Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells.

Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo AII (LpAI) and particles containing apo AI and apo AII (LpAI:AII) isolated from native human plasma, and from HDL2 or HDL3, showed that only LpAI were able to promote cholesterol efflux, despite the fact that both kinds of particles were able to bind to receptor sites within the same range of concentrations (apparent Kd values between 10 and 25 micrograms/ml). During this long-term exposure, LpAI:AII demonstrated a concentration-dependent inhibition (10-60 micrograms/ml) of LpAI-mediated cholesterol efflux from adipose cells under conditions where LpAI:AII did not deliver cholesterol to the cells and where no net change in the distribution of apo AI between LpAI and LpAI:AII was observed. The antagonizing and modulating role of LpAI:AII in preventing cholesterol efflux mediated by LpAI appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo AI in LpAI particles and apo AII in LpAI:AII particles. The actual concentrations of these particles in the interstitial fluid bathing peripheral cells might be critical for the in vivo occurrence of cholesterol efflux.

[Influence of alcohol on plasma lipids and atherogenesis. Epidemiological and biochemical aspects]. / Influence de l'alcool sur les lipides plasmatiques et l'athérogenèse. Aspects épidémiologique et biochimique.

The results of epidemiological and clinical studies published since 1980 concerning the effects of alcohol intake on coronary artery disease are rather contradictory. Although some protective action of alcohol, notably of wine against atherosclerosis, has been described by some authors, the methodological limitations of these studies make it impossible to establish a cause-effect relationship in this matter. Biochemical studies have provided a more precise approach of the effect of alcohol on the mechanism of atherosclerosis. An increase of HDL has been shown in patients who regularly consume alcoholic drinks. However, a detailed analysis of HDL subfractions (and notably HDL2 regarded as an antiatherogenic lipoprotein) has given equally contradictory results. When the antiatherogenic lipoprotein particles present in HDL are accurately identified, the physiopathological consequences of regular alcohol consumption will be more clearly determined. Biochemical and epidemiological information is still insufficient for us to attribute an antiatherogenic effect to alcohol.

Purification of an apolipoprotein A binding protein from mouse adipose cells.

A protein recognizing apolipoproteins AI, AII and AIV was purified from cultured mouse adipose cells of the Ob17MT18 clonal line. Apolipoprotein A binding sites were solubilized in the presence of proteinase inhibitors using the non-denaturating detergent CHAPS. Chromatography of the soluble extract on DEAE-Trisacryl was followed by immunoaffinity chromatography of the complex apolipoprotein AI-binding proteins on anti-(apolipoprotein AI) coupled to Sepharose 4B and then by h.p.l.c. on an RP-Select B column. A 1400-fold purification over the starting crude homogenate was achieved. The purified material contained two proteins that were both able to bind apolipoproteins AI, AII and AIV, but not low-density lipoprotein. Glycopeptidase F treatment showed the existence of a single protein bearing either N-linked high-mannose or complex oligosaccharide chains. The purified material showed an apparent molecular mass of 80 +/- 9 kDa by h.p.l.c. on a TSKG 3000 SW column. Rabbit polyclonal antibodies directed against the purified material revealed two protein bands of 80 and 92 kDa after SDS/PAGE under reducing conditions and immunoblotting. These bands were undetectable in growing Ob17PY cells previously shown not to bind the various apolipoproteins A and not to undergo cholesterol efflux, whereas they were conspicuous in growth-arrested Ob17PY cells which have recovered these properties.

Effect of alcohol intake on human apolipoprotein A-I-containing lipoprotein subfractions.

High-density lipoprotein comprises two main types of lipoprotein particles: (1) those that contain apolipoproteins A-I and A-II, designated LpA-I:A-II, and (2) those that contain apolipoprotein A-I but not apolipoprotein A-II, designated LpA-I. Both have been extensively studied and are believed to represent distinct metabolic entities that may confer differing protection against coronary artery disease risk. We have previously suggested that LpA-I might represent the antiatherogenic effect, which has been ascribed mainly to its effect on high-density lipoprotein cholesterol; we set out to investigate, in 344 men, the relation between LpA-I:A-II and LpA-I levels and alcohol consumption. As the alcohol intake rose, LpA-I:A-II levels increased, while LpA-I levels fell. On the assumption that LpA-I is the antiatherogenic fraction of high-density lipoprotein, the putative protective action of alcohol consumption against coronary artery disease should be reconsidered.

Influence of serum amyloid A on cholesterol esterification in human plasma.

Lecithin-cholesterol acyltransferase (EC 2.3.1.43, LCAT) is the enzyme responsible for the formation of the bulk of cholesteryl ester in human plasma. The LCAT-reaction takes place mainly on high-density lipoproteins and requires an apolipoprotein as activator. Besides apolipoprotein (apo) A-I several other potent activator apolipoproteins (AIV, E and CI) were identified, furthermore apo A-II was shown to be a modulator of the enzyme's reaction in the presence of apo A-I. Serum amyloid A, an apolipoprotein mainly associated with high-density lipoprotein, massively accumulates in plasma upon acute phase reactions. We therefore studied the possible influence of this acute phase reactant on cholesterol esterification in human plasma. There was a significant decrease of esterified cholesterol in plasma during acute phase reaction. We found a highly significant correlation between the unesterified part of plasma cholesterol and serum amyloid A levels (r = 0.694, P = 0.0001). Also, plasma LCAT activity was negatively correlated with serum amyloid A levels. Lipoproteins containing apo A-I and A-II (LpA-I: A-II) and lipoproteins containing apo A-I but no A-II (LpA-I) decreased significantly with the appearance in plasma of serum amyloid A. To study the influence of serum amyloid A on the LCAT reaction, artificial substrates were prepared either by a detergent dialysis procedure or by addition of apolipoprotein to a sonicated aqueous dispersion of lipid. In addition two different molar ratios of apolipoprotein/phospholipid (PC) (1:50 and 1:310) were chosen at a constant molar ratio of total cholesterol/PC of 1:20. The various substrates were incubated with purified LCAT enzyme. DMPC - or egg yolk phosphatidylcholine - cholesterol-[4-14C]cholesterol-serum amyloid A complexes per se did not stimulate LCAT activity significantly. However, apo serum amyloid A incorporated together with apo A-I by a detergent dialysis procedure lead at low concentrations of serum amyloid A to a marked increase in cholesteryl ester formation as compared to apo A-I alone but inhibited the cholesteryl ester formation at high concentrations. Thus, the low levels of esterified cholesterol in acute phase plasma are to some extent due to decreased plasma enzyme activity and in part may be due to interference of apo serum amyloid A with the natural substrate complexes of plasma HDL.

Apolipoprotein B metabolism in homozygous familial hypercholesterolemia.

This report describes the metabolism of apolipoprotein B-containing lipoproteins in seven familial hypercholesterolemic (FH) homozygotes and compares the results to the values obtained from five healthy control subjects. The concentration, composition, and metabolism of large, triglyceride-rich very low density lipoproteins (VLDL1, Sf 60-400) were the same in the control and FH groups, indicating that this component of the VLDL delipidation cascade ws unaffected by the absence of receptors. In contrast, familial hypercholesterolemic small VLDL2 (Sf 20-60) was enriched with cholesterol and depleted in triglyceride. Moreover, its plasma concentration was elevated as a result of an increase in its synthesis and a defect in the removal of a remnant population within this density interval. The latter accounted for up to 50% of the total mass of the fraction. Onward transfer of apolipoprotein B (apoB) from small VLDL through intermediate density lipoprotein (IDL) to low density lipoprotein (LDL) was retarded, suggesting that receptors were involved in this supposedly lipase-mediated event. IDL and LDL concentrations increased up to fourfold above normal in the plasma of the FH patients due partly to the delay in maturation and partly to defective direct catabolism. We conclude that the LDL receptor plays multiple and important roles in the metabolism and transformation of apoB-containing particles in the Sf 0-400 flotation interval.

Binding characteristics of high density lipoprotein subclasses to porcine liver, adrenal and skeletal muscle plasma membranes.

1. We compared binding characteristics of 125I-labeled high density lipoprotein (HDL) subclasses to porcine liver, adrenal and skeletal muscle plasma membranes. 2. HDL subclasses were discriminated by their buoyant densities (HDL2 and HDL3) or by their apolipoprotein (apo) content (Lp-AI (particles containing apoA-I but no apoA-II) and LpA-I/A-II (particles containing both apoA-I and apoA-II)). 3. HDL2 and HDL3 showed saturable binding to the three types of membrane preparations. 4. No differences were found in the Kds within one HDL subclass. 5. Kds and maximal binding of HDL2 were lower than these of HDL3. Unlabeled HDL2 and HDL3, but not LDL, effectively displaced 125I-HDL2 and 125I-HDL3. 6. Binding of HDL was independent of the concentration of NaCl and did not require calcium. 7. These results suggest a process mediated by a single specific receptor in porcine liver, adrenal and skeletal muscle plasma membranes. 8. We also studied binding characteristics of HDL subclasses Lp-AI and LpA-I/A-II to porcine liver membranes. LpA-I showed the highest Kd and maximal binding. 9. All types of HDL subclasses studied (i.e. HDL2, HDL3, LpA-I and LpA-I/A-II) effectively competed for binding of both Lp-AI and LpA-I/A-II, suggesting that the HDL subclasses studied bind to the same receptor by their apoA-I moiety.

[Quantitative analysis of apolipoproteins and lipoprotein particles in patients with head injuries]. / Analyse quantitative des apolipoprotéines et des particules lipoprotéiniques chez les traumatisés crâniens.

By head-injured patients, apo A-I and apo A-II concentrations were more decreased in HDL3 than in HDL2. Then, the plasmatic concentrations of the main lipoprotein particles present in HDL fraction were modified. For example, a significant decrease of Lp A-I: A-II particles was observed and this variation was similar to that of total apo A-I (r = 0.78). On the other hand, the concentration of Lp A-I particles was slightly modified, apo C-III concentration was markedly decreased whereas apo E concentration was significantly increased (p less than 0.05); in plasma samples obtained 10 days after a severe head injury, apo E reached three times the normal value.

Enzyme-linked immunosorbent assay for serum amyloid A apolipoprotein with use of specific antibodies against synthetic peptides.

We describe a noncompetitive enzyme-linked immunosorbent assay for amyloid A apolipoprotein in human serum (apo SAA) in which specific antibodies against synthetic peptides are used. Microtiter plates were used as solid phase and coated with affinity-purified antibodies raised against SAA1-(95-104) peptide. After incubation with delipidated plasmas, the bound apo SAA was revealed by labeled antibodies raised against SAA1-(58-69) peptide. The assay offers several advantages over existing techniques: sensitivity, specificity, simplicity, and non-use of radioisotopes. Results correlate well with those by a nephelometric method in which polyclonal antibodies are used.

Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.

Apolipoprotein A-I (apoA-I), the major protein component of human high density lipoprotein, appears intracellularly as an intermediate precursor (proapoA-I) with a hexapeptide extension (Arg-His-Phe-Trp-Gln-Gln) at its amino terminus. To investigate the regulation of processes that regulate plasma apoA-I levels, a sensitive and simple assay for proapoA-I is required. We describe a specific enzyme-linked immunosorbent assay (ELISA) for quantification of proapoA-I using monospecific rabbit antibodies raised against the peptide: Arg-His-Phe-Trp-Gln-Gln-Asp-Glu-Pro. The monospecificity of antibodies to propeptide has been checked and no cross-reaction with mature apoA-I has been found although three first mature apoA-I amino acids (Asp-Glu-Pro) were included in the immunizing peptide. The assay is a non-competitive sandwich ELISA in which polystyrene microtiter plates were used with antibodies to propeptide adsorbed on the wells. After incubation with plasma samples, the bound proapoA-I was revealed by labeled rabbit polyclonal antibodies directed against mature apoA-I. The working range was 10 to 100 ng/ml, recovery of proapoA-I added to plasma was 94.6 to 106.5%, and the intra- and interassay coefficients of variation were 3.8% and 7.9%, respectively. A delipidation step using diisopropylether-n-butanol was necessary to expose antigen sites of proapoA-I in native lipoproteins. Mean level of proapoA-I in normal subjects was 87 +/- 15 micrograms/ml. It represented 7.1% of total apoA-I while in Tangier serum it represented 29%.

[Receptors and molecular heterogeneity of lipoprotein particles containing apolipoprotein A-I]. / Récepteurs et hétérogénéité moléculaire des particules lipoprotéiques contenant l'apolipoprotéine A-I.

High density lipoproteins (HDL) are heterogeneous, with respect to their hydrated density (HDL2, HDL3), and to their apolipoprotein composition (Lp A-I : A-II contains both apolipoprotein A-I and A-II, Lp A-I contains apolipoprotein A-I but not apolipoprotein A-II). Lp A-I and Lp A-I and Lp A-I : A-II particles have different metabolic functions. Only Lp A-I particles seem to be involved in the antiatherogenic role of HDL. Alcohol consumption raises Lp A-I : A-II level but not Lp A-I. Different tissue possess specific binding sites for HDL: steroidogenic tissue, hepatocytes peripheral cells. Apolipoprotein A-I and/or A-II are possible ligands. HLD, after binding to the receptor, can provide the cells with cholesterol, or promote an efflux of cholesterol from the cells and the "reverse cholesterol transport" from the peripheral cells to the liver. The HDL subfractions possess different metabolic roles: binding of Lp A-I to mouse adipose cell receptors promotes cholesterol efflux. Apo A-II are antagonists for this effect.

Effect of different APO A-I containing lipoprotein particles on reverse cholesterol transport in fat cells.

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with LDL cholesterol. Long term exposure to LpA-I particles isolated from HDL2 and HDL3 promoted cholesterol efflux while LpA-I:A-II particles isolated from both fractions did not. However, LpA-I and LpA-I:A-II particles isolated from both HDL subfractions were effective in competing for the binding of 125I-HDL3 to apo A-I/A-II cell surface receptor sites. These results underline the role of LpA-I particles in the promotion of cholesterol efflux and suggest that the concentrations and/or the relative proportions of LpA-I and LpA-I:A-II particles might be critical for the regulation of cholesterol efflux from adipose cells.

Relationship in adipose cells between the presence of receptor sites for high density lipoproteins and the promotion of reverse cholesterol transport.

The role of a high-affinity receptor site for high-density lipoproteins (HDL) has been investigated in parental Ob1771 adipose cells and their transformed counterparts after transfer of the complete early region of polyoma virus (Ob17PY cells). Binding of ApoAI, ApoAII and HDL3 occurs in Ob1771 cells and derived membranes, whereas no binding is observed in Ob17PY cells and derived membranes. After thymidine block, growth-arrested Ob17PY cells become able to bind ApoAI, ApoAII and HDL3; this recovery is prevented in actinomycin D- or cycloheximide-treated cells. In contrast to ApoAI, ApoAII or HDL3 binding, both growing and growth-arrested Ob17PY cells do show receptor activities for low density lipoproteins and transferrin, respectively, which are similar in affinity and maximal capacity. Following cholesterol accumulation which takes place in the presence of LDL cholesterol, subsequent exposure to HDL3 or ApoAI promotes cholesterol efflux from Ob1771 cells and growth-arrested Ob17PY cells but not from growing Ob17PY cells. These results show that the presence of a high-affinity receptor site for HDL in intact adipose cells is required for the promotion of reverse cholesterol transport.

Variations in lipoproteins during aminoglutethimide therapy.

Plasma lipids, apolipoproteins, and gamma-glutamyl-transpeptidase (GGT) were measured in 28 patients receiving aminoglutethimide (500 mg) and hydrocortisone (30 or 40 mg) for advanced breast cancer. A rise in cholesterol (CHOL), LDL-CHOL, apoprotein B, apoprotein CIII, and GGT was observed after 45 days. When the patients were divided in two groups according to lipid basal plasma levels, those with high CHOL and triglyceride did not experience any modification of lipid parameters (only GGT were elevated). Conversely normolipidaemic patients experienced an increase in CHOL, triglycerides, LDL-CHOL, apoproteins B and CIII, and GGT.

[Multivariate analysis of biological markers of excessive consumption of alcohol. Value of lipoprotein parameters]. / Analyse multivariée d'indicateurs biologiques de consommation excessive d'alcool. Intérêt des paramètres lipoprotéiques.

In order to investigate the usefulness of lipid, lipoprotein and apolipoprotein parameters for detection of intemperate drinkers, 90 adult male subjects with an alcohol consumption above 400 g weekly were compared to 90 matched (age, body weight index) male subjects with an alcohol consumption below 250 g weekly. GGT and MCV were also evaluated. Univariate analysis showed GGT, apolipoprotein A-II and phospholipid-LP no B levels to be parameters the most significantly different between drinkers and controls. Discriminant analysis using six parameters in addition to apolipoprotein A-II allowed to classify correctly 80% of subjects (efficacy) with respect to their alcohol consumption. However, sensitivity of the parameters remains relatively low and a good efficacy is obtained only with a composite index of several parameters.

Apolipoprotein A-I containing lipoproteins in coronary artery disease.

At least 2 main types of lipoprotein particles are identified within HDL. Those which contain apo A-I and apo A-II (LpA-I:A-II) and those which contain apo A-I but not apo A-II (LpA-I). This study was designed to elucidate to what degree the HDL cholesterol decrease observed in coronary artery disease affects these 2 types of lipoprotein particles. Concentrations of LpA-I:A-II and LpA-I were measured in plasma from 100 normolipidemic male subjects with angiographically defined coronary artery disease (CAD(+)) or without CAD (CAD(-)) and from 50 control subjects, matched for age. CAD(+) subjects had significantly lower levels of HDL cholesterol, total apo A-I, and LpA-I than controls. When compared to CAD(-) subjects, only their levels of HDL cholesterol and LpA-I were found lower. In both cases (CAD(+) vs CAD(-) and CAD(+) vs controls), LpA-I levels were decreased while LpA-I:A-II levels were unchanged. Even, when the levels of their total plasma lipids and lipoproteins are normal, atherosclerotic patients are characterized by a different distribution of apo A-I between LpA-I and LpA-I:A-II. These data support the view that LpA-I might represent the "antiatherogenic" fraction of HDL.