METABOLIC ROLE OF HUMAN APOPROTEIN A-IV

METABOLIC ROLE OF HUMAN APOPROTEIN A-IV

INTRODUCTION

Compared with the other human apoproteins, physiological variations and metabolic functions of apo A-IV remain obscure. The apo A-IV has been discovered in rat HDL and has been later found in human plasma, mesenteric lymph and interstitial fluid. Apo A-IV is synthetized by intestine and liver and is catabolized by liver and kidneys. As apo A-IV has been suspected to play a role in triglyceride transport, we undertook the study of its plasma concentration in a population of subjects with various plasma triglyceride levels. Moreover, recent studies brought some evidence for an implication of apo A-IV in the reverse cholesterol transport. Because recent reports suggested that HDL was the major lipoprotein class involved in this metabolic pathway, we investigated the structural and metabolic relation between apo A-IV and HDL.

RQL[ OF APOPROT[IN A-IV IN TRIGLYC[RID[ TRANSPORT

By using an apo A-IV competitive enzyme immunoassay we found a significant positive correlation between apo A-IV and triglyceride concentrations in human sera (1) <Figure1). These data were in good agreement with previous reports suggesting a relation between apo A-IV and triglyceride secretion. An increase of plasma apo A-IV concentration has been found after lipid feeding (2,3) and in circumstances associated with accumulation of remnants of triglyceride-rich lipoproteins, such as chronic renal failure treated by peritoneal dialysis or hemodialysis (4,5). Moreover, a relation between the apo A-IV mRNA synthesis and the triglyceride secretion has been observed in intestine and liver (6,7).

As this correlation between apo A-IV triglycerides was found in non­ chylomicronemic sera from fasting subjects, we can assume that triglycerides were contained mainly in VLDL from hepatic origin and that the liver, in fasted subjects, could'contribute significantly to the plasma apo A-IV pool.

ROLE OF APOPROTEIN A-IV IN REVERSE CHOLESTEROL TRANSPORT

Distribution of apoprotein A-IV in normolipidemic human serum

As apo A-IV has been suspected to play a role in reverse cholesterol transport. its structural relation with HDL was studied. Whereas it is known that in rat most of apo A-IV associates with HDL, in human very little apo A-IV has been found on lipoprotein particles isolated by traditional ultracentrifugation methods. However. the distribution of apo A-IV between lipoprotein free and lipoprotein fractions varies widely and appears to be dependent on the techniques used to fractionate sera. About 20 to 35 Y. of apo A-IV were found associated with HDL after agarose gel permeation chromatography of total plasma (2,3,8), while more than 90 Y.

of human apo A-IV localized in the lipoprotein-free fraction after ultra­ centrifugation of plasma at density 1.21 (2,9).

By using a high performance Superose 12-HR column (Pharmacia), we were able not only to confirm the presence of apo A IV in the HDL fraction but to demonstrate that, in fasting human sera, a majority of apo A-IV eluted with the HDL fraction, mainly within the HDL2 size range (10>. Moreover, the gel filtration procedure allowed us to evidence the potential disruptive effect of ultracentrifugation on the apo A-IV distribution in total serum <Figure 2>. This distribution of apo A-IV could be attributable to the combined effects of high ionic strength and high sheering forces of ultracentrifugation procedure.

Concurrently, the coprecipitation of the most part of apo A-IV after incubation of total serum with anti-apo A-I antibodies indicated that about 70 to 80 Y. of serum apo A-IV were carried by apo A-I containing HDL

<Figure 3). This proportion of apo A-IV coprecipitated with HDL is

considerably lowered (less than 10 %) when HDL particles were dissociated by addition of Tween to the serum dilutions prior to the precipatation step.

These results provided evidence that, in fasted normal human sera. apo A-IV preferably associated with HDL and that this apo A-IV-HDL asso­ ciation is a weak one, easily disrupted. The remodeling of lipoprotein surfaces can modify the equilibrium between free and bound apo A-IV. Particularly, LCAT activity can induce a displacement of apo A-IV from lipoprotein-free fraction to the HDL particles.

Apoprotein A-IV and HDL conversion

Beyond its structural relation with HDL, apo A-IV can also play a dynamic role in the intravascular metabolism of HDL particles, especially in the size redistribution or conversion induced by the Cholesterol [ster Transfer Protein CCKTP> (11>. By using gradient gel electrophoresis, it was shown that the incubation of total lipoproteins in the presence of CKTP at physiological levels induced a general displacement of HDL towards large size particles, a decrease in the HDL3 subpopulation and the appearance of small conversion products with mean diameters of 7.8 and 7.4 nm (Figure 4).

This phenomenon can be significantly altered by apo A-IV. Compared with incubation with CETP alone. incubation with CETP and apo A-IV increased the size redistribution of HDL and particularly favored the formation of the very small sized lipoprotein particles (mean diameter 7.4 nml. Incubation of total lipoproteins and apo A-IV, in the absence of CETP, do not modify the HDL distribution profiles.

DESCRIPTION 0 THE MULTIPOTENTIAL INVOLVEMENT 0 APO A-IV IN THE REVERSE CHOLESTEROL TRANSPORT

The results presented above, together with previously reported data, show that apo A-IV is involved at different levels of HDL metabolism and suggest that this apoprotein could play a major role in cholesterol transport from peripheral tissues to the liver. More precisely, the loca­lization of apo A-IV within HDL. its implication in the formation of very small sized lipoprotein particles, its activating role in plasma choles­terol esterification by LCAT and its potential involvement in the cholesterol movements between intra- and extra-cellular media suggest that this apoprotein could play a major role in the reverse cholesterol transport (Figure 5).

By its combined effects with C[TP, apo A-IV can promote the formation of very small sized HDL particles. Such particles are susceptible to mi­ grate easily in the interstitial space where they could participate to the cell cholesterol efflux <11). Moreover, as apo A-IV is relatively stable in aqueous solution, the free apoprotein itself could also participate directly to the peripheral cholesterol uptake. The particles issued from interstitial space, enriched with free cholesterol and phospholipids can then interact with plasma LCAT. The small HDL, enriched with unesterified cholesterol <UC> and phospholipids <PL>, are known to be good substrates

for this enzyme <12) which can be furthermore activated by apo A-IV <13, 14). These uptake and esterification of cholesterol, which constitute a key step in the Reverse Cholesterol Transport, lead to the formation of large sized HDL2-like particles enriched in esterified cholesterol. In vitro studies on cell cultures have indicated that apo A-IV could specifi­ cally interact with rat hepatocytes <15,16). As we observed that apo A-IV

in human serum localized mainly in large sized HDL particles, it can be postulated that apo A-IV could facilitate specific uptake of HDL by the liver.

In conclusion, by promoting efflux of peripheral cholesterol, by activating cholesterol esterification in plasma, and by facilitating cholesterol uptake by the liver, apo A-IV could play a central role in the Reverse Cholesterol Transport.

REFERENCES

1. L. Lagrost, P. Gambert, S. Meunier, P.Morgado, J. Degres, P. d'Athis, and C. Lallemant, Correlation between apolipoprotein A-IV and tri­ glyceride concentrations in human sera, J. Lipid Res., 30:701 <1989).

2. P. H. Green, R.P. Glickman, J.W. Riley, and [. Quinet, Human apolipoprotein A-IV : intestinal origin and distribution in plasma, J. Clin. Invest., 65:911 <1980).

3. C. L. Bisgaier, 0. P. Sachdev, I. L. Megna, and R. M. Glickman, Distri­ bution of apolipoprotein A-IV in human plasma, J. Lipid Res., 26:11 (1985).

4. P. J. Nestel, N. H. Fidge, and M. H. Tan, Increased lipoprotein-remnant formation in chronic renal failure, N. [ngl. J. Med., 307:329 <1982).

5. M. Seishira, andY. Muto, An increased apo A-IV serum concentration of patients with chronic renal failure on hemodialysis, Clin. Chim. Acta, 167:303 <1987).

6. N. A. [lshourbagy, M. S. Boguski, W. S. L. Liao, L. S. Jefferson, J. I. Gordon, and J. M. Taylor, [xpression of rat apolipoprotein A-IV and A-I genes : mRNA induction during development and in response to glucocorticoids and insulin, Proc. Natl. Acad. Sci. USA, 82:8242 (1985).

7. M. Pessah, C. Salvat, S. R. Wang, and R. Infante, In vitro synthesis of apo A-IV and apo C by liver and intestinal mRNAs from lean and obese Zucker rats. Biochem. Biophys. Res. Commun, 142:78 <1987).

8. M. Rosseneu, G. Michiels, W. De Keersgieter, J. Bury, J. P. De Slypere, H. Dieplinger, and G. Utermann, Quantification of human apolipoprotein A-IV by"sandwich"-type enzyme-linked immunosorbent assay, Clin. Chern., 34:739 <1988).

9. G. Utermann, and U. Beisiegel, Apolipoprotein A-IV : a protein occur­ ring in human mesenteric lymph chylomicron and free in plasma. Isolation and quantification, [ur. J. Biochem., 99:333 <1979).

10. L. Lagrost, P. Gambert. M. Boquillon, and C. Lallemant. Evidence for high density lipoprotein as the major apolipoprotein A-IV containing fraction in normal human serum, J. Lipid Res., 30:1525 <1989).

11. P. Gambert, L. Lagrost, A. Athias, S. Bastiras, and C. Lallemant, Role of apolipoprotein A-IV in the interconversion of HDL subclasses, Advances in Experimental Medicine and Biology, 243:263 <1988).

12. P. J. Barter, G. J. Hopkins, and L. Gorjatschko, Lipoprotein substrates for plasma cholesterol esterification. Influence of particle size and composition of the high-density lipoprotein subfraction 3, Atherosclerosis, 58:97 <1985).

13. A. Steinmetz, and G. Utermann, Activation of lecithin : cholesterol acyltransferase by human apolipoprotein A-IV, J. Biol. Chern., 260:2258 (1985).

14. C. H. Chen, and J. J. Albers, Activation of lecithin : cholesterol

acyltransferase by apolipoprotein E-2, E-3, and A-IV isolated from

plasma, Biochim. Biophys. Acta, 836:279 (1985)

15. E. Dvorin, N. L. Gorder, D. M. Benson, and A.M. Gotto, Jr., Apolipoprotein A-IV. A determinant for binding and uptake of high density lipoproteins by rat hepatocytes. J. Biol. Chern .. 261:15714 (1986).

16. Y. B. Mitchel. V. A. Rifici. and H. A. Eder. Characterization of the specific binding of rat apolipoprotein E-deficiency HDL to rat hepatic plasma membranes, Biochim. Biophys. Acta, 917:324 <1987).

Read More

SYMMETRY OF THE SURFACE, AND STRUCTURE OF THE CENTRAL CORE OF HUMAN LDL PARTICLES, ANALYZED BY X-RAY SMALL ANGLE SCATTERING.

SYMMETRY OF THE SURFACE, AND STRUCTURE OF THE CENTRAL CORE OF HUMAN LDL PARTICLES, ANALYZED BY X-RAY SMALL ANGLE SCATTERING

Although there exists a widely accepted model of the general structure of the LDL particle, there are still some important open questions, the most interesting being the three dimensional structure of the apoB molecule. There is very little knowledge of the exact conformation of this large polypeptide chain, containing many hydrophobic residues. Despite this hydrophobicity it was shown by several techniques (MABs, NMR, FTIR) that many domains of this protein are localized at the particle surface or are at least accessible to the solvent. An elaborate evaluation of X-ray scattering data from three LDL subtractions shows that a) The apoB molecule has to cover at least 47% of the surface of small LDL particles, and 37% of the surface of large LDL.

b) Most of the mass of the apoB molecule is located within a small shell of 2.2nm width directly at the particle surface.

c) There is no evidence for symmetries other than spherical, which means, that there are no marked 'spikes' of protein at the surface.

An other uncertain point is the organization of the central cholesterol ester (CE) core below the phase transition temperature. Our X-ray data confirms those models in which the cholesterol moieties of the CE-molecules are located at two concentric shells of 3.2 and 6.4 nm radius. In contrast to previous models we propose, based on detailed space filling calculations, that the CE molecules are arranged .in an alternating orientation in such a way that about half of the acyl chains of the CE-molecu!es in each shell point towards the center of the particle, the other half pointing to the surface. This model facilitates an interdigitation of acyl chains of cholesterol esters with each other and with surface phospholipids. The interdigitation of acyl chains of core and surface lipids has also been proposed in the case of HDL and protein free models of LDL, and seems to be a general feature of lipoprotein structure.

Our current knowledge on the molecular structure of low density lipoproteins (LDL) originates to a large extent from X-ray and neutron small-angle studies that have been published by several groups. For references see 1 2 3 4 • In all of these studies evaluation theories assuming mono disperse, radially symmetric particles were used. Only Luzzati et al.6 considered deviations from spherical symmetry. While still assuming monodispersity, deviations from spherical symmetry turned out to be essential in Luzzati's interpretation of the data. Our evaluation theory 8 7 predicts the exact scattering intensity of a "polydisperse ensemble, of quasi radially symmetric particles", and consequently needs neither to assume monodispersity nor perfect radial symmetry. It has to be emphasized that assuming monodisperse particle populations is in obvious contrast to biochemical and metabolic features of lipoproteins.

In this paper we address mainly two questions: the structure of the apoB molecule and the structure of the central lipid core. Differences between LDL subtractions are discussed in detail in4 • The aim of our study was to find a model of the LDL structure which reproduces the scattering intensity of the sample within the experimental error (noise band) of the X-ray scattering data. None of the models published up to now has been shown to be consistent with this requirement. Since both, polydispersity and deviations from radial symmetry are quantitatively treated in our model, our method is well suited for investigating the question of whether the assumption of a multipole component, as proposed by6•8

is in fact necessary to explain the experimental data. In addition, our approach allows to give an estimate of the random errors of the resulting electron density profiles. New aspects arise from the precisely determined electron density profiles and from the fact that we determined the electron density profiles of three different LDL subtractions. Based on our molecular model and chemical analysis, we present a detailed comparison of volumes, areas, and radii given by the electron density profiles to the space requirement of the molecular components.

MATERIALS AND METHODS

Samples

Serum was obtained from freshly drawn blood (50 ml without anticoagulant) of male, clinically healthy donors. LDL (d = 1.006-1.063 g/ml) was isolated by standard methods8 • LDL subtractions were prepared as described previously4 • After centrifugation the material of subtractions LDL-1 (d=1.006-1.031 g/ml), LDL-3 (d=1.034-1.037 g/ml), and LDL-6 (d=1.044-1.063 g/ml) was dialyzed against a buffer containing 0.196 mol/kg NaCI, 0.5 g/1 NaN3 and 0.1 g/1 EDTA. An immersible CX-30000 ultrafilter (Milliporel was used to concentrate the samples to total cholesterol concentrations of up to 50 mg/ml.

Chemical analysis

In all subtractions total cholesterol, free cholesterol, triacylglycerol (all Boehringer, Mannheim), and phospholipids (bioM6rieux, Nurtingen) were determined. CE concentrations were calculated as (total cholesterol - free cholesterol) x 1.68. All tests were standardized according to the manufacturers' instructions. ApoB concentrations were determined by kinetic rate nephelometry using an automated Beckman ICS Analyzer II. This test was carefully standardized using amino acid analysis (for details see4). The given protein concentrations therefore represent amino acid concentrations and do not include the carbohydrate bound to apoB. For that reason in all subsequent calculations a molecular weight of 513,000 based on the amino acid sequence 10 11 is

used. The results of the chemical analysis are given in Tab. 1.

X- ray small-angle scattering

X-ray small-angle diagrams were recorded as described in detail in a previous publication 12• We used temperatures of 4°C and 37°C to record the scattering diagrams. These temperatures are below and above the phase transition temperature 13 of the cholesterol ester molecules inside the LDL particle. The actual phase transition temperatures Tm of our LDL preparations (LDL-1: 21.1 °C, LDL-3: 29.7°C, LDL-6: 29.9°C) were monitored by differential scanning calorimetry (DSC).

The method for evaluating the X-ray small-angle scattering diagrams was described previously4•8 7• 12• In brief, the particle structure is described by a set of parameters, which are adjusted by a non-linear least-square fit procedure to give an optimum fit to the experimentally determined X-ray scattering curve.

RESULTS AND DISCUSSION

Radial symmetry

To test the hypothesis of radial symmetry we tried to obtain fits of the scattering intensities by using the model "polydisperse ensemble of radially symmetric particles". No deviations from radial symmetry were allowed. It was possible to obtain satisfactory fits without any difficulty (Fig. 1a) in the case of scattering intensities recorded at 37°C. This holds for all types of LDL subtractions investigated in this study. Therefore there is no need to introduce symmetries other than radial to explain the observed scattering intensities of the LDL particles at 37°C.

Fits of the intensities recorded at 4°C were not as perfect as those of the high temperature form. In particular, the width of the scattering maximum at 1/3.7 nm'1 could not be reproduced completely satisfactorily by our model (Fig. 1b). A detailed analysis of this very small deviation turned out to be outside the precision of our scattering data. Although an asymmetry of the LDL particle at 4°C cannot be completely excluded on the basis of our data, there is no evidence to assume the existence of such deviations from radial symmetry. Other deviations from our model could exist and are even more probable. For example one could speculate that the ordered, liquid­ crystal-like core is of the same size in all particles within the polydisperse ensemble. In fact such a "monodisperse" core would produce a sharper maximum at 1/3.7 nm·1 than predicted by our model.

We conclude that with the assumption of a polydisperse ensemble of radially symmetric particles it was possible to obtain fits of the scattering curves that were within the precision of the experimental data, and therefore our data does not support a shape of the LDL particle with marked 'spikes' of protein as proposed by6•8• It should be noted that this was not the case with HDL particles12 where quadrupole-like deviations from radial symmetry had to be assumed to explain the measured scattering curves. The radial symmetry of the LDL particle together with the electron density profile implies a spread out conformation of the protein. Although apoB contains many hydrophobic domains 10 11, it must be localized predominantly inside the outer surface shell of high electron density, which has a width of about 2.2nm. This conformation is further supported by the fact that a large area of the particle surface has to be covered by protein since the surface of the LDL particle cannot be covered by phospholipid headgroups and free cholesterol alone2• 14• An area of at least 470 nm2 has to be covered by apoB, even if one assumes relatively

large and additive areas of 0.7nm2 per phospholipid and 0.41nm2 per cholesterol molecule, which are areas observed at the air-water interface for single compounds 16 Areas observed in mixed bilayers are usually smaller 16 and would suggest a larger apoB area covered by apoB. It is remarkable that the calculated area covered by apoB is nearly the same for all three LDL subtractions, which strongly supports our conclusion . The view that the apoB molecule covers a large amount of the particle surface is supported by many studies : it was shown by tryptic digestion 11 and by binding studies with monoclonal antibodies 17 (reviews: 18 18 that domain s inmany regions of apoB are located at the particle surface . Using nuclear magnetic resonance 20 •21 it could be demonstrated that about 20 % of the phospholipid headgroups are immobilized by interaction with apoB . Fast Fourier infrared spectroscopy 22 indicated that the a-helical, random coil and B-turn structures may be situated on or near the surface of the LDL particle, while the B-strands have no or only restricted contact with the external solution . It has been hypothesized that amphiphilic B-strands23 are the major lipid binding structure of apo810 •

High and low temperature form of the LDL particle

We now want to compare the determined electron density profiles of the low temperature form with the high temperature form of the LDL particle: As shown in Fig. 2 a common feature of all profiles measured at 4°C and at 37°C is the outer double shell consisting of a shell of high electron density (HEDS) at the particle surface, and a shell of low electron density (LEOS) just below this shell. Both, HEDS and LEOS are similar in all particles. Great differences exist inside the core region. For the high temperature form of the core only comparably small oscillations were found which did not cross the zero level. In contrast there exists a pronounced and reproducible shell structure inside the core of the LDL particle below the phase transition temperature. We find two peaks of high electron density at average radii of 6.37 ± 0.29 nm and 3.23 ± 0.21 nm (average of LDL-1, LDL-3, and LDL-6, at 4°C). In accordance with 24• 26 these two peaks in the electron density profile can well be explained by a localization of the steroid moiety of the cholesterol esters on two concentric shells (Fig. 3) at radii of 6.37 and 3.23 nm, respectively. Such a model results in a cross-sectional area of 0.33 to 0.38 nm2 per steroid system, a value close to that obtained for cholesterol esters in crystal packing, where values of 0.35 nm2 26 and 0.366 nm2 27 have been reported. These values are smaller than the one measured at the air-water interface (0.405 nm2) 16•

Details on the arrangement of the cholesterol ester acyl chains can be deduced from the electron density profile by a careful comparison of the measured dimensions with the volumes required to arrange the cholesterol esters in a certain way. The corresponding calculation shall be shown in detail for the case of an average LDL-3 particle which, in average, contains 1886 cholesterol ester molecules: We assume that the steroid shells are centered at radii of 3.2 and 6.4 nm, and that they have a width of 1.7 nm, corresponding to the length of a cholesterol molecule 27 •

According to the available surface in each shell 377 cholesterol ester molecules are located at the inner steroid shell and 1509 molecules at the outer steroid shell. If a volume of 0.46 nm3 per acyl chain is assumed 28, 118 chains can be packed inside the inner shell (below 3.2 - 1.7/2 = 2.35 nm). Consequently the remaining 259 acyl chains have to be localized between the two steroid shells. This shell (4.05 to 5.55 nm) has room for further 693 acyl chains. The remaining 816 acyl chains from the second steroid shell are localized at a radius of >7.25 nm, and are pointing towards the particle surface. The volumes required for the cholesterol moieties alone (238 and 951 nm3 correspond well with the volumes of the steroid shells given by the model, which are 224 nm3 (2.35 to 4.05 nm) and 880 nm3 (5.5 to 7.25 nm). Although the above calculations contain some simplifications, they undoubtedly show that some sort of alternating orientation of the acyl chains has to exist. One simple model is that about half of the acyl chains of each shell point radially towards the center of the particle, the other half pointing to the surface (Fig. 3). Such an arrangement is further supported by neutron small-angle scattering data since it perfectly reproduces the radii of gyration (R ) measured by Laggner et al.29 : Laggner published a mean radius of gyration of 6.0 ± 0.2 nm for the fully deuterated acyl chain, and a value of ± 0.3 nm for the deuterated C-25 isopropyl groups7 • From our model we calculate average values of 5.95 nm for the acyl chains and 7.03 nm for the C-25 isopropyl groups. Laggner's preferred model (all acyl chains pointing to the center of the particle) gives values of 5.0 and 6.9 nm, respectively.

In addition, our model facilitates an interdigitation of acyl chains of cholesterol esters and surface phospholipids. This interdigitation of acyl chains of core and surface lipids was proposed in the case of the particles3 12 30 , as well as protein free models of LDL31 , and seems to be an essential principle in lipoprotein structure.

At 37°C, above the phase transition, the arrangement of the cholesterol esters appears to be much less ordered. The oscillations of the electron density are of only half the amplitude compared to those at 4°C. Model calculations showed that they might originate from cut-off effects of the Fourier series used to represent Pm(r). Furthermore, it should be mentioned that the electron density near the very center of the particle is poorly defined, since the errors in determining the electron density profile are proportional to 1/r12 32 •

Comparing the size of LDL particles at 4 and 37°C, one finds slightly larger particle sizes for LDL below the phase transition point. This larger radius at 4°C is almost completely explained by an increase of the thickness of the outer surface shell (HEDSw,dth). Only in the case of the smallest LDL particles (LDL-6) a larger core is additionally measured. The small increase of HEDSw,dth• consistently found for all three subtractions, indicates that the phase transition of the core is accompanied by structural changes of the particle surface. Such changes are most probably related to a conformational change of the apoB molecule, as proposed by Laggner and Kostner 33, based on ESR spectroscopic data. Interestingly circular dichroism spectra of native LDL are identical at 4 and 50°C34 •

In conclusion we were able to demonstrate that our procedure of X-ray data evaluation is able to resolve differen es between human LDL subtractions, and the corresponding high and low temperature forms of the particle core. Besides new information on structural differences between LDL subtractions and conformation of apoB a novel interpretation of the arrangement of the cholesterol esters in the ordered state is proposed.

This work was supported by the Deutsche Forschungsgemeinschaft, SFB 60, Teilprojekt D8.

REFERENCES

1. D. Atkinson, and D. M. Small, 1986, Recombinant lipoproteins: implications for structure and assembly of native lipoproteins, Ann. Rev. Biophys. Chern., 15:403.

2. D. Atkinson, D. M. Small, and G. G. Shipley, 1980, X-ray and neutron scattering studies of plasma lipoproteins, in: "Lipoprotein structure, • A. M. Scanu, and F. R. Landsberger, eds., Annals of the New York Academy of Sciences, Vol. 348:284.

3. P. Laggner, and K. W. Muller, 1978, The structure of serum lipoproteins as analysed by X-ray small-angle scattering, 0. Rev. Bioohvs. 11:371.

4. M. W. Baumstark, W. Kreutz, A. Berg, I. Frey, and J. Keul, 1990, Structure of human low-density lipoprotein subtractions, determined by X-ray small-angle scattering, Biochim. Biophys. Acta 1037:48.

5. V. Luzzati, A. Tardieu, and L. P. Aggerbeck, 1979, Structure of serum low-density lipoprotein I. Solution X-ray scattering study of a hyperlipidemic monkey low-density lipoprotein, J. Mol. Bioi. 131:435.

6. M. W. Baumstark, W. Welte, and W. Kreutz, 1982, A theory for the evaluation of small-angle scattering diagrams of quasi radially symmetric particles considering polydispersity and deviations from radial symmery, Acta Cr'LSL A38:835.

7. M. W. Baumstark, and W. Kreutz, 1982, Iterative Deconvolution Method for Evaluating X-Ray Small Angle Scattering Diagrams., in: "Deconvolution Reconvolution, • M. Bouchy, ed., E.N.S.I.C. - I.N.P.L., Nancy.

8. T. G. Gulik-Krzywicki, M. Yates, and L. P. Aggerbeck, 1979, Structure of serum low-density lipoprotein. II. A freeze- etching electron microscopy study, J. Mol. Bioi. 131:475.

9. F. T. Lindgren, Preparative ultracentrifugal laboratory procedures and suggestions for lipoprotein analysis, 1975, ln.: •Analysis of lipids and lipoproteins, • E. G. Perkins, ed., American Oil Chemists' Society, Champaign, Ill.

10. T. J. Knott, R. J. Pease, L. M. Powell, S. C. Wallis, S. C. Rail jr, T. L. lnnerarity, B. Blackhart, W. H. Taylor, Y. Marcel, R. Milne, D. Johnson, M. Fuller, A. J. Losis, B. J. McCarthy, R. W. Mahley, B. Levy-Wilson, and J. Scott, 1986, Complete protein sequence and identification of structural domains of human apolipoprotein B, Nature 323:734.

11. C. Y. Yang, S. H. Chen, S. H. Gianturco, W. A. Bradley, J. T. Sparrow, M. Tanimura, W. H. Li, D. A. Sparrow, H. DeLoof, M. Rosseneu, F. S. Lee, Z. W. Gu, A. M. Gotto jr, and L. Chan, 1986, Sequence, structure, receptor-binding domains and internal repeats of human apolipoprotein B-100, Nature 323:738.

12. M. W. Baumstark, W. Welte, and W. Kreutz, 1983, Electron-density determination of three high-density lipoprotein subtractions, considering polydispersity and deviations from radial symmetry, Biochim. Bioohvs. Acta 751:108.

13. R: J. Deckelbaum, G. G. Shipley, and D. M. Small, 1977, Structure and interactions of lipids in human plasma low density lipoproteins, J. Bioi. Chern. 252:744.

14. P. Laggner, G. M. Kestner, U. Rakusch, and D. Worcester, 1981, Neutron small angle scattering on selectively deuterated human plasma low density lipoproteins, J. Bioi. Chern. 256:11832.

15. G. L. Gaines jr, 1966, "Insoluble monolayers at lipid-gas interface, • lnterscience, New York.

16. H. Lecuyer, D. G. Dervichian, 1969 , J. Mol. Bioi. 45:39.

17. Y. L. Marcel, T. L. lnnerarity, C. Spilman, R. W. Matiley, A. A. Protter, and R. W. Milne, 1987, Mapping of human apolipoprotein B antigenic determinants, Arteriosclerosis 7:166.

18. W.- H. Li, M. Tanimura, C.- C. Luo, S. Datta, and L. Chan, 1988, The apolipoprotein multigene family: biosynthesis, structure, structure-function relationships, and evolution, ,!, Lipid Res. 29:245.

19. G. Schonfeld, and E. S. Krul, 1986, Immunologic approaches to lipoprotein structure, J. Lipid Res. 27:583.

20. E. G. Finer, R. Henry, R. B. Leslie, and R.N. Robertson, 1975, NMR studies of pig low- and high-density serum lipoproteins. Molecular motions and morphology, Biochim. Biophys. Acta 380:320.

21. P. L. Yeagle, R. G. Langdon, and R. B. Martin, 1977, Phospholipid-protein interactions in human low density lipoprotein detected by 31P nuclear magnetic resonance: Biochemistry 16:3487.

22. E. Herzyk, D. C. Lee, R. C. Dunn, K. R. Bruckdorfer, and D. Chapman, 1987, Changes in the secondary structure of apolipoprotein B-100 after Cu2 + -catalysed oxidation of human low-density lipoproteins monitored by Fourier transform infrared spectroscopy, Biochim. Biophys. Acta 922:145.

23. D. Osterman, R. Mora, F. J. Kezdy, E. T. Kaiser, and S. C. Meredith, 1984, A synthetic amphiphilic B-strand tridecapeptide: a model for apolipoprotein B, J. Am. Chern. Soc. 106:6845.

24. D. Atkinson, R. J. Deckelbaum, D. M. Small, and G. G. Shipley, 1977, Structure of human plasma low-density lipoproteins: Molecular organisation of the central core, Proc. Natl. Acad. Sci. USA 74:1042.

25. P. Laggner, G. Degovics, K. W. Muller, 0. Glatter, 0. Kratky, G. Kestner, and A. Holasek, 1977, Molecular packing and fluidity of lipids in human serum low densiy lipoproteins, Hoppe Seyler's Z. Physiol. Chern. 358:771.

26. R. P. Rand, and V. Luzzati, 1968, X-ray diffraction study in water of lipids extracted from human erythrocytes, Biophys. J. 8:125.

27. B. M. Craven, and G. T. De Titta, 1976, Cholesteryl myristate: structure of the crystalline and mesophases, J. Chern. Soc .. Perkin Trans. 27:814.

28. C. Tanford, 1980, "The hydrophobic effect: Formation of micelles & biological membranes, •

2nd edition, John Wiley & Sons Inc., New York.

29. P. Laggner, G. M. Kestner, G. Degovics, and D. L. Worcester, 1984, Structure of the cholesteryl ester core of human plasma low density lipoproteins: Selective deuteration and neutron small-angle scattering, Proc. Natl. Acad. Sci. USA 81:4389.

30. W. Stoffel, 0. Zierenberg, B. Tunggal, and E. Schreiber, 13C Nuclear magnetic resonance spectroscopic evidence for hydrophobic lipid-protein interactions in human high density lipoproteins, Proc. Nat. Acad. Sci. USA 71:3696.

31. G. S. Ginsburg, D. M. Small, and D. Atkinson, 1982, Microemulsions of phospholipids and cholesterol esters. Protein-free models of low density lipoprotein, J. Bioi. Chern. 257:8216.

32. A. Tardieu, L. Mateu, C. Sardet, B. Weiss, V. Luzzati, L. Aggerbeck, and A. M. Scanu, 1976, Structure of human serum lipoproteins in solution. II. Small-angle X-ray scattering study of HDL3 and LDL, J. Mol. Bioi. 101:129.

33. P. Laggner, and G. M. Kestner, 1978, Thermotropic changes in the surface structure of lipoprotein B from human-plasma low-density lipoproteins, Eur. J. Biochem. 84:227.

34. G. S. Ginsburg, M. T. Walsh, D. M. Small, and D. Atkinson, 1984, Reassembled plasma low density lipoproteins. Phospholipid-cholesterol ester-apoprotein B complexes, J. Bioi. Chern. 259:6667.

Read More

REGRESSION OF ATHEROMA AND PUTATIVE ROLE OF CETP IN CHOLESTERYL ESTER REMOVAL.

REGRESSION OF ATHEROMA AND PUTATIVE ROLE OF CETP IN CHOLESTERYL ESTER REMOVAL

Evidence for regression of atherosclerosis induced by cholesterol feeding has been provided by several investigators [1-3]. However, since the extent of atherosclerotic involvement is quite variable, quantitative evaluation of regression is difficult. We have used 3H-cholesteryl linoleyl ether (3H-CLE), a nonhydrolyzable analog of cholesteryl easter as a stable marker for the quantitation of atherosclerotic involvement [4] and evaluat­ ed the potential usefulness of 3H-CLE in the evaluation of regression of atheromatosis [5]. To that end, 20 rabbits were kept on a purina diet en­ riched with 1% cholesterol for 1 month and then on alternate weeks for an additional 2 months. The animals were randomized into two groups according to their plasma cholesterol levels and injected with autologous plasma labeled with 3H-CLE [5]. The baseline group was killed 10-12 days after injection, while the regression group was fed purina fortified with 3% cholestyramine and killed 8-11 months after injection of the 3H-CLE. We investigated the following: Will the 3H-CLE remain in the aorta during the 11-month period of regression? If 3H-CLE is retained, then the specific activity expressed as 3H-CLE/CE mass should rise with CE loss during regression; 3. Is the loss of CE during regression similar from the different parts of the aorta?

At the end of the cholesterol feeding period, the mean plasma chol­ esterol was 1298 mg/dl. The amount of labeled 3H-CLE in the aorta varied markedly among the individual rabbits, but was highly correlated (r =0.875) with the amount of aortic cholesteryl ester determined in the base- line group (Fig. 1). The results presented in Fig. 2 compare the total and esterified cholesterol in the entire aorta of the baseline (10-12 days) and regression groups (11 months). In the baseline group, the mean total cholesterol was 13.2 ± 2.1 mg/aorta and the esterified cholesterol was 7.6 ± 1.3 mg/aorta. After 11 months of the regression regimen, the total cholesterol was 9.5 ± 1.9 mg/aorta, while cholesteryl ester decreased to

3.1 ± 0.7 mg/aorta. The loss of cholesteryl ester was significant (p < 0.01). On the other hand, the amount of 3H-CLE in the regression group was not different from that seen in the baseline group (Fig. 2). The mean specific activity of 3H-CLE/CE was compared in the arch, thoracic and abdominal aorta of the baseline and regression groups (Fig. 3). As can be seen in the baseline group, the specific activity in the three regions examined was quite similar. A much higher specific activity of 3H-CLE/CE was found in the regression group, the highest being seen in the region of the aortic arch.

These results permitted us to conclude that 3H-CLE injected into cholesterol fed rabbits was retained in the aorta for up to 11 months of the regression period, while cholesteryl ester content decreased. The retention of 3H-CLE in the rabbit aorta, in face of high plasma CETP, would not favour the role of CETP in CE removal from the aorta. This could have been due to several possibilities, among them that the 3H-CLE was not accessible to the transfer protein. Indeed, in a model system in culture [6], we have shown that while 3H-CLE present in lipoproteins and bound to

extracellular matrix was accessible to CETP and could be released into the culture medium; once the lipoprotein had become ingested by a cell such as a macrophage it became inaccessible to CETP [6]. Recently, Morton [7] has presented evidence that CETP is able to remove cholesteryl ester from intact macrophages. We proposed to test the putative role of CETP in CE egress from reticuloendothelial cells in vivo in an animal model in which one can modulate plasma CETP levels by dietary means. Son and Zilversmit [8] have shown that cholesterol feeding in rabbits is accompanied by an increase in plasma CETP. We looked, therefore, for a smaller animal which would respond to cholesterol feeding with a rise of plasma CETP in analogy to the rabbit. In view of the studies of Dietschy et al. [9, 10], the hamster appeared to be a suitable model, but there were no data in the literature with respect to plasma CETP in the hamster. Therefore, we have examined hamsters for CETP activity and found measurable activity under

control dietary conditions. We were able to modulate this activity by feeding diets enriched in cholesterol and fat [11]. As seen in Table 1, the hamsters responded to a high fat-high cholesterol diet with a significant increase in CETP activity. Therefore, we decided to use hamsters to evaluate the role of CETP in cholesteryl ester efflux from cells in vivo. The approach was based on our previous findings [12] that when acetylated LDL is labeled with 3H-CLE and injected into rats, it disappears from the liver at a very slow rate. Since the rat does not have measurable CETP

under normal dietary conditions or even after feeding of high fat and cholesterol [11], a comparison of loss of 3H-CLE labeled acetylated LDL from rat and hamster liver could provide some information with respect to the role of CETP in cholesteryl ester removal from cellular elements in vivo. These experiments are now in progress and preliminary results suggest that under these experimental conditions, loss of 3H-CLE from the liver is not increased by CETP.

REFERENCES

1. D.Vesselinovitch, R.W.Wissler, K.Fisher-Dzoga, R.Hughes, and L.Dubien, Regression of atherosclerosis in rabbits. Part 1. Treatment with low-fat diet, hyperoxia and hypolipidemic agents, Atherosclerosis 19:259 (1974).

2. R.W.St.Clair, Atherosclerosis regression in animal models: Current concepts of cellular and biochemical mechanisms, Prog.Cardiovasc. Dis. 26: 109 (1983).

3. M.R.Malinow, Experimental models of atherosclerosis regression.

Atherosclerosis 48: 105 (1983).

4. Y.Stein, O.Stein, and G.Halperin, Use of 3H- holesteryl linoleyl ether for the quantitation of plasma cholesteryl ester influx into the aortic wall in hypercholesterolemic rabbits, Arteriosclerosis 2:281 (1982).

5. O.Stein, G.Hollander, Y.Dabach, G.Halperin, and Y.Stein, Use of 3H­ cholesteryl linoleyl ether as a quantitative marker for loss of cholesteryl ester during regression of cholesterol-induced aortic atheromas in rabbits, Arteriosclerosis 9:247 (1989).

6. O.Stein, G.Halperin, and Y.Stein, Cholesteryl ester efflux from extra­ cellular and cellular elements of the arterial wall. Model systems in culture with cholesteryl linoleyl ether. Arteriosclerosis 6:70 (1986).

7. R.E.Morton, Interaction of plasma-derived lipid transfer protein with macrophages in culture, J.Lipid Res. 29:1367 (1988).

8. Y.-S.C.Son and D.B.Zilversmit, Increased Lipid Transfer Activities in hyperlipidemic rabbit plasma, Arteriosclerosis, 6:345 (1986).

9. D.K.Spady, and J.M.Dietschy, Dietary saturated triacylglycerols suppress hepatic low density lipoprotein receptor activity in the hamster, Proc.Natl .Acad.Sci.USA, 82: 4526 (1985).

10. D.K.Spady, and J.M.Dietschy, Interaction of dietary cholesterol and triglycerides in the regulation of hepatic low density lipoprotein transport in the hamster, J.Clin.Invest. 81:300 (1988).

11. Y.Stein, Y.Dabach, G.Hollander, and O.Stein, Cholesteryl ester transfer activity in hamster plasma: increase by fat and chol­ esterol rich diets, Biochim.Biophys.Acta 1042: 138 (1990).

12. Y.Stein, Y.Kleinman, G.Halperin, and O.Stein, Hepatic retention and elimination of cholesteryl linoleyl ether after injection of labeled acetylated LDL or chylomicrons, Biochim.Biophys.Acta 750:300 (1983).

Read More

DIS1RIBUTION OF CHOLESTEROL WITHIN HIGH DENSITY LIPOPROTEINS FRACTIONATED BY IMMUNOAFFINITY CHROMATOGRAPHY.

DIS1RIBUTION OF CHOLESTEROL WITHIN HIGH DENSITY LIPOPROTEINS FRACTIONATED BY IMMUNOAFFINITY CHROMATOGRAPHY

INTRODUCTION

Cholesterol enjoys notoriety as a pro or anti-atherogenic lipid by virtue of the lipoprotein with which it is associated. This simplistic interpretation is most evident in the positive and negative correlations with the atherosclerotic process manifested by cholesterol associated with the most abundant lipoprotein species, respectively low density (LDL) and high density (HDL) lipoproteins 1,2. The statement requires qualification, however, when lipoprotein sub-populations are contemplated. Thus, within the HDL density spectrum, the protective influence is largely believed to reside within the ultracentrifugally­ defined lower density HDL-2 subclass 3. HDL-3 enjoys much less support as an anti-atherogenic lipoprotein particle, a somewhat unsatisfactory state of affairs when considering mechanistic explanations of the function of HDL. Notably, particles of the size of HDL-3 are the principal acceptors of cellular cholesterol, the initiating step in reverse cholesterol transport 4. In addition, they can act as a repository for lipids shed from triglyceride-rich lipoproteins, greatly facilitating the catabolic removal of these potentially atherogenic lipoprotein particles 5. Further subfractionation of lipoproteins into, hopefully, metabolically homogenous sub-populations is one approach which should yield more satisfying explanations. This is the rationale behind the studies described in the present report. HDL subClasses 2 and 3, as defined by the physico-chemical criteria of ultracentrifugation, have been further fractionated using an immunoaffinity approach 6,7 targetting the predominant HDL apolipoprotein (apo) components, apos A-I and A-II. We have examined the cholesterol distribution of such immunoaffinity-defined fractions within HDL-2 and 3 isolated from healthy male and female populations. Furthermore, the distribution has been analysed in sub-groups corresponding to the lowest and highest total HDL-cholesterol quartiles of the same populations.

MATERIALS AND METHODS

Study populations

Healthy male (n=35) and female (n=33) subjects were recruited from the university hospital and medical research centre in Geneva.

Basic clinical characteristics of these groups have been described previously 8. Average HDL-cholesterol values were 1.22±0.19mm/l for men and 1.55±0.33mm/l for women.

The populations were also segregated into quartiles based on values of total HDL-cholesterol. For the male population, quartiles 1 and IV averaged HDL-cholesterol levels of 0.99±0.09mm/l (range 0.80- 1.07mm/l) and 1.49±0.13mm/l (range 1.29-1.60mm/l). Corresponding values for female quartiles I and IV were 1.19±0.1Omm/1 (range 0.98- 1.28mm/l) and 2.03±0.26mm/l (range 1.76-2.46mm/l).

Lipoprotein fractionation

Serial fractionation of high density lipoproteins from fasting plasma by ultracentrifugation and immunoaffinity chromatography was achieved as described 8. The immunoaffinity procedure gave rise to two types of lipoprotein particle, described according to the presence of apos A-1 and A-11. Thus LpAI,AII contains both apos, whereas LpAI contains apo A-1 but no apo A-11. The particles are further defined by their subclass source ie HDL-2 or HDL-3.

Other analyses

Lipid and protein measurements and statistical analyses were performed as described previously 8,9,10

RESULTS

Fig. 1 shows the distribution of cholesterol within the subfractions in both the male and female populations. Cholesterol was principally associated with HDL3-LpAI,AII and concentrations were not significantly different between the males and females. In contrast,

highly significant differences (p<0.0001 for HDL-2 derived subfractions ; p=0.0001 for HDL3-LpAI) between populations were observed for the remaining subfractions . These differences were particularly marked for HDL2-LpAI and HDL2-LpAI,AII, being respectively 100% and 60%

higher in the female group. Of the cholesterol associated with (AI,AII), 31% was within the HDL-2 density range in men, compared to 39% for women. For (AI), the male group had 54% of associated cholesterol within HDL-2, whereas the female group had 65% within the lower density subclass. Total HDL-cholesterol (measured after phosphotungstate precipitation) correlated strongly with HDL2-LpAI and HDL2-LpAI,AII cholesterol, with coefficients of +0.66 and +0 .67 (men) and +0.83 and +0.82 (women) respectively. These contrast with

the coefficients observed for the quantitatively major fraction, HDL3- LpAI,AII cholesterol, with values of +0.53 (men) and +0.46 (women).

Cholesterol of both HDL-2 derived fractions showed negative correlations with plasma triglyceride levels. For HDL2-LpAI these were -0.53 for men and -0.36 for women: corresponding correlations for HDL2-LpAI,AII were -0.39 and -0.30 respectively.

The lipoprotein association of cholesterol was also examined in the first and fourth quartiles of both populations. For the female population, significantly lower cholesterol levels were found in quartile I in both HDL-2 and 3 density ranges (25.9±5.0 v 58.1±9.4mg/dl (p<O.OOOI) and 41.5±4.9 v 50.7±6.5mg/dl (p=0.015)). Further subfractionation (Fig. 2) showed that differences at the HDL-3 level were due to the (AI) lipoproteins (p=0.0005), there being similar concentrations of HDL3-LpAI,AII (p=0.09). Both immunoaffinity defined fractions from HDL-2 were highly significantly increased in quartile IV (LpAI, p=0.0002; LpAI,AII, p=0.0001).

When cholesterol associated with the subfractions was correlated with plasma triglyceride levels (Table 1) some interesting differences between the two quartiles emerged. Thus, for HDL-2, negative correlations were observed for LpAI for quartile I, but with LpAI,AII for quartile IV. Contrasting results w·ere also evident for HDL-3 derived immunoaffinity-derived particles (Table 1): triglycerides were correlated in a positive manner with HDL3-LpAI,AII from quartile I, but negatively with the same fraction from quartile IV.

With respect to quartiles established for the male population, qualitatively similar conclusions could be drawn from an analysis of the lipoprotein association of cholesterol. Namely, quartile I had significantly lower cholesterol concentrations within subclasses HDL-2 (14.8±3.1 v 29.4±5.7mg/dl; p=0.0007) and HDL-3 (37.7 ±6.0 v 48.1±2.6mg/dl; p=0.005). Likewise, HDL2-LpAI (p=0.002) and HDL2- LpAI,AII (p=0.0004) were also significantly lower in quartile 1 (Fig. 2). Within HDL-3, LpAI,AII cholesterol was significantly higher (p=0.004) in quartile IV, but there were no significant differences (p=0.12) in cholesterol concentrations of LpAI (Fig. 2).

As with the female population, the male quartiles differed somewhat when examined in terms of triglyceridaemia. Whereas negative correlations were observed with fractions originating from HDL-2 of both quartiles, divergent correlations were found for HDL-3 derived fractions (Table 1). Both fractions of quartile I were positively correlated with triglyceride levels, in contrast to negative correlations exhibited for the same fractions from quartile IV.

The dual fractionation procedure adopted herein provides a more precise definition of the cholesterol distribution within the high density lipoprotein spectrum. The results demonstrate the importance of both HDL2-LpAI and HDL2-LpAI,AII in determining total plasma levels of HDL-cholesterol. This is evident from the strength of the correlation coefficients when comparing total HDL-cholesterol with cholesterol in each subfraction. Moreover, male-female differences in HDL levels largely reside in the concentrations of HDL2-LpAI and HDL2-LpAI,AII (Fig. 1). Finally, within the same population, it is these two subfractions that essentially differentiate subjects in the first and fourth quartiles of HDL plasma concentrations. It suggests that physiological events giving rise to both fractions are important in determining HDL-cholesterol levels.

Although the major proportion of HDL-2 cholesterol is present in LpAI,AII in both males and females, it would appear that HDL2-LpAI is the more sensitive indicator of HDL-cholesterol levels. Thus, concentrations of HDL2-LpAI cholesterol are 2.5 to 3 fold higher in quartile IV as compared to quartile I. In contrast, levels of HDL2- LpAI,AII cholesterol in quartile IV are less than double those in quartile I. Further, in quartile I of both populations, only HDL2-LpAI cholesterol shows a strong correlation with total HDL-cholesterol. Interestingly, subjects in quartile IV also showed strong correlations between total HDL-cholesterol and HDL2-LpAI,AII cholesterol, again in both sexes. The latter is one observation that differentiates the two quartiles. Another is the correlation between triglyceridaemia and cholesterol levels. Notably, HDL3-LpAI,AII cholesterol showed a positive correlation with triglycerides in quartile I, but a negative correlation in quartile IV. Other differences were also evident (Table 2), although there was less of a parallel in the response of the male and female quartiles.

Overall, the results suggest that the combination of distinct fractionation procedures can be helpful in further defining the association of cholesterol with high density lipoproteins. It should provide information useful in determining the relative importance of the different subfractions to the anti-atherogenic effect of HDL.

ACKNOWLEDGEMENTS

The work reported herein was supported by grants 3.999-0.86 and 32.9484-88 from the Swiss National Research Fund.

REFERENCES

1. NIH Consensus Development Conference, Lowering blood cholesterol to prevent heart disease, JAMA., 253: 2080 (1985)

2. Study group, European Atherosclerosis Society, The recognition and management of hyperlipidaemia in adults: A policy statement of the European Atherosclerosis Society, Europ. Heart L 9: 571 (1988)

3. N .E. Miller, Association of high density lipoprotein subclasses with ischaemic heart disease and coronary atherosclerosis, Am. Heart J., 113: 589 (1987)

4. J .F. Oram, Effects of high density lipoprotein subfractions on cholesterol homeostasis in human fibroblasts and arterial smooth muscle cells. Arteriosclerosis, 3: 420 (1983)

5. S. Eisenberg, High density lipoprotein metabolism, J. Lipid Res.,

25: 1017 (1984)

6. P. Alaupovic, The physicochemical and immunological heterogeneity of human plasma high density lipoproteins, in: 'Clinical and metabolic aspects of high density lipoproteins,' N.E. Miller and G.J. Miller, eds., Elsevier, Amsterdam (1984)

7. M.C. Cheung and J .J. Albers, Characterisation of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-1 and A-II and particles containing A-1 but no A­ II, J. Biol. Chern., 259: 12201 (1984)

8. R.W. James and D. Pometta, Immunofractionation of high density lipoprotein subclasses 2 and 3. Similarities and differences of fractions isolated from male and female populations, Atherosclerosis., In Press

9. R.W. James, A. Proudfoot and D. Pometta, Immunoaffinity fractionation of high density lipoprotein subclasses 2 and 3 using anti-apolipoprotein A-1 and A-II immunosorbent gels, Biochim. Biophys. Acta. 1002: 292 (1989)

10. R.W. James and D. Pometta, Differences in lipoprotein subfraction composition and distribution between diabetic patients and controls. A study in male, type I (insulin­ dependent) diabetes, Diabetes. In Press

Read More

LIPOPROTEIN A-I CONTAINING PARTICLES

LIPOPROTEIN A-I CONTAINING PARTICLES

INTRODUCTION

Many epidemiological studies have indicated that the plasma level of high density lipoproteins (HDL) is inversely correlated with the risk for coronary artery disease1. It has been hypothesized that HDL exerts this protective effect by the "reverse" transport of excess cholesterol from peripheral tissues to the liver2.

Nevertheless, in spite of its role in anti-atherogenesis, the true mechanisms of HDL uptake of peripheral cholesterol and subsequent delivery to the liver are still under investigation.

Conventionally HDL are isolated by ultracentrifugation in the density range of 1.063 to 1. 1 g/ml. HDL represents a heterogenous population of particles which differ in size, in lipid and protein composition and overall in their metabolic functions.

Although nearly all the apoproteins that have been characterized thus far can be found in HDL in variable proportions, apo A-I and apo together comprise 85-90% of the total HDL protein.

Ultracentrifugation has been an invaluable tool for subfractionation of lipoprotein particles but it has been shown that this procedure alters the structure and composition of the particles3.

we have been using immunological procedures to isolate the different particles of the HDL fraction. Particles containing apo A-I and apo A-II (LpA-I:A-II) and particles that do not contain apo A-II (LpA-I).

Studies published by our group in collaboration with Ailhaud's group have shown that on long-term exposure to LpA-I and LpA-I:A-II particles ; only the LpA-I particles were able to promote cholesterol efflux from cholesterol preloaded, differentiated OB 1771 adipose cells4.

More recently5 it has been shown that particles containing apo A-IV are equally effective in producing cholesterol efflux.

Taking into account these results we decided to further study the different HDL particles ; to do so we prepared directly from whole plasma LpA-I and LpA-I:A-II particles and simultaneously we isolated from the same plasma sample, the particles containing apo A-IV (LpA-IV) and the particles containing apo A-I, A-IV and A-II (LpA-I:A-IV:A-II).

The present studies were undertaken with two major goals :

- to define the lipid and protein composition of the different particles ;

- to correlate their composition to their ability to promote the cholesterol efflux from adipocytes in culture.

In order to understand the intracellular IOOchanism of cholesterol efflux and to appreciate the different roles of apo A-I and apo A-II, we decided to test the PKC involvement in this phenomenon.

It was recently described6 that binding of HDL3 to 3H-phosphatidyl­ choline (PC) labelled platelets stimulated a transient biphasic increase in diacylglycerol (DAG). We have analysed this stimulatory effect using HDL3 and proteoliposomes containing apo A-I or A-II on incubation with the adipose cells 08 1771.

MATERIAL AND METHODS

The study was carried out on the plasma of five normolipidemic male subjects ; the blood was drawn into tubes containing EDTA and a mixture of protease inhibitors. The plasma was promptly separated by low-speed centrifugation at 4°C and immediately used for isolation of the parti­ cles. All manipulations were performed at 4°C.

The particles were prepared by immunoaffinity chromatography as outlined before1 ,2. On each fraction, we determined :the lipid composi­ tion by enzymatic methods ; the apoprotein composition by ELISA.

We measured the concentration of the two major phospholipids phosphatidylcholine and shingomyelin isolated by thin layer chromato­ graphy?. We estimated the proportion of the molecular species of fatty acids on the lipid extract purified by thin layer chromatography. The fatty acids I).Ydrolyzed and methylated were measured on gas liquid chromatography<>.

The activity of the lecithin cholesterol acyl transferase (LCAT) was measured by the proteoliposome method of Chen and Albers9. Cholesterol efflux was determined on differentiated 08 1771 cells preloaded with 3H-cholesteryl ester LDL prepared by the method of Craig et al10.

3H-diacylglycerol was measured after separation on thin layer chromatography. The apo A-I and apo A-II containing liposomes were prepared by the cholate dialysis procedure9, molar ratio DMPC to protein 150:1.

Quantitation of apo A containing lipoprotein particles

In order to quantify LpA-I and LpA-I:A-II, two tests have been developed. TO directly determine LpA-I:A-II, we have used an enzyme­ linked differential antibody bnmunosorbent assay11. TO directly determine LpA-I we have developed a simpler procedure using differential electro­ immunoassay12. By using a large excess of anti A-II, LpA-I:A-II particles are retained in the first peak and LpA-I migrates as a second peak. A monoclonal anti A-I labelled with peroxidase revealed the two peaks while a monoclonal anti A-II revealed only one peak.

RESULTS

Figure 1 shows the representation of the composition by weight percentage of the different particles.

The four types of particles have about the same proportion of protein, but they show significant differences in cholesterol and trigly­ ceride content. LpA-I and LpA-I:A-II particles contained more cholesterol (11% of the total mass) than LpA-IV and LpA-I:A-IV:A-II which contain 6%, conversely the particles containing apo A-IV have more triglycerides (12%).

Apolipoprotein analysis showed that LpA-I and LpA-IV contain a single apolipoprotein, 97 and 98.6% respectively, LpA-I:A-II contain 53% of apo A-I and 45% of apo A-II. The LpA-I:A-IV:A-II contain 65% of apo A-I, 18% of apo A-IV and 14% of apo A-II.

We determined the proportion of the different molecular species of fatty acids, the most striking results were found in the phospholipid fractions. The LpA-IV particles contain a high proportion of saturated fatty acids (76%) significatively different from LpA-I (58%) and LpA-I:A-II (47%).

Figure 2 shows the results of the determination of the activity of LCAT. Taking the value for the LpA-I particle as one hundred percent activity, we found that the LpA-IV particles have the most activity, followed by LpA-IV:A-I:A-II and the LpA-I : the LpA-I:A-II particles have very little LCAT activity.

The phospholipid analysis revealed differences among the particles in the type of phospholipid constituents. The phosphatidylcholine/sphin­ gomyelin ratios were 3.9, 5.3 for LpA-I, LpA-I:A-II respectively and around 1 for the particles containing apo A-IV.

Incubation of 3a-PC prelabelled adipose cells in the presence of HDL3 or liposome containing apo A-I results in 3a-DAG production with a maximum at 5 min. On the other hand, tetranitromethane modified HDL3 TNM-HDL3) which is not recognized by the HDL receptor, did not induce C breakdown and DAG release. Moreover despite their ability to be effective competitors for apo A-I binding sites, apo A-II containing liposomes were ineffective in stimulating DAG production by phospholipase activation. It seems likewise that only the binding of apo A-I to cell surface receptors is able to promote DAG generation (Figure 4).

The presence of phorbol esters in the incubation medium enhanced the cholesterol efflux efficiency in LDL-cholesterol loaded 0817 adipose cells (Figure 5). This data strongly suggests that PC-breakdown, DAG production and protein kinase C activation are involved in the cholesterol efflux.

DISCUSSICN

Our results show that factors other than the lipid composition of the particles studied are the major determinant of the ability to promote cholesterol efflux.

The compositional data of LpA-I and the two apo A-IV containing particles reveals that these lipoproteins are very different, but on incubation with adipocytes they are equally effective in facilitating the efflux of cholesterol. On the other hand LpA-I:A-II are very similar in composition to LpA-I but are unable to promote cholesterol efflux. These results confirm the antagonist role proposed for apo A-II.

This antagonist role of apo A-II is demonstrated in our studies on the production of DAG and correlates very well with the results obtained in epidemiological studies.

We have shown recently that the lower apo A-I levels for patients with significant coronary artery disease were reflecting, in fact, a decrease in LpA-I particles13. Other data obtained in octogenarians14 supports, also, the view that LpA-I might represent the "anti-athero­ genic" fraction of HDL.

Apo A-I in fenales and apo A-II in males were lower in octogenarians while apo A-I in males and apo A-II in females were similar in octogenarian and control subjects. However, LpA-I was significantly elevated in octogenarian males and females by comparison with younger control subjects.

Recently it has been observed that the level of LpA-I in children whose patients suffer from premature coronary heart disease (CHD) was lower than that of a cnntrol group without any familial history of CHD15.

The clinical interest in the quantification of LpA-I and LpA-I:A-II is illustrated by the effect of moderate alcohol consumption on HDL16. We have measured HDL cholesterol, apo A-I, apo A-II, LpA-I and LpA-I:A-II in plasma from three hundred and fifty male subjects matched for age and clinical data and divided into five groups according to their alcohol consumption. Results confirm that alcohol consumption increases LpA-I:A-II and decreases LpA-I. These opposite variations are dose dependent and the differences are highly significant. Our findings indicate that an increase in HDL cholesterol can reflect an increase in LpA-I :A-II and a decrease in LpA-I. r.bre011er, assuming that LpA-I is the "anti-atherogenic" subfraction, alcohol " <A::mld oot have any anti-athero­ genic effect through the increase in HDL.

REFERENCES

1. T. Gordon, W.P. Castelli, M.C. Hjortland, W.B. Kannel, T.R. Dawber, High density lipoprotein as protective factor against coronary heart disease, Am. J. Med. 62:707 (1977).

2. J. A. Glomset, The plasma lecithin-cholesterol acyl transferase reaction, J. Lipid Res. 9:155 (1968).

3. G. R. Castro, and C.F. Fielding, Evidence for the distribution of apolipoprotein E between lipoprotein classes in human normo­ cholesterolemic plasma and for the origin of unassociated apolipo­ protein E (LpE), J. Lipid Res. 25:58 (1984).

4. R. Barbaras, P. Puchois, J.C. Fruchart, and G. Ailhaud, Cholesterol efflux fran cultured adipose cells is mediated by LpA-I particles but not by LpA-I:A-II particles, Biochem. Biophys. Res. Oommun. 142:63 (1987).

5. A. Steinmetz, R. Barbaras, N. Ghalim, V. Clavey, J .C. Fruchart, and G. Ailhaud, Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from  adipose cells, J. Biol. Chern. 265:7859 (1990).

6. C. Delbart, N. Theret, G. Ailhaud, J.C. Fruchart, Phosphatidylcholine breakdown during receptor binding of HDL3, 62nd Scientific Sessions of American Heart Association, New-Orleans, 13-16 novembre 1989, Circulation, 80/4, supplement II, II-487, abstract 1938 (1989).

7. A. Nouvelot, G. Sezille, P. Dewailly, J.C. Fruchart, Chranatographie monodimensionnelle des lipides polaires sur couche mince de gel de silice en gradient discontinu d'humidite, Revue Francaise des Cbrps Gras 7:251 (1977).

8. L. Hagenfelt, A gas chromatography method for the determination on individual free fatty acids in plasma, Clin. Chim. Acta 13:266 (1966).

9. C. H. Chen and J.J. Albers, Characterization of proteoliposomes containing apolipoprotein A-I :a new substrate for the measurement of lecithin cholesterol acyl transferase activity, J. Lipid Res. 23:680 (1982).

10. J. F. Craig, D.P. Via, B.C. Sharril, L.A. Labar, W.W. Mantulin, A.M. Gotto, and L. Smith, Incorporation of defined cholesteryl esters into lipoproteins using cholesterol ester-rich microemulsions, J. Biol. Chern. 257:330 (1982).

11. E. Koren, P. Puchois, P. Alaupovic, J. Fesmire, A. Kandoussi, J.C. Fruchart, Quantification of two different types of apolipoprotein A-I containing lipoprotein particles in plasma by enzyme linked differential antibody immunosorbent assay, Clin. Chern. 33:38 (1987).

12. H. J. Parra, H. Mezdour, N. Ghalim, J.M. Bard, J.C. Fruchart, Differential electroimmunoassay on ready-to-use plates for human LpA-I lipoprotein particles, Clin. Chern. in press (1990).

13. P. Puchois, A. Kandoussi, P. Fievet, J.L. Fourrier, M. Bertrand, E. Koren, J .C. Fruchart, Apolipoprotein A-I containing lipoproteins in coronary artery disease, Atherosclerosis 68:35 (1987).

14. G. Luc, J.M. Bard, s. Lussier-Cacan, H.J. Parra, J.C. Fruchart, J. Davignon, High density lipoprotein particles in octogenarians, Metal:x>lism in press (1990)•

15. P. Amouyel, H.J. Parra, D. Boute, J.M. Bard, D. Isorez, D. Barbier,

J.C. Fruchart, G. Zylberberg, Inlfuence des antecedents cardio­

vasculaires familiaux sur le profil lipoproteique d'une population issue de la metropole lilloise, XVeme Reunion de l'ADELF, Bordeaux, 12-15 septembre 1989.

16. N. Ghalim, P. Puchois, G. Zylberberg, P. Fievet, C. Demarquilly, J.C. Fruchart, Effect of alcohol intake on human apolipoproteins A-I containing lipoproteins subfractions, Arch. Int. Med. in press (1990).

Read More

BINDING OF APOLIPOPROTEINS A TO ADIPOSE CELLS : ROLE OF RECEPTOR SITES IN CHOLESTEROL EFFLUX AND PURIFICATION OF BINDING PROTEIN(S).

BINDING OF APOLIPOPROTEINS A TO ADIPOSE CELLS : ROLE OF RECEPTOR SITES IN CHOLESTEROL EFFLUX AND PURIFICATION OF BINDING PROTEIN(S)

Epidemiological studies have shown a relationship between low concentrations of high density lipoprotein (HDL) cholesterol and the incidence risk of cardiovascular diseases 1'2 . Recent pharmacological studies3 have clearly demonstrated the protective role of HDL and their involvment in reverse cholesterol transport in vi vo4, 5. In that respect apo E-free HDL has been long known to bind to a variety of cells and to promote cholesterol efflux6. Among peripheral tissues, adipose tissue is recognized both in man and rodents for its ability to accumulate, store and, when needed, mobilize a large pool of unesterified cholesterol?' 8. Thus adipose cells represent a cell type suitable to study the first step in reverse cholesterol transport, i.e. cholesterol efflux. Unfortunately adipocytes isolated from adipose tissue loose their viability within a few hours, preventing the analysis of middle-term and long-term responses. During the last decade have been established in our laboratory preadipocyte cell lines from adipose tissue of genetically­ obese ob/ob mice9 and their lean counterpartlO. The validity of these cellular models is supported by i) the biochemical properties of differentiated cells which are similar, if not identical, to those of adipocytes isolated from fat tissue and ii) the ability of undifferentiated cells to differentiate in vivo within a few weeks into fully mature fat cells after their injection into athymic mice,· under conditions where these cells could be unambiguously demonstrated not to be fat cells originating from the host animalll. Most of the studies, if not otherwise stated, were performed with Obl771 cells, a subclone of Obl7 cells established from ob/ob mice12, 13.

CHARACTERIZATION OF LDL AND HDL BINDING SITES AND CHOLESTEROL FLUX/EFFLUX IN OB1771 CELLS

The binding of human apo AI, apo AI! and apo AIV to mouse adipose cells and the study of their functional properties were made feasible owing to extensive homologies existing between rat, mouse and human apolipoproteins 14-16. In addition important homologies do exist between rat (and likely mouse) and human apo B, including the consensus region of apo B and apo E which should be involved in the binding to the apo B,E receptor17-20. The binding of 125r-LDL was competitively inhibited by LDL > VLDL > total HDL ; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apo E-free HDL were not competitors. In contrast, the binding of 125r-apo E-free HDL was competitively inhibited by apo E­ free HDL > total HDL and that of 125r-HDL3 by mouse HDL. Thus mouse adipose cells possess distinct apo B,E and apo E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. Further studies of apo E-free HDL binding sites revealed that the binding of 125r-HDL3 was competitively inhibited by apo AI/dimyristoylphosphatidyl­

choline complexes > mouse HDL > HDL3. To explore the possibility that apo AI, apo AI! and apo AIV bind to the same sites, competition experiments were performed in which binding of either of the three apolipoproteins was performed in the presence of the two other unlabeled apolipoproteins. The results suggest strongly that apo AI, apo AI! and apo AIV bind to common receptor sites21. This hypothesis is supported by the finding that a highly purified protein from Ob1771 cells remains able to bind the three apolipoproteins (ref.22 and vide infra) . The observation that - 1 mol of apo AI! is bound per 2 mol of apo AI or apo AIV (Table I) could be explained if one assumes that the receptor site is a dimeric structure (see Fig.l) which recognizes each monomer of dimeric apo AI! in the same way that it recognizes two molecules of monomeric apo AIV or apo AI. In any event, the stoichiometry of apo AI (or apo AIV) versus apo AI! binding has been consistently observed in intact cells and in homogenates after detergent solubilization as well as after extensive purification of binding proteins of 80 and 92 kDa (ref.22 and vide infra).

During the course of these studies, it was observed that the endogenous cholesterol synthesis was nil12 but the most striking observation was the fact that long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apo B,E receptor sites and that of apo E-free HDL receptor sites. In other words, the "buffering" capacity of adipose cells seems limited with respect to the regulation of cholesterol content. This lack of cholesterol homeostasis would explain the rather unique ability of adipose tissue in vivo to accumulate and mobilize a large pool of unesterified cholesterol 7,B. Since differentiated Ob1771 cells were able to find, internalize and degrade LDL12, it appeared that adipose cells did not show an efficient cholesterol homeostasis in vitro and thus, as a first prediction, should accumulate cholesterol. The second prediction was that cholesterol­ preloaded cells should mobilize cholesterol when exposed to appropriate lipoprotein particles. Both predictions were fullfilled. As shown in Table I, it is of interest to note

that comparisons between apparent Kct values for binding of apo AI and apo AIV and the ECso values for cholesterol efflux are within the same range of concentrations2l-2 4 . These results suggest that specific binding to these distinct sites was a pre-requisite to cholesterol accumulation and subsequently to cholesterol mobilization. It is also of interest to note that cholesterol accumulation was taking place in the presence of LDL under the form of unesterified cholesterol only, in agreement with the fact that, at least in rat fat tissue, the majority (75-95%) of adipocyte cholesterol is unesterified and associated with central oil (triacylglycerol) droplet7,8.

RELATIONSHIPS IN ADIPOSE CELLS BETWEEN RECEPTOR SITES FOR APO AI, APO AI I AND PROMOTION OF CHOLESTEROL EFFLUX

In order to establish whether receptor sites for HDL were indeed required for the promotion of cholesterol efflux, use was made of Obl7 cells in which have been induced genetically defined alterations of the growth control mechanism by transferring cloned oncogenes25. Obl 7PY cells were obtained after transfer of the complete early region of polyoma virus whereas Obl 7MT cells were obtained after transfer of a modified genome encoding only the middle T protein. The broad range of phenotypes thus generated has also offered us unique opportunities to study cholesterol efflux in adipose cells as cells of the Obl7MT18 subclone had a 3-fold higher number of HDL receptor sites than cells of the parental Obl7 clone whereas growing Obl 7PY cells did not have any detectable sites (see below) .

As a pre-requisite to study the critical role, if any, of HDL receptor sites and to undertake their purification, conditions for their visualisation were searched and found using bivalent cross-linking reagent discuccimidyl suberate at 4°C in the presence of apo AI-containing liposomes and intact Obl771 cells or derived crude membranes26. The existence of two specific cell-surface protein components of Mr 100,000 and 130,000 was demonstrated. It is possible that two different proteins of Mr -70,000 and -100,000 able to bind one molecule of proteins of apo AI of Mr 28,000 are indeed present in adipose cells. Alternatively the possibility of either a single glycoprotein able to bind one molecule of apo AI but having different degrees of glycosylation, or a single glycoprotein able to cross-link one or two molecules of apo AI, could be envisionned. The key observation in our study on the role of HDL receptor sites in the promotion of cholesterol efflux was that no binding of HDL3, apo AI, apo AI! or apo AIV was observed in growing Obl7PY cells and derived crude membranes, in contrast to growing or growth­ arrested Obl771 cells (see Table I) or Obl7MT18 cells (not shown). After thymidine block, growth-arrested Obl7PY cells became able to recover in parallel binding activities for HDL3, apo AI, apo AI! and apo AIV. The possibility that this recovery was an event common to various cell surface receptors is not very likely since apo B,E and transferrin receptor sites were both present in growing and growth-arrested Obl 7PY cells as well as in Obl 771 cells. The recovery of HDL receptor sites in growth-arrested Obl 7PY cells was rapid (16 h) and prevented in actinomycin D- or cycloheximide­ treated cells, adding further support to the conclusion that these sites are protein component (s) . When experiments of cholesterol efflux were performed, the results showed that, after cholesterol accumulation taking place in the presence of LDL cholesterol, subsequent exposure to HDL3 or apo AI (but again not apo AI!) led to cholesterol efflux from Obl771 cells and growth- arrested Obl7PY cells but not from growing Obl7PY cells26. Thus it appears that the presence of high-affinity receptor sites for HDL in intact adipose cells is required for the promotion of cholesterol efflux. The existence of cell surface binding sites which recognize apolipoproteins A is supported by recent experiments showing that the binding of apo AI/DMPC complexes to intact Obl771 cells was followed within 1-2 minutes by the formation of diacylglycerol from phosphatidylcholine as substrate ; it is of interest that apo AII/DMPC complexes were inactive in that respect, supporting the view that apo AI! was playing the role of an antagonist21,24,27,28. Altogether, these observations led us to attempt in purifying apo A binding proteins by using Obl7MT18 cells, a transformed cell line enriched 3-fold in apo A binding sites as compared to the parental Obl7 cells.

The purification scheme is shown in Figure 2. An 1,400- fold purification over the starting crude homogenate was achieved22. The purified material contained two proteins that were both able to bind apolipoproteins AI, AI! and AIV but not LDL. 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 high-pressure liquid chromatography on TSKG 3000 SW column. Rabbit polyclonal antibodies directed against the purified material revealed two protein bands of 80 and 92 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting. These bands were undetectable in growing Obl7PY cells previously shown not to bind the various apo As or not to undergo cholesterol efflux, whereas they were conspicuous in growth-arrested Obl7PY cells which recovered these properties. It is of utmost importance to recall that these binding sites are present at the cell surface of intact cells but more than 90% of apo A and apo B,E (LDL) binding sites were shown to be present intracellularlyl2. This situation is similar to that observed in skin fibroblasts where a large proportion of LDL binding activity is also present within the cells. Therefore both cell surface and intracellular binding sites were purified in the present study, but it must be recalled that the affinities of these binding sites for their ligands were very similar in intact Obl7 cells and derived crude membranesl2 and that the binding parameters were found to be very similar for intact Obl7MT18 cells and the fraction purified from these cells by DEAE­ Trisacryl chromatography (Fig.2). Thus it is assumed that cell surface binding sites and intracellular binding sites are identical and that a receptor recognizing apolipoprotein A has been purified. Figure 1 summarizes our working hypothesis : it is possible that the functional apolipoprotein A receptor, required for cholesterol efflux but not for

binding activity (Fig.1), is a dimer of two single polypeptide chains. This dimeric structure would be able to recognize either one mole of the dimeric apo AI! or two moles of the monomeric apo AI! or AIV. If so, we envision that the binding of one molecule of apo AI (or apo AIV), but not that of apo AII, might induce a conformational change allowing the binding of a second molecule of apo AI (or AIV) and the formation of an activated receptor. It is suggested that, within the apo AI (or apo AIV)/DMPC complexes, the apolipoprotein plays the role of a ligand triggering the PKC pathway (cholesterol translocation to the cell surface) whereas the liposomal structure per se plays the role of a cholesterol acceptor (cholesterol efflux from the cell surface) . Recent experiments indicate that it is indeed the case and that a distinction between both events can be made experimentaly (N. Theret-Bidoui et al., unpublished work).

ACKNOWLEDGEMENTS

The authors wish to thank Miss V. Boivin and Mrs. B. Barhanin for expert technical help, Dr. J. Barhanin for helpful advice in cross-link experiments and Mrs.G. Oillaux for expert secretarial assistance. This work was supported by the "Centre National de la Recherche Scientifique" (CNRS UPR 7300), by the "Fondation pour la Recherche Medicale Francaise" (Nice) and by "Institut Pasteur" (Lille).

REFERENCES

1. T. Gordon, W.P. Castelli, M. C. Hjortland, W. B. Kannel and T. R. Dawber, High density lipoprotein as a protective factor agains coronary heart disease: the

Frammingham Study, Am· J. Med, 62:707 (1977).

2. N. E. Miller, 0. H. Forde, D. S. Thelle and 0. D. Mjos, The Tromso Heart Study: high-density lipoprotein and coronary heart disease: a prospective cas control study, Lancet 1:965 (1977).

3. M. Heikki Frick et al., Helsinki heart study: Primary­ prevention trial with gemfibrozil in middle-aged men with dyslipidiema: safety or treatment, changes in risk factors, and incidence of coronary heart disease, N. Engl. J Med. 317:1237 (1987).

4. N. E. Miller, A. La Ville and D. Crook, Direct evidence that reverse cholesterol transport is mediated by high­ density lipoprotein in rabbit, Nature 314:109 (1985).

5. Th. J.C. van Berkel, H. F. Bakkeren, F. Kuipers and R. J. Vonk : Proceedings of Xth International Symposium on Drugs Affecting Metabolism, p.47 (1989).

6. A. R. Tall and D. M. Small, Body cholesterol removal: role of plasma high-density proteins, Adv. Lipid Res. 17:1 (1980).

7. R. K. Krause and A. D. Hartman, Adipose tissue and cholesterol metabolism, J. LiPid Res. 25:97 (1984).

8. A. Angel and B. Fang, Lipoprotein interactions and cholesterol metabolism in human fat cells, in: "The Adipocyte and Obesity: Cellular and Molecular Mechanisms", A. Angel, C. H. Hollenberg and D. A. K. Roncari, eds., Raven Press, New York (1983).

9. R. Negrel, P. Grimaldi and G. Ailhaud, Establishment of preadipocyte clonal line from epididymal fat pad ob/ob mouse that responds to insulin and to lipolytic hormones, Proc. Natl. Acad. Sci. USA 75:6054 (1978).

10. C. Forest, A. Doglio, L. Casteilla, D. Ricquier and G. Ailhaud, Expression of the mitochondrial uncoupling protein in brown adipocytes. Absence in brown preadipocytes and BFC-1 cells. Modulation by isoproterenol in adipocytes, Exp. Cell Res. 168:233 (1987).

11. D. Gaillard, P. Poli and R. Negrel, Characterization of ouabain-resistant mutants of the preadipocyte Obl 7 clonal line. Adipose conversion in vitro and in vivo, Exp Cell Res. 156:513 (1985).

12. R. Barbaras, P. Grimaldi, R. Negrel, and G. Ailhaud, Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells, Biochim. Biophys. Acta 845:492 (1985).

13. R. Barbaras, P. Grimaldi, R. Negrel, and G. Ailhaud, Characterization of high-density lipoprotein binding and cholesterol efflux in cultured mouse adipose cells, Biochim. BioPh Acta 888:143 (1986).

14. P. Forgez, M. J. Chapman, S. C. Rall Jr. and M. C. Camus, The lipid transport system in the mouse, Mus musculus: isolation and characterization of apolipoproteins B, AI, AII, and CIII, J. Lipid Res. 25:954 (1984).

15. C. G. Miller, T. D. Lee, R. C. LeBoeuf and J. E. Shively, Primary structure of apolipoprotein AI! from inbred mouse strain BALB/c, J. Lipid Res. 28:311 (1987)

16. S. C. Williams, S. M. Bruckheimer, A. J. Lusis, R. C. LeBoeuf and A. J. Kinniburgh, Mouse apolipoprotein AIV gene: nucleotide sequence and induction by a high-lipid diet, Mol. Cell. Bjol. 6:3807

(1986).

17. T. L. Innerarity, E. J. Friedlander, S. C. Rall, K. H. Weisgarber and R. W. Mahley, The receptor-binding domain of human apolipoprotein E, J. Biol. Chern.

258:12341 (1983).

18. T. J. Knott, R. J. Pease, L. M. Powell, S. C. Wallis, S. C. Rall Jr., T. L. Innerarity, B. Blankhart, W. H. Taylor, Y. Marcel, R. W. Mahley, B. Levy-Wilson and J. Scott, Complete protein sequence and identification of structural domains of human apolipoprotein B, Nature 323:734 (1986).

19. C. Y. Yang, S. H. Chen, S. H. Gianturco, W. A. Bradley, J. T. Sparrow, M. Tanimura, W. K. Li, D. A. Sparrow, H. DeLoof, M. Rosseneu, F. S. Lee, Z. W. Gu, A. M. Gotto Jr. and L. Chan, Sequence, structure, receptor binding domains and internal repeats of human apolipoprotein B-100, Nature 323:738 (1986).

20. A. J. Lusis, R. West, M. Mehrabian, M. A. Reuben, R. C. LeBoeuf, J. S. Kaptein, D. F. Johnson, V. N. Schumaker, M. P. Yuhasz, M. C. Schatz and J. Elovson, Cloning and expression of apolipoprotein B, the major protein of low and very low density lipoproteins, Natl. Acad. Sci. USA 82:4597 (1985).

21. A. Steinmetz, R. Barbaras, N. Ghalim, V. Clavey, J.C. Fruchart, and G. Ailhaud, Human apolipoprotein AIV binds to apolipoprotein AI/AI! receptor sites and promotes cholesterol efflux from adipose cells, J. Biol. Chern. 265:7859 (1990)

22. R. Barbaras, P. Puchois, J.C. Fruchart, A. Pradines­ Figueres and G. Ailhaud, Purification of the apolipoprotein A receptor from mouse adipose cells, Biochem. J., in press.

23. R. Barbaras, P. Puchois, J.C. Fruchart and G. Ailhaud, Cholesterol efflux from cultured adipose cells is mediated by LpAI particles and not by Lpai :AI! particles, Biochem. Bjophys Res. Commun. 142:63 (1987) .

24. R. Barbaras, P. Puchois, P. Grimaldi, A. Barkia, J.C. Fruchart and G. Ailhaud, .in.: "Eicosanoids, Apolipoproteins, Lipoprotein Particles, and Atherosclerosis", C. L. Malmendier and P. Alaupovic, eds., Plenum Press, New York (1988).

25. P. Grimaldi, D. Czerucka, M. Rassoulzadegan, F. Cuzin and G. Ailhaud, Obl 7 cells transformed by middle-T-only gene of polyoma virus differentiate in vitro and in vivo into adipose cells, Proc Natl Acad. Sci. USA 81:5440 (1984).

26. R. Barbaras, P. Puchois, P. Grimaldi, A. Barkia, J.C. Fruchart and G. Ailhaud, Relationship in adipose cells between the presence of receptor sites for high density lipoproteins and the promotion of reverse cholesterol transport, Biochem. Biophys. Res. Commun.

149:545 (1987).

27. C. Delbart, N. Theret, G. Ailhaud, J.C. Fruchart, Phosphatidylcholine breakdown during receptor binding of HDL3, Circulation 80:487 (1989).

28. A. Barkia, P. Puchois, N. Ghalim, R. Barbaras, G. Ailhaud and J.C. Fruchart, Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells, submitted.

Read More