Comparison of the LDL-receptor binding of VLDL and LDL from apoE4 and apoE3 homozygotes

1999 ◽  
Vol 276 (3) ◽  
pp. E553-E557 ◽  
Author(s):  
Cyril D. S. Mamotte ◽  
Marian Sturm ◽  
Jock I. Foo ◽  
Frank M. van Bockxmeer ◽  
Roger R. Taylor
Keyword(s):  
Plasma Lipids ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Cultured Fibroblasts ◽  
Ldl Receptors ◽  
G2 Cells

Compared with apolipoprotein E3 (apoE3), apoE2 is less effective in mediating the binding of lipoproteins to the low-density lipoprotein (LDL) receptor. The influence of the E4 isoform, which is associated with adverse effects on plasma lipids and coronary heart disease, is less clear. We compared the ability of very low density lipoprotein (VLDL) and LDL from paired E4/4 and E3/3 subjects to compete against125I-labeled LDL for binding with the LDL receptor on cultured fibroblasts and Hep G2 cells. The concentrations of VLDL or LDL required to inhibit binding of125I-LDL by 50% (IC50, μg apoB/ml) were determined, and results were assessed in terms of an IC50 ratio, E4/4 IC50 relative to E3/3 IC50, to reduce the influence of interassay variability. In Hep G2 cells, E4/4 VLDL was more effective than E3/3 VLDL in competing for the LDL receptor, the IC50 ratio being lower than unity (0.73 ± 0.31, P < 0.05, two-tailed t-test). IC50 values themselves were marginally lower in E4/4 than E3/3 subjects (3.7 ± 1.3 vs. 6.1 ± 3.7, P < 0.08). However, there was no difference between E4/4 and E3/3 VLDL in competing for the LDL receptor on fibroblasts or between E4/4 and E3/3 LDL in competing for the LDL receptor on either cell type. These results suggest that inheritance of apoE4 is associated with an increased affinity of VLDL particles for LDL receptors on hepatocytes and may partly explain the influence of the E4 isoform on lipid metabolism.

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

1987 ◽  
Vol 247 (3) ◽  
pp. 739-746 ◽  
Author(s):  
L M Havekes ◽  
E C M de Wit ◽  
H M G Princen
Keyword(s):  
Free Cholesterol ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Activity ◽  
Low Density ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Acat Activity ◽  
G2 Cells

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.

Differences in metabolism of three low density lipoprotein subfractions in Hep G2 cells

1990 ◽  
Vol 1047 (3) ◽  
pp. 212-222 ◽  
Author(s):  
Dorine W. Swinkels ◽  
Jan C.M. Hendriks ◽  
Pierre N.M. Demacker ◽  
Anton F.H. Stalenhoef
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Lipoprotein Subfractions ◽  
G2 Cells

Interaction of apolipoprotein B-containing lipoprotein secreted by Hep G2 cells with receptors for low-density lipoprotein

1991 ◽  
Vol 1086 (2) ◽  
pp. 237-240 ◽  
Author(s):  
Ilia V. Fuki ◽  
Georgy B. Menschikov ◽  
Mario Menschikowski ◽  
Irina Yu. Adamova ◽  
Vadim S. Repin
Keyword(s):  
Apolipoprotein B ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
G2 Cells

scholarly journals Characterization of the low-density-lipoprotein-receptor-independent interaction of β-very-low-density lipoprotein with rat and human parenchymal liver cells in vitro

1992 ◽  
Vol 282 (1) ◽  
pp. 41-48 ◽  
Author(s):  
R De Water ◽  
J A A M Kamps ◽  
M C M Van Dijk ◽  
E A M J Hessels ◽  
J Kuiper ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Liver Cells ◽  
Recognition Site ◽  
Low Density ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Parenchymal Liver ◽  
Parenchymal Cells

beta-Migrating very-low-density lipoprotein (beta-VLDL) is a cholesteryl-ester-enriched lipoprotein which under normal conditions is rapidly cleared by parenchymal liver cells. In this study the characteristics of the interaction of beta-VLDL with rat parenchymal cells, Hep G2 cells and human parenchymal cells are evaluated. The binding of beta-VLDL to these cells follows saturation kinetics (Bmax. respectively 117, 106 and 103 ng of beta-VLDL apoliprotein/mg of cell protein), with a relatively high affinity (Kd respectively for beta-VLDL of 10.7, 5.1 and 8.4 micrograms/ml). Competition studies of unlabelled beta-VLDL, low-density lipoprotein (LDL) or acetylated LDL with the binding of radiolabelled beta-VLDL indicate that a LDL-receptor-independent, Ca(2+)-independent, specific recognition site for beta-VLDL is present on rat and human parenchymal cells, whereas with Hep G2 cells or mouse macrophages beta-VLDL recognition is performed by the LDL receptor. The binding of beta-VLDL to Hep G2 cells was down-regulated by 89% by prolonged exposure to beta-VLDL, whereas for human parenchymal and rat parenchymal cells down-regulation of 44% and 20% respectively was observed. Studies with antibodies against the LDL receptor support the presence of a LDL-receptor-independent specific beta-VLDL recognition site on rat and human parenchymal cells. It is concluded that a LDL-receptor-independent recognition site for beta-VLDL is present on rat and human parenchymal liver cells. The presence of a LDL-receptor-independent recognition site on human parenchymal cells may mediate in vivo the uptake of beta-VLDL during consumption of a cholesterol-rich diet, when LDL receptors are down-regulated, thus protecting against the extrahepatic accumulation of the atherogenic beta-VLDL constituents.

scholarly journals Evidence for sterol-independent regulation of low-density lipoprotein receptor activity in Hep-G2 cells

1991 ◽  
Vol 279 (1) ◽  
pp. 175-187 ◽  
Author(s):  
J L Ellsworth ◽  
C Chandrasekaran ◽  
A D Cooper
Keyword(s):  
Low Density Lipoprotein ◽  
Calf Serum ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Cholesteryl Esters ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Ldl Uptake ◽  
Stimulation Of

The relationship between the serum factor(s)-mediated induction of low-density lipoprotein (LDL) receptor activity and changes in cellular cholesterol metabolism was examined in the human hepatoma cell line Hep-G2. Relative to incubation with serum-free media [Eagle's minimal essential medium (MEM) control], short-term (less than 8 h) incubation with medium containing 15% of either calf serum (MEM + serum) or the d greater than 1.25 fraction of calf serum (MEM + d greater than 1.25) produced a time- and concentration-dependent increase in the uptake of 125I-LDL. Immunoblotting with anti-(LDL receptor) antibodies demonstrated that this was correlated with a 2-fold increase in the amount of the mature 136,000 Da LDL receptor protein in detergent-solubilized Hep-G2 cell membranes. Incubation with MEM + serum, but not MEM + d greater than 1.25, increased the efflux of radiolabelled cholesterol from Hep-G2 cells. However, the induction of 125I-LDL uptake by MEM + d greater than 1.25 (2.3-fold) and MEM + serum (2.2-fold) was virtually identical. Addition of the d less than 1.063 lipoproteins of calf serum to MEM + d greater than 1.25 at their original or three times their serum concentration decreased the induction of 125I-LDL uptake by MEM + d greater than 1.25 by only 20-30%. Together, these results suggest that the stimulation of 125I-LDL uptake was not due to the presence of high-density lipoprotein, the absence of LDL or the stimulation of cholesterol efflux. MEM + serum stimulated 125I-LDL uptake in cells cholesterol-loaded by incubation with rat very-low-density lipoprotein with beta electrophoretic mobility (beta-VLDL). Compared to incubation with the MEM control, either MEM + serum or MEM + d greater than 1.25 produced time-dependent increases in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase which also occurred in cholesterol-loaded cells. However, cholesterol biosynthesis, whether measured from 3H2O, [14C]acetate or [3H]mevalonic acid, was not increased. Incubation with MEM + serum or MEM + d greater than 1.25 did not affect [3H]oleate incorporation into cellular cholesteryl esters, hydrolysis of intracellular [3H]cholesteryl esters or the cellular mass of unesterified or esterified cholesterol. Incubation with MEM + serum or MEM + d greater than 1.25 produced a transient increase in the level of LDL receptor mRNA, reaching a maximum of 5-10-fold by 2 h and decreasing to near baseline levels by 4 h. Actinomycin D blocked the serum-factor-mediated induction of LDL receptor mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)

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