Acrolein impairs ATP binding cassette transporter A1-dependent cholesterol export from cells through site-specific modification of apolipoprotein A-I

Acrolein is a highly reactive alpha,beta-unsaturated aldehyde, but the factors that control its reactions with nucleophilic groups on proteins remain poorly understood. Lipid peroxidation and threonine oxidation by myeloperoxidase are potential sources of acrolein during inflammation. Because both pathways are implicated in atherogenesis and high density lipoprotein (HDL) is anti-atherogenic, we investigated the possibility that acrolein might target the major protein of HDL, apolipoprotein A-I (apoA-I), for modification. Tandem mass spectrometric analysis demonstrated that lysine 226, located near the center of helix 10 in apoA-I, was the major site modified by acrolein. Importantly, this region plays a critical role in the cellular interactions and ability of apoA-I to transport lipid. Indeed, we found that conversion of Lys-226 to N(epsilon)-(3-methylpyridinium)lysine by acrolein associated quantitatively with decreased cholesterol efflux from cells via the ATP-binding cassette transporter A1 pathway. In the crystal structure of truncated apoA-I, Glu-234 lies adjacent to Lys-226, suggesting that negatively charged residues might direct the modification of specific lysine residues in proteins. Finally, immunohistochemical studies with a monoclonal antibody revealed co-localization of apoA-I with acrolein adducts in human atherosclerotic lesions. Our observations suggest that acrolein might interfere with normal reverse cholesterol transport by HDL by modifying specific sites in apoA-I. Thus, acrolein might contribute to atherogenesis by impairing cholesterol removal from the artery wall.     

Role of leucine zipper motif in apoE3 N-terminal domain lipid binding activity

The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant "leucine to alanine" (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity.     

Acrolein Impairs the Cholesterol Transport Functions of High Density Lipoproteins

High density lipoproteins (HDL) are considered athero-protective, primarily due to their role in reverse cholesterol transport, where they transport cholesterol from peripheral tissues to the liver for excretion. The current study was designed to determine the impact of HDL modification by acrolein, a highly reactive aldehyde found in high abundance in cigarette smoke, on the cholesterol transport functions of HDL. HDL was chemically-modified with acrolein and immunoblot and mass spectrometry analyses confirmed apolipoprotein crosslinking, as well as acrolein adducts on apolipoproteins A-I and A-II. The ability of acrolein-modified HDL (acro-HDL) to serve as an acceptor of free cholesterol (FC) from COS-7 cells transiently expressing SR-BI was significantly decreased. Further, in contrast to native HDL, acro-HDL promotes higher neutral lipid accumulation in murine macrophages as judged by Oil Red O staining. The ability of acro-HDL to mediate efficient selective uptake of HDL-cholesteryl esters (CE) into SR-BI-expressing cells was reduced compared to native HDL. Together, the findings from our studies suggest that acrolein modification of HDL produces a dysfunctional particle that may ultimately promote atherogenesis by impairing functions that are critical in the reverse cholesterol transport pathway.     

Replacement of helix 1' enhances the lipid binding activity of apoE3 N-terminal domain

The N-terminal domain of human apolipoprotein E (apoE-NT) harbors residues critical for interaction with members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid free apoE-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational adaptation is required for manifestation of LDLR binding ability. To investigate the structural basis for this conformational change, the short helix connecting helix 1 and 2 in the four-helix bundle was replaced by the sequence NPNG, introducing a beta-turn. Recombinant helix-to-turn (HT) variant apoE3-NT was produced in Escherichia coli, isolated and characterized. Stability studies revealed a denaturation transition midpoint of 1.9 m guanidine hydrochloride for HT apoE3-NT vs. 2.5 M for wild-type apoE3-NT. Wild-type and HT apoE3-NT form dimers in solution via an intermolecular disulfide bond. Native PAGE showed that reconstituted high-density lipoprotein prepared with HT apoE3-NT have a diameter in the range of 9 nm and possess binding activity for the LDLR on cultured human skin fibroblasts. In phospholipid vesicle solubilization assays, HT apoE3-NT was more effective than wild-type apoE3-NT at inducing a time dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. In lipoprotein binding assays, HT apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent that wild-type apoE3-NT. The results indicate that a mutation at one end of the apoE3-NT four-helix bundle markedly enhances the lipid binding activity of this protein. In the context of lipoprotein associated full-length apoE, increased lipid binding affinity of the N-terminal domain may alter the balance between receptor-active and -inactive conformational states.     

ELISA quantitation of apolipoproteins in plasma lipoprotein fractions: ApoE in apoB-containing lipoproteins (Lp B:E) and apoB in apoE-containing lipoproteins (Lp E:B)

Growing clinical evidence suggests that metabolic behavior and atherogenic potential vary within lipoprotein subclasses that can be defined by apolipoprotein variation. Variant constituency of apolipoproteins B and E (apoB and apoE) may be particularly important because of the central roles of these apolipoproteins in the endogeneous lipid delivery cascade. ApoB is the sole protein of low-density lipoprotein (LDL), and like LDL cholesterol, the plasma apoB level has been positively correlated with risk for atherosclerotic disease. ApoE is a major functional lipoprotein in the triglyceride-rich lipoproteins, and may be crucial in the conversion of very low density lipoprotein (VLDL) to LDL. Based on work by others that enabled the quantititation of apoB-containing particles by content of up to two other types of apolipoprotein, we have developed a method for determining the amount of apoE in apoB-containing lipoproteins (Lp B:E) and the amount of apoB in apoE-containing lipoproteins (Lp E:B). From the Lp B:E and Lp E:B concentrations, the molar ratio of apoE to apoB in lipoproteins containing apoB and/or apoE in plasma can be determined. The methodology is fast, specific, and sensitive and should prove extremely useful in further categorizing lipoproteins and characterizing their behavior. In applying this method to clinical groupings of normo- and hyperlipidemia, we found that the plasma triglyceride level correlated with the apoE and Lp B:E concentrations in plasma, while the total cholesterol level correlated with the apoB and Lp E:B levels.     

Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain

This study was undertaken to determine if apolipoprotein (apo) E-containing lipoproteins and their receptors could provide a system for lipid transport and cholesterol homeostasis in the brain, as they do in other tissues. To accomplish this goal, the lipoproteins in human and canine cerebrospinal fluid (CSF) were characterized, and rat brain and monkey brain were examined for the presence of apoB,E(LDL) receptors. Apolipoprotein E and apoA-I were present in human and canine CSF, but apoB could not be detected. Apo-lipoprotein E and apoA-I were both present on lipoproteins with a density of approximately 1.09 to 1.15 g/ml. In human CSF, the lipoproteins were primarily spherical (approximately 140 A), whereas in canine CSF the lipoproteins were a mixture of discs (200 x 65 A) and spheres (approximately 130 A). Apolipoproteins E and A-I were contained primarily in separate populations of lipoproteins. Although the apoE of CSF was more highly sialylated than plasma apoE, the apoE-containing lipoproteins in canine CSF competed as effectively as canine plasma apoE HDLc for binding of 125I-LDL to the apoB,E(LDL) receptors on human fibroblasts. The presence of apoB,E(LDL) receptors in both rat and monkey brain was demonstrated by immunocytochemistry. Astrocytes abutting on the arachnoid space and pial cells of the arachnoid itself, both of which contact CSF, expressed apoB,E(LDL) receptors. Relatively few receptors were present in the cells of the gray matter of the cortex. Receptors were more prominent on the astrocytes of white matter and in the cells of the brain stem. The expression of apoB,E(LDL) receptors by brain cells and the presence of apoE- and apoA-I-containing lipoproteins in CSF suggest that the central nervous system has a mechanism for lipid transport and cholesterol homeostasis similar to that of other tissues.     

Provision of cholesterol to lymphocytes by high density and low density lipoproteins. Requirement for low density lipoprotein receptors

The capacity of lipoprotein fractions to provide cholesterol necessary for human lymphocyte proliferation was examined. When endogenous synthesis of cholesterol was blocked, proliferation of mitogen-stimulated normal human lymphocytes was markedly inhibited unless an exogenous source of sterol was supplied. All lipoprotein fractions with the exception of high density lipoprotein subclass 3 were able to provide cholesterol for lymphocyte proliferation. Each of the lipoprotein subfractions capable of providing cholesterol was also able to regulate endogenous sterol synthesis in cultured human lymphocytes. Provision of cholesterol by lipoproteins required the interaction of apolipoprotein B or apolipoprotein E with specific receptors on normal lymphocytes. Apolipoprotein modification by acetylation or methylation, which markedly reduced the ability to regulate sterol biosynthesis, also diminished the capacity of lipoproteins to provide cholesterol. In addition, depletion of apolipoprotein B- and apolipoprotein E-containing particles from high density lipoprotein decreased its ability to suppress cholesterol synthesis and prevented it from providing cholesterol to proliferating lymphocytes. Monoclonal antibodies directed against the receptor-recognition sites on apolipoprotein B and apolipoprotein E were used to define the specific apolipoproteins required for the provision of cholesterol to lymphocytes by the various lipoprotein fractions. The antibody to apolipoprotein B inhibited cholesterol provision by both low density lipoprotein (LDL) and other lipoprotein fractions. The antibody to apolipoprotein E did not decrease provision of cholesterol by LDL but did inhibit the capacity of other fractions to provide cholesterol. In addition, a monoclonal antibody against the ligand binding site on the LDL receptor inhibited provision of cholesterol to normal lymphocytes by all lipoproteins. Finally, lymphocytes lacking LDL receptors were unable to obtain cholesterol from any lipoprotein fraction. These studies demonstrate that LDL receptor-mediated interaction with apolipoprotein B or apolipoprotein E is essential for the provision of cholesterol to normal human lymphocytes from all lipoprotein sources.     

Acrolein, the toxic endogenous aldehyde, induces neurofilament-L aggregation

Acrolein is a highly reactive by product of lipid peroxidation and individuals with neurodegenerative disorders have been shown to contain elevated concentrations of this molecule in the brain. In the present study, we examined the pattern of neurofilament-L (NF-L) modification elicited by acrolein. When NF-L was incubated with acrolein, protein aggregation occurred in a acrolein concentration-dependent manner. Exposure of NF-L to acrolein also led to the generation of protein carbonyl compounds. Through the addition of free radical scavengers we observed a significant decrease in acrolein-mediated NF-L aggregation. These results indicate that free radicals may be involved in the modification of NF-L by acrolein. In addition, dityrosine crosslink formation was observed in acrolein-mediated NF-L aggregates and these aggregates displayed thioflavin T reactivity, reminiscent of amyloid. This study suggests that acrolein-mediated NF-L aggregation might be closely related to oxidative reactions, thus these reactions may play a critical role in neurodegenerative diseases.     

Mechanism of the HDL2 stimulation of progesterone secretion in cultured placental trophoblast

Lipoprotein cholesterol (C) supports the high rate of progesterone production by the human placenta as endogenous cholesterol synthesis is low. To study underlying mechanisms whereby lipoproteins, including high density lipoprotein-2 (HDL2), stimulate progesterone secretion, trophoblast cells were isolated from human term placentas and maintained in primary tissue culture. Lipoproteins were added at several concentrations and medium progesterone secretion was determined. HDL2 (d 1.063-1.125 g/ml) as well as low density lipoproteins (LDL) (d 1.019-1.063 g/ml) but not HDL3 (d 1.125-1.21 g/ml) stimulated progesterone secretion in a dose-dependent manner, with HDL2 cholesterol entering the cell and serving as substrate for progesterone synthesis. Conversely, LDL and HDL2 produced a significant decrease in [2-14C]acetate incorporation into cell cholesterol. Cholesterol-depleted lipoproteins did not stimulate progesterone secretion. The stimulating effect of LDL was abolished by apolipoprotein modification by cyclohexanedione or reductive methylation and by the addition of anti-LDL receptor antibody or 10 microM chloroquine to the medium. [14C]acetate conversion into cholesterol was accelerated by these procedures. However, HDL2 stimulation of progesterone secretion and reduction of [14C]acetate incorporation into cholesterol was not blocked by chemical modification of apolipoproteins, anti-LDL receptor antibody, or chloroquine. Treatment of HDL2 with tetranitromethane or dimethylsuberimidate also did not block the stimulation of progesterone. To determine whether the capacity of HDL2 to deliver cholesterol to the trophoblast cells was restricted to subfractions differing in apoE content, HDL2 was chromatographed on heparin-Sepharose and three fractions (A, B, and C) were obtained. Fraction A was poorest in apoE and free cholesterol, fraction B contained the majority of cholesterol, and fraction C was the richest in apoE and free cholesterol. When added to trophoblast cells, fraction A stimulated little progesterone secretion, fraction B stimulated moderately, and fraction C did so greatly. Modification of these subfractions with cyclohexanedione or reductive methylation did not inhibit these effects. In conclusion, HDL2 stimulated progesterone secretion in human trophoblast cell culture. Contrary to LDL, the HDL effect was not mediated by apolipoproteins or the LDL receptor pathway. The ability of HDL2 to stimulate progesterone secretion is consistent with the passive transfer of free cholesterol to the cell membrane from a physicochemically specific subfraction of HDL. This mechanism may be an auxiliary source of cholesterol for human steroidogenic cells.     

Semisynthesis and segmental isotope labeling of the apoE3 N-terminal domain using expressed protein ligation

Apolipoprotein E (apoE) is an exchangeable apolipoprotein that functions as a ligand for members of the LDL receptor family, promoting lipoprotein clearance from the circulation. Productive receptor binding requires that apoE adopt an LDL receptor-active conformation through lipid association, and studies have shown that the 22 kDa N-terminal (NT) domain (residues 1–183) of apoE is both necessary and sufficient for receptor interaction. Using intein-mediated expressed protein ligation (EPL), a semisynthetic apoE3 NT has been generated for use in structure-function studies designed to probe the nature of the lipid-associated conformation of the protein. Circular dichroism spectroscopy of EPL-generated apoE3 NT revealed a secondary structure content similar to wild-type apoE3 NT. Likewise, lipid and LDL receptor binding studies revealed that EPL-generated apoE3 NT is functional. Subsequently, EPL was used to construct an apoE3 NT enriched with ¹⁵N solely and specifically in residues 112–183. ¹H-¹⁵N heteronuclear single quantum correlation spectroscopy experiments revealed that the ligation product is correctly folded in solution, adopting a conformation similar to wild-type apoE3-NT. The results indicate that segmental isotope labeling can be used to define the lipid bound conformation of the receptor binding element of apoE as well as molecular details of its interaction with the LDL receptor.     

Severe hypercholesterolemia,impaired fat tolerance,and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E

LDL receptor-related protein 5 (LRP5) plays multiple roles, including embryonic development and bone accrual development. Recently, we demonstrated that LRP5 is also required for normal cholesterol metabolism and glucose-induced insulin secretion. To further define the role of LRP5 in the lipoprotein metabolism, we compared plasma lipoproteins in mice lacking LRP5, apolipoprotein E (apoE), or both (apoE;LRP5 double knockout). On a normal chow diet, the apoE;LRP5 double knockout mice (older than 4 months of age) had approximately 60% higher plasma cholesterol levels compared with the age-matched apoE knockout mice. In contrast, LRP5 deficiency alone had no significant effects on the plasma cholesterol levels. High performance liquid chromatography analysis of plasma lipoproteins revealed that cholesterol levels in the very low density lipoprotein and low density lipoprotein fractions were markedly increased in the apoE;LRP5 double knockout mice. There were no apparent differences in the pattern of apoproteins between the apoE knockout mice and the apoE;LRP5 double knockout mice. The plasma clearance of intragastrically loaded triglyceride was markedly impaired by LRP5 deficiency. The atherosclerotic lesions of the apoE;LRP5 double knockout mice aged 6 months were approximately 3-fold greater than those in the age-matched apoE-knockout mice. Furthermore, histological examination revealed highly advanced atherosclerosis, with remarkable accumulation of foam cells and destruction of the internal elastic lamina in the apoE;LRP5 double knockout mice. These data suggest that LRP5 mediates both apoE-dependent and apoE-independent catabolism of plasma lipoproteins.     

Modification and inactivation of Cu,Zn-superoxide dismutase by the lipid peroxidation product,acrolein

Acrolein is the most reactive aldehydic product of lipid peroxidation and is found to be elevated in the brain when oxidative stress is high. The effects of acrolein on the structure and function of human Cu,Zn-superoxide dismutase (SOD) were examined. When Cu,Zn-SOD was incubated with acrolein, the covalent crosslinking of the protein was increased, and the loss of enzymatic activity was increased in a dose-dependent manner. Reactive oxygen species (ROS) scavengers and copper chelators inhibited the acrolein-mediated Cu,Zn-SOD modification and the formation of carbonyl compound. The present study shows that ROS may play a critical role in acrolein-induced Cu,Zn-SOD modification and inactivation. When Cu,Zn-SOD that has been exposed to acrolein was subsequently analyzed by amino acid analysis, serine, histidine, arginine, threonine and lysine residues were particularly sensitive. It is suggested that the modification and inactivation of Cu,Zn-SOD by acrolein could be produced by more oxidative cell environments. [BMB Reports 2013; 46(11): 555-560]     

Recycling of apolipoprotein E is not associated with cholesterol efflux in neuronal cells

After receptor-mediated endocytosis of apolipoprotein E (apoE)-containing lipoproteins in hepatocytes, the isoform apoE3 is efficiently recycled in a process which is associated with cholesterol efflux. Recycling and cholesterol efflux are greatly reduced when apoE4 is the only isoform present. ApoE is the main apolipoprotein in cerebrospinal fluid, and it plays a pivotal role in maintaining cholesterol homeostasis in the brain. The isoform apoE4 is associated with an increased risk of Alzheimer's disease and it has been postulated that high intracellular cholesterol levels promote the amyloidogenic processing of amyloid precursor protein. Therefore we investigated the cellular processing of different apoE isoforms as well as the associated cholesterol efflux in the murine neuronal cell line HT-22. Uptake of apoE3-containing lipoproteins resulted in the expected recycling while, as seen in non-neuronal cells, recycling of apoE4 was significantly reduced. However, despite these differences in apoE recycling, there was no difference in rates of cholesterol efflux. Therefore we conclude that in this neuronal cell model the reduced recycling of apoE4 does not affect cellular cholesterol metabolism.     

Apolipoprotein E Structure and Substrate and Receptor-Binding Activities of Triglyceride-Rich Human Plasma Lipoproteins in Normo- and Hypertriglyceridemia

Cysteine-arginine interchanges along the primary sequence of human plasma apolipoprotein E (apoE) play an important role in determining its biological functions due to a high mutation frequency of cytosine in CGX triplet that codes 33 of 34 apolipoprotein arginine residues. The contribution of apoE secondary structure to apolipoprotein-lipid interaction is described. The significance of apolipoprotein in triglyceride synthesis, lipoprotein lipolysis, and receptor-mediated clearance of lipolytic remnants of triglyceride-rich lipoproteins is discussed as well. The metabolic flow of lipoproteins in normo- and hypertriglyceridemia can be described by separate compartments that contribute to lipoprotein interaction with at least six different receptors: 1) low density lipoprotein (LDL) receptor; 2) LDL receptor-related protein (LRP); 3) apoB(48) macrophage receptor for hypertriglyceridemic very low density lipoproteins (VLDL); 4) scavenger receptors; 5) VLDL receptor; 6) lipolysis-stimulated receptor. The contribution of the exposure of apoE molecules on the surface of triglyceride-rich particles sensitive both to lipolysis and plasma triglyceride content to the interaction with LDL receptor and LRP is emphasized.     

Apolipoprotein E distribution among human plasma lipoproteins: role of the cysteine-arginine interchange at residue 112

Human apolipoprotein (apo) E occurs as three common isoforms (apoE4, E3, and E2), all of which influence plasma cholesterol levels. Although both apoE4 and E3 bind with equal effectiveness to the low density lipoprotein receptor, they associate preferentially with different classes of plasma lipoproteins: apoE4 with very low density lipoproteins, apoE3 with high density lipoproteins. The primary structure of apoE3 differs from that of apoE4 at only a single site; apoE3 has its sole cysteine residue at position 112, while apoE4 contains arginine at position 112 and completely lacks cysteine. The present study investigated how this structural difference between apoE4 and E3 determines their distribution among plasma lipoproteins, and analyzed the role of the disulfide-linked heterodimer apoE-A-II (which apoE4 cannot form) in determining the distribution. Human plasma was incubated with 125I-labeled apoE, and lipoproteins were separated by agarose chromatography. Both apoE3 that had been reduced and alkylated with iodoacetamide and apoE3-A-II distributed with high density lipoproteins, indicating that a combination of an inherent property of the monomeric apoE3 structure and apoE-A-II formation account for distribution of apoE3 to the high density lipoproteins. Cysteamine modification of apoE3 resulted in an apoE4-like distribution, demonstrating that a positive charge at position 112 determined the apoE4 distribution and that the effect was not exclusively due to the presence of arginine at this position.(ABSTRACT TRUNCATED AT 250 WORDS)     

LDL receptor deficiency or apoE mutations prevent remnant clearance and induce hypertriglyceridemia in mice

We have used adenovirus-mediated gene transfer and bolus injection of purified apolipoprotein E (apoE) in mice to determine the contribution of LDL receptor family members in the clearance of apoE-containing lipoproteins in vivo and the factors that trigger hypertriglyceridemia. A low dose [5 x 10(8) plaque-forming units (pfu)] of an adenovirus expressing apoE4 did not normalize plasma cholesterol levels of apolipoprotein E-deficient (apoE(-/-)) x low density lipoprotein receptor-deficient (LDLr(-/-)) mice and induced hypertriglyceridemia. A similar phenotype of combined dyslipidemia was induced in apoE(-/-) or apoE(-/-) x LDLr(-/-) mice after infection with a low dose (4 x 10(8) pfu) of an adenovirus expressing the apoE4[R142V/R145V] mutant previously shown to be defective in receptor binding. In contrast, a low dose of 5 x 10(8) pfu of the apoE4-expressing adenovirus corrected hypercholesterolemia in apoE(-/-) mice and did not trigger hypertriglyceridemia. Bolus injection of purified apoE in apoE(-/-) x LDLr(-/-) mice did not clear plasma cholesterol levels and induced mild hypertriglyceridemia. In contrast, similar injection of apoE in apoE(-/-) mice cleared plasma cholesterol and caused transiently mild hypertriglyceridemia. These findings suggest that a) the LDL receptor alone can account for the clearance of apoE-containing lipoproteins in mice, and the contribution of other receptors is minimal, and b) defects in either the LDL receptor or in apoE that affect its interactions with the LDL receptor, increase the sensitivity to apoE-induced hypertriglyceridemia in mice.     

ApoB-containing lipoproteins in apoE-deficient mice are not metabolized by the class B scavenger receptor BI

The scavenger receptor class B type I (SR-BI) recognizes a broad variety of lipoprotein ligands, including HDL, LDL, and oxidized LDL. In this study, we investigated whether SR-BI plays a role in the metabolism of cholesterol-rich lipoprotein remnants that accumulate in apolipoprotein E (apoE)(-/-) mice. These particles have an unusual apolipoprotein composition compared with conventional VLDL and LDL, containing mostly apoB-48 as well as substantial amounts of apoA-I and apoA-IV. To study SR-BI activity in vivo, the receptor was overexpressed in apoE(-/-) mice by adenoviral vector-mediated gene transfer. An approximately 10-fold increase in liver SR-BI expression resulted in no detectable alterations in VLDL-sized particles and a modest depletion of cholesterol in intermediate density lipoprotein/LDL-sized lipoprotein particles. This decrease was not attributable to altered secretion of apoB-containing lipoproteins in SR-BI-overexpressing mice. To directly assess whether SR-BI metabolizes apoE(-/-) mouse lipoprotein remnants, in vitro assays were performed in both CHO cells and primary hepatocytes expressing high levels of SR-BI. This analysis showed a remarkable deficiency of these particles to serve as substrates for selective lipid uptake, despite high-affinity, high-capacity binding to SR-BI. Taken together, these data establish that SR-BI does not play a direct role in the metabolism of apoE(-/-) mouse lipoprotein remnants.     

Induction of neonatal tolerance to oxidized lipoprotein reduces atherosclerosis in ApoE knockout mice

BACKGROUND: In the course of atherosclerosis, humans and apolipoprotein (apoE) Knockout (KO) mice exhibit an active cell-mediated and humoral immune process, both at the systemic level and within atheromata. Low density lipoproteins (LDL) infiltrate the vascular wall, where they are oxidatively modified. This oxidative modification may generate new epitopes for which tolerance is not achieved during ontogenesis. Such epitopes could constitute new targets for autoreactive immune responses that may have a physiopathological role in disease development. MATERIALS AND METHODS: Exposing mice to high dose of antigens during thymic T-cell education induces immunological tolerance to the administered antigens. We injected newborn apoE KO mice with oxidized LDL. They were fed a cholesterol-rich diet and aortic atherosclerosis, cell-mediated immune response, and T-cell repertoire were analyzed after 5 months. RESULTS: Injection of oxidized LDL at birth reduced not only the immune response to oxidized LDL, but also susceptibility to atherosclerosis in apoE mice. Injection of oxidized LDL induced T-cell tolerance due to clonal deletion, rather than anergy of the reactive T cells. The T-cell repertoire of apoE KO mice was affected by the development of the disease, whereas tolerization normalized it. CONCLUSIONS: This study demonstrates that the immune response against oxidized LDL has a deleterious role in atherogenesis and that a fine-tuning of this response could modify the course of the disease.     

Secretion of lipids, apolipoproteins, and lipoproteins by human hepatoma cell line, HepG2: effects of oleic acid and insulin

The aim of this study was to determine the effect of oleic acid and insulin on the secretion of lipoproteins by HepG2 cells grown in minimum essential medium. Triglycerides were the major neutral lipid (57% of total) and apoB was the predominant apolipoprotein (56% of total) secreted by these cells. The addition of oleate resulted in a two-fold increase in the concentration of neutral lipids but only a slight to moderate increase in the apolipoprotein (A-I, A-II, B, and E) levels. The secretion of very low density lipoproteins (VLDL) was stimulated by 425%, low density lipoproteins (LDL) by 77%, and high density lipoproteins (HDL) by 68%. Whereas neutral lipid composition of LDL was unchanged, the VLDL particles contained a significantly higher percentage of triglyceride and lower percentages of cholesterol and cholesteryl esters compared with VLDL secreted in the absence of oleate. Oleate had no significant effect on the composition of apolipoproteins in VLDL, LDL and HDL. In basal medium, insulin caused a significant decrease in the secretion of neutral lipids and apolipoproteins, particularly triglycerides and apoB. In addition to a 60-68% reduction in the total concentration of VLDL and LDL, insulin altered their composition by producing particles that had a significantly lower content of triglycerides, contained less apoB, and were deficient in apoE. There were no major changes in the concentration or composition of HDL particles. Insulin had a similar but less pronounced effect on the concentration and composition of lipoproteins secreted in the presence of oleate. The increased accumulation of triglycerides in the HepG2 cells concomitant with their reduced levels in the medium suggests that insulin may affect the secretion rather than synthesis of triglyceride-rich lipoproteins.