Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions

The regulation of the LDL receptor activity in the human hepatoma cell line Hep G2 was studied. In Hep G2 cells, in contrast with fibroblasts, the LDL receptor activity was increased 2.5-fold upon increasing the concentration of normal whole serum in the culture medium from 20 to 100% by volume. Incubation of the Hep G2 cells with physiological concentrations of LDL (up to 700 micrograms/ml) instead of incubation under serum-free conditions resulted in a maximum 2-fold decrease in LDL receptor activity (10-fold decrease in fibroblasts). Incubation with physiological concentrations of HDL with a density of between 1.16 and 1.20 g/ml (heavy HDL) resulted in an approximately 7-fold increase in LDL receptor activity (1.5-fold increase in fibroblasts). This increased LDL receptor activity is due to an increase in the number of LDL receptors. Furthermore, simultaneous incubation of Hep G2 cells with LDL and heavy HDL (both 200 micrograms/ml) resulted in a 3-fold stimulation of the LDL receptor activity as compared with incubation in serum-free medium. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was also stimulated after incubation of Hep G2 with heavy HDL (up to 3-fold). The increased LDL receptor activity in Hep G2 cells after incubation with heavy HDL was independent of the action of lecithin:cholesterol acyltransferase during that incubation. However, previous modification of heavy HDL by lecithin:cholesterol acyltransferase resulted in an enhanced ability of heavy HDL to stimulate the LDL receptor activity. Our results indicate that in Hep G2 cells the heavy HDL-mediated stimulation of the LDL receptor activity overrules the LDL-mediated down-regulation and raises the suggestion that in man the presence of heavy HDL and the action of lecithin:cholesterol acyltransferase in plasma may be of importance in receptor-mediated catabolism of LDL by the liver.     

Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A-I mRNA in Hep G2 cells

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A-I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA-I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA-I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA-I gene expression and regulation.     

Effect of alcohol on lipoprotein metabolism. I. High density lipoprotein binding

The effects of ethanol upon the binding of [125I]-labelled human high density lipoprotein 3 (HDL3) was examined in rat liver microsomes and monolayer cultures of human hepatoma (Hep G2) cells. Alcohol feeding to rats (35% caloric content) caused a significant (p less than 0.05) increase in serum cholesterol concentrations relative to pair-fed controls, but HDL3 binding to rat liver microsomes was unaffected by alcohol consumption. By contrast, addition of 10 mM ethanol to Hep G2 cells increased HDL3 binding, and this increase was observed after 14, 28 and 40 days of exposure. This alcohol-dependent rise in HDL3 binding was associated with a 2.3- to 5-fold rise in receptor number (Bmax), and a 2- to 6-fold increase in the dissociation constant (Kd). The data suggest that the net effect of increased receptor number and lower receptor affinity is to increase the capacity of hepatocytes to metabolize circulating high density lipoproteins, and that this increase in the face of elevated plasma high density lipoprotein cholesterol consequent upon alcohol consumption would facilitate greater mobilization of cholesterol from peripheral tissues to the liver.     

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.     

Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity.

We have previously shown that in Hep G2 cells and human hepatocytes, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor activity is only weakly down-regulated after incubation of the cells with LDL, whereas incubation with high-density lipoproteins (HDL) of density 1.16-1.20 g/ml (heavy HDL) strongly increased the LDL-receptor activity. To elucidate this difference between hepatocytes and fibroblasts, we studied the cellular cholesterol homoeostasis in relation to the LDL-receptor activity in Hep G2 cells. (1) Interrupting the cholesteryl ester cycle by inhibiting acyl-CoA: cholesterol acyltransferase (ACAT) activity with compound 58-035 (Sandoz) resulted in an enhanced LDL-mediated down-regulation of the receptor activity. (2) The stimulation of the receptor activity by incubation of the cells with cholesterol acceptors such as heavy HDL was not affected by ACAT inhibition. (3) Incubation of the Hep G2 cells with LDL, heavy HDL or a combination of both grossly affected LDL-receptor activity, but did not significantly change the intracellular content of free cholesterol, suggesting that in Hep G2 cells the regulatory free cholesterol pool is small as compared with the total free cholesterol mass. (4) We used changes in ACAT activity as a sensitive (indirect) measure for changes in the regulatory free cholesterol pool. (5) Incubation of the cells with compactin (2 microM) without lipoproteins resulted in a 4-fold decrease in ACAT activity, indicating that endogenously synthesized cholesterol is directed to the ACAT-substrate pool. (6) Incubation of the cells with LDL or a combination of LDL and heavy HDL stimulated ACAT activity 3-5 fold, whereas incubation with heavy HDL alone decreased ACAT activity more than 20-fold. Our results suggest that in Hep G2 cells exogenously delivered (LDL)-cholesterol and endogenously synthesized cholesterol are primarily directed to the cholesteryl ester (ACAT-substrate) pool or, if present, to extracellular cholesterol acceptors (heavy HDL) rather than to the free cholesterol pool involved in LDL-receptor regulation.     

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.     

Regulation of scavenger receptor class B type I in hamster liver and Hep3B cells by endotoxin and cytokines

Multiple changes in HDL metabolism occur during infection and inflammation that could potentially impair the antiatherogenic functions of HDL. Scavenger receptor class B type I (SR-BI) promotes cholesterol efflux from peripheral cells and mediates selective uptake of cholesteryl ester into hepatocytes, thereby playing a pivotal role in reverse cholesterol transport. We studied the effect of endotoxin (lipopolysaccharide, LPS) and cytokines [tumor necrosis factor (TNF) and interleukin 1 (IL-1)] on hepatic SR-BI mRNA and protein levels in Syrian hamsters. LPS significantly decreased SR-BI mRNA levels in hamster liver. This effect was rapid and sustained, and was associated with a decrease in hepatic SR-BI protein levels. High cholesterol diet did not change hepatic SR-BI mRNA levels, and LPS was able to decrease SR-BI mRNA levels during high cholesterol feeding. TNF and IL-1 decreased SR-BI mRNA levels in the liver, and the effects of TNF and IL-1 were additive. TNF and IL-1 also decreased SR-BI levels in Hep3B hepatoma cells. More importantly, TNF and IL-1 decreased the uptake of HDL cholesteryl ester into Hep3B cells. In addition, we studied the effect of LPS on SR-BI mRNA in RAW 264.7 cells, a macrophage cell line. LPS rapidly decreased SR-BI mRNA levels in RAW 264.7 cells, but the effect was not sustained and did not lead to a reduction in SR-BI protein levels. Our results suggest that the decrease in hepatic SR-BI levels due to LPS and cytokines during infection and inflammation may decrease selective uptake of cholesteryl ester into the liver and result in impaired reverse cholesterol transport.     

Phosphatidylinositol promotes cholesterol transport and excretion

Administration of phosphatidylinositol (PI) to New Zealand White rabbits increases HDL negative charge and stimulates reverse cholesterol transport. Intravenously administered PI (10 mg/kg) associated almost exclusively with the HDL fraction in rabbits. PI promoted an increase in the hepatic uptake of plasma free cholesterol (FC) and a 21-fold increase in the biliary secretion of plasma-derived cholesterol. PI also increased cholesterol excretion into the feces by 2.5-fold. PI directly affects cellular cholesterol metabolism. In cholesterol-loaded macrophages, PI stimulated cholesterol mass efflux to lipid-poor reconstituted HDL. PI was about half as effective as cAMP at stimulating efflux, and the effects of cAMP and PI were additive. In cultured HepG2 cells, PI-enriched HDL also enhanced FC uptake from HDL by 3-fold and decreased cellular cholesterol synthesis and esterification. PI enrichment had no effect on the selective uptake of cholesterol esters or on the internalization of HDL particles. PI-dependent metabolic events were efficiently blocked by inhibitors of protein kinase C and the inositol signaling cascade.The data suggest that HDL-PI acts via cell surface ATP binding cassette transporters and signaling pathways to regulate both cellular and intravascular cholesterol homeostasis.     

Regulation of apoprotein synthesis and secretion in the human hepatoma Hep G2. The effect of exogenous lipoprotein

The regulation of lipoprotein assembly and secretion at a molecular level is incompletely understood. To begin to identify the determinants of apoprotein synthesis and distribution among lipoprotein classes, we have examined the effects of chylomicron remnants which deliver triglyceride and cholesterol, and beta very low density lipoprotein (beta VLDL), which deliver primarily cholesterol, on apolipoprotein synthesis and secretion by the human hepatoma Hep G2. Hep G2 cells were incubated with remnants or beta VLDL for 24 h, the medium was changed and the cells then incubated with [35S]methionine. The secreted lipoproteins were separated by gradient ultracentrifugation and the radiolabeled apoproteins were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and counted. Remnants caused a 14-fold, and beta VLDL a 7-fold, increase in VLDL apoprotein (apo) secretion; the apoB/apoE ratio in this class was unchanged. Preincubation with either of the lipoproteins also stimulated low density lipoprotein apoB secretion. Preincubation with beta VLDL, but not with remnants, significantly increased apoE and apoA-I secreted in high density lipoprotein (HDL). In addition, the apoE/apoA-I ratio precipitated from the HDL of beta VLDL-treated cells by anti-apoE was 2.2-fold higher than that precipitated by anti-apoA-I. There was no difference in the ratios precipitated from control HDL. This was due to the secretion of a lipoprotein, subsequently isolated by immunoaffinity chromatography, that contained predominantly apoE. When Hep G2 cells were preincubated with oleic acid alone, total apoprotein secretion was not altered. However, cholesterol-rich liposomes stimulated secretion of newly synthesized apoE, but not apoB, while apoA-I secretion was variably affected. Cholesterol-poor liposomes had no effect. Thus, lipid supply is a determinant of apoprotein synthesis and secretion, and cholesterol may be of particular importance in initiating apoprotein synthesis.     

Leukotriene uptake by hepatocytes and hepatoma cells.

The uptake of tritiated cysteinyl leukotrienes (LTC4, LTD4, LTE4) and LTB4 was investigated in freshly isolated rat hepatocytes and different hepatoma cell lines under initial-rate conditions. Leukotriene uptake by hepatocytes was independent of an Na+ gradient and a K+ diffusion potential across the hepatocyte membranes as established in experiments with isolated hepatocytes and plasma membrane vesicles. Kinetic experiments with isolated hepatocytes indicated a low-Km system and a non-saturable system for the uptake of cysteinyl leukotrienes as well as LTB4 under the conditions used. AS-30D hepatoma cells and human Hep G2 hepatoma cells were deficient in the uptake of cysteinyl leukotrienes, but showed significant accumulation of LTB4. Moreover, only LTB4 was metabolized in Hep G2 hepatoma cells. Competition studies on the uptake of LTE4 and LTB4 (10 nM each) indicated inhibition by the organic anions bromosulfophthalein, S-decyl glutathione, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, probenecid, docosanedioate, and hexadecanedioate (100 microM each), but not by taurocholate, the amphiphilic cations verapamil and N-propyl ajmaline, and the neutral glycoside ouabain. Cholate and the glycoside digitoxin were inhibitors of LTB4 uptake only. Bromosulfophthalein, the strongest inhibitor of leukotriene uptake by hepatocytes, did not inhibit LTB4 uptake by Hep G2 hepatoma cells under the same experimental conditions. Leukotriene-binding proteins were analyzed by comparative photoaffinity labeling of human hepatocytes and Hep G2 hepatoma cells using [3H]LTE4 and [3H]LTB4 as the photolabile ligands. Predominant leukotriene-binding proteins with apparent molecular masses in the ranges of 48-58 kDa and 38-40 kDa were labeled by both leukotrienes in the particulate and in the cytosolic fraction of hepatocytes, respectively. In contrast, no labeling was obtained with [3H]LTE4 in Hep G2 cells. With [3H]LTB4 a protein with a molecular mass of about 48 kDa was predominantly labeled in the particulate fraction of the hepatoma cells, whereas in the cytosolic fraction a labeled protein in the range of 40 kDa was detected. Our results provide evidence for the existence of distinct uptake systems for cysteinyl leukotrienes and LTB4 at the sinusoidal membrane of hepatocytes; however, some of the inhibitors tested interfere with both transport systems. Only LTB4, but not cysteinyl leukotrienes, is taken up and metabolized by the transformed hepatoma cells.     

Possible involvement of protein kinase C and calcium in GSH efflux from Hep G2 cells.

We investigated the effects of protein kinase C modulations and calcium mobilization on GSH efflux in Hep G2 cells. GSH efflux from Hep G2 cells was increased by a phorbol ester. Staurosporine, an inhibitor of protein kinase C, diminished phorbol ester-stimulated GSH efflux from the cells. GSH efflux was negatively correlated with extracellular calcium concentrations. Verapamil enhanced GSH efflux, whereas ATP decreased GSH efflux. The latter effect was diminished in the absence of extracellular calcium. Protein kinase C and calcium mobilization may be crucial factors in GSH efflux from human hepatocytes.     

The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.     

The role of high density lipoproteins in rat adrenal cholesterol metabolism and steroidogenesis

Addition of rat or human high density lipoproteins (HDL) or human low density lipoproteins (LDL) to rat adrenocortical cells in vitro was found to enhance steroid production and increase cell cholesterol content. These effects of HDL were not observed in cultured mouse Y-1 adrenal cells, suggesting that rat adrenal cells possess a specific mechanism for uptake of HDL cholesterol not found in Y-1 cells. The effects of HDL were most marked on cells previously stimulated with adrenocorticotropin (ACTH) and depleted of their endogenous cholesterol stores. Such cells were prepared either by treatment in vivo with 4-aminopyrazolopyrimidine or in vitro with ACTH (10(-7) M) in lipoprotein-poor media. Steroid production by treated cells exhibited a saturable dependence on media HDL concentration. In addition to enhancing ACTH stimulated steroid production, addition of HDL also resulted in a saturable concentration-dependent increase in cell cholesterol content. Both aminoglutethimide and cycloheximide were found to inhibit HDL-enhanced steroid production. Finally, addition of HDL to short term incubations (5 1/2 h) of ACTH-treated cells caused no change in the rate of incorporation of 14C-acetate into cholesterol or corticosterone. These results indicate that rat adrenocortical cells possess a specific, saturable, ACTH-dependent mechanism for uptake of HDL cholesterol. Moreover, cellular uptake of HDL cholesterol exceeded by at least 4-fold the amount of cholesterol associated with HDL apoprotein degraded by the cells, suggesting that utilization of HDL cholesterol does not require endocytosis and lysosomal degradation of the entire HDL particle.     

槲皮素诱导Hep G2自噬与胞内游离Ca2+浓度的变化相关

槲皮素具有诱导细胞自噬、抑制肿瘤细胞增殖等抗癌功能,但其诱导细胞自噬的分子机制还不太清楚. 本文通过激光共聚焦显微镜观察槲皮素对Hep G2细胞自噬的影响; Fluo-3 AM和Cyto-IDTM Green Detection Reagent染色标记, 流式细胞术测定了槲皮素对Hep G2细胞内游离钙离子浓度［Ca2+］_i 及Ca2＋螯合剂BAPTA-AM对自噬水平的影响. 探讨了槲皮素诱导人肝癌细胞 Hep G2自噬过程中［Ca2+］i的变化. 结果表明, 在槲皮素较低浓度范围内(0 ～ 50 μg/mL), 可明显抑制Hep G2细胞增殖, 并以剂量依赖方式诱导细胞自噬. 同时发现,槲皮素刺激Hep G2细胞可使［Ca2+］i明显增加, 进而促进自噬. 而当胞内Ca2＋螯合剂 BAPTA-AM存在时, 细胞的自噬水平受到一定的抑制. 这些结果表明,细胞内［Ca2+］i的升高可促进自噬, ［Ca2+］_i 的降低可能会抑制自噬. Hep G2细胞自噬与细胞内游离钙离子浓度的变化有关系.     

Cholesterol metabolism. Effect of 26-thiacholesterol and 26-aminocholesterol, analogues of 26-hydroxycholesterol, on cholesterol synthesis and low-density-lipoprotein-receptor binding.

1. The effects of 26-aminocholesterol and 26-thiacholesterol on cholesterol synthesis and LDL (low-density lipoprotein)-receptor activity were compared with naturally occurring 26-hydroxycholesterol utilizing both human fibroblasts and hepatoma (Hep G2) cells. 2. At equimolar concentrations (0.625 microM), down-regulation of LDL-receptor activity and cholesterol synthesis was greater with human fibroblasts than with Hep G2 cells. 3. At much higher concentrations (5-20 microM) the 26-thia analogue had little effect on either cholesterol synthesis or LDL-receptor activity.     

Effect of 25-hydroxycholesterol and bile acids on the regulation of cholesterol metabolism in Hep G2 cells.

The effect of 25-hydroxycholesterol (25-OH-cholesterol) and chenodeoxycholic (CDC) acid on apoprotein secretion, low-density lipoprotein receptor activity, and [3H]triacylglycerol secretion in Hep G2 cells was studied. Both 25-OH-cholesterol and CDC acid increased the secretion of apolipoprotein (apo) E by Hep G2 cells. The secretion of apo A-I was slightly lowered (less than 10% disease). The maximal increase in apo E secretion was observed in culture medium containing 2 micrograms of 25-OH-cholesterol/ml or 10 micrograms of CDC acid/ml plus 10% fetal calf serum. Cholesterol, 7-OH-cholesterol and other bile acids were ineffective in inducing increases in apo E secretion. Another cholesterol synthesis inhibitor, mevinolin, was also ineffective in generating an increase in apoprotein secretion. The data indicated a specific interaction between 25-OH-cholesterol or CDC acid and apo E secretion in Hep G2 cells. Cholesterol synthesis, as measured by the incorporation of [14C]acetic acid into sterols, was repressed in Hep G2 cells in the presence of 25-OH-cholesterol (17% of control value). CDC acid, on the other hand, increased [14C]acetic acid incorporation (156% of control value). The number of LDL receptors in Hep G2 cells was decreased after incubation with 25-OH-cholesterol (62% of control value), but increased significantly after incubation with CDC acid (149% of control value). The secretion of [3H]triacylglycerol by Hep G2 cells incubated with 25-OH-cholesterol was greatly increased (248% of control value). On the contrary, CDC acid did not cause any increase in [3H]triacylglycerol secretion. The above results suggest that 25-OH-cholesterol and CDC acid have different effects on lipid metabolism in Hep G2 cells. The mRNA levels of apo E increased in cells preincubated with 25-OH-cholesterol and CDC acid, which suggested that the increase in apo E secretion is at least partly due to an increase in synthesis.     

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells

Summary. Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.     

Human plasma cholesteryl ester transfer protein enhances the transfer of cholesteryl ester from high density lipoproteins into cultured HepG2 cells

The role of human plasma cholesteryl ester transfer protein (CETP) in the cellular uptake of high density lipoprotein (HDL) cholesteryl ester (CE) was studied in a liver tumor cell line (HepG2). When HepG2 cells were incubated with [3H]cholesteryl ester-labeled HDL3 in the presence of increasing concentrations of CETP there was a progressive increase in cell-associated radioactivity to levels that were 2.8 times control. The CETP-dependent uptake of HDL-CE was found to be saturated by increasing concentrations of both CETP and HDL. The CETP-dependent uptake of CE radioactivity increased continuously during an 18-h incubation. In contrast to the effect on cholesteryl ester, CETP failed to enhance HDL protein cell association or degradation. Enhanced uptake of HDL cholesteryl ester was shown for the d greater than 1.21 g/ml fraction of human plasma, partially purified CETP, and CETP purified to homogeneity, but not for the d greater than 1.21 g/ml fraction of rat plasma which lacks cholesteryl ester transfer activity. HDL cholesteryl ester entering the cell under the influence of CETP was largely degraded to free cholesterol by a process inhibitable by chloroquine. CETP enhanced uptake of HDL [3H]CE in cultured smooth muscle cells and to a lesser extent in fibroblasts but did not significantly influence uptake in endothelial cells or J774 macrophages. These experiments show that, in addition to its known role in enhancing the exchange of CE between lipoproteins, plasma CETP can facilitate the in vitro selective transfer of CE from HDL into certain cells.     

Isolation and characterization of lipoproteins produced by human hepatoma-derived cell lines other than HepG2

A total of six established human hepatoma-derived cell lines, including Hep3B, NPLC/PRF/5 (NPLC), Tong/HCC, Hep 10, huH1, and huH2, were screened for their ability to accumulate significant quantities of lipoproteins in serum-free medium. Only two cell lines, Hep3B and NPLC, secreted quantitatively significant amounts of lipoproteins. In a 24-h period the accumulated mass of apolipoproteins (apo) A-I, A-II, B, and E and albumin for Hep3B cells was 1.96, 1.01, 1.96, 1.90, and 53.2 micrograms/mg cell protein per 24 h, respectively. NPLC cells secreted no detectable albumin but the 24-h accumulated mass for apolipoproteins A-I, A-II, B, and E was 0.45, 0.05, 0.32, and 0.68 micrograms/mg cell protein per 24 h, respectively. Twenty four-hour serum-free medium of Hep3B cells contained lipoproteins corresponding to the three major density classes of plasma; percent protein distribution among the lipoprotein classes was 4%, 41%, and 56% for very low density lipoprotein ("VLDL"), low density lipoprotein ("LDL"), and high density lipoprotein ("HDL"), respectively. NPLC was unusual since most of the lipoprotein mass was in the d 1.063-1.235 g/ml range. Hep3B "LDL", compared with plasma LDL, contained elevated triglyceride, phospholipid, and free cholesterol. Nondenaturing gradient gel electrophoresis revealed that Hep3B "LDL" possessed a major component at 25.5 nm and a minor one at 18.3 nm. Immunoblots showed that the former contained only apoB while the latter possessed only apoE. Like plasma VLDL, Hep3B "VLDL" particles (30.5 nm diameter) isolated from serum-free medium contained apoB, apoC, and apoE. "HDL" harvested from Hep3B and NPLC medium were enriched in phospholipid and free cholesterol and poor cholesteryl ester which is similar to the composition of HepG2 "HDL." "HDL" from Hep3B and NPLC culture medium on gradient gel electrophoresis had peaks at 7.5, 10, and 11.9 nm which were comparable to major components found in HepG2 cell medium. Hep3B cells, in addition, possessed a particle that banded at 8.2 nm which appeared to be an apoA-II without apoA-I particle by Western blot analysis. The cell line also produced a subpopulation of larger-sized "HDL" not found in HepG2 medium. NPLC "HDL" had a distinct peak at 8.3 nm which by Western blot was an apoE-only particle. Electron microscopy revealed that "HDL" harvested from Hep3B and NPLC medium consisted of discoidal and small, spherical particles like those of HepG2. The "HDL" apolipoprotein content of each cell line was distinct from that of HepG2. ApoA-II at 35% of apolipoprotein distinguishes Hep3B "HDL" from HepG2, which contains only 10%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hepatic lipase promotes the selective uptake of high density lipoprotein-cholesteryl esters via the scavenger receptor B1.

Hepatic lipase (HL) plays a major role in high-density lipoprotein (HDL) metabolism both as a lipolytic enzyme and as a ligand. To investigate whether HL enhances the uptake of HDL-cholesteryl ester (CE) via the newly described scavenger receptor BI (SR-BI), we measured the effects of expressing HL and SR-BI on HDL-cell association as well as uptake of 125I-labeled apoA-I and [3H]CE-HDL, by embryonal kidney 293 cells. As expected, HDL cell association and CE selective uptake were increased in SR-BI transfected cells by 2- and 4-fold, respectively, compared to controls (P < 0.001). Cells transfected with HL alone or in combination with SR-BI expressed similar amounts of HL, 20% of which was bound to cell surface proteoglycans. HL alone increased HDL cell association by 2-fold but had no effect on HDL-CE uptake in 293 cells. However, in cells expressing SR-BI, HL further enhanced the selective uptake of CE from HDL by 3-fold (P < 0.001). To determine whether the lipolytic and/or ligand function of HL are required in this process, we generated a catalytically inactive form of HL (HL-145G). Cells co-transfected with HL-145G and SR-BI increased their HDL cell association and HDL-CE selective uptake by 1.4-fold compared to cells expressing SR-BI only (P < 0.03). Heparin abolished the effect of HL-145G on SR-BI-mediated HDL-CE selective uptake.Thus, the enhanced uptake of HDL-CE by HL is mediated by both its ligand role, which requires interaction with proteoglycans, and by lipolysis with subsequent HDL particle remodeling. These results establish HL as a major modulator of SR-BI mediated selective uptake of HDL-CE.