Identification of a functional receptor differing from the LDL receptor that catabolizes chylomicron remnant in Hep 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.

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Effect of ketoconazole on cholesterol synthesis and on HMG-COA reductase and LDL-receptor activities in Hep G2 cells

Biochemical Pharmacology ◽

10.1016/0006-2952(87)90077-3 ◽

1987 ◽

Vol 36 (8) ◽

pp. 1245-1249 ◽

Cited By ~ 31

Author(s):

Herman Jan Kempen ◽

Kees Van Son ◽

Louis H. Cohen ◽

Marieke Griffioen ◽

Hans Verboom ◽

...

Keyword(s):

Cholesterol Synthesis ◽

Ldl Receptor ◽

Hep G2 ◽

Hep G2 Cells ◽

Hmg Coa Reductase ◽

G2 Cells ◽

Coa Reductase

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Hormonal regulation of low-density lipoprotein (LDL) receptor activity in human hepatoma Hep G2 cells. Insulin increases LDL receptor activity and diminishes its suppression by exogenous LDL

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1988.tb14084.x ◽

1988 ◽

Vol 174 (1) ◽

pp. 213-218 ◽

Cited By ~ 61

Author(s):

David P. WADE ◽

Brian L. KNIGHT ◽

Anne K. SOUTAR

Keyword(s):

Hormonal Regulation ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Receptor Activity ◽

Low Density ◽

Human Hepatoma ◽

Hep G2 ◽

Hep G2 Cells ◽

G2 Cells

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Porcine smooth muscle cell-conditioned medium stimulates LDL receptor activity in Hep G2 cells

Journal of Cell Science ◽

10.1242/jcs.103.2.541 ◽

1992 ◽

Vol 103 (2) ◽

pp. 541-549 ◽

Cited By ~ 1

Author(s):

C.D. Moorby ◽

E. Gherardi ◽

D. Riddell ◽

D.E. Bowyer

Keyword(s):

Smooth Muscle ◽

Conditioned Medium ◽

Mdck Cells ◽

Ldl Receptor ◽

Receptor Activity ◽

Relative Molecular Mass ◽

Hep G2 ◽

Hep G2 Cells ◽

Paracrine Factors ◽

G2 Cells

Paracrine factors may modulate low density lipoprotein (LDL) receptor activity in hepatocytes. To study this the effect of conditioned medium prepared from a range of cell types on the binding and internalisation of 125I-LDL in Hep G2 cells was studied. Seven of the fourteen conditioned media tested, including those from P388D1, U937, porcine smooth muscle (Pc SMC) Swiss 3T3, STO, = 48 and MDCK cells, were found to increase the binding and internalisation of 125I-LDL at 37 degrees C by Hep G2 cells (P < 0.01). The largest increase in LDL receptor activity was produced by conditioned medium from Pc SMC cells and was, therefore, selected for further analysis. The Pc SMC-conditioned medium increased LDL receptor number in Hep G2 cells by three-fold but had no effect on LDL receptor activity in human skin fibroblasts. DNA synthesis and cholesterol synthesis by Hep G2 cells were inhibited by Pc SMC-conditioned medium. Preliminary characterisation of the Pc SMC-derived factor(s) suggests that it is a protein(s) of low relative molecular mass.

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Kinetics of low-density lipoprotein receptor activity in Hep-G2 cells: derivation and validation of a Briggs–Haldane-based kinetic model for evaluating receptor-mediated endocytotic processes in which receptors recycle

Biochemical Journal ◽

10.1042/bj3230649 ◽

1997 ◽

Vol 323 (3) ◽

pp. 649-659 ◽

Cited By ~ 21

Author(s):

H. James HARWOOD ◽

Lorraine D. PELLARIN

Keyword(s):

Steady State ◽

Rate Constant ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Rate Equations ◽

Cell Protein ◽

Hep G2 ◽

Hep G2 Cells ◽

Receptor Mediated Endocytosis ◽

G2 Cells

The process of receptor-mediated endocytosis for receptors that recycle to the cell surface in an active form can be considered as being kinetically analogous to that of a uni-substrate, uni-product enzyme-catalysed reaction. In this study we have derived steady-state initial-velocity rate equations for this process, based on classical Briggs–Haldane and King–Altman kinetic approaches to multi-step reactions, and have evaluated this kinetic paradigm, using as a model system the low-density lipoprotein (LDL)-receptor-mediated endocytosis of the trapped label [14C]sucrose-LDL in uninduced, steady-state Hep-G2 cells. Using the derived rate equations, together with experimentally determined values for Bmax (123 fmol/mg of cell protein), Kd (14.3 nM), the endocytotic rate constant ke (analogous to kcat; 0.163 min-1), Km (80 nM) and maximal internalization velocity (26.4 fmol/min per mg), we have calculated the ratio ke/Km (0.00204 nM-1·min-1), the bimolecular rate constant for LDL and LDL-receptor association (0.00248 nM-1·min-1), the first-order rate constant for LDL–LDL-receptor complex dissociation (0.0354 min-1), the total cellular content of LDL receptors (154 fmol/mg of cell protein), the intracellular LDL receptor concentration (30.7 fmol/mg of cell protein) and the pseudo-first-order rate constant for LDL receptor recycling (0.0653 min-1). Based on this mathematical model, the kinetic mechanism for the receptor-mediated endocytosis of [14C]sucrose-LDL by steady-state Hep-G2 cells is one of constitutive endocytosis via independent internalization sites that follows steady-state Briggs–Haldane kinetics, such that LDL–LDL-receptor interactions are characterized by a rapid-high-affinity ligand–receptor association, followed by ligand–receptor complex internalization that is rapid relative to complex dissociation, and by receptor recycling that is more rapid than complex internalization and that serves to maintain 80% of cellular LDL receptors on the cell surface in the steady-state. The consistency with which these quantitative observations parallel previous qualitative observations regarding LDL-receptor-mediated endocytosis, together with the high correlation between theoretical internalization velocities (calculated from determined rate constants) and experimental internalization velocities, underscore the validity of considering receptor-mediated endocytotic processes for recycling receptors in catalytic terms.

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Regulation of LDL Receptor, apoB, and apoE Protein and mRNA in Hep G2 Cells

DNA and Cell Biology ◽

10.1089/dna.1992.11.291 ◽

1992 ◽

Vol 11 (4) ◽

pp. 291-300 ◽

Cited By ~ 6

Author(s):

HANS GEORG KRAFT ◽

STEPHEN J. DEMOSKY ◽

KURT SCHUMACHER ◽

H. BRYAN BREWER ◽

JEFFREY M. HOEG

Keyword(s):

Ldl Receptor ◽

Hep G2 ◽

Hep G2 Cells ◽

Apoe Protein ◽

G2 Cells

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Transforming growth factor-β1 and interleukin-1β stimulate LDL receptor activity in Hep G2 cells

Atherosclerosis ◽

10.1016/0021-9150(92)90047-k ◽

1992 ◽

Vol 97 (1) ◽

pp. 21-28 ◽

Cited By ~ 21

Author(s):

Catriona D. Moorby ◽

Ermanno Gherardi ◽

Lynda Dovey ◽

Cheryl Godliman ◽

David E. Bowyer

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Ldl Receptor ◽

Transforming Growth Factor Β1 ◽

Receptor Activity ◽

Interleukin 1Β ◽

Hep G2 ◽

Hep G2 Cells ◽

G2 Cells

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

Biochemical Journal ◽

10.1042/bj2470739 ◽

1987 ◽

Vol 247 (3) ◽

pp. 739-746 ◽

Cited By ~ 83

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.

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