Estrogens induce low-density lipoprotein receptor activity and decrease intracellular cholesterol in human hepatoma cell line Hep G2

Administration of estrogens in pharmacologic doses to rats and rabbits induces hepatic low-density lipoprotein (LDL) receptor activity. To determine if estrogens can regulate LDL receptor activity in human cells, 125I-LDL binding and ligand blotting studies were performed with the cell line Hep G2, well-differentiated cells derived from a human hepatoma, and with normal human fibroblasts. Addition of estradiol to Hep G2 cells growing in lipoprotein-deficient medium increased cell surface receptor activity by 141%, whereas fibroblast receptors were slightly reduced. Measurement of LDL internalization and degradation showed that estradiol induced the entire LDL receptor pathway and not simply surface receptors for LDL. Scatchard analysis of specific binding data in Hep G2 cells revealed that increased LDL receptor activity was due to high-affinity binding. When Hep G2 cells were incubated with LDL as well as estradiol, estradiol induction of LDL receptor activity did not occur. Estrogen treatment reduced Hep G2 free cholesterol content by 24% as determined by gas-liquid chromatography but had no significant effect on fibroblast free cholesterol, suggesting that estrogens may induce Hep G2 LDL receptor activity indirectly by lowering intracellular cholesterol. LDL receptor activity in Hep G2 cells grown in the absence of estradiol was resistant to down-regulation by LDL; incubation of cells with LDL for 48 h reduced receptor activity by only 25.8% in Hep G2 cells compared to 80.3% in fibroblasts. The Hep G2 LDL receptor was shown to be biochemically similar to the fibroblast receptor by ligand blotting and immunoblotting with IgG-C7, a monoclonal antibody to the extrahepatic LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The metabolism in vitro of human low-density lipoprotein by the human hepatoma cell line Hep G2.

The human hepatoma cell line Hep G2 was studied with respect to metabolism of human low-density lipoprotein (LDL). The Hep G2 cells bind, take up and degrade human LDL with a high-affinity saturable and with a low-affinity non-saturable component. The high-affinity binding possesses a KD of 25 nM-LDL and a maximal amount of binding of about 70 ng of LDL-apoprotein/mg of cell protein. The high-affinity binding, uptake and degradation of LDL by Hep G2 cells is dependent on the extracellular Ca2+ concentration and is down-regulated by the presence of fairly high concentrations of extracellular LDL. Incubation of the Hep G2 cells with LDL results in suppression of the intracellular cholesterol synthesis. It is concluded that the human hepatoma cell line Hep G2 possesses specific LDL receptors similar to the LDL receptors demonstrated on extrahepatic tissue cells.     

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.     

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.     

Crosstalk of sterol-dependent and non-sterol-dependent signaling in human monocytes after in vitro addition of LDL

The aim of the present study was to investigate low density lipoprotein (LDL)-induced, non-sterol-dependent signaling and its possible role in cholesterol balance. LDL in 10 microg ml(-1) concentration could induce inositol trisphosphate (IP3) and Ca2+ signal generation through a pertussis toxin (PT) sensitive G protein in human monocytes. The increase in [Ca2+]i was derived from the intracellular pools. LDL also induced activation and translocation of protein kinase C (PKC) into the cell membrane, by processes, which were significantly inhibited in the first 20 min by preincubation with PT and PKC-inhibitor H-7. The PKC-activating phorbol-12-myristate-13-acetate (PMA), differently from LDL, enhanced the LDL-receptor (LDL-R)-mediated binding and degradation of [125I]LDL, but inhibited endogenous cholesterol synthesis, and both effects were inhibited by H-7. The LDL-induced inhibition of binding and degradation of [125I]LDL was not affected by H-7, whereas decreased cholesterol synthesis was counteracted by H-7. These results suggest the existence of a non-sterol-dependent signal pathway of LDL-Rs, by which endogenous cholesterol synthesis, that is, the [14C]acetate incorporation, is regulated through PKC activation.     

Taurine enhances low density lipoprotein binding. Internalization and degradation by cultured Hep G2 cells

To evaluate the impact of taurine on hepatic cholesterol catabolism low density lipoprotein (LDL) binding, internalization and degradation were measured in cultured Hep G2 cells. Preincubation of cells with 0.1-10 mM taurine for 24 h stimulated LDL receptor activity by as much as 100%. Only the high affinity LDL receptor activity (specific) was increased by taurine preincubation, whereas the low affinity receptor activity (nonspecific) remained unchanged. Scatchard analysis of the binding data revealed that taurine doubled the number of LDL receptors without affecting receptor affinity. Taurine-enhanced LDL receptor activity was most pronounced when LDL concentrations exceeded 100 micrograms/ml, but was noted at taurine concentrations as low as 0.1 mM (plasma level). Interestingly, taurine had no effect on LDL receptor activity when it was added simultaneously with 125I-LDL to Hep G2 cells, or when non-bile acid-producing human skin fibroblasts were tested. Stimulation of LDL receptor activity was also obtained with 10 mM cysteine, a taurine precursor, but not with glycine. Increased cellular concentrations of taurine and cysteine were associated with an elevated rate of bile acid synthesis and a reduced cellular free cholesterol concentration. The data suggest that taurine enhanced LDL receptor activity by sparing cysteine, a known sulfhydryl group donor and stimulator of 7 alpha-hydroxylase activity, and that the latter stimulated bile acid production leading to increased utilization of cellular free cholesterol and enhanced LDL uptake.     

蛋白激酶C抑制剂对U937细胞清道夫受体功能的影响

为了解细胞内蛋白质磷酸化水平对清道夫受体功能的影响,用蛋白激酶Ｃ抑掉剂形孢菌素（ｓｔａｕｒｏｓｐｏｒｉｎｅ,ＳＴＡ）处理人Ｕ９３７细胞,分别测定对照组和处理组细胞对碘标记的氧化低密度脂蛋白（＾１２５Ｉ）ｏｘ－ＬＤＬ的降解,结合,细胞表面受体复合物的内移以及细胞内脂质蓄积的程度,并利用放射自显影方法观察药物对细胞表面受体表达的影响,结果发现ＳＴＡ可以促进细胞结合（＾１２５Ｉ）ｏｘ－ＬＤＬ增加细胞表面     

Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells

We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins.     

Regulation of low-density lipoprotein receptors in cultured bovine adrenocortical cells

Bovine adrenocortical cells in monolayer culture produce cortisol and respond to corticotropin (ACTH) by an increase in cortisol secretion. Several lines of evidence are indicative that much of the cholesterol that serves as precursor for steroid hormone biosynthesis by these cells is derived from low-density lipoprotein (LDL) cholesterol that is taken up endocytotically by means of specific receptors localized in bovine adrenocortical plasma membranes. ACTH stimulated this process concomitant with an increase in steroid production. In the absence of LDL, ACTH had no effect on steroid biosynthesis. ACTH action in bovine adrenocortical cells resulted in an increase in the number of LDL receptor sites in the membrane fractions, whereas the dissociation constant for LDL binding was not changed. Chloroquine and NH₄Cl, considered to be inhibitors of lysosomal degradative activity, caused an increase in the number of [¹²⁵I]iodoLDL binding sites in the plasma membrane but the effect of ACTH was still apparent in the presence of these agents. These results are suggestive that the lifetime of the LDL receptor is increased when lysosomal activity is inhibited. When aminoglutethimide was added to block cholesterol side-chain cleavage activity and inhibit steroid production, the number of [¹²⁵I]iodoLDL binding sites in the membrane fractions prepared from bovine adrenocortical cells cultured in the presence of ACTH was reduced to 50% of that in cells maintained in aminoglutethimide-free medium. However, under these conditions the number of binding sites was still significantly greater than in cells maintained in the absence of ACTH. The effects of aminoglutethimide on uptake and degradation of [¹²⁵I]iodoLDL were similar to the effects on the number of [¹²⁵I]iodoLDL binding sites. Based on these results, we conclude that the action of ACTH to stimulate LDL metabolism in bovine adrenocortical cells results from an increase in the number of LDL binding sites in the plasma membranes. This action of ACTH appears to be, at least in part, independent of cholesterol utilization for cortisol biosynthesis. However, the effect of aminoglutethimide is indicative that changes in the intracellular cholesterol concentration might modulate the action of ACTH to increase the number of LDL binding sites and therefore to stimulate LDL degradation.     

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.     

Complete down-regulation of low-density-lipoprotein-receptor activity in the human hepatoma cell line Hep G2 by beta-migrating very-low-density lipoprotein and non-lipoprotein cholesterol. Different cellular regulatory pools of cholesterol.

Regulation of low-density-lipoprotein-receptor activity by low-density lipoprotein (LDL), cholesteryl-ester-rich beta-migrating very-low-density lipoprotein (beta-VLDL) and non-lipoprotein cholesterol was investigated in the human hepatoma cell line Hep G2. Competition studies indicate that LDL and beta-VLDL are bound to the same recognition site, tentatively the LDL receptor. The regulatory response of the LDL receptor upon prolonged incubation with LDL or beta-VLDL was, however, markedly different. 22 h preincubation of Hep G2 cells with excess LDL caused a partial down regulation to 31% of the initial level of the high-affinity association of LDL and 26% of the high-affinity degradation of LDL, while with beta-VLDL a complete down regulation of the LDL-receptor activity is observed. Preincubation of Hep G2 cells with beta-VLDL for 22 h led to a fourfold increase in intracellular cholesterol esters and a twofold increase in acyl-coA:cholesterol acyltransferase activity. With LDL, the amount of intracellular cholesterol esters is increased 1.6-fold. The more effective down regulation of LDL receptors by beta-VLDL as compared to LDL can be explained by the more effective intracellular cholesterol delivery with beta-VLDL than with LDL. Preincubation of Hep G2 cells for 22 h with acetylated LDL hardly influenced the LDL-receptor activity. Non-lipoprotein cholesterol, however, caused a complete down regulation of LDL-receptor activity at even lower extracellular cholesterol concentrations than with beta-VLDL. The complete down regulation of LDL receptors by non-lipoprotein cholesterol is not accompanied by a significant increase in acyl-coA:cholesterol acyltransferase activity, while the intracellular cholesterol ester concentration is only increased 1.6-fold. It is suggested that the effectiveness of non-lipoprotein cholesterol to regulate LDL receptors is caused by its efficiency to reach the sterol regulatory site. The inability of LDL to down regulate its receptor completely can thus be explained by the inability of LDL to deliver cholesterol adequately at the intracellular regulatory site of the LDL receptor. The observed complete down regulation of the LDL receptor by beta-VLDL may be responsible for the cholesterol-rich-diet induced, complete down regulation of LDL-receptor-mediated clearance of LDL in vivo.     

Regulation of the selective uptake of high density lipoprotein-associated cholesteryl esters by human fibroblasts and Hep G2 hepatoma cells

We have previously shown that the liver and steroidogenic tissues of rats in vivo and a wider range of cells in vitro, including human cells, selectively take up high density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles. This process is regulated in tissues of rats and in cultured rat cells according to their cholesterol status. In the present study, we examined regulation of HDL selective uptake in cultured human fibroblasts and Hep G2 hepatoma cells. The cholesterol content of these cells was modified by a 20-hr incubation with either low density lipoprotein (LDL) or free cholesterol. Uptake of HDL components was examined in a subsequent 4-6-hr assay using intracellularly trapped tracers: 125I-labeled N-methyl-tyramine-cellobiose-apoA-I (125I-NMTC-apoA-I) to trace apoA-I, and [3H]cholesteryl oleyl ether to trace cholesteryl esters. In the case of fibroblasts, pretreatment with either LDL or free cholesterol resulted in decreased selective uptake (total [3H]cholesteryl ether uptake minus that due to particle uptake as measured by 125I-NMTC-apoA-I). In contrast, HDL particle uptake increased with either form of cholesterol loading. The amount of HDL that was reversibly cell-associated (bound) was increased by prior exposure to free cholesterol, but was decreased by prior exposure to LDL. In the case of Hep G2 cells, exposure to free cholesterol only slightly increased HDL particle uptake; selective uptake decreased after both forms of cholesterol loading, and reversibly bound HDL increased after exposure to free cholesterol, but either did not change or decreased after exposure to LDL. It was excluded that either LDL carried over into the HDL uptake assay or that products secreted by the cultured cells influenced these results. Thus, selective uptake by cells of both hepatic and extrahepatic origin was down-regulated by cholesterol loading, under which conditions HDL particle uptake increased. Total HDL binding was not directly correlated with either the rate of selective uptake or the rate of HDL particle uptake or the cholesterol status of the cells, suggesting more than one type of HDL binding site.     

Calcium channel blockers stimulate LDL receptor synthesis in human skin fibroblasts

Human skin fibroblasts incubated in lipoprotein-deficient medium in the presence of 50-100 microM of the calcium channel blockers verapamil or diltiazem incorporated up to 2.5 times more [35S]methionine into immunoprecipitable LDL receptor protein than did control cells. Verapamil was found to be more potent in this regard than diltiazem. The calcium channel blockers did not influence the overall synthesis of cellular proteins or the half-life of the LDL receptor, and they were not able to prevent the suppression of LDL receptor synthesis caused by exogenous LDL or 25-hydroxycholesterol. The calcium channel blocker-induced stimulation of LDL receptor synthesis was accompanied by a corresponding increase in binding and internalization of [125I]LDL, but the degradation of internalized lipoprotein was slightly decreased. The results suggest that intracellular Ca2+ levels modulate LDL receptor metabolism in human skin 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.     

Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.     

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.     

The effect of L-carnitine on cholesterol metabolism in rat (Rattus bubalus) hepatocyte cells.

1. This paper concerns the study of the effect of L-carnitine on cholesterol metabolism in rat hepatocyte cells BRL-3A. In this research the binding of [125I]human low density lipoprotein (LDL) to BRL-3A cells and 3-hydroxy 3-methylglutaryl CoA reductase activity (HMG-CoA reductase activity) after L-carnitine incubation were studied. 2. It was found that L-carnitine is able to increase either the [125I]LDL binding or inhibit the HMG-CoA reductase activity in BRL-3A cells. 3. These results indicate that L-carnitine affects the cholesterol metabolism through an inhibition of HMG-CoA reductase activity that could be responsible for the increased [125I]LDL binding in rat hepatocytes.     

Defective internalization of low density lipoprotein in epidermoid cervical cancer cells

Cells of an epidermoid cancer cell line of human uterine cervix, which possessed a high-affinity, specific receptor for low density lipoprotein (LDL), internalized and degraded [125I]iodo-LDL at a very low rate. In these cells, LDL did not stimulate cholesteryl ester synthesis, nor did it suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase to the same extent as in the control cells. The binding of [125I]iodo-LDL by these cells was not decreased by preincubation of the cells in medium containing LDL. Using ferritin-labeled LDL (F-LDL) and electron microscopy, it was determined that at 4 degrees C the cells bound F-LDL in the same way as other cancer cell lines that did not have a defect in internalization. When these cells were warmed to 37 degrees C the F-LDL remained on the surface, whereas in cells from control cancer cell lines the F-LDL was internalized and was no longer observed on the cell surface. On the basis of the results of these studies it is concluded that cells of this epidermoid cancer cell line have a defective ability to internalize LDL.     

Effects of prostaglandins on LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes

The effects of prostaglandin (PG) E1, PGE2, the stable prostacyclin analogue Iloprost, and PGF2 alpha on low density lipoprotein (LDL) receptor activity and cholesterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 45 hr in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of 125I-labeled LDL. Addition of PGE1 in increasing concentrations to the incubation medium inhibited cholesterol synthesis and the specific accumulation and degradation of 125I-labeled LDL; at a concentration of 10 microM, the inhibitions were 61%, 70%, and 67%, respectively, after an incubation of 20 hr. The effects of PGE2 and Iloprost were similar. The action of the prostaglandins on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in the binding affinity. The prostaglandins yielded sigmoidal log concentration-effect curves. In contrast, PGF2 alpha had no influence on cholesterol synthesis or LDL receptor activity up to a concentration of 10 microM. PGE1, PGE2, and Iloprost, but not PGF2 alpha, led to an increase in the concentration of intracellular cyclic AMP. Dibutyryl cyclic AMP mimicked the effects of the E-prostaglandins and Iloprost on the LDL receptor activity. The results suggest that PGE1, PGE2, and prostacyclin affect LDL receptor activity and cholesterol synthesis and, therefore, may play a role in the regulation of cholesterol homeostasis and in the development of atherosclerosis.