Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17α-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.     

Overexpression of SR-BI by adenoviral vector promotes clearance of apoA-I,but not apoB,in human apoB transgenic mice

Scavenger receptor BI (SR-BI) is a multi-ligand lipoprotein receptor that mediates selective lipid uptake from HDL, and plays a central role in hepatic HDL metabolism. In this report, we investigated the extent to which SR-BI selective lipid uptake contributes to LDL metabolism. As has been reported for human LDL, mouse SR-BI expressed in transfected cells mediated selective lipid uptake from mouse LDL. However, LDL-cholesteryl oleoyl ester (CE) transfer relative to LDL-CE bound to the cell surface (fractional transfer) was approximately 18-fold lower compared with HDL-CE. Adenoviral vector-mediated SR-BI overexpression in livers of human apoB transgenic mice ( approximately 10-fold increased expression) reduced plasma HDL-cholesterol (HDL-C) and apolipoprotein (apo)A-I concentrations to nearly undetectable levels 3 days after adenovirus infusion. Increased hepatic SR-BI expression resulted in only a modest depletion in LDL-C that was restricted to large LDL particles, and no change in steady-state concentrations of human apoB. Kinetic studies showed a 19% increase in the clearance rate of LDL-CE in mice with increased SR-BI expression, but no change in LDL apolipoprotein clearance. Quantification of hepatic uptake of LDL-CE and LDL-apolipoprotein showed selective uptake of LDL-CE in livers of human apo B transgenic mice. However, such uptake was not significantly increased in mice over-expressing SR-BI. We conclude that SR-BI-mediated selective uptake from LDL plays a minor role in LDL metabolism in vivo.     

Decomposition of lipid hydroperoxides enhances the uptake of low density lipoprotein by macrophages.

This study examined the roles of low-density lipoprotein (LDL) lipid oxidation and peroxide breakdown in its conversion to a form rapidly taken up by mouse peritoneal macrophages. Oxidation of the LDL without decomposition of the hydroperoxide groups was performed by exposure to gamma radiation in air-saturated solutions. Virtually complete decomposition of the hydroperoxides was achieved by treatment of the irradiated LDL with Cu2+ under strictly anaerobic conditions. No uncontrolled LDL uptake by macrophages occurred when the lipoprotein contained less than 150 hydroperoxide groups per particle. More extensively oxidized LDL was taken up and degraded by mouse macrophages significantly faster than the native lipoprotein. The uptake was greatly enhanced by treatment of the oxidized LDL with Cu2+. A significant proportion of the LDL containing intact or copper-decomposed LDL hydroperoxide groups accumulated within the macrophages without further degradation. Treatment of the radiation-oxidized LDL with Cu2+ was accompanied by aggregation of the particles. Competition studies showed that the oxidized LDL was taken up by macrophages via both the LDL and the scavenger receptors, whereas the copper-treated lipoprotein entered the cells only by the scavenger pathway. Phagocytosis also played an important role in the metabolism of all forms of the extensively modified LDL. Our results suggest that minimally-oxidized LDL is not recognized by the macrophage scavenger receptors unless the lipid hydroperoxide groups are decomposed to products able to derivatize the apo B protein.     

Apolipoprotein B-32: a new truncated mutant of human apolipoprotein B capable of forming particles in the low density lipoprotein range.

We have identified a new species of apolipoprotein (apo) B in an individual with heterozygous hypobetalipoproteinemia. The new apo B (apo B-32) is the result of a single point mutation (1450 Gln----Stop) in the apo B gene that prevents full length translation. Apo B-32 is predicted to contain the 1449 amino-terminal amino acids of apo B-100 and is associated with a markedly decreased low density lipoprotein (LDL) cholesterol level. The density distribution of apo B-32 in the plasma lipoproteins makes it unique amongst other truncated apo B species. Normally, apo B-100 is found in both very low density lipoprotein (VLDL) and LDL particles. However, the majority of the apo B-32 protein was found in the high density lipoprotein (HDL) and lipoprotein-deplete (d greater than 1.21 g/ml) fractions, suggesting that it was mainly assembled into abnormally dense lipoprotein particles. A small amount of apo B-32 was also found in the LDL, making it the shortest known apo B variant capable of forming particles in this density range. Apo B-32 was undetected in VLDL. The apo B-32 mutation further defines the minimum length of the apo B protein that is required for the assembly of LDL.     

Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ～400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.     

Enhanced macrophage uptake of elastase-modified high-density lipoproteins

Incubation of human HDL (d = 1.063-1.21 g/ml) with monocyte-derived elastase causes selective proteolysis of apoA-II and apoA-I apolipoproteins. We have found that elastase-digested HDL (ED-HDL) bind to J774-A1 murine macrophages with enhanced affinity and are internalized and degraded at a rate threefold higher than that of native HDL. Unlike oxidized LDL and HDL and proteolytically modified LDL, the uptake of ED-HDL lipoproteins does not affect the cellular lipid biosynthesis nor modify the cell lipid content. The cell surface binding of (125)I-ED-HDL can be competed by native HDL but not by acetylated LDL, consistent with the idea that ED-HDL are recognized by the class B type I scavenger receptor. The liberation of elastase by lipid-engorging macrophages is regarded as an important event during atherogenesis. By enhancing the cellular uptake of HDL this process can lead to a local decrease of antiatherogenic HDL particles.     

Lipoprotein secretion by the human hepatoma cell line Hep G2: differential rates of accumulation of apolipoprotein B and lipoprotein lipids in tissue culture media in response to albumin, glucose and oleate

Serum low-density lipoprotein (LDL) concentration is a major determinant of susceptibility to the development of atherosclerosis. A major component of the protein moiety of LDL and its precursor very-low-density lipoprotein is apolipoprotein B (apo B). The human hepatoma cell line, Hep G2, was used as a model for the investigation of mechanisms which control hepatic secretion of the apo B and lipid components of lipoproteins. Using a sensitive immunoradiometric assay for apo B developed in this laboratory, we showed that bovine serum albumin inhibited and glucose, and fatty acids enhanced the rate of accumulation of apo B in the culture medium of Hep G2 cells. However, these substances did not necessarily affect LDL lipids in the same way as apo B. This finding appeared to be due to Hep G2 cells expressing lipase activities which led to triacylglycerol and phospholipid hydrolysis and lipid reuptake. Reuptake of apo B also occurred, but its rate of accumulation in the culture medium suggested it was a closer reflection of its true secretory rate.     

Structure of the lipopolysaccharide/human plasma low density lipoprotein complex. 31P-NMR and fluorescence spectroscopy studies

Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) containing various amounts of LPS were prepared in vitro. The 31P-NMR spectra showed that in the LDL-LPS complexes as well as in native LDL all phosphate groups of phospholipids are accessible to the paramagnetic shift reagent, Pr3+. Besides, the low frequency mobility of phospholipid phosphates in the complex is diminished. It was supposed that the phospholipid molecules in the LDL/LPS complex as in native LDL form a monolayer structure on the surface of LDL. The intrinsic fluorescence spectra of tryptophan residues of the apoprotein (apo B-100) revealed that the incorporation of LPS molecules into LDL particles is accompanied by minor changes in the conformation and orientation of the apo B molecule. As a result of these changes, certain fragments become exposed to a more hydrophilic environment and become more accessible to fluorescence quenchers. The use of charged (I-, Cs+) and uncharged (acrylamide) quenchers permitted to identify in the apo B molecule different tryptophan residues, some of which are localized in the vicinity of negatively charged groups, whereas others are neighbouring positively charged groups. It is suggested that the LPS molecules incorporated into LDL particle do not screen the apo B molecule to such an extent that it would hinder the LDL/LPS complex binding to apo B/E cellular receptors.     

Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E.

Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.     

Extent of copper LDL oxidation depends on oxidation time and copper/LDL ratio: chemical characterization

The aim of our study was to determine, as a function of [Cu(2+)]/[LDL] ratios (0.5 and 0.05) and of oxidation phases, the extent of LDL oxidation by assessing the lipid and apo B oxidation products. The main results showed that: (i) kinetics of conjugated diene formation presented four phases for Cu(2+)/LDL ratio of 0.5 and two phases for [Cu(2+)]/[LDL] ratio of 0.05; (ii) oxidation product formation (cholesteryl ester and phosphatidylcholine hydroperoxides, apo B carbonyl groups) occurred early in the presence of endogenous antioxidants, under both copper oxidation conditions; (iii) apo B carbonylated fragments appeared when antioxidants were totally consumed at [Cu(2+)]/[LDL] ratio of 0.5; and (iv) antioxidant concentrations were stable, oxysterol formation was negligible, and no carbonylated fragment was detected at [Cu(2+)]/[LDL] ratio of 0.05. Depending on the copper/LDL ratio, oxidized LDL differ greatly in the nature of lipid peroxidation product and the degree of apo B fragmentation.     

Dynamic behavior of apoproteins of human plasma very low density lipoproteins and lipolysis regulation

Using the dynamic fluorescence quenching method, it was shown that very low density (VLDL) apoproteins (apo B, E and C) tryptophanyls exhibit a lower accessibility towards water-soluble quenchers as compared to apo B LDL chromophores. The efficiency of proteolytic degradation by trypsin of VLDL-associated apo E and apo C was much lower than that of apo B. These results may be due to the cluster arrangement of amphipatic apo E and apo C on the VLDL surface and/or to their partial shielding by apo B. Treatment of VLDL particles with sub-lytic concentrations of the detergent, Tween-20, did not change the relaxation characteristics of amphipatic apoprotein tryptophanyl microenvironment, but resulted in a reversible structural transition registered by a "red" shift of the emission spectrum maximum as well as by change of the iodine quenching pattern. The detergent-induced increase of the VLDL tryptophanyl accessibility to acrylamide and the decrease of the quenching constant at the partial and complete particle solubilization were related to a change of the apo B molecular package. Treatment of VLDL with Tween-20 or cow milk lipoprotein lipase resulted in the appearance of tryptophanyl population that was not involved in the resonance energy transfer to the lipid phase-localized fluorescent probe pyrene, which is indicative of the protein dissociation. Treatment of VLDL particles with sub-lytic concentrations of Tween-20 revealed a lower (compared to apo C) relative affinity of apo E for the VLDL lipid surface. Inhibition of the lipoprotein lipase activity by apoprotein C-III was found to be non-competitive. It was concluded that lipolysis is a self-regulatory process which involves changes in the effector apoprotein concentration on the surface of triglyceride-rich particles.     

Apolipoprotein-lipid association in oxidatively modified HDL and LDL

We investigated the effect of Cu²⁺ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation of HDL with Cu²⁺ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (λ_max). LDL incubated with Cu²⁺ also showed a rapid decrease in Trp fluorescence intensity with time, but with no associated increase in λ_max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in λ_max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in λ_max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in λ_max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.     

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.     

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

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.     

Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 (congruent to 450,000). The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.     

Endocytosis is not required for the selective lipid uptake mediated by murine SR-BI

The scavenger receptor class B, type I (SR-BI) mediates the cellular selective uptake of cholesteryl esters and other lipids from high-density lipoproteins (HDL) and low-density lipoproteins (LDL). This process, unlike classical receptor-mediated endocytosis, does not result in lipoprotein degradation. Instead, the lipid depleted particles are released into the medium. Here we show that selective lipid uptake mediated by murine SR-BI can be uncoupled from the endocytosis of HDL or LDL particles. We found that blocking selective lipid uptake by incubating cells with the small chemical inhibitors BLT-1 or BLT-4 did not affect endocytosis of HDL. Similarly, blocking endocytosis by hyperosmotic sucrose or K+ depletion did not prevent selective lipid uptake from HDL or LDL. These findings suggest that mSR-BI-mediated selective uptake occurs at the cell surface upon the association of lipoproteins with mSR-BI and does not require endocytosis of HDL or LDL particles.     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.     

A synthetic low density lipoprotein particle capable of supporting U937 proliferation in vitro.

A synthetic LDL (sLDL) has been prepared by combining a lipid microemulsion with amphipathic peptides containing the apoprotein B receptor domain. The biological properties of sLDL have been investigated using the U937 in vitro cell proliferation assay. sLDL exhibits a concentration dependent and saturable stimulation of U937 proliferation. By utilizing different amphipathic peptides, variable proliferation is achieved, indicating a specific interaction between sLDL and the U937 LDL receptor are possible. U937 proliferation is reduced by the addition of an anti-LDL receptor antibody, indicating that sLDL is assimilated via the LDL receptor pathway.The behavior of sLDL mimics that of native LDL, and this approach represents a viable technique for the production of an sLDL particle on a large scale for research and general application.     

Investigation of Lipid Peroxidation in Human Low Density Lipoprotein

Human plasma low density lipoprotein (LDL) exposed to oxygen saturated buffer becomes depleted of alpha-tocopherol within 3 to 6 hours. Thereafter, lipid peroxidation commences as evidenced by the loss of 18:2 (67nmol/mg LDL) and 20:4 (12nmol/mg LDL) and the concomitant formation of 4-hydroxy-nonenal (0.28 nmol/mg LDL) and fluorescent compounds. The major fluorophor in apo B of oxidized LDL has an excitation maximum at 355 nm and an emission maximum at 430 nm. A fluorophor with the same spectral properties is produced in apo B, if LDL is incubated with 4-hydroxynonenal, whereas malonal-dehyde gives a fluorophor with excitation and emission maxima at 400/470nm. Three-dimensional fluorescence spcetroscopy proved to be an useful tool in analysing the complex fluorescence of apo B.