scholarly journals Measurement of the absolute number of functioning low-density lipoprotein receptors in vivo using a monoclonal antibody

1995 ◽  
Vol 305 (3) ◽  
pp. 897-904 ◽  
Author(s):  
C Fitzsimmons ◽  
R Bush ◽  
D Hele ◽  
C Godliman ◽  
E Gherardi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Body Weight ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Absolute Number ◽  
Low Density ◽  
Ldl Receptors ◽  
The Absolute

MAC188 S/S is a monoclonal antibody which can be used in vivo to measure the absolute number of functioning low-density lipoprotein (LDL) receptors in a rabbit. The antibody binds to the extra-cellular domain of the LDL receptor and binding is not blocked by the presence of LDL. When the antibody-receptor complex is internalized, receptor recycling is inhibited for several hours. Thus when saturating doses of MAC188 S/S are administered intravenously, the amount of antibody removed from the blood (minus non-specific removal) is determined solely by the total number of LDL receptors in an animal. In this study MAC188 S/S was used to measure the number of LDL receptors in control rabbits and in animals treated with 17 alpha-ethinyl oestradiol. After treatment (which caused a 47% decrease in plasma cholesterol), receptor-mediated removal of MAC188 S/S from the blood was saturated in both groups following injection of 3.0 mg of antibody per kg body weight. Based on the amount of antibody removed via the LDL receptor at this dose, the total number of accessible LDL receptors was calculated as (2.0 +/- 0.3) x 10(15) receptors per kg body weight in control rabbits and (4.0 +/- 0.4) x 10(15) receptors per kg body weight in oestrogen-treated animals. The number of receptors in various organs was also determined. The monoclonal antibody approach therefore, allows accurate determination of LDL receptor numbers in animals with markedly different concentrations of circulating LDL, conditions in which the use of endogenous ligand would be subject to significant errors.

scholarly journals Interaction of 4-hydroxynonenal-modified low-density lipoproteins with the fibroblast apolipoprotein B/E receptor

1986 ◽  
Vol 234 (1) ◽  
pp. 245-248 ◽  
Author(s):  
W Jessup ◽  
G Jurgens ◽  
J Lang ◽  
H Esterbauer ◽  
R T Dean
Keyword(s):  
Apolipoprotein B ◽  
Negative Charge ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipid Peroxidation Product ◽  
Modified Low Density Lipoproteins ◽  
Peroxidation Product

The incorporation of the lipid peroxidation product 4-hydroxynonenal into low-density lipoprotein (LDL) increases the negative charge of the particle, and decreases its affinity for the fibroblast LDL receptor. It is suggested that this modification may occur in vivo, and might promote atherogenesis.

Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

1986 ◽  
Vol 6 (9) ◽  
pp. 3268-3277
Author(s):  
R D Sege ◽  
K F Kozarsky ◽  
M Krieger
Keyword(s):  
Gamma Irradiation ◽  
Cho Cells ◽  
Gamma Ray ◽  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Ldl Receptors

The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

scholarly journals Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene.

1986 ◽  
Vol 102 (5) ◽  
pp. 1567-1575 ◽  
Author(s):  
K F Kozarsky ◽  
H A Brush ◽  
M Krieger
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Wild Type ◽  
Ovary Cells ◽  
Ldl Receptors

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild-type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.

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.

Increase in In Vivo Low-Density Lipoprotein (LDL) Receptor Binding after PGE1 and 13,14-Dihydro-PGE1 Treatment in Rabbits

1993 ◽  
Vol 21 (3) ◽  
pp. 503-506 ◽  
Author(s):  
H. Sinzinger ◽  
Irene Virgolini ◽  
S. R. Li ◽  
A. Gerakakis ◽  
P. Fitscha ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density

scholarly journals Regulation of plasma LDL: the apoB paradigm

2009 ◽  
Vol 118 (5) ◽  
pp. 333-339 ◽  
Author(s):  
Allan D. Sniderman ◽  
Jacqueline De Graaf ◽  
Patrick Couture ◽  
Ken Williams ◽  
Robert S. Kiss ◽  
...  
Keyword(s):  
Apolipoprotein B ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Critical Aspect ◽  
Lipoprotein Particles ◽  
Ldl Particles ◽  
Plasma Compartment

The objectives of this analysis are to re-examine the foundational studies of the in vivo metabolism of plasma LDL (low-density lipoprotein) particles in humans and, based on them, to reconstruct our understanding of the governance of the concentration of plasma LDL and the maintenance of cholesterol homoeostasis in the hepatocyte. We believe that regulation of cholesterol homoeostasis within the hepatocyte is demonstrably more complex than envisioned by the LDL receptor paradigm, the conventional model to explain the regulation of plasma LDL and the fluxes of cholesterol into the liver, a model which was generated in the fibroblast but has never been fully validated in the hepatocyte. We suggest that the LDL receptor paradigm should be reconfigured as the apoB (apolipoprotein B) paradigm, which states that the rate at which LDL particles are produced is at least an important determinant of their concentration in plasma as the rate at which they are cleared from plasma and that secretion of cholesterol within VLDL (very-low-density lipoprotein) particles is an important mechanism of maintaining cholesterol homoeostasis within the hepatocyte. These two paradigms are not mutually exclusive. The LDL receptor paradigm, however, includes only one critical aspect of the regulation of plasma LDL, namely the rate at which LDL particles are cleared through the LDL receptor pathway, but ignores another – the rate at which LDL particles are added to the plasma compartment. The apoB paradigm includes both and points to a different model of how the hepatocyte achieves cholesterol homoeostasis in a complex metabolic environment.

Low-density lipoprotein receptors in rat adipocytes: regulation with fasting

1994 ◽  
Vol 266 (1) ◽  
pp. E26-E32 ◽  
Author(s):  
F. B. Kraemer ◽  
C. Laane ◽  
B. Park ◽  
C. Sztalryd
Keyword(s):  
Adipose Tissue ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Expression ◽  
Low Density ◽  
Fat Cell ◽  
Ldl Receptors ◽  
Rat Adipocytes ◽  
Epididymal Fat

Adipose tissue metabolism is exquisitely sensitive to caloric intake. With increasing adiposity more triglyceride and cholesterol are stored within increasingly large adipocytes, whereas less triglyceride and cholesterol are stored as the size of the fat cell decreases. A portion of the uptake of cholesterol by adipocytes is mediated by low-density lipoprotein (LDL) receptors. The present studies addressed whether LDL receptors are differentially regulated in adipose tissue and the liver during fasting in the rat. Two days of fasting caused a reduction in body weight with an approximately 40% decrease in the epididymal fat depot and fat cell size. No changes in serum cholesterol were noted, but serum triglycerides fell approximately 55% with fasting. LDL receptors detected by immunoblotting decreased progressively with fasting to levels that were 95% below controls in adipocytes isolated from epididymal fat pads by 2-3 days. In contrast, hepatic LDL receptor expression was unaltered by fasting. After 2 days of fasting, the rate of synthesis of LDL receptors in isolated adipose cells was decreased approximately 35%, whereas levels of LDL receptor mRNA were diminished approximately 55%. It is concluded that the expression of LDL receptors in rat adipocytes is markedly downregulated during fasting through transcriptional and posttranscriptional mechanisms. Furthermore, LDL receptor expression is differentially regulated in adipose tissue and liver during fasting in the rat.

scholarly journals The influence of particle size and multiple apoprotein E-receptor interactions on the endocytic targeting of beta-VLDL in mouse peritoneal macrophages.

1991 ◽  
Vol 115 (6) ◽  
pp. 1547-1560 ◽  
Author(s):  
I Tabas ◽  
J N Myers ◽  
T L Innerarity ◽  
X X Xu ◽  
K Arnold ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Peritoneal Macrophages ◽  
Low Density ◽  
Cholesterol Acyl Transferase ◽  
Ldl Receptors ◽  
Apo E ◽  
Partial Neutralization ◽  
Mouse Peritoneal Macrophages

Low density lipoprotein (LDL) and beta-very low density lipoprotein (beta-VLDL) are internalized by the same receptor in mouse peritoneal macrophages and yet their endocytic patterns differ; beta-VLDL is targeted to both widely distributed and perinuclear vesicles, whereas LDL is targeted almost entirely to perinuclear lysosomes. This endocytic divergence may have important metabolic consequences since beta-VLDL is catabolized slower than LDL and is a more potent stimulator of acyl-CoA/cholesterol acyl transferase (ACAT) than LDL. The goal of this study was to explore the determinants of beta-VLDL responsible for its pattern of endocytic targeting. Fluorescence microscopy experiments revealed that large, intestinally derived, apoprotein (Apo) E-rich beta-VLDL was targeted mostly to widely distributed vesicles, whereas small, hepatically derived beta-VLDL was targeted more centrally (like LDL). Furthermore, the large beta-VLDL had a higher ACAT-stimulatory potential than the smaller beta-VLDL. The basis for these differences was not due to fundamental differences in the means of uptake; both large and small beta-VLDL were internalized by receptor-mediated endocytosis (i.e., not phagocytosis) involving the interaction of Apo E of the beta-VLDL with the macrophage LDL receptor. However, large beta-VLDL was much more resistant to acid-mediated release from LDL receptors than small beta-VLDL. Furthermore, partial neutralization of the multiple Apo Es on these particles by immunotitration resulted in a more perinuclear endocytic pattern, a lower ACAT-stimulatory potential, and an increased sensitivity to acid-mediated receptor release. These data are consistent with the hypothesis that the interaction of the multivalent Apo Es of large beta-VLDL with multiple macrophage LDL receptors leads to a diminished or retarded release of the beta-VLDL from its receptor in the acidic sorting endosome which, in turn, may lead to the widely distributed endocytic pattern of large beta-VLDL. These findings may represent a physiologically relevant example of a previously described laboratory phenomenon whereby receptor cross-linking by multivalent ligands leads to a change in receptor targeting.

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