Arherosclerosis, 87 (1991) 135-146 A 1991 Elsevier Scientific Publishers ADONIS 0021915091000921

ATHERO

135 Ireland,

Ltd. 0021-9150/91/$03.50

04612

Differential role of apolipoprotein A *-containing particles in cholesterol efflux from adipose cells Ahmed Barkia ‘, Pascal Puchois ‘, Nordine Ghalim I, Gerard Torpier ‘, Ronald Barbaras 2,3, GCrard Ailhaud 2 and Jean-Charles Fruchart ’ ’Instirur Pasteur, SERLIA & INSERM U-325, I rue du Proj: Calmette, 59019 Lille CPdex (France), .’Centre de Biochimie (CNRS UPR 7300). Faculte des Sciences, Parr Valrose, 06034 Nice CPdex (France), and J Laborarorres Fournier, 50 rue de Dijon, 21 I21 Daix (France) (Received 7 June, 1990) (Revised, received 9 November, 1990) (Accepted 29 November, 1990)

Summary

Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo A ,, (LpA , ) and particles containing apo A I and apo A ,, (LpA , : A ,r) isolated from native human plasma, and from HDL, or HDL,, showed that only LpA, 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 K, values between 10 and 25 pgg/ml). During this long-term exposure, LpA, : An demonstrated a concentration-dependent inhibition (lo-60 pg/ml) of LpA *-mediated cholesterol efflux from adipose cells under conditions where LpA, : A,, did not deliver cholesterol to the cells and where no net change in the distribution of apo A, between LpA, and LpA, : A,, was observed. The antagonizing and modulating role of LpA , : A ,, in preventing cholesterol efflux mediated by LpA, appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo A, in LpA, particles and apo A,, in LpA, : An 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.

Key words:

LpA ,; LpA , : At,; Cholesterol

efflux

Correspondence IO: Dr. G. Ailhaud, Centre Biochimie (CNRS UPR7300), Part Valrose, F-06034 Nice, France. Telex UNINICE 970 281 F; Fax: (93) 52 99 17.

Abbreuiations: HDL, high-density lipoprotein; LDL, lowdensity lipoprotein; LCAT, 1ecithin:cholesterol acyltransferase: CETP, cholesterol ester transfer protein; SDS, sodium dodecyl sulfate: PAGE, polyacrylamide gel electrophoresis.

136 Introduction Several studies have reported that high-density lipoproteins (HDL) can promote both a bidirectional flux of cholesterol and a net unidirectional cholesterol efflux in a variety of cells (1,2). In adipose tissue, 90% of tissue cholesterol was found in adipocytes and to a very large extent (75-90%) as unesterified cholesterol [3]. This tissue is able to store and, when needed, to mobilize a large pool of unesterified cholesterol. Cultured mouse adipose cells, which possess both distinct apo B, E [4] and cell surface receptor sites [4,5] recognizing apo A ,, apo An [4,5] and apo A ,v [6] are unable to synthesize cholesterol, do not exhibit efficient cholesterol homeostasis and can, as observed in vivo for adipocytes, accumulate or mobilize unesterified cholesterol (5). As previously shown, long-term exposure of 0b1771 adipose cells to increasing concentrations of low density lipoproteins (LDL) leads to an increased accumulation of cellular unesterified cholesterol but cholesterol esters remain undetectable [5]. The capacity of adipose cells to regulate their cholesterol content is therefore limited. As a consequence, cholesterol loading via LDL cholesterol mimics the physiological situation and cultured adipose cells seem to be a suitable model of peripheral cells to study the first step in reverse cholesterol transport, i.e. cholesterol efflux. The process of cholesterol accumulation is reversible and saturable: cholesterol efflux takes place as a function of time and concentration by exposure of cholesterol-preloaded cells to HDL, or apo A,containing liposomes whereas apo An-containing liposomes do not promote this efflux [5]. The use of a heterologous system, i.e. human (apo)lipoproteins and mouse adipose cells, is valid in light of several observations made in 0b1771 cells (i) mouse LDL compete with human LDL for binding to apo B, E receptor sites [4]; (ii) mouse HDL (containing mainly apo A, and apo An) compete with human HDL, for the binding to apo A,, A,, receptor sites [4,5]; (iii) mouse apo A, and apo A,, resemble their human counterparts in amino acid composition [7,8] and, moreover, aminoacid sequence analysis demonstrates strong homology for apo A, [7] or apo An [8] between human and mouse. The ‘agonist’ role of apo A,

and the ‘antagonist’ role of apo A,, in cholesterol efflux, have been demonstrated in the two populations of apo A,-containing lipoprotein particles present in plasma: those that also contain apo A,, (LpA, :A,,) and those that do not (LpA,), and which together represent the bulk of the particles present in the HDL fraction; LpA, but not efflux from choLpA, : An promotes cholesterol lesterol-preloaded 0b1771 cells [9]. The present study was undertaken to compare the binding of LpA, and LpA, : An to the cell surface receptor sites of Ob1771 cells, and to determine whether apo E-free LpA, and apo E-free LpA, : A ,, particles isolated from native plasma, and from HDL, and HDL, fractions (which represent lipoprotein particles of different densities) behave similarly. The possible modulation by LpA, : An of the LpA,-mediated cholesterol efflux was also was investigated. Materials and methods Materials Culture media and bovine serum were from Flow Laboratories (Bethesda, U.S.A.). Na”‘I and [ 3H]cholesterol oleate were obtained from Amersham International (Buckinghamshire, U.K.). All other products were purchased from Sigma Chemical Co. (St. Louis, U.S.A.). Cells 0b1771 cells (a subclone of 0b17 cells) were plated at 2.5 X lo3 cells per cm2 in multi-well plates (Limbro) or 60-mm diameter (Falcon). Cells were grown in Dulbecco’s modified Eagle’s medium containing 7% (v/v) bovine serum and maintained after confluence in the same medium supplemented with insulin, triiodothyronine, putrescine and methylglyoxal bis(guanylhydrazone), as previously described [4]. Media were changed every 2 days. Differentiated cells showed both the morphological and biochemical properties of rodent adipose cells [4]. Under the culture conditions used, the cells accumulated small lipid droplets, but the triacylglycerol content of differentiated cells remained low, i.e. at least 2 orders of magnitude lower than that of mature fat cells [lo]. The experiments reported below were performed between day 10 and day 12 after confluence. Cells

137 were exposed at 37°C to a medium containing 10% lipoprotein-deficient bovine serum (referred to as lipoprotein-deficient medium) for 24 h before performing assays of binding at 4°C (vide infra) and assays of cholesterol influx or efflux at 37°C [4]. Plasma Blood of normolipemic blood donors (after an overnight fast) was collected into EDTA-containing tubes (1.5 mg/ml) and promptly centrifuged at 4°C for 15 min at 1500 X g to separate cells from plasma. Plasma samples were kept at 4°C for no more than 24 h and analyzed for lipoproteins, apolipoproteins and lipids. Isolation of lipoproteins and quantitation of apolipoproteins and lipids Human LDL (d = 1.006-1.063 g/ml), HDL, (d = 1.063-1.15 g/ml), HDL, (d = 1.15-1.21 g/ml) were separated at 4°C by sequential ultracentrifugation of plasma [ll], in the absence of LCAT inhibitor. Removal of apo E and apo B from native plasma was performed by heparinSepharose column chromatography [12]. Removal of apo E from HDL, and HDL, was performed by immunoaffinity chromatography, as previously described [13]. Native plasma, HDL, and HDL, depleted of apo E and apo B were then used for isolation of LpA, and LpA, : A,, particles by immunoaffinity chromatography, as previously described [14]. Briefly, isolation of LpA, : A,, particles was performed on anti-apo An immunosorber prepared by coupling polyclonal anti-apo A ,, antibodies to cyanogen-bromide Sepharose 4B. For isolation of LpA, particles, the unretained fraction on anti-apo A,, immunosorber was checked for the absence of apo An by enzyme immunoassay and subsequently chromatographied on anti-apo A, immunosorber. In both cases, the retained fractions were eluted with 3 M NaSCN. To minimize contact between eluted lipoprotein particles and the dissociating agent, immunosorbers were constructed with a layer of Sephadex G-25 which allowed immediate separation of the particles from the agent. LpA,, particles were isolated from plasma of a patient with Tangier’s disease and also from pooled plasma of blood donors (vide supra). Each plasma was chromato-

graphed on a column of Sepharose 4B coupled to anti-apo A,, antibodies. The retained fraction, after elution with 3 M NaSCN and dialysis against phosphate-buffered saline (pH 7.4) was put onto a column of Sepharose 4B coupled to anti-apo A, antibodies. The unretained fraction was concentrated using an Amicon membrane filter (30 000 mol. wt. cut-off). This fraction which, based on protein content, represented 2-3% of all apo A,,containing particles in blood donor plasma and showed only apo A,, when analyzed by SDSPAGE. Human apo A,, apo An, apo B, apo C,,, apo C,,,, and apo E were measured in each fraction using specific enzyme immunoassays [14]. Protein was measured by the method of Lowry et al. [15]. Cholesterol (total and unesterified), phospholipids and triglycerides were determined enzymatically [16-181. Electron microscopy LpA, and LpA, : A,, particles were dialyzed against a large excess of buffer (0.125 M ammonium acetate, 2.6 mM ammonium hydrogenocarbonate and 0.26 mM tetrasodium EDTA, pH 7.4) and negatively stained with 1% many1 formate. Electron micrographs were taken with a Philips EM 420 transmission electron microscope. The primary magnification was usually 52000 diameters. Labeling of high-density lipoproteins The procedure of Bilheimer et al. [19] was used for the radioiodination of human HDL,, LpA, and LpA, : A,,. The specific activities varied between 130 and 200 cpm per ng of protein for HDL, and between 120 and 760 cpm per ng of protein for LpA, and LpA, : A,,. Since lipoprotein particles did not contain apo E, no binding by means of the apo B, E receptor was likely to occur. However, in most experiments, any interference of apo B, E receptor sites was prevented by prior exposure of intact Ob1771 cells (16-mm multi-well plates) for 2 h at 4°C to 1 ml of a lipoprotein-deficient medium containing LDL at a concentration of 100 pg protein/ml. Cells were then washed twice at 4°C with 1 ml of ice-cold phosphate-buffered saline (pH 7.4). Competition experiments were subsequently performed at 4°C and at a fixed concentration of [lZ51]HDL, (30

138 pg/ml) added simultaneously to increasing concentrations of the various unlabeled lipoprotein particles (see Fig. 2). After 2 h, the radioactivity bound to the cells was determined as previously described [4]. Binding experiments of labeled LpA , and LpA, : A,, to intact cells were performed for 2 h at 4°C by increasing concentrations of lipoprotein particles (Fig. 2). The non-specific binding was measured in the presence of a 15-fold excess of HDL,; it varied between 45 and 55% of the total binding. All assays were performed on duplicate dishes. Variability between duplicate values was not more than 10%. Cholesterol

accumulation

and effrux

As previously reported, exposure to LDL cholesterol proved to be critical in order to achieve cholesterol loading of 0b1771 cells [5]. Only with this “physiological” procedure was it possible to obtain reproducible and meaningful results for the subsequent determination of cholesterol efflux. To promote cholesterol accumulation in the cells, differentiated 0b1771 cells in 60-mm diameter dishes were first maintained at 37°C for 24 h in 5 ml of lipoprotein-deficient medium and then exposed for 48 h to unlabeled LDL at a fixed concentration of 100 pg protein/ml in the same medium. Subsequently, the cells were rinsed 3 times with phosphate-buffered saline (pH 7.4) at 37°C and then maintained in 1 ml of lipoprotein-deficient medium with or without various unlabeled lipoprotein particles. Another series of experiments was performed by first maintaining differentiated Ob1771 cells (35-mm diameter dishes) at 37°C for 48 h in the presence of 2 ml lipoprotein-deficient serum containing or not LDL at a concentration of 100 pg protein/ml. Subsequently the cells were rinsed as above and then maintained in 1 ml Dulbecco’s modified Eagle’s medium containing 1 mg/ml fatty acid free-bovine serum albumin and 50 pg protein/ml of the various particles (see Fig. 3A). The cholesterol efflux was then determined by high-pressure liquid chromatography [5] either as a function of time (see Fig. 3) or as a function of increasing concentrations of these lipoprotein particles after 6 h incubation (see Fig. 4). The concentration of cholesterol in Ob1771 cells was determined by HPLC as described previously [5] after rinsing the cells with ice-cold phosphate-

buffered saline (pH 7.4) and solubilization with 2 ml of 0.1 M NaOH per dish. Under these conditions l-50 pg of injected cholesterol could be routinely determined. Since separation of cholesterol and cholesterol esters from lipid extracts of these cells by HPLC (using a different solvent system) had previously shown the absence of detectable cholesterol esters [5], cholesterol was directly determined without prior hydrolysis. A different method was used to determine cholesterol balance between cells and medium. The direct estimates of the efflux and influx components of cholesterol transport were determined by loading the cells with LDL containing [3H]cholesterol oleate (G. Castro et al., unpublished data) and prepared according to Craig et al. [20] who showed that the biological properties of these labeled LDL were normal. In that case, cholesterol loading was performed by exposing the cells at 37°C to labeled LDL (400 cpm/pg of cholesterol; 150 pg cholesterol/ml) in the presence of 7% lipoprotein-deficient bovine serum (1 m1/35-mm dish). After 48 h, cells were rinsed as described above and then maintained in 1 ml lipoprotein-deficient bovine serum containing increasing concentrations of HDL,. The measurement of cholesterol mobilized from the cells was performed by recovering the incubation medium from various dishes as a function of time and counting the radioactivity after lipid extraction [5]. After recovery of the incubation medium, the cholesterol content of the cells was determined by rinsing the cells as described above and counting the radioactivity after lipid extraction. Control experiments showed that measurements of cellular cholesterol by HPLC and radioactive assays did not differ by more than 10% whereas the total cholesterol recovered (cells plus medium) ranged between 86 and 99% of that present in the cells at time zero of efflux. Results Analysis of apolipoproteins, lipids and size distribution in LpA, and LpA, : A,, As shown in Table 1, both LpA, and LpA, : A,,

isolated by immunoaffinity chromatography from plasma, HDL, or HDL, were contained 61-67s protein by weight. The protein content of LpA, and LpA, : A,, from plasma, 63 and 67%, respec-

139 TABLE

1

PROTEIN

AND

LIPID

COMPOSITION

The protein and lipid composition isolated from the same plasma, determined.

LpA, (plasma) LPA, (HDL,) LPA I WDL,) LpA, : A ,, (plasma) LpA,:A,, (HDL,) LPAI:A,, (HDL,)

OF LpA,

AND

LpA,

: A,,

are given as % mass; the values are mean k range of two preparations for LpA, and LpA, and from two preparations of HDL, and HDL, obtained from the same plasma. n.d.,

Protein

Cholesteryl esters

Unesterified cholesterol

Phospholipids

Triglycerides

Diameters

63.4 k1.56 50.99* 1.59 63.91+ 2.0 67.25 f 0.91 53.04+ 2.77 61.22 f 3.05

9.26 i 0.55 14.66 + 0.69 11.18 +0.74 8.65 kO.11 13.67 + 0.25 11.84k0.52

2.51 f 1.27 3.78 +0.21 2.17 + 0.24 1.67k0.31 2.94 + 0.41 2.21+ 0.04

19.95 + 0.85 26.06+1.73 16.92 + 0.36 17.06 f 1.47 26.52 + 1.36 20.82 f 3.95

4.84+ 1.13 4.48 f 0.39 5.79 k 0.65 5.29 + 0.74 3.79kO.74 3.88+1.45

n.d. 108.4& 19.5 107.3 + 23.6 nd. 106 k18.3 96.6 + 17.8

tively, are at variance with those of Cheng et al. [21] who reported values ranging from 46 to 52% whereas their lipid composition showed similar values, the percentages of total cholesterol, phospholipid and triglycerides being approximately 12, 20 and 5 for LpA,, and lo,17 and 5 for LpA, : At,. The results of Table 1 indicate also that LpA, and LpA, : At, from HDL, contained more cholesterol than LpA, and LpA, : At, from HDL,. The cholesterol ester/unesterified cholesterol ratio was lower in LpAr and LpA, : A,, from HDL, than in LpA , and LpA, : A,, from HDL,; it was also lower in LpA, than in LpA, : A tt, both isolated from plasma. When LpA, and LpA, : A,, were analyzed for their apolipoprotein composition it appears, as shown in Table 2, that both kinds of particles, isolated by immunoaffinity chromatography from plasma, HDL, or HDL,, were largely devoid of apo B and apo C,, whereas apo E was undetecta-

TABLE

1

: A,, not

(A)

ble. As expected, no apo A,, could be detected in LpA, from plasma [LpA,(P)], HDL, [LpA, (HDL2)] or HDL, [LpA,(HDL,)]. The molar ratio of apo A, to apo A,, was close to unity in LpA I : A ,t isolated from plasma [LpA , : A t,(P)], HDL, [LpA, :A,,(HDL,)] and HDL, [LpA,: A,,(HDL,)]. The data indicate that LpA, and from HDL, or HDL, and LpA, : A,,, isolated thus exhibiting different densities, and also those isolated from plasma, contained apo C,,, as the major additional apolipoprotein among the ones quantitated by immunoassays. The determination of the apo A,, content was not made, but if we assume that most apo A,, associated with the HDL fraction should have been lost during ultracentrifugation [22], it is reasonable to consider LpA, as particles containing apo A, as the main constituent and LpA, : A,, as particles containing apo A,, and apo A, as main constituents. Electron micrographs of LpA, and LpA, : A,,

2

APOLIPOPROTEIN

LpA, (plasma) LPA, (HDL,) LPA, (HDL,) LpA , : A ,, (plasma) LPA, :A,, (HDL,) LpA,:A,, (HDL,)

AND LIPID

COMPOSITION

OF LpA , AND

LpA,

: A ,,

APO A,

APO A,,

Apo B

APO C I,

APO C,,,

Apo E

96.6 f0.9 93.39* 1.74 95.93 f 1.76 52.88 kO.95 49.77 &-1.43 53.5 k1.45

und. und. und. 41.6 +O.ll 47 +1.92 42.26+0.73

0.055 0.045 0.029 0.067 0.024 0.013

0.74+0.13 0.77 + 0.38 0.75kO.11 0.78 + 0.05 0.3 *0.008 0.59 * 0.35

2.55 kO.73 5.78 + 1.35 3.28 f 1.65 4.66 i_ 1 2.89 * 0.49 3.69+1.83

und. und. und. und. und. und.

The apoliprotein composition mean 5 range and correspond

f 0.038 + 0.008 + 0.002 +O.Ol + 0.007 + 0.003

is given by taking as 100% the total apolipoproteins to the same fractions of Table 1; und., undetectable.

determined

by immunoassays;

the values

are

140 from the same samples of either lipoprotein subspecies were prepared and morphometric analyses were carried out (Table 2). The average pOarticle size (mean f SD) was 108.4 + 19.5 A for LpA,(HDL,), 107.3 f 23.6 A for LpA,(HDL,), 100.6 + 18.3 A for LpA,: A,,(HDL,) and 96.6 + 17.8 A for LpA,:A,,(HDL,); it was not determined for LpA,(P) and LpA, : A,,(P). Fig. 1 shows the profiles of the size distribution of the various lipoprotein particles. These profiles indicate that there exists for either lipoprotein subspecies some particles whose average diameter is larger than those reported above. ,The morphographic analysis of particle diameters suggests a bimodal size distribution for LpA,(HDL,), LpA, :A,, from HDL, and HDL, but not for LpA,(HDL,); more detailed studies with statistical data would be required to draw firm conclusions in that respect. Competitive binding of LpA, or LpAI: A,, with HDL, to intact Obl771 cells The relative binding activities of LpA, and LpA, : An from native plasma, HDL, or HDL,

were examined in competition assays with [‘251]HDL,. As shown in Fig. 2, LpA, (Fig. 2A) and LpA, : An particles (Fig. 2B) prevented the [‘251]HDL, binding to adipose cells at 4°C. The concentrations required to prevent 50% of the binding were between 10 and 20 pg/ml of protein. Considering the range of Kd values, it is likely that LpA, and LpA, : A,, bind with similar affinities to the unique receptor sites which have been already described as recognizing both apo A, and apo An [5] as well as apo An, [6]. By direct binding assays, the apparent Kd values for labeled LpA, and LpA,:A,, from HDL, and HDL, ranged also between 10 and 25 pg/ml (Fig. 2C). Cholesterol efflux from cholesterol-preloaded Obl771 cells exposed to various apo AI-containing particles Differentiated 0b1771 cells were exposed as a function of time to LpA, and LpA, : Au after prior exposure or not to LDL. The curves of Fig. 3A show that in cholesterolnonloaded cells maintained in the absence of serum, LpA, and LpA, : Au were able to promote

20-

20.

[Lpi

10

200 : 0

E5.656f.665A

,A.,

50

100

160

1Lp/

iL'1143,133A

20.

zoo

s

. DIAMETER

L

(A)

Fig. 1. Morphographic analysis of particle diameters. LpA, and LpA, : A,, isolated from HDL, and HDL, (see Table 1 for apolipoprotein and lipid composition) were analyzed for size by negative staining. For each particle, n = 500. Particle diameter means (x) and standard deviation (+) are displayed on each graph: x,, smaller mode; xL, larger mode.

I

0

20 40

60 80 100120

Lipoprotein (pg of protein /ml) with 100 Fig. 2. Competitive inhibition of [ ‘251]HDL, binding to intact 0b1771 cells. A and B: 0b1771 cells were preincubated pg/ml of LDL as described in Materials and Methods and then incubated at 4°C for 2h with 30pg/ml [‘251]HDL, and increasing concentrations of (A) LpA,(P) (O), LpA,(HDL,) (o), LpA,(HDL,) (A), HDL, (m) and (B) LpA,:A,,(P) (A), and LpA,:A,, (0). 100% binding corresponds to the value obtained in the absence of competitor. The mean (HDL,) (0) and LpA,: A,,(HDLs) values from duplicate dishes, which did not differ by more than lo%, are reported. Similar values have been obtained in another independent experiment performed in the presence of LpA,(HDL,) and LpA, : A,,(HDL,) and using a different series of cells. A separate batch of native plasma was used to directly prepare LpA,(P) and LpA, : A,,(P); C: Ob1771 cells were incubated at 4°C for 2 h with increasing concentrations of labeled LpA,(HDL,) (o), LpA,(HDL,) (A). LpA, :A,,(HDL,) (0) and LpA, :A,,(HDL,) (0). as described in Materials and Methods. The curves show the specific binding (total binding minus non-specific binding) ; the values are representative of two independent experiments performed on different series of cells.

0

0.25

0.5

1

3

6

24

0

3

6

0

3

6

0

3

6

TIME (hours) Fig. 3. Cholesterol efflux in Ob1771 cells exposed to LpA, and LpA, : A ,, particles. A: Ob1771 cells in duplicate 35mm dishes were maintained for 72 h at 37°C in the presence of 2 ml lipoprotein-deficient serum and exposed (1 ml final volume) to 50 wg protein/ml LpA, (A) or LpA,: A,, (0) from native plasma depleted of apo E; the medium contained 1 mg/ml of fatty acid poor-bovine serum albumin instead of serum in parallel experiments cells were exposed at 37°C for 24 h to lipoprotein-deficient medium and then for 48 h to 100 ag protein/ml LDL; cells were then exposed to lipoprotein-deficient medium containing 50 ug protein/ml LpA, (A) or LpA, : A,, (0). Zero time corresponds to the addition of LpA, or LpA, : A,, particles. Duplicate dishes were pooled for the determination of cellular cholesterol content by HPLC. The curves are representative of two experiments performed on two different series of cells. B-D: cholesterol preloading was performed as described in Materials and Methods. Cells were then maintained in the absence (m) or presence of 50 pg protein/ml LpA, (A) or LpA, : A,, (0) from native plasma (B), HDL, (C) or HDL, (D). Duplicate dishes were assayed separately for cellular cholesterol content. Median f range values from duplicate dishes are reported: they are representative of the values obtained with two independent series of cells. The cholesterol content of cells maintained in lipoprotein-deficient medium expressed in ug/mg of cell protein was 11.4 f 0.4 in A, 22 f 1.2 in B and 17 + 0.8 in C and D.

142 neither cholesterol accumulation above nor cholesterol efflux below a basal cellular cholesterol content for times ranging from 15 min to 24 h. In contrast, and as described previously [9], LpA, was able to promote cholesterol efflux from cholesterol-preloaded cells maintained in the presence of serum whereas LpA, : A,, brought neither an increase of cholesterol efflux nor an increase of the cellular cholesterol content. It must be pointed out that, in the presence of LpA,, the final cholesterol content of cholesterol-preloaded (serum-exposed) cells was very similar to that measured in cholesterol-nonloaded (serum-free exposed) cells. Cholesterol-preloaded Ob1771 cells were subsequently exposed in serum-containing medium to 50 pg of protein/ml of LpA, and LpA, : A tt isolated from native plasma (Fig. 3B), HDL, (Fig. 3C) and HDL, (Fig. 3D). A rapid decrease in the cellular cholesterol content occurred within 3 h and reached a lower value within 6 h. This value was similar to that determined in control cells before exposure to LDL cholesterol. No decrease was observed in the absence of LpA, or in the presence of LpA, : A,, from all three sources. The lack of effect of LpA, : A,, cannot be due to the lack of binding of LpA, : A,, to the receptor sites as the concentration of LpA, :A,, (50 pg/ml)

being used was near sufficient sites (Fig. 2).

to saturate these

Modulation by LpA, : A,, of LpA,-mediated cholesterol effrux in Obl771 cells For that purpose LpA, and LpA, : A,, isolated

from HDL, or HDL, and depleted of apo E were used. The respective concentrations of LpA, and LpA, : At, were chosen to mimic the actual plasma concentrations of these particles in HDL, and HDL, [23,24]. As shown in Fig. 4, both LpA, :A,,(HDL,) were able to inhibit and LpA, : Au (HDL,) cholesterol efflux mediated by LpA,(HDL,) or LpA,(HDL,). LpA, : A,,(HDL,) was more inhibitory than LpA, : A,,(HDL,) in the presence of either LpA, species. The concentration required to prevent 50% of LpA,-mediated cholesterol efflux ranged for LpA, : An(HDL,) from 15 pg/ml (Fig. 4B) to 20 pg/ml (Fig. 4A). These values are in fairly good agreement with the concentrations required to prevent 50% of [‘251]HDL, binding to intact Ob1771 cells (see Fig. 2B). However, some unexplained discrepancy occurred in the case of LpA, : A,, (HDL,), the respective figures being 40 pg/ml for the inhibition of cholesterol efflux and only 10 pg/ml for the inhibition of binding. Dur-

(HDL2)

(HDLS)

20

40

60 Lp AI:AII

0

10

20 30

(Wml)

cholesterol efflux in 0b1171 cells. Cholesterol-preloaded Ob1771 cells were Fig. 4. Net decrease by LpA, : A tt of LpAr-mediated exposed to LpA, (40 pg/ml) isolated from HDL, (A) or HDL, (B), in the presence of increasing concentrations of LpA, : A,, isolated from HDL, (0) or HDL, (0).After 6 h at 37”C, cellular cholesterol content was determined as described in Materials and Methods. Control cells were continuously exposed to LDL cholesterol and contained 62 pg cholesterol per mg cell protein whereas cholesterol-preloaded cells subsequently exposed for 6 h to LpAt(HDL,) or LpA,(HDL,) had a remaining content of cellular cholesterol content of 32 pg cholesterol per mg cell protein; 100% decrease corresponds to the difference between both values, i.e. 30 pg cholesterol per mg cell protein. The curves are representative of 2 independent experiments performed with two different series of cells.

143 and apo An distribution when the incubation temperature was raised from 4 to 37’C as the concentrations of total apo A, and that of apo A, in LpA, : A,, in the incubation medium remained similar whereas that of apo A, in LpA, actually increased. In the presence of cells, the concentrations of total apo A, and apo A I in LpA, : An decreased to some extent compared to those measured in the absence of cells whereas that of apo A, in LpA, further increased. Taken together, these results appear to exclude the possibility of a randomization of the lipoprotein particles with respect to their apolipoprotein composition. However a redistribution of LCAT between LpA, and LpA, : A,, cannot be excluded, as recently shown by Cheung and Wolf [26].

ing the course of these experiments, it was noted, as previously reported for cholesterol efflux in human fibroblasts [l], that HDL,, in contrast to HDL,, did not promote cholesterol efflux from cholesterol-preloaded cells. The stronger inhibithan tion of efflux caused by LpA, : A,,(HDL,) by LpA,: A,,(HDL,) was striking. It was hypothesized that HDL, but not HDL, might contain lipoprotein particles composed of apo An but not apo A I (LpA ,, ), in addition to LpA I : A ,, present in both HDL, and HDL,. Preliminary evidence shows that LpA,, were indeed isolated from HDL, but not from HDL, (not shown). LpA,, isolated from plasma of a patient with Tangier’s disease and LpA,, isolated from native plasma of normolipemic blood donors were able to inhibit the LpA,-mediated cholesterol efflux: for instance, in the presence of 40 pg/ml of LpA, from native plasma, 25 pg/ml of LpA,, from normolipemic blood donors inhibited cholesterol efflux by 68%. Further experiments will be required to assess this point.

Discussion The lipoprotein particles used in this study were isolated either from native plasma or from HDL, and HDL, recovered from plasma after ultracentrifugation; these particles were free of apo B and apo E. Recent reports have indicated that, among the apo A,-containing lipoprotein particles, LpA, are less stable than LpA, : An upon ultracentrifugation [27]. Because of the small extent of pertubation induced by this technique on the apolipoprotein composition of either LpA, : An species shown in Table 2, it is likely that LpA, : A,,(HDL,) and LpA, : A,i(HDL,) are similar to LpA, : A ,, isolated from native plasma

Lack of remodeling of lipoprotein particles during incubation The possibility of remodeling of particles was considered by measuring apo A, bound to apo A ,, and apo A, not bound to apo A,, by enzyme linked immunoassay [24,25], in the absence of cells, both at 4°C and 37°C and in the presence of cells at 37°C. The results of Table 3 indicate, in the absence of cells, no net change in the apo A,

TABLE

3

LACK OF REMODELING CELLS

Cells

_ +

OF LIPOPROTEIN

PARTICLES

DURING

INCUBATION

Incubation time(h)

Temperature

Total apo A,

Apo A, in

(“C)

pg/ml

LPA,

24 6 6

4 31 31

8.8 8.4 1.4

5.8 5.0 3.8

IN THE ABSENCE

: A,, (ag/mO

OR PRESENCE

OF

Apo A, in LPA, ag/d

(W)

3.0 (34.1) 3.4 (40.5) 3.6 (48.7)

When present, differentiated Ob 1771 cells in 35-mm dishes were maintained for 48 h at 37’C in the presence of 2 ml medium containing 7% lipoprotein-deficient bovine serum and then exposed to the same incubation medium supplemented with 150 gg protein/ml LDL. After 3 washes at 37°C with phosphate-buffered saline, LpA, and LpA, : An particles, first depleted of apo E by immunoaffinity chromatography, were added to the incubation medium. At the indicated times, the quantitative determination of was performed using an enzyme linked differential antibody immunosorbent technique which has been LpA, and LpA,:A,, described in details elsewhere [24]. The difference between total apo A, and apo At which was in particles without apo A,,(LpA, : At,) represents apo A, which was in particles without apo A,,(LpA,).

144 [23,24]. The situation may be different for LpA, particles which have been reported to be more labile upon ultracentrifugation [27]. In any event, it is clear that intact LpA,(P) or “remodeled” LpA, isolated from HDL, or HDL, behave similarly and quite efficiently in promoting cholesterol efflux from cholesterol-preloaded adipose cells. Nevertheless, since the cellular cholesterol content was determined when efflux reached a plateau, it cannot be excluded that various LpA, particles behaved distinctly different from a kinetic point of view. The characterization of a subpopulation of apo A ,-containing particles of apparent M, = 70 kDa in native plasma, which appears to behave as the first acceptor of cholesterol from human skin fibroblasts preloaded with cholesterol [28], may well be relevant on this point. In contrast to LpA, particles, LpA, : An particles isolated from native plasma, HDL, or HDL, are inactive with respect to cholesterol efflux (Fig. 3) despite the fact that they were recognized by receptor sites showing similar apparent affinities for both LpA, and LpA, : An (Fig. 2). Among hypotheses to explain the antagonist role of LpA, : A,, already described in the process of cholesterol efflux [9], it must be recalled that studies with apo A, and apo A,, proteoliposomes, containing or not unesterified cholesterol, have indicated that apo An binds with a higher affinity than apo A, to intact 0b17 cells and that apo A, only promotes cholesterol efflux both in serumsupplemented [5] and serum-free medium [29], thus excluding a critical role of LCAT and CETP in this initial event. It is unlikely that the lack of effect of LpA, : An is due to their lipid composition since the latter does not appear to be widely different for LpA, and LpA, : A,, isolated from either plasma or HDL,. Moreover LpAt : An particles appear never to be active despite the fact that particles isolated from plasma, HDL, or HDL, showed different lipid composition, arguing also against this possibility. Another possibility could also be considered, that is the delivery of cholesterol to cells by LpA, : An. As shown in Fig. 3A, this hypothesis was also excluded as LpA I : At, could promote neither an accumulation nor a mobilization of unesterifed cholesterol whether the cells had been or not pre-loaded with cholesterol, both on a short-term (15 min) and long-term basis

(24 h). In most cases, a 6 h exposure to the various lipoprotein particles was chosen since it was sufficient to decrease cellular cholesterol content to control values (Fig. 3B-D) whereas no extensive remodeling of particles with respect to apo A, distribution between LpA, and LpA, : An seemed to occur (Table 2). Taken together, it is reasonable to assume from the results that only apo A, is involved in the agonist role played by LpAt and that only apo An is involved in the antagonist role played by LpA, : A,,. The fact that LpA, and LpA, : An appeared to compete for the same receptor sites recognizing apo A, and apo An (see Fig. 2 and ref. 5) led us to study the modulation by LpA I : An of LpA,mediated cholesterol efflux (Fig. 4). This modulation occurs in vitro at concentrations similar to those required for binding (from 10 to 40 pg protein/ml; see Fig. 2). This observation is consistent with a direct role played by the apo A,, A,, receptor sites in this process [30,31]. The possibility that the results of Fig. 4 could be explained by LpA, : A,, transferring unesterified cholesterol to LpA,, thus diminishing their ability to remove cholesterol, is unlikely since data of Table 1 would indicate that such cholesterol movement should take place in the opposite direction. Moreover it is rather unlikely that LpA, : Au particles, which do not deliver cholesterol to intact cells to which they bind with a high affinity, should do so ‘at random’ when mixed with LpA, particles. The possibility that remodeling of particles takes place during incubation was also envisaged. The results of Table 3 do not show any significant ‘randomization’ process of the particles, in agreement with recently published work [26]. Thus it appears that the presence of apo An in a lipoprotein particle is critical for inhibition of cholesterol efflux. The significance in vivo of our observations remains to be established, but it is tempting to postulate that the magnitude of cholesterol efflux from peripheral cells might be adjusted according to the actual concentrations of both kinds of particles in the interstitial fluid bathing the cells. Acknowledgements

The help of Dr. A. Doglio and Miss M. CazalQ for growing 0b1771 cells and the secretarial assis-

145 tance of Mrs. G. Oillaux are gratefully acknowledged. The authors are grateful to Prof. D. Goldberg (Toronto, Canada) and Dr. A. Steinmetz (Marburg, F.R.G.) for careful reading of the manuscript and helpful suggestions. The authors wish to thank the “Institut Pasteur” (INSERM U325 and U167/CNRS 624, Lille, France) and the “Centre National de la Recherche Scientifique” (CNRS UPR 7300, Nice, France) for financial support. Thanks are also due to the “ Fondation pour la Recherche Medicale Francaise” (Paris, France).

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