scholarly journals The low-density-lipoprotein pathway of native and chemically modified low-density lipoproteins isolated from plasma incubated in vitro

1984 ◽  
Vol 224 (2) ◽  
pp. 569-576 ◽  
Author(s):  
R Zechner ◽  
H Dieplinger ◽  
A Roscher ◽  
G M Kostner
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Fold Increase ◽  
Low Density ◽  
Apo B ◽  
Modified Ldl ◽  
Apo E ◽  
Lower Extent ◽  
Ldl Catabolism

Normal fasting human plasma was incubated for 24 h at 37 degrees C in the presence or absence of lecithin:cholesterol acyltransferase (LCAT) inhibitors. The low-density lipoprotein (LDL) fractions of incubated plasma (control LDL and LCAT-modified LDL) were studied with respect to their chemical and functional properties. LCAT-modified LDL differed from control LDL by a decreased phospholipid and free-cholesterol content, but increased cholesteryl esters. Furthermore, an increase of the relative protein content in LDL by 16-20% was found. Apolipoproteins of LCAT-modified LDL exhibited a 10-fold increase of apo AI, a 4-5-fold increase of apo E, and a 2-fold increase of apo C. All these apolipoproteins resided together with apo B on the same particles. LCAT-modified LDL displayed a higher electrophoretic mobility, a higher hydrated density, a decreased flotation constant and a smaller diameter. Cultured human fibroblasts bound and internalized LCAT-modified LDL to a lower extent than control LDL. The degradation, however, was faster. Modified LDL suppressed 3-hydroxy-3-methylglutaryl-CoA reductase activity to a lower extent than did control LDL. Our results demonstrate that LCAT action, together with lipid transfer and exchange processes, markedly alters the chemical and physiochemical properties of LDL. This in turn significantly influences LDL catabolism in vitro.

scholarly journals Development of Capture Assays for Different Modifications of Human Low-Density Lipoprotein

2005 ◽  
Vol 12 (1) ◽  
pp. 68-75 ◽  
Author(s):  
Gabriel Virella ◽  
M. Brooks Derrick ◽  
Virginia Pate ◽  
Charlyne Chassereau ◽  
Suzanne R. Thorpe ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Gas Chromatography Mass Spectrometry ◽  
Low Density ◽  
Modified Ldl ◽  
Capture Assay ◽  
Carboxymethyl Lysine

ABSTRACT Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), N ε(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r = 0.706, P < 0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.

scholarly journals Low-density lipoprotein susceptibility to in vitro oxidation in healthy smokers and nonsmokers

1996 ◽  
Vol 42 (4) ◽  
pp. 524-530 ◽  
Author(s):  
R Siekmeier ◽  
P Wülfroth ◽  
H Wieland ◽  
W Gross ◽  
W März
Keyword(s):  
Body Mass ◽  
Ldl Cholesterol ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Hdl Cholesterol ◽  
Thiobarbituric Acid ◽  
Low Density ◽  
Apo B ◽  
Lag Times

Abstract We analyzed the susceptibility of low-density lipoproteins (LDL) to oxidation in 17 healthy smokers (43.3 +/- 16.8 pack-years) and 19 healthy nonsmokers, matched for age (smokers: 52 +/- 7 years; nonsmokers: 53 +/- 7 years), gender, and relative body mass. Cholesterol, triglycerides, LDL cholesterol, HDL cholesterol, and apolipoprotein (apo) B were not different between smokers and nonsmokers; apo A-I was slightly lower in smokers (one-tailed P = 0.066). To study whether LDL from smokers were prone to in vitro oxidation than LDL from nonsmokers, we measured the time kinetics of diene formation and the production of malondialdehyde during oxidation of LDL in vitro. In smokers and nonsmokers, respectively, the mean (+/-SD) lag times (tinh) of diene formation were 111 +/- 26 and 100 +/- 27 min, the peak rates of diene formation (Vmax) were 5.99 +/- 2.34 and 6.34 +/- 2.30 mmol x min-1 x g-1, and the amounts of dienes produced during the propagation phase (dmax) were 250 +/- 264 and 248 +/- 56 mmol x g-1. Neither the malondialdehyde content of LDL (measured as thiobarbituric acid-reactive substances) before oxidation nor the amount of malondialdehyde generated during oxidation (smokers: 57.0 +/- 14.2 micromol x g-1; nonsmokers: 63.2 +/- 15.2 micromol x g-1 indicated any statistically significant effect of smoking. When nonsmokers and smokers were considered together, the amount of malondialdehyde generated during oxidation correlated with age (nonparametric rs = 0.405), body mass index (r2 = 0.573), and concentrations of apo B (rs = 0.480), cholesterol (rs = 0.448), triglycerides (rs = 0.436), and LDL cholesterol (rs = 0.398). Our data show that smoking is not associated with increased oxidizability of LDL in healthy men and women at ages 42-63 years.

scholarly journals Scavenger Receptors Class A-I/II and CD36 Are the Principal Receptors Responsible for the Uptake of Modified Low Density Lipoprotein Leading to Lipid Loading in Macrophages

2002 ◽  
Vol 277 (51) ◽  
pp. 49982-49988 ◽  
Author(s):  
Vidya V. Kunjathoor ◽  
Maria Febbraio ◽  
Eugene A. Podrez ◽  
Kathryn J. Moore ◽  
Lorna Andersson ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Gas Chromatography Mass Spectrometry ◽  
Scavenger Receptors ◽  
Low Density ◽  
Lipid Uptake ◽  
Modified Ldl ◽  
Ldl Uptake

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptakein vitroand atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75–90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.

scholarly journals The metabolic conversion of very-low-density lipoprotein into low-density lipoprotein by the extrahepatic tissues of the rat

1979 ◽  
Vol 178 (2) ◽  
pp. 455-466 ◽  
Author(s):  
B S Suri ◽  
M E Targ ◽  
D S Robinson
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Quantitative Information ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Apo B ◽  
Extrahepatic Tissues

1. The work reported was designed to provide quantitative information about the capacity of the extrahepatic tissues of the rat to degrade injected VLD lipoproteins (very-low-density lipoproteins, d less than 1.006) to LD lipoproteins (low-density lipoproteins, d 1.006–1.063) and to study the fate of the different VLD-lipoprotein apoproteins during the degradative process. 2. Rat liver VLD lipoproteins, radioactively labelled in their protein moieties, were produced by the perfusion of the organ and were either injected into the circulation of the supradiaphragmatic rats or incubated in rat plasma at 37 degrees C. At a time (75 min) when approx. 90% of the triacylglycerol of the VLD lipoproteins had been hydrolysed the supradiaphragmatic rats were bled and VLD lipoproteins, LD lipoproteins and HD lipoproteins (high-density lipoproteins, d 1.063–1.21) were separated from their plasma and from the plasma incubated in vitro. The apoproteins of each of the lipoprotein classes were resolved by gel-filtration chromatography into three main fractions, designated peaks I, II and III. 3. Incubation of the liver VLD lipoproteins in plasma in vitro led to the transfer of about 30% of the total protein radioactivity to the HD lipoproteins. The transfer mainly involved the peak-II (arginine-rich and/or apo A-I) and peak-III (apo C) proteins. There was also a small transfer of radioactivity (about 5% of the total) to the LD lipoproteins. 4. Injection of the liver VLD lipoproteins into the circulation of the supradiaphragmatic rat resulted in the transfer of about 15% of the total VLD-lipoprotein radioactivity to the LD lipoproteins. The transfer involved mainly the peak-I (apo B) proteins and accounted for about 20% of the total apo B protein radioactivity of the injected VLD lipoproteins. When the endogenous plasma VLD lipoprotein was taken into account the transfer of apo B protein was about 35%. 5. The transfer of peak-II protein radioactivity from the VLD to the HD lipoproteins was greater in the plasma of the supradiaphragmatic rat than in the incubated plasma suggesting that there was a net transfer of peak-II apoproteins during the VLD lipoprotein degradation. The transfer of peak-III protein radioactivity was not greater in the plasma of the supradiaphragmatic rat, but there was a loss of this radioactivity from the circulation.

scholarly journals Plasma clearance and net uptake of α-tocopherol and low-density lipoprotein by tissues in WHHL and control rabbits

1992 ◽  
Vol 287 (1) ◽  
pp. 247-254 ◽  
Author(s):  
W Cohn ◽  
M A Goss-Sampson ◽  
H Grun ◽  
D P R Muller
Keyword(s):  
Plasma Clearance ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Alpha Tocopherol ◽  
Low Density ◽  
Apo B ◽  
Net Uptake ◽  
Lipoprotein Composition

The mechanism(s) of uptake of vitamin E (alpha-tocopherol) by tissues is poorly understood. It has, however, been suggested from studies in vitro that the apolipoprotein B/E (apo B/E) receptor pathway for low-density lipoprotein (LDL) may be involved. To investigate the role of the apo B/E receptor pathway in vivo, we have studied the transport and uptake of alpha-tocopherol by tissues in Watanabe Heritable Hyperlipidaemic (WHHL) rabbits, which lack functional LDL (apo B/E) receptors, and controls. [3H]alpha-Tocopherol incorporated within LDL labelled with [14C]sucrose was used in these studies, as this enabled the uptake of both alpha-tocopherol and LDL to be studied independently. The principal findings were as follows. (1) Concentrations of the circulating lipids (including alpha-tocopherol) and LDL were increased and the plasma fractional disappearance rates of alpha-tocopherol and LDL decreased in the WHHL rabbits. (2) The WHHL rabbits clear more LDL and alpha-tocopherol from the circulation than controls do, because of their increased pool sizes of alpha-tocopherol and LDL. (3) The lipoprotein composition of the WHHL rabbits differed from that of the controls, and there was exchange of alpha-tocopherol between the lipoprotein fractions in vivo and in vitro. (4) High-affinity apo B/E receptors were not essential for the uptake of alpha-tocopherol by tissues. (5) Evidence from the plasma-clearance and tissue data suggest that alpha-tocopherol can be taken up by tissues in association with, and also independent of, LDL. We conclude that there are several different mechanisms for the uptake of alpha-tocopherol by tissues, which include receptor-dependent and receptor-independent pathways, independent transport and co-transport of alpha-tocopherol and LDL, and uptake from a number of different lipoproteins.

scholarly journals Low-density-lipoprotein binding by mast-cell granules. Demonstration of binding of apolipoprotein B to heparin proteoglycan of exocytosed granules

1987 ◽  
Vol 241 (2) ◽  
pp. 583-589 ◽  
Author(s):  
J O Kokkonen ◽  
P T Kovanen
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Major Protein ◽  
Low Density ◽  
Apo B ◽  
Apo E ◽  
Lipoprotein Binding ◽  
Positively Charged

To study the interaction between low-density lipoprotein (LDL) and granules from rat serosal mast cells in vitro, mast cells were stimulated with the degranulating agent 48/80 to induce exocytosis of the secretory granules. Subsequent incubation of the exocytosed granules with 125I-LDL resulted in binding of the labelled LDL to the granules. When increasing amounts of agent 48/80 were added to mast-cell suspensions, a dose-dependent release of granules was observed and a parallel increase in the amount of 125I-LDL bound to granules resulted. 125I-LDL bound to a single class of high-affinity binding sites on the granules. At saturation, 105 ng of LDL were bound per microgram of granule protein. The lipoprotein binding to mast-cell granules was apolipoprotein(apo)-B + E-specific. Thus 125I-LDL binding to the granules was effectively compared for by LDL (apo-B) or by dimyristoyl phosphatidylcholine vesicles containing apo-E, but not by high-density lipoprotein (HDL3) containing apo-AI as their major protein component. Neutralization by acetylation of the positively charged amino groups of apo-B of LDL or presence of a high ionic strength in the incubation medium prevented LDL from binding to the granules, indicating the presence of ionic interactions between the positively charged amino acids of LDL and negatively charged groups of the granules. It could be demonstrated that LDL bound to the negatively charged heparin proteoglycan of the granules. Thus treatment of granules with heparinase resulted in loss of their ability to bind LDL, and substances known to bind to heparin, such as Toluidine Blue, avidin, lipoprotein lipase, fibronectin and protamine, all effectively competed with LDL for binding to the granules. The results show that LDL is efficiently bound to the heparin proteoglycan component of mast-cell granules once the mast cells are stimulated to release their granules into the extracellular space.

scholarly journals Stimulation of apolipoprotein secretion in very-low-density and high-density lipoproteins from cultured rat hepatocytes by dexamethasone

1990 ◽  
Vol 271 (3) ◽  
pp. 575-583 ◽  
Author(s):  
P Martin-Sanz ◽  
J E Vance ◽  
D N Brindley
Keyword(s):  
Low Density Lipoprotein ◽  
Lipoprotein Metabolism ◽  
Density Lipoprotein ◽  
Rat Hepatocytes ◽  
High Density ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Apo B ◽  
Apo E ◽  
The Masses

The effects of dexamethasone (a synthetic glucocorticoid) and insulin on the secretion of very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) were investigated. Rat hepatocytes in monolayer culture were preincubated for 15 h in the presence or absence of combinations of 100 nM-dexamethasone and 2 nM-, 10 nM- or 50 nM-insulin. Dexamethasone increased [3H]oleate incorporation into secreted triacylglycerol by 2.7-fold and the mass of triacylglycerol secreted by 1.5-fold. Insulin alone decreased these parameters and antagonized the effect of dexamethasone. Dexamethasone increased the secretion of [3H]leucine in apolipoprotein (apo) E, and in the large (BH) and small (BI) forms of apo B in VLDL by about 7.1-, 3.6- and 4.0-fold respectively. Insulin alone decreased the secretion of these 3H-labelled apolipoproteins in VLDL. However, 2 nM-insulin with dexamethasone increased the secretion of 3H-labelled apo BH and apo BL by a further 0.8- and 3.2-fold respectively; 50 nM-insulin decreased the secretions of apo E, apo BH and apo BL in VLDL. Similar effects for dexamethasone or insulin alone were also obtained for the masses of apo E and apo BL + H secreted in VLDL. Albumin secretion was not significantly altered by either dexamethasone or insulin alone, but in combination they stimulated by 2.1-2.6-fold. Insulin or dexamethasone alone had little effect on the secretion of apolipoproteins in the HDL fraction. However, dexamethasone plus 2 nM-insulin increased the incorporation of [3H]leucine into apo AI, apo AH plus apo C, apo AIV and apo E of HDL by about 1.8-, 1.6-, 1.7- and 2.0-fold respectively. The apo E in the bottom fraction represented about 69% of the total 3H-labelled apo E secreted. The responses in the total secretion of apo E from the hepatocytes resembled those seen in HDL. The interactions of insulin and dexamethasone are discussed in relation to the general regulation of lipoprotein metabolism, the development of hyperlipidaemias and the predisposition to premature atherosclerosis.

Activity of low-density lipoprotein receptors as estimated from concentrations of apolipoprotein B and C-II in serum.

1988 ◽  
Vol 34 (11) ◽  
pp. 2224-2227 ◽  
Author(s):  
H Ito ◽  
C Naito ◽  
H Hayashi ◽  
M Kawamura
Keyword(s):  
Serum Cholesterol ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Receptor Activity ◽  
Total Serum ◽  
Total Serum Cholesterol ◽  
Low Density ◽  
Apo B ◽  
Apo E

Abstract The correlation between low-density lipoprotein (LDL) receptor activity and concentrations of lipids and apolipoproteins in serum was examined in 12 subjects with heterozygous familial hypercholesterolemia (FH) and in four with non-FH type II hyperlipoproteinemia. Concentrations of high-density lipoprotein cholesterol and of apolipoproteins (apo) A-I, C-II, and C-III were significantly positively correlated with LDL receptor activity, whereas LDL receptor activity was significantly inversely correlated with LDL cholesterol and apo B concentrations, and with apo ratios B/A-I and B/A-II. Neither total serum cholesterol, triglyceride, phospholipid, apo A-I, nor apo E concentrations correlated significantly with LDL receptor activity. Multiple regression analysis, with LDL receptor activity as the dependent variable, revealed concentrations of apo B and apo C-II to be the principal determinant factors. To confirm this, we subsequently calculated the LDL receptor activities before and after administration of CS-514, an inhibitor of hydroxymethylglutaryl-CoA reductase (EC 1.1.1.88), which increases the hepatic LDL receptor activity and decreases the concentration of cholesterol in serum. This drug increased calculated LDL receptor activities significantly, with a significant decrease in serum cholesterol.

scholarly journals The p.Leu167del Mutation in APOE Gene Causes Autosomal Dominant Hypercholesterolemia by Down-regulation of LDL Receptor Expression in Hepatocytes

2016 ◽  
Vol 101 (5) ◽  
pp. 2113-2121 ◽  
Author(s):  
Ana Cenarro ◽  
Aitor Etxebarria ◽  
Isabel de Castro-Orós ◽  
Marianne Stef ◽  
Ana M. Bea ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Fold Increase ◽  
Receptor Expression ◽  
Apoe Gene ◽  
University Hospital ◽  
Low Density ◽  
Down Regulation ◽  
Apo E

Abstract Context: The p.Leu167del mutation in the APOE gene has been associated with hyperlipidemia. Objectives: Our objective was to determine the frequency of p.Leu167del mutation in APOE gene in subjects with autosomal dominant hypercholesterolemia (ADH) in whom LDLR, APOB, and PCSK9 mutations had been excluded and to identify the mechanisms by which this mutant apo E causes hypercholesterolemia. Design: The APOE gene was analyzed in a case-control study. Setting: The study was conducted at a University Hospital Lipid Clinic. Patients or Other Participants: Two groups (ADH, 288 patients; control, 220 normolipidemic subjects) were included. Intervention: We performed sequencing of APOE gene and proteomic and cellular experiments. Main Outcome Measure: To determine the frequency of the p.Leu167del mutation and the mechanism by which it causes hypercholesterolemia. Results: In the ADH group, nine subjects (3.1%) were carriers of the APOE c.500_502delTCC, p.Leu167del mutation, cosegregating with hypercholesterolemia in studied families. Proteomic quantification of wild-type and mutant apo E in very low-density lipoprotein (VLDL) from carrier subjects revealed that apo E3 is almost a 5-fold increase compared to mutant apo E. Cultured cell studies revealed that VLDL from mutation carriers had a significantly higher uptake by HepG2 and THP-1 cells compared to VLDL from subjects with E3/E3 or E2/E2 genotypes. Transcriptional down-regulation of LDLR was also confirmed. Conclusions: p.Leu167del mutation in APOE gene is the cause of hypercholesterolemia in the 3.1% of our ADH subjects without LDLR, APOB, and PCSK9 mutations. The mechanism by which this mutation is associated to ADH is that VLDL carrying the mutant apo E produces LDLR down-regulation, thereby raising plasma low-density lipoprotein cholesterol levels.

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