated at the lowest concentration of heparin (a concentration which precipitates LDL) were the most effective in competing with 125I-LDL for binding, internalization, and degradation. A striking characteristic of these lipoproteins was the occurrence of a prominence of the arginine-rich apoprotein. The HDL1 or HDLc subfractions which were not precipitated by heparin/managanese lacked detectable arginine-rich apoprotein and did not compete significantly with the 125I-LDL for binding and degradation. Furthermore, the lipid to protein ratio differed in the precipitable and nonprecipitable lipoproteins, with those which were most efficiently bound and degraded containing more cholesterol. Specific lipoprotein interaction with heparin and with the cell surface receptors may occur by a common mechanism; namely, through a positively charged region on the lipoprotein surface which may reside with the B and arginine-rich apoproteins.     

Biochemical and morphological effects of gentamicin in human proximal tubular cells: effects on lipid and lipoprotein metabolism

The effects of gentamicin, an antibiotic used extensively for antimicrobial therapy on the ultrastructure, binding, internalization, degradation, and cholesterol esterification of low-density lipoproteins, were investigated in cultured human proximal tubular cells. Cells were incubated with 0.3 mM gentamicin for 21 days with the following observations. Cells treated with gentamicin contained numerous "myeloid bodies." The binding, internalization, and degradation of 125I-labeled low-density lipoproteins ([125I]LDL) in cells treated with gentamicin was twofold lower than control cells. Pulse-chase experiments demonstrated that gentamicin did not impair the internalization of receptor-bound LDL and their subsequent transport to the lysosome. The relative amounts of [125I]LDL displaced by increasing concentrations of unlabeled LDL were the same in both gentamicin-treated and control cells. This pattern was reflected in the cell surface binding, internalization, and degradation of [125I]LDL. Gentamicin did not alter the degradation of [125I]LDL in cell homogenates at 4.0. The data suggest that gentamicin decreases the receptor-mediated endocytosis of LDL and subsequent lipid metabolism.     

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.     

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.     

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 DLD at concentrations to 1 mg LDL protein/ml and times to 24 h. After a 3 h (37 degrees C) incubation with 50 microgram 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 degrees C) were similar to amounts for fibroblasts, but DLD internalization and degradation were considerably less. Rabbit hyperlipidemic 125I-labelled DLD 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 O 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 degrees 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.     

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 lipoprotein-deficient serum subsequently bound, internalized and degraded 125I-labeled high-density lipoproteins2 (HDL2). The rate of degradation of HDL2 was constant in incubations from 3 to 25 h. As the concentration of HDL2 in the incubation medium was increased, binding reached saturation. At 37 degrees C, half-maximal binding (Km) was achieved at a concentration of 7.3 micrograms of HDL2 protein/ml (4.06 X 10(-8)M) and the maximum amount bound was 476 ng of HDL2 protein/mg of cell protein. At 4 degrees C, HDL2 had a Km of 18.6 micrograms protein/ml (1.03 X 10(-7)M). Unlabeled low-density lipoproteins (LDL) inhibited only at low concentrations of 125I-labeled HDL2. Quantification of 125I-labeled HDL2 binding to a specific receptor (based on incubation of cells at 4 degrees C with and without a 50-fold excess of unlabeled HDL) yielded a dissociation constant of 1.45 X 10(-7)M. Excess HDL2 inhibited the binding of both 125I-labeled HDL2 and 125I-labeled HDL3, but excess HDL3 did not affect the binding of 125I-labeled HDL3. Preincubation of hepatocytes in the presence of HDL resulted in only a 40% reduction in specific HDL2 receptors, whereas preincubation with LDL largely suppressed LDL receptors. HDL2 and LDL from control and hypercholesterolemic rabbits inhibited the degradation of 125I-labeled HDL2, but HDL3 did not. Treatment of HDL2 and LDL with cyclohexanedione eliminated their capacity to inhibit 125I-labeled HDL2 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 degrees C and was inhibited by EDTA or excess LDL. The binding of 125I-labeled HDL2 was much lower than that of 125I-labeled LDL and was less inhibited by unlabeled lipoproteins. The binding of 125I-labeled HDL3 was not inhibited by any unlabeled lipoproteins. EDTA did not affect the binding of either HDL2 or HDL3 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.     

Rat high density lipoprotein subfraction (HDL3) uptake and catabolism by isolated rat liver parenchymal cells.

Rat liver parenchymal cell binding, uptake, and proteolytic degradation of rat 125I-labeled high density lipoprotein (HDL) subfraction, HDL3 (1.10 less than d less than 1.210 g/ml), in which apo-A-I is the major polypeptide, were investigated. Structural and metabolic integrity of the isolated cells was verified by trypan blue exclusion, low lactic dehydrogenase leakage, expected morphology, and gluconeogenesis from lactate and pyruvate. 125I-labeled HDL3 was incubated with 10 X 10(6) cells at 37 degrees and 4 degrees in albumin and Krebs-Henseleit bicarbonate buffer, pH 7.4. Binding and uptake were determined by radioactivity in washed cells. Proteolytic degradation was determined by trichloroacetic acid-soluble radioactivity in the incubation medium. At 37 degrees, maximum HDL3 binding (Bmax) and uptake occurred at 30 min with a Bmax of 31 ng/mg dry weight of cells. The apparent dissociation constant of the HDL3 receptor system (Kd) was 60 X 10(-8) M, based on Mr = 28,000 of apo-A-I, the predominant rat HDL3 protein. Proteolytic degradation showed a 15-min lag and then constant proteolysis. After 2 hours 5.8% of incubated 125I-labeled HDL3 was degraded. Sixty per cent of cell radioactivity at 37 degrees was trypsin-releasable. At 37 degrees, 125I-labeled HDL3 was incubated with cells in the presence of varying concentrations of native (cold) HDL3, very low density lipoproteins, and low density lipoproteins. Incubation with native HDL3 resulted in greatest inhibition of 125I-labeled HDL3 binding, uptake, and proteolytic degradation. When 125I-labeled HDL3 was preincubated with increasing amounts of HDL3 antiserum, binding and uptake by cells were decreased to complete inhibition. Cell binding, uptake, and proteolytic degradation of 125I-labeled HDL3 were markedly diminished at 4 degrees. Less than 1 mM chloroquine enhanced 125I-labeled HDL3 proteolysis but at 5 mM or greater, chloroquine inhibited proteolysis with 125I-labeled HDL3 accumulation in cells. L-[U-14C]Lysine-labeled HDL3 was bound, taken up, and degraded by cells as effectively as 125I-labeled HDL3. These data suggest that liver cell binding, uptake, and proteolytic degradation of rat HDL3 are actively performed and linked in the sequence:binding, then uptake, and finally proteolytic degradation. Furthermore, there may be a specific HDL3 (lipoprotein A) receptor of recognition site(s) on the plasma membrane. Finally, our data further support our previous reports of the important role of liver lysosomes in proteolytic degradation of HDL3.     

Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.     

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.     

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins 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.