Liver X receptor activation controls intracellular cholesterol trafficking and esterification in human macrophages.

Liver X receptors (LXRs) are nuclear receptors that regulate macrophage cholesterol efflux by inducing ATP-binding cassette transporter A1 (ABCA1) and ABCG1/ABCG4 gene expression. The Niemann-Pick C (NPC) proteins NPC1 and NPC2 are located in the late endosome, where they control cholesterol trafficking to the plasma membrane. The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant governing its availability for efflux to extracellular acceptors. Here we investigated the influence of LXR activation on intracellular cholesterol trafficking in primary human macrophages. Synthetic LXR activators increase the amount of free cholesterol in the plasma membrane by inducing NPC1 and NPC2 gene expression. Moreover, ABCA1-dependent cholesterol efflux induced by LXR activators was drastically decreased in the presence of progesterone, which blocks postlysosomal cholesterol trafficking, and reduced when NPC1 and NPC2 mRNA expression was depleted using small interfering RNA. The stimulation of cholesterol mobilization to the plasma membrane by LXRs led to a decrease in cholesteryl ester formation and Acyl-coenzyme A cholesterol acyltransferase-1 activity. These data indicate that LXR activation enhances cholesterol trafficking to the plasma membrane, where it becomes available for efflux, at the expense of esterification, thus contributing to the overall effects of LXR agonists in the control of macrophage cholesterol homeostasis.

Peroxisome proliferator-activated receptor alpha controls cellular cholesterol trafficking in macrophages.

The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant that governs its availability for efflux to extracellular acceptors. NPC1 and NPC2 are proteins localized in the late endosome and control cholesterol transport from the lysosome to the plasma membrane. Here, we report that NPC1 and NPC2 gene expression is induced by oxidized LDL (OxLDL) in human macrophages. Because OxLDLs contain natural activators of peroxisome proliferator-activated receptor alpha (PPARalpha), a fatty acid-activated nuclear receptor, the regulation of NPC1 and NPC2 by PPARalpha and the consequences on cholesterol trafficking were further studied. NPC1 and NPC2 expression is induced by synthetic PPARalpha ligands in human macrophages. Furthermore, PPARalpha activation leads to an enrichment of cholesterol in the plasma membrane. By contrast, incubation with progesterone, which blocks postlysosomal cholesterol trafficking, as well as NPC1 and NPC2 mRNA depletion using small interfering RNA, abolished ABCA1-dependent cholesterol efflux induced by PPARalpha activators. These observations identify a novel regulatory role for PPARalpha in the control of cholesterol availability for efflux that, associated with its ability to inhibit cholesterol esterification and to stimulate ABCA1 and scavenger receptor class B type I expression, may contribute to the stimulation of reverse cholesterol transport.

Peroxisome proliferator-activated receptor alpha reduces cholesterol esterification in macrophages.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor activated by fatty acid derivatives and hypolipidemic drugs of the fibrate class. PPARalpha is expressed in monocytes, macrophages, and foam cells, suggesting a role for this receptor in macrophage lipid homeostasis with consequences for atherosclerosis development. Recently, it was shown that PPARalpha activation promotes cholesterol efflux from macrophages via induction of the ABCA1 pathway. In the present study, the influence of PPARalpha activators on intracellular cholesterol homeostasis was investigated. In human macrophages and foam cells, treatment with fibrates, synthetic PPARalpha activators, led to a decrease in the cholesteryl ester (CE):free cholesterol (FC) ratio. In these cells, PPARalpha activation reduced cholesterol esterification rates and Acyl-CoA:cholesterol acyltransferase-1 (ACAT1) activity. However, PPARalpha activation did not alter ACAT1 gene expression, whereas mRNA levels of carnitine palmitoyltransferase type 1 (CPT-1), a key enzyme in mitochondrial fatty acid catabolism, were induced. Finally, PPARalpha activation blocked CE formation induced by TNF-alpha, possibly due to the inhibition of neutral sphingomyelinase activation by TNF-alpha. In conclusion, our results identify a role for PPARalpha in the control of cholesterol esterification in macrophages, resulting in an enhanced availability of FC for efflux through the ABCA1 pathway.

Fh-Souassi: a founder frameshift mutation in exon 10 of the LDL-receptor gene, associated with a mild phenotype in Tunisian families.

Familial hypercholesterolemia (FH) has a higher prevalence in central Tunisia together with a milder clinical expression than in western countries. The molecular basis of FH in Tunisia remains unknown. Our aim was to identify FH-causing mutations in three unrelated families (21 subjects) from the area of Souassi (central Tunisia). In probands with a presentation of homozygous FH, the promoter and 18 exons of the low density lipoprotein (LDL)-receptor gene were sequenced in both orientations. A novel complex frameshift mutation was identified in exon 10, nucleotides 1477-1479 (TCT) at Serine 472 were replaced by an insertion of seven nucleotides (AGAGACA), producing a premature termination codon 43 amino acids downstream. Binding of 125I-labelled LDL at 4 degrees C to cultured fibroblasts from two probands showed <2% normal LDL-receptor activity. AvaII digestion of PCR amplified genomic DNA identified this unique mutation in all families; homozygotes n=11, heterozygotes n=10. All mutation carriers shared the same haplotype (7 RFLPs), suggesting that they had a common ancestor. Despite high plasma LDL levels (m=16.0+/-3.0 mmol/l) and extravascular cholesterol deposits, most homozygotes were diagnosed after puberty and had a delayed onset of cardiovascular complications. Moreover, most heterozygotes were free of clinical signs and had plasma LDL cholesterol in the normal range (4.7+/-1.3 mmol/l) without taking any lipid-lowering medication. This mild clinical phenotype which contrasted with the severity of the mutation, could not be explained by specific apolipoprotein E or lipoprotein lipase alleles.

Lack of toxic effects of F 12511, a novel potent inhibitor of acyl-coenzyme A: cholesterol O-acyltransferase, on human adrenocortical cells in culture.

Inhibition of acyl-coenzyme A: cholesterol O-acyltransferase (EC 2.3.1.26; ACAT) reduces intracellular cholesteryl esters that are substrates for steroidogenesis in adrenal cells. The adrenal side effects of ACAT inhibitors remain a key point for their development as antiatherosclerotic agents. The aim of this study was to characterize the effects of a novel and powerful ACAT inhibitor, F 12511 (S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-phenylacetanilide, on the NCI-H295R cell line, which has functional properties comparable to those of normal human adrenal cells. F 12511 incubated with cultured cells for 4-72 hr strongly inhibited cholesteryl oleate formation. The concentrations required to produce 50% inhibition (IC50) values) ranged from 20 to 50 nM; in the presence of low-density lipoproteins (LDL), this effect was paralleled by a decrease in cholesteryl ester mass and an increase in intracellular free cholesterol. At concentrations 100-fold larger than the IC(50) value for up to 48 hr, F 12511 reduced neither the basal release of cortisol and aldosterone nor the production of cortisol stimulated by forskolin. F 12511 did not modify the mRNA levels of the steroidogenic enzyme genes cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17), or cytochrome P450 21-hydroxylase (P450c21) or those of the LDL receptor and high-density lipoprotein scavenger receptor class B, type I (SR-BI) genes, either in the presence or absence of adenosine 3',5'-cyclic monophosphate stimulation for 24 hr. Exposure to F 12511 at up to 3 microM for 24 or 48 hr did not result in significant change in morphological and ultrastructural characteristics; the cytoplasm contained large numbers of mitochondria with intact crystae, and the same typical features of secretory activity were observed in NCI-H295R control cells. Exposure to 3 microM of F 12511 for 96 hr also did not affect cell viability. These data demonstrate that reduction of the substrate for steroidogenesis by the ACAT inhibitor F 12511 impairs neither steroid production nor transcription of genes involved in steroidogenesis and lipoprotein uptake in the pluripotent human adrenal cell line NCI-H295R.

PPAR-alpha and PPAR-gamma activators induce cholesterol removal from human macrophage foam cells through stimulation of the ABCA1 pathway.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate lipid and glucose metabolism and cellular differentiation. PPAR-alpha and PPAR-gamma are both expressed in human macrophages where they exert anti-inflammatory effects. The activation of PPAR-alpha may promote foam-cell formation by inducing expression of the macrophage scavenger receptor CD36. This prompted us to investigate the influence of different PPAR-activators on cholesterol metabolism and foam-cell formation of human primary and THP-1 macrophages. Here we show that PPAR-alpha and PPAR-gamma activators do not influence acetylated low density lipoprotein-induced foam-cell formation of human macrophages. In contrast, PPAR-alpha and PPAR-gamma activators induce the expression of the gene encoding ABCA1, a transporter that controls apoAI-mediated cholesterol efflux from macrophages. These effects are likely due to enhanced expression of liver-x-receptor alpha, an oxysterol-activated nuclear receptor which induces ABCA1-promoter transcription. Moreover, PPAR-alpha and PPAR-gamma activators increase apoAI-induced cholesterol efflux from normal macrophages. In contrast, PPAR-alpha or PPAR-gamma activation does not influence cholesterol efflux from macrophages isolated from patients with Tangier disease, which is due to a genetic defect in ABCA1. Here we identify a regulatory role for PPAR-alpha and PPAR-gamma in the first steps of the reverse-cholesterol-transport pathway through the activation of ABCA1-mediated cholesterol efflux in human macrophages.

Apolipoprotein AII enrichment of HDL enhances their affinity for class B type I scavenger receptor but inhibits specific cholesteryl ester uptake.

Apolipoproteins of high density lipoprotein (HDL) and especially apolipoprotein (apo)AI and apoAII have been demonstrated as binding directly to the class B type I scavenger receptor (SR-BI), the HDL receptor that mediates selective cholesteryl ester uptake. However, the functional relevance of the binding capacity of each apolipoprotein is still unknown. The human adrenal cell line, NCI-H295R, spontaneously expresses a high level of SR-BI, the major apoAI binding protein in these cells. As previously described for murine SR-BI, free apoAI, palmitoyl-oleoyl-phosphatidylcholine (POPC)-AI, and HDL are good ligands for human SR-BI. In vitro displacement of apoAI by apoAII in HDLs or in Lp AI purified from HDL by immunoaffinity enhances their ability to compete with POPC-AI to bind to SR-BI and also enhances their direct binding capacity. The next step was to determine whether the higher affinity of apoAII for SR-BI correlated with the specific uptake of cholesteryl esters from these HDLs. Free apoAII and, to a lesser extent, free apoAI that were added to the cell medium during uptake experiments inhibited the specific uptake of [(3)H]cholesteryl esters from HDL, indicating that binding sites on cells were the same as cholesteryl ester uptake sites. In direct experiments, the uptake of [(3)H]cholesteryl esters from apoAII-enriched HDL was highly reduced compared with the uptake from native HDL. These results demonstrate that in the human adrenal cell line expressing SR-BI as the major HDL binding protein, efficient apoAII binding has an inhibitory effect on the delivery of cholesteryl esters to cells.

Cell culture conditions determine apolipoprotein CIII secretion and regulation by fibrates in human hepatoma HepG2 cells.

Fibrates are widely used drugs which lower triglycerides and increase HDL concentrations in serum. Recent findings from our laboratory have shown that fibrates repress apolipoprotein (apo) CIII gene expression, an effect that explains partially the triglyceride-lowering activity of these drugs. The goal of the present study was to compare the effect of various fibrates on apo CIII gene expression in the human hepatoblastoma cell line HepG2. First, we demonstrate that the level of apo CIII secretion by HepG2 cells is controlled by serum factors whereas apo CIII mRNA levels are not and even increase under conditions when apo CIII secretion dramatically decreases. Twelve different fetal calf serum batches were tested during this study and apo CIII secretion in cell medium could only be detected with three of them. The effect of serum on apolipoprotein secretion was more pronounced for apo CIII whereas other apolipoproteins (apo E, apo B, apo AII and apo AI) were affected to a lesser extent. Under serum conditions allowing apo CIII secretion, treatment with the peroxisome-proliferator activated receptor (PPAR)alpha activators fenofibrate, gemfibrozil and Wy-14643 result in a marked lowering of apo CIII secretion and gene expression, this effect being most pronounced with Wy-14643. Comparison of the activity of a PPARgamma-specific ligand, the antidiabetic thiazolidinedione, BRL-49653 and a PPARalpha ligand Wy-14643 showed a marked decrease of apo CIII secretion and gene expression after activation of PPARalpha but not PPARgamma. In conclusion, fibrates down-regulate apo CIII gene expression in human HepG2 cells, most likely via PPARalpha but not via PPARgamma. However, these effects are only observed in HepG2 cells cultured under appropriate conditions.

Comparison of expression and regulation of the high-density lipoprotein receptor SR-BI and the low-density lipoprotein receptor in human adrenocortical carcinoma NCI-H295 cells.

In rodents, cholesterol for adrenal steroidogenesis is derived mainly from high-density lipoproteins (HDL) via the HDL receptor, scavenger receptor-BI (SR-BI). In humans cholesterol for steroidogenesis is considered to be derived from the low-density lipoprotein (LDL) receptor pathway, and the contribution of SR-BI to that is unknown. In the present study SR-BI expression and regulation by steroidogenic stimuli was analysed in human adrenocortical cells and compared with LDL receptor expression. In addition, the functional contribution of both receptors for cholesteryl ester delivery to human adrenocortical cells was compared. Northern blot and reverse transcription-PCR amplification and sequence analysis demonstrated the presence of SR-BI mRNA in foetal and adult human adrenal cortex. Furthermore, SR-BI mRNA was expressed to similar levels in human primary adrenocortical and adrenocortical carcinoma NCI-H295 cells, indicating its presence in the steroid-producing cells. Treatment of NCI-H295 cells with 8Br-cAMP, a stimulator of glucocorticoid synthesis via the protein kinase A second messenger signal transduction pathway, resulted in an increase of both SR-BI and LDL receptor mRNA levels in a time- and dose-dependent manner. The induction of SR-BI and LDL receptor by cAMP was independent of ongoing protein synthesis and occurred at the transcriptional level. Ligand blot experiments indicated that a protein of similar size to SR-BI is the major HDL-binding protein in NCI-H295 cells. Western blot analysis demonstrated that cAMP treatment increased the levels of LDL receptor and, to a lesser extent, SR-BI protein in NCI-H295 cells. Binding and uptake of cholesterol was quantitatively smaller from HDL than from LDL, both in basal as well as in cAMP-stimulated cells. Scatchard analysis under basal conditions indicated that NCI-H295 cells express twice as many specific binding sites for LDL than for HDL. Dissociation constant values (Kd; in nm) were approximately five times higher for HDL than for LDL, indicating a lower affinity of HDL compared with LDL. The combined effects of these two parameters and the low cholesteryl ester content of HDL subfraction 3 (HDL3) contributes to a lower cholesteryl ester uptake from HDL than from LDL by the NCI-H295 cells. In conclusion, both the SR-BI and LDL receptor genes are expressed in the human adrenal cortex and coordinately regulated by activators of glucocorticoid synthesis. In contrast to rodents, in human adrenocortical cells the HDL pathway of cholesterol delivery appears to be of lesser importance than the LDL pathway. Nevertheless, the SR-BI pathway may become of major importance in conditions of functional defects in the LDL receptor pathway.

Branched synthetic peptide constructs mimic cellular binding and efflux of apolipoprotein AI in reconstituted high density lipoproteins.

This study investigates the suitability of the trimeric apolipoprotein (apo)AI(145-183) peptide that we recently described, to serve as a model to probe the relationship between apoAI structure and function. Three copies of the apoAI(145-183) unit, composed each of two amphipathic alpha-helical segments, were branched onto a covalent core matrix and the construct was recombined with phospholipids. A similar construct was made with the apoAI(102-140) peptide and used as a comparison with dimyristoylglycerophosphocholine (DMPC)-apoAI complexes. The DMPC-trimeric-apoAI(145-183) complexes had similar immunological reactivity with monoclonal antibodies directed against the 149-186 apoAI sequence (A44), suggesting that the A44 epitope is exposed similarly in both the synthetic peptide and the native apoAI complexes. The complexes generated with the trimeric-apoAI(145-183) bind specifically to HeLa cells with comparable affinity to the DMPC apoAI complexes; they are a good competitor for binding of apoAI to both HeLa cells and Fu5AH rat hepatoma cells; finally, these complexes promote cholesterol efflux from Fu5AH cells with an efficiency comparable with the apo AI/lipid complexes. To study LCAT activation by the trimeric apo AI(145-183) construct, complexes were prepared with dipalmitoylphosphatidylcholine (DPPC), cholesterol (C) and either the trimeric construct or apoAI. LCAT activation by the trimeric construct was much lower than by apo AI, possibly because the conformation of the trimeric 145-183 peptide in DPPC/C/peptide complexes does not mimic that of apoAI in the corresponding complexes. In comparison, the complexes generated with the multimeric apoAI(102-140) construct had a poor capacity to mimic the physico-chemical and biological properties of apoAI. The apoAI(102-140) construct had low affinity for lipid compared with the (145-183) construct. After association with lipids, it was a poor competitor of DMPC-apoAI complexes for cellular binding and had only limited capacity to promote cholesterol efflux. These results suggest trimeric constructs can serve as an appropriate models for apoAI, enabling further investigations and new experimental approaches to determine the structure-function relationship of apoAI.

Mutation screening of the LDLR gene and ApoB gene in patients with a phenotype of familial hypercholesterolemia and normal values in a functional LDL receptor/apolipoprotein B assay.

Mutations in the LDL receptor (LDLR) or the apolipoprotein B-100 genes causing familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB), two of the most frequent inherited diseases, are the underlying genetic defects in a small proportion of patients suffering from premature atherosclerotic heart disease. Consequently, secure diagnostic tools for these conditions allowing early preventive measures are needed. Since clinical and biochemical diagnosis often is inaccurate, assays analyzing patient LDLR function and LDL affinity have been established. These assays are, however, not able clearly to differentiate between suspected FH/FDB samples and normal controls. To evaluate if this may be caused by other hitherto undescribed genetic defects or to failure of the functional assays, we undertook denaturing gradient gel electrophoresis based mutation screening of the LDLR gene and the codon 3456 3553 region of the apolipoprotein B gene in six French FH/FDB patients with normal outcomes on functional assays. In all six patients, pathogenic LDLR mutations were found, including three previously undescribed mutations, suggesting that failure of the functional assays explains the normal results found in some phenotypic FH/FDB patients and illustrating the need for DNA based screening techniques for routine genetic diagnosis in FH/FDB.

In vivo glucosylated LpA-I subfraction. Evidence for structural and functional alterations.

This study compared the structural and functional properties of glucosylated and non-glucosylated LpA-I particle subfractions (GLpA-I and NGLpA-I, respectively) isolated from patients with poorly controlled type 1 (insulin-dependent) diabetes. Compared with NGLpA-I, GLpA-I showed an enrichment in triglycerides (P < .05) and a depletion in phospholipid (P < .05) content. Moreover, the triglycerides-to-cholesteryl esters ratio was increased (P < .05), suggesting an increased cholesteryl ester transfer protein activity and a possible transport defect that accelerates atherogenesis. The surface-to-core constituents ratio, an indirect estimate of particles size, is lower in GLpA-I (P < .01) than in NGLpA-I, correlating well with a larger median size (P < .05) as seen by electron microscopy. The apolipoprotein (apo) A-I conformation was evaluated through determination of the immunological accessibility of three different domains defining specific epitopes for anti-apo A-I monoclonal antibodies. We observed a marked decreased accessibility for two of these regions, which interestingly have already been implicated in the interaction with cells. Cell culture data suggest that nonenzymatic glycosylation occurring on apo A-I can modify lipoprotein function, since it results in a decreased binding of GLpA-I to HeLa cells and impaired cholesterol efflux from Fu5AH rat hepatoma cells.

HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways.

Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.

High-density-lipoprotein subfraction 3 interaction with glycosylphosphatidylinositol-anchored proteins.

To elucidate further the binding of high-density-lipoprotein subfraction 3 (HDL3) to cells, the involvement of glycosylphosphatidylinositol-anchored proteins (GPI-proteins) was studied. Treatment of cultured cells, such as fibroblasts or SK-MES-1 cells, with a phosphatidylinositol-specific phospholipase C (PI-PLC) significantly decreases specific HDL3 binding. Moreover, PI-PLC treatment of cultured cells or cellular plasma membrane fractions results in releasing proteins. These proteins have a soluble form and can also bind HDL3, as revealed by ligand blotting experiments with HDL3. In order to obtain enriched GPI-proteins, we used a detergent-free purification method to prepare a caveolar membrane fraction. In the caveolar fraction, we obtained, by ligand blotting experiments, the enrichment of two HDL3-binding proteins with molecular masses of 120 and 80 kDa. These proteins were also revealed in a plasma membrane preparation with two other proteins, with molecular masses of 150 and 104 kDa, and were sensitive to PI-PLC treatment. Electron microscopy also showed the binding of Au-labelled HDL3 inside the caveolar membrane invaginations. In SK-MES-1 cells, HDL3 are internalized into a particular structure, resulting in the accumulation and concentration of such specific membrane domains. To sum up, a demonstration has been made of the implication of GPI-proteins as well as caveolae in the binding of HDL3 to cells.

[Serum lipids and lipoproteins in a population from Oran. Comparison with a population from Lille]. / Lipides et lipoprotéines sériques dans une population oranaise. Comparaison avec une population lilloise.

Lipids, lipoproteins and apolipoproteins were analyzed in plasma of a city living Algerian population (Oran) free of obvious pathology. 129 individuals of this population were compared to a sample of subjects living in the surroundings of Lille (France). Several parameters were significantly different between the two populations. Cholesterol and phospholipids were lower in the population of Oran when compared with the population of Lille (4.90 mmol/l versus 5.47 mmol/l; 2.73 mmol/l versus 3.22 mmol/l respectively). Lipoproteins LpAI and LpAI:All were also lower in the population of Oran in comparison with the population of Lille (0.38 g/l versus 0.52 g/l; 0.68 g/l versus 1.00 g/l respectively), while LpCIII:B, LpE:B and Lp(a) were higher in the population of Oran when compared with the population of Lille (0.09 g/l versus 0.06 g/l; 0.28 g/l versus 0.16 g/l; 0.1 g/l versus 0.048 g/l) respectively. Several results argue in favor of the use of these lipoprotein parameters to better define the atherosclerotic risk. This was the main reason for characterizing the lipoprotein profile of such a population with a still unknown cardiovascular disease incidence.

Apo B-containing lipoprotein particles in poorly controlled insulin-dependent diabetes.

The goal of this study was to compare the structural and biological characteristics of apolipoprotein (apo) B-100-containing particle subfractions isolated from poorly controlled diabetic patients with insulin-dependent diabetes (IDDM), and healthy controls matched for sex, age and body mass index (BMI). Different apo B-containing particles were isolated by sequential immunochromatography and were free of apo A-I, apo A-II, apo A-IV and apo(a). Particles lipoprotein (Lp) B/C-III contained apo B and apo C-III. They were free of apo E. Particles Lp B/E contained apo B and apo E. They were free of apo C-III. Particles Lp B were devoided of apo C-III and apo E. All these particles could contain other known apolipoproteins not cited here, as for example apo C-II and/or apo C-I. The plasma levels of cholesterol, triglycerides, phospholipids, apo A-I, B-100, C-III, E, total Lp B/C-III, total Lp B/E were not different between patients and controls. The physico-chemical properties of Lp B/C-III and Lp B/E were similar in both groups. Only Lp B from patients exhibited some changes, an increase in the size and a decrease in the cholesterol and cholesteryl ester levels. The conformational properties of the lipoproteins were studied through their immunoreactivity against four different anti-apo B-100 monoclonal antibodies (MAb) for which sequential epitopes have been located on the protein, and one MAb for which the epitope is conformationally expressed. Again, minor changes were observed between patients and controls, and only a slight decrease in the immunoreactivity of the epitope encompassing amino-acid residues 405 to 539 of Lp B and of the conformationally expressed epitope of Lp B/C-III were found in patients. Nevertheless, whatever these conformational and/or physico-chemical modifications may be, they were not sufficient to induce functional alterations in the binding of the particles from the patients to the LDL-receptor of HeLa cells. This study shows that IDDM is not associated with any significant abnormalities in the apo-containing lipoprotein particles. The excessive occurrence of coronary heart disease (CHD) and other atherosclerotic vascular disease in patients with IDDM must have other causes.

Lipoproteins containing apolipoprotein B isolated from patients with abetalipoproteinemia and homozygous hypobetalipoproteinemia: identification and characterization.

Abetalipoproteinemia (ABL) and homozygous hypobetalipoproteinemia (HBL) are inherited disorders which are classically characterized by progressive retinal and spinocerebellar disease, fat-soluble vitamin deficiency, and absence of apolipoprotein (apo) B from the plasma. Using immunoaffinity chromatography with an anti-apo B antiserum, we isolated apo B-containing lipoprotein (LpB) particles from the plasma of 4 ABL and 2 HBL patients. The LpB particles were characterized and compared with low density lipoprotein (LDL) and LpB isolated from normal plasma. The ABL/HBL LpB particles were similar in size and charge to normal LpB particles but were relatively enriched in several other apolipoproteins. They contained alpha-tocopherol in a ratio to cholesterol that was proportionately much higher than the very low ratio of alpha-tocopherol to cholesterol in plasma. They bound saturably to fibroblasts and were internalized and degraded similarly to LDL. Hence, the molecular defects in ABL and HBL permit the secretion of a very small number of apo B-containing lipoproteins which may be important for transport of alpha-tocopherol to peripheral tissues.

Modulation of lipoprotein B binding to the LDL receptor by exogenous lipids and apolipoproteins CI, CII, CIII, and E.

We have recently shown that apo B-containing lipoproteins isolated by immunoaffinity chromatography bind to the LDL receptor with an affinity dependent on their apo E or apo CIII content. However, these lipoproteins--LpB:E, LpB:CIII, and LpB:CIII:E--isolated from whole plasma have variable lipid and apolipoprotein contents, and it is difficult to consider each parameter separately, particularly because an increase in the apo CIII content is always associated with an increase in the content of other C apolipoproteins. Therefore, we used affinity-purified LpB free of other apolipoproteins. Lipid content of LpB was increased by incubation with a lipid emulsion, and this triglyceride-enriched LpB was named TG-LpB. Free apo CI, apo CII, apo CIII, and apo E were added to LpB and TG-LpB and their associations to the lipoprotein were assessed by gel filtration, nondenaturing electrophoresis, and immunoblotting. Molar ratios of 6 (apo E), 30 (apo CII), 20 (apo CIII), and 30 (apo CI) for 1 apo B were obtained. The association of apo CII to LpB and TG-LpB induced modifications to the LpB structure and a redistribution of lipids and apolipoproteins on the lipoprotein particles. The binding of these LpBs and TG-LpBs with and without added apo CI, CII, CIII, and E was tested at 4 degrees C on the LDL receptors of HeLa cells. The increased content of lipids reduced TG-LpB binding to the LDL receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein pattern and plasma lecithin cholesterol acyl transferase activity in children with Alagille syndrome.

Alagille syndrome is frequently associated with hyperlipidemia and xanthoma. The aim of the study was to assess the lipid profile (plasma lipoproteins, apolipoproteins (apo)) and lecithin cholesterol acyl transferase (LCAT) activity, with and without treatment with cholestyramine in Alagille syndrome. Five children (mean age = 6 +/- 4 years) with Alagille syndrome were studied at two different times while receiving no treatment, and while receiving cholestyramine. They were compared with 12 normal controls, who were not different from patients for age and sex. In Alagille syndrome, total serum cholesterol, triglycerides and phospholipids were elevated compared with the controls (P < 0.008). VLDL-cholesterol, LDL-cholesterol, HDL-triglycerides, LDL-triglycerides and VLDL-phospholipids were higher, whereas HDL-cholesterol was lower than controls (P < 0.03). Apo B, CIII, E and lipoprotein particles Lp AI were higher (P < 0.001), whereas Lp AI:AII was lower than controls (P < 0.03). Lipoprotein-X was present in the 5 children with Alagille syndrome and explained in part the elevation of plasma cholesterol, phospholipids, and apo CIII. LCAT activity was decreased (P < 0.01) and might cause some abnormalities of HDL with lower cholesterol, higher triglycerides, apo E and apo CIII contents than controls, and abnormalities of VLDL and LDL with higher cholesterol, triglycerides, phospholipids and apo B contents than controls. Some of the risk factors of atherosclerosis were found in Alagille syndrome, namely high levels of plasma cholesterol, LDL cholesterol, apo B, apo B/apo AI. Treatment with cholestyramine resulted in a few modifications to the lipid profile, while lipoprotein-X and the decrease of LCAT activity persisted.