Sodium oleate promotes a redistribution of cholesteryl esters from high to low density lipoproteins

Atherosclerosis, 84 (1990) 13-24 Elsevier Scientific Publishers Ireland,

ATHERO

13 Ltd.

04512

Sodium oleate promotes a redistribution of cholesteryl esters from high to low density lipoproteins P.J. Barter, L.B.F. Chang and O.V. Rajaram Baker Medical Research Institute,

Melbourne (Australia)

(Received 19 January, 1990) (Revised, received 17 April, 1990) (Accepted 19 April, 1990)

Cholesteryl esters readily exchange between the low density lipoproteins (LDL) and high density lipoproteins (HDL) in human plasma in a process of equilibration catalysed by the cholesteryl ester transfer protein (CETP). In the present studies, in which mixtures of human LDL and HDL have been incubated in vitro with partially pure CETP, it has been found that Na oleate disrupts the CETP-mediated equilibrium between LDL and HDL and promotes a concentration dependent redistribution of cholesteryl esters from HDL to LDL. The end result of the redistribution is the appearance of a cholesteryl ester enriched LDL fraction and an HDL fraction which is protein-rich, lipid-depleted and markedly reduced in particle size.

Key words: Sodium oleate; Cholesteryl lipoproteins

ester transfer protein;

Introduction

The importance of understanding factors which influence the partitioning of cholesterol between

Correspondence to: Dr. Philip J. Barter, Baker Medical Research Institute, Commercial Road, Prahran, Victoria 3181, Australia. Abbreviations: apo A-I, apolipoprotein A-I; apo A-II, apolipoprotein A-II; apo B, apolipoprotein B; CETP, cholesteryl ester transfer protein; HDL, high density lipoproteins; LDL, low density lipoproteins; VLDL, very low density lipoproteins; TBS, Tris-buffered saline; LCAT, lecithin : cholesterol acyltransferase. 0021-9150/90/$03.50

0 1990 Elsevier

Scientific

Publishers-Ireland,

High density lipoproteins;

Low density

different plasma lipoprotein fractions is highlighted by the observation that the risk of developing coronary heart disease correlates positively with the concentration of cholesterol in low density lipoproteins (LDL) [l] and negatively with that in high density lipoproteins (HDL) [2]. Most of the cholesterol in plasma exists as cholesteryl esters which reside with triacylglycerol in the hydrophobic core of lipoproteins. In human plasma, cholesteryl esters exchange between all lipoprotein fractions in a process of equilibration catalysed by the cholesteryl ester transfer protein (CETP) [3,4]. Since the rate of the CETP-mediated exchange between LDL and HDL in human plasma is rapid relative to the rate of catabolism of each lipoproLtd.

14

tein fraction [5], the cholesteryl esters in these two lipoproteins must be close to equilibrium in viva. Thus, in terms of regulating the partitioning of cholesteryl esters between LDL and HDL, it is probable that the level of activity of CETP is not normally rate limiting. We now report, however, that the CETP-mediated equilibrium between LDL and HDL can be dismpted by Na oleate which, as a consequence, promotes a shift in the partitioning of cholesteryl esters from the non-atherogenic HDL to the atherogenic LDL. Materials and Methods Lipoproteins

Blood from healthy male and female subjects aged 24-48 years was collected after a light breakfast into tubes containing disodium EDTA (1 mg/ml) and placed immediately on ice. The plasma was separated by centrifugation at 4O C and then adjusted to appropriate densities by adding solid KBr [6]. The combined LDL and HDL fraction (d = 1.019-1.21 g/ml) was isolated by sequential ultracentrifugation [7] using a Beckman Ti 50 rotor at a speed of 48000 r-pm, with a single 24 h spin at the lower density and two successive 40 h spins at the higher density. The isolated lipoproteins were dialysed against 0.01 M Tris-HCl (pH 7.4) containing 0.15 M NaC1/0.005 M disodium EDTA/0.003 M NaN, (T&-buffered saline, TBS). Isolation of the cholestevl ester transfer protein

Cholesteryl ester transfer protein was purified from titrated human plasma. Ammonium sulphate precipitation of proteins, ultracentrifugation to remove the lipoproteins, hydrophobic interaction chromatography on phenyl-Sepharose CL4B and cation-exchange chromatography on CM-52 cellulose were performed as described by Pattnaik et al. [3] except that the plasma proteins were precipitated with ammonium sulphate between 35% and 55% saturation. The subsequent purification was performed by anion-exchange chromatography on a Mono Q HR 5/5 column (PharmaciaLKB, Uppsala, Sweden). In terms of cholesteryl ester transfer activity, this partially pure preparation had a specific activity of about 50 transfer units per mg protein compared with 0.01 units per

mg protein in human lipoprotein-free plasma; i.e. it was purified about 5000 fold compared with lipoprotein-free plasma. Assay of cholesteryl ester transfer activity

Transfer activity in a given sample was measured as the capacity of the sample to facilitate transfer of [3H]cholesteryl ester from LDL to HDL during an incubation at 37’ C [3]. Transfer activity is expressed as units per ml or per mg protein, the number of units being the rate constant, k, for the transfer of LDL tracer to HDL per 3 h using the formula of Pattnaik et al. [3]. Incubations AU incubations

were carried out in stoppered tubes in a shaking water bath at 37’C. Non-incubated control samples were stored at 4O C. In some of the experiments, the incubation mixtures were supplemented with Na oleate (Sigma Chemical Co., St. Louis, MO, U.S.A.) [8]. Other experiments were performed in the presence of human serum albumin (Sigma Chemical Co.). Two different preparations of albumin were used: one, Sigma catalogue #A8763, is not depleted of fatty acid; the other, Sigma catalogue #A3782, differs only in that it is essentially free of fatty acids. These two preparations of human serum albumin are denoted in this report as fatty acid-rich and fatty acid-poor albumin respectively. Preparation of HDL esters

labelled with [3H]cholesteryl

HDL labelled in the cholesteryl ester moiety was prepared essentially as described elsewhere [9]. In brief, [1,2-3H]cholesterol (Amersham Australia Pty Ltd, 40 Ci/mmol) was dissolved in ethanol and added to a preparation of the freshly isolated and dialysed plasma fraction of d > 1.125 g/ml. The mixture was then incubated for 24 h at 37” C to allow cholesterol esterification in the reaction catalysed by lecithin : cholesterol acyltransferase (LCAT). The HDL fraction was re-isolated as described [9] and found to contain labelled cholesteryl esters which accounted for more than 95% of the total radioactivity. Processing of samples

Following incubation, samples were separated by various techniques. In some experiments the

15 LDL and HDL fractions were separated by 16 h of ultracentrifugation at 1.063 g/ml in a Beckman TL-100 table top ultracentrifuge using a Beckman TLA-100.2 rotor at a speed of 100000 rpm; completeness of the separation was established by the consistent observation that more than 96% of the apolipoprotein (apo) B in the mixtures was recovered in the supernatant, while apo A-I was measurable only in the infranatant fractions. In other experiments, mixtures were subjected to 16 h of ultracentrifugation (Beckman TLA-100.2 rotor at a speed of 100000 rpm) at a density of 1.25 g/ml in the table top ultracentrifuge. The supernatant fractions were then subjected to gradient gel electrophoresis on non-denaturing 4-30% polyacrylamide gels (Pharmacia-LKB, Uppsala, Sweden) to define the particle size distribution of HDL [lo]. In some studies, the incubation mixtures were separated by size exclusion chromatography on a column of Superose 6 HR lo/30 (Pharmacia-LKB) [ll] without prior ultracentrifugation of the samples. Sample recovery from the column, as assessed by the recovery of total cholesterol, was always greater than 90%.

Other analyses All chemical assays were performed on a Cobas-Bio centrifugal analyser (Roche Diagnostics, Zurich, Switzerland). Concentrations of total and unesterified cholesterol, triacylglycerol and phospholipids were measured using enzymatic kits (Boehringer Mannheim GmbH, F.R.G.). The concentration of esterified cholesterol was calculated as the difference between the concentrations of total and unesterified cholesterol. Concentrations of apo A-I, apo A-II and apo B were determined by immunoturbidometric assay [12]. Apolipoprotein standards were purchased from Behring Werke AG (Marburg, F.R.G.) and antibodies to these apolipoproteins were from Boehringer Mannheim, F.R.G. Protein concentration was assayed by the method of Lowry et al. [13] adapted for use in the Cobas-Bio centrifugal analyser [14]. Since more than 95% of the ‘H added to incubations was recovered in the cholesteryl ester moiety in all cases, [ 3H]cholesteryl esters were equated with the total 3H activity which was assayed as described elsewhere [ 151.

Results Distribution of cholestetyl esters between high and low density lipoproteins (Table I) Samples of the plasma fraction of density 1.019-1.21 g/ml containing a mixture of HDL and LDL were isolated from each of 5 subjects and incubated at 37 QC for 24 h in the presence of various additions. By comparison with incubations performed in the presence of buffer alone, incubation with CETP at a concentration of 2.4 units/ml resulted in the net mass transfer of 40% (mean value) of the HDL cholesteryl esters to the LDL fraction. When, however, identical mixtures of HDL, LDL and CETP were supplemented with TABLE

1

DISTRIBUTION HDL AND LDL

(1) (2) (3) (4) (5) (6)

OF CHOLESTERYL

ESTERS

BETWEEN

Addition to incubation mixture

Cholesteryl esters (nmol/fraction) LDL

HDL

Buffer CETP CETP HSA (FA-poor) CETP HSA (FA-rich) CETP sodium oleate sodium oleate

228+65 281+69 259*66

134+22 81k19 104*18

277rt68

85f19

37*9

316 f 68

46+15

66k8’

127*19

5f4

236k65

HDL cholesteryl esters transferred to LDL (%) 4Ok9 21f5 *

*

The plasma fraction of density 1.019-1.21 g/ml (LDL+ HDL) was isolated from each of 5 subjects. In non-incubated samples the distribution of cholesteryl esters was: LDL 225 &66 nmol/fraction and HDL 136 &-18 nmol/fraction. Samples were then incubated at 37’ C for 24 h in the presence of: (1) buffer alone, (2) CETP (2.4 units/ml), (3) CETP (2.4 units/ml) plus fatty acid-poor human serum albumin (40 mg/ml), (4) CETP (2.4 units/ml) plus fatty acid-rich human serum albumin (40 mg/ml), (5) CETP (2.4 units/ml) plus sodium oleate (0.12 mM) and (6) sodium oleate (0.12 mM). Final incubation volumes were 300 pl. After incubation the LDL and HDL were separated by ultracentrifugation at d = 1.063 g/ml. The percentage of HDL cholesteryl esters transferred to LDL was calculated by reference to the control incubation (1) which was performed in the presence of buffer alone. Values represent means* SD of 5 separate experiments each of which was performed in duplicate. * Significantly different from the transfer in incubations containing CETP alone (P < 0.05 by Student’s t-test for paired samples).

16 CETP, addition of sodium oleate at a concentration up to 0.24 mM did not promote measurable net mass transfers of cholesteryl esters from HDL to LDL (result not shown). In the presence of CETP, however, the net mass transfer of cholesteryl esters from HDL to LDL was enhanced in a concentration dependent fashion by the addition of sodium oleate (Fig.1). 6

066 Sodun

oi2 oleate

Oi8

0.24

(mM)

Fig. 1. Enhancement of the CETP-mediated transfer of cholesteryl ester mass from HDL to LDL by sodium oleate: concentration dependence of sodium oleate. The plasma fraction of d = 1.019-1.21 g/ml (containing a mixture of LDL and HDL) was isolated and supplemented with CETP. Aliquots of this mixture (final incubation concentrations of LDL cholestetyl esters 0.83 mM, HDL cholesteryl esters 0.45 mM and CETP 2.4 units/ml) were further supplemented with sodium oleate at concentrations as shown and incubated at 37 o C for 24 h. Final incubation volumes were 300 cl. Incubations were stopped by placing the samples on ice; the LDL and HDL were then separated by ultracentrifugation at density 1.063 g/ml. The points represent the percentage of the HDL cholesteryl esters transferred to LDL; a sample incubated in the absence of CETP and sodium oleate served as a no-transfer control. Each point represents the mean of duplicate estimations.

fatty acid-poor human serum albumin at a concentration of 40 mg/ml, there was an approximate 50% inhibition of the net mass transfer, with only 21% of the HDL cholesteryl esters being redistributed to LDL in these incubations. By contrast, addition of fatty acid-rich human serum albumin did not inhibit the CETP-mediated net mass transfer of cholesteryl esters from HDL to LDL. When the mixtures of HDL, LDL and CETP were supplemented with Na oleate at a concentration of 0.12 mM, the CETP-mediated net mass transfers were markedly increased, with 66% of the HDL cholesteryl ester mass being transferred to LDL during the 24 h of incubation. Addition of Na oleate in the absence of CETP, on the other hand, produced results which were not significantly different from those of incubation in the presence of buffer alone (Table 1). Concentration

dependence

of sodium oleate (Fig. 1)

A mixture of HDL and LDL was incubated at 37’C for 24 h in the presence of varying concentrations of sodium oleate. In the absence of

Relationship between net mass transfers and the transfers of isotopically labelled cholesteryl esters from HDL to LDL (a) Incubations with CETP and albumin (Fig. 2).

When mixtures of HDL and LDL were incubated for up to 24 h in the presence of buffer alone (Fig. 2A) or in the presence of fatty acid-poor human serum albumin alone (result not shown), there was no demonstrable redistribution of cholesteryl esters from HDL to LDL whether measured in terms of isotopic transfer (upper panel) or net mass transfer (lower panel). When the lipoproteins were incubated in the presence of CETP, there were obvious isotopic and net mass transfers of cholesteryl esters from HDL to LDL (Fig. 2B). The time courses of the two processes, however, were markedly different. The isotopic transfers indicated that equilibration of the pools of cholesteryl esters in HDL and LDL was complete after l-3 h of incubation (Fig. 2B, upper panel). The net mass transfer, on the other hand, was progressive over the 24 h of incubation (lower panel). Fig. 2C shows the effects of adding fatty acid-poor albumin to the mixture of HDL, LDL and CETP. While the presence of albumin had no effect on the rate of equilibration of isotopically labelled cholesteryl esters between HDL and LDL (Fig. 2C, upper panel), the CETP-mediated net mass transfer of cholesteryl esters was reduced by more than 60% by the addition of albumin (Fig. 2C, lower panel). (b) Incubations with CETP and sodium oleate (Fig. 3). Incubation of mixtures of HDL and

LDL for up to 24 h in the presence of 0.12 mM sodium oleate alone (Fig. 3A) had no apparent effect on the distribution of cholesteryl esters whether measured as isotopic transfer (upper panel) or net mass transfer (lower panel). Incubation of the lipoproteins with CETP alone (Fig. 3B) produced a result identical to that described above

17 C Buffer

5

CETP

loooo+

CETP + HSA

iHDL

LDL

.‘-

LDL

.

0

I

0

12 Hours

2h

b

12

24

Of lncubatlon

Fig. 2. Transfers of cholesteryl esters from HDL to LDL promoted by CETP: effects of human serum albumin. The plasma fraction of d = 1.019-1.21 g/ml was isolated and supplemented with a tracer amount of HDL labelled with ‘H in the cholesteryl ester moiety. Aliquots of this mixture (final incubation concentrations of LDL cholesteryl esters 0.75 mM, HDL cholesteryl esters 0.50 mM and [3H]cholesteryl esters 320000 cpm/ml) were further supplemented with Tris-buffered saline alone (panel A) , CETP (2.4 units/ml) (panel B) , and CETP (2.4 units/ml) plus fatty acid-poor human serum albumin (40 mg/ml) (panel C) and incubated at 37OC for periods up to 24 h. Final incubation volumes were made up to 300 pl by the addition, if necessary, of Tris-buffered saline. Incubations were stopped by placing the samples on ice and the LDL and HDL were then separated by ultracentrifugation at d = 1.063 g/ml. LDL and HDL were assayed for cholesteryl ester mass and for [3H]cholesteryl eaters. The upper panels show the specific activities (cpm/nmol) of cholesteryl esters in LDL and HDL as a function of time. The lower panels show the percentage of the HDL cholesteryl ester mass which had been transferred to LDL at each time point. Each point represents the mean of duplicate estimations.

(see Fig. 2B). When the mixture of HDL, LDL and CETP was further supplemented with sodium oleate at a concentration of 0.12 mM (Fig. 3C) there was no additional effect on the rate of transfer of isotopically labelled cholesteryl esters (upper panel) but there was a 70% increase in the net mass transfer of cholesteryl esters from HDL to LDL (lower panel). Other changes during incubation of HDL and LDL in the presence of CETP and sodium oieate

To define the effects of CETP and sodium oleate on lipoprotein constituents other than cholesteryl esters, the plasma fraction of density 1.019-1.21 g/ml was isolated from each of two

subjects and incubated at 37 o C for varying times in the presence of CETP (3.2 units/ml) and sodium oleate (0.24 mM). Samples were subsequently subjected to size exclusion chromatography without prior ultracentrifugation to define the distribution of lipids and apolipoproteins across the lipoprotein spectrum and to non-denaturing gradient gel electrophoresis of the ultracentrifuged 1.25 g/ml supematant to define the particle size distribution of HDL. Since the changes observed in the two experiments were qualitatively very similar, results’ are presented for only one of the studies. (a) Distribution of lipid constituents between HDL and LDL (Figs. 4 and 5). Incubation of the mix-

ture of HDL and LDL at 37” C for 24 h in the

18 had been transferred to the LDL fraction; at this stage, there was at most only minimal redistribution of the surface lipid constituents, unesterified cholesterol (Fig. 5A) and phospholipids (Fig. 5B). Beyond 3 h, the net mass transfers of cholesteryl esters and triacylglycerol from HDL to LDL continued progressively until, by 24 h the HDL fraction was markedly depleted of each constituent (Fig. 4). After 12 h of incubation there was also a demonstrable transfer of unesterified cholesterol and, to a lesser extent, phospholipid from HDL to LDL (Fig. 5). In the case of each of these surface constituents, the elution of the HDL peaks was retarded in samples which had been incubated for 12 or more hours in the presence of CETP and sodium oleate: this is consistent with the forma-

absence of CETP and sodium oleate or in the presence of sodium oleate alone had no effect on the recovery of lipid constituents in the total incubation mixture nor on the distribution of any of the constituents between HDL and LDL (result not shown). After incubation for up to 24 h in the presence of CETP and sodium oleate, there was again no change in the concentration of cholesteryl esters, triacylglycerol, unesterified cholesterol or phospholipids in the total incubation mixture (result not shown). Under these conditions, however, there were obvious redistributions of constituents between HDL and LDL. After 3 h of incubation in the presence of CETP and sodium oleate, about 40% of the HDL cholesteryl esters (Fig. 4A) and about 30% of the HDL triacylglycerol (Fig. 4B)

A Sodium

B oleate

CETP

CETP

+ Sodium

oleate

F

HDL

HDL

I

.g...a

ipA i -

. .. .. .. ...

,.o...o

9

LDL

:-

. .. .. ... .

LDL

t-

1.2 Hours

ot

2b

4 0

12

24

Incubation

Fig. 3. Transfers of cholesteryl esters from HDL to LDL promoted by CETP: effects of sodium oleate. The plasma fraction of d =1.019-1.21 g/ml was isolated and supplemented with a tracer amount of HDL labelled with 3H in the cholesteryl ester moiety. Aliquots of this mixture (final incubation concentration of LDL cholesteryl esters 0.73 mM, HDL cholesteryl esters 0.50 mM and [‘H]cholesteryl esters 420000 cpm/ml) were supplemented with sodium oleate (0.12 mh4) (panel A), CETP (2.4 units/ml) (panel B) and CETP (2.4 units/ml) plus sodium oleate (0.12 mM) (panel C) and incubated at 37OC as indicated. Final incubation volumes were 300 pl. Following incubation, samples were processed as described in the legend to Fig. 2. The upper panels show the specific activities (cpm/nmol) of cholesteryl esters in LDL and HDL as a function of tune. The lower panels show the percentage of the HDL cholesteryl ester mass which had been transferred to LDL at each time point. Each point represents the mean of duplicate estimations.

19 and sodium oleate there was an appearance of a minor population of very small particles of radius 3.7 nm, although the original populations of particles of Stoke’s radius 5.2 nm and 4.3 nm remained prominent. By 6 h, the particles of radius 4.3 run were markedly reduced and the very small particles of radius 3.7 nm were now the major population; the HDL, (Stoke’s radius 5.2 nm), however, were still prominent at this stage. After 12 h of incubation, both the original HDL, and HDL, had disappeared and by 24 h, the HDL consisted of a single population of very small

tion of HDL which, as a consequence of becoming depleted of core lipids, had been reduced in particle size. (b) HDL particle size (Fig. 6). In a non-incubated sample of the mixture of HDL and LDL, the HDL consisted of two main populations of particles as revealed by gradient gel electrophoresis: one of Stoke’s radius 5.2 nm (HDL,) and the other of Stoke’s radius 4.3 nm (HDL,). Incubation for 24 h in the presence of sodium oleate alone produced no change (result not shown). After incubation for 3 h in the presence of CETP

A

B

Cholesteryl

ester

Tnacylglycerol 40

LDL

HDL

I

I I

Oh

20

0 40 E ;

20

a

E

6 0 $ -

b E s!

0

200

‘ii

100

x _” 20

40

t

c 0

s 5 ~

E 5

0

\

0

:

200

0 40

100

20

P v 0

0 200

40

100

20

0

0 15

20

25

30

35

Fracton

15

20

25

30

35

number

Fig. 4. Changes to the distribution of cholesteryl esters and triacylglycerol between LDL and HDL during incubation with CETP and sodium oleate. The plasma fraction of d=1.019-1.21 g/ml was isolated and supplemented with CETP (final incubation concentration 3.2 units/ml) and sodium oleate (final concentration 0.24 mM) and incubated at 37 o C for 0, 3, 6, 12 and 24 h as indicated. Incubations were stopped by placing the samples on ice. Mixtures were then subjected to size exclusion chromatography on a Superose-6 column connected to a high pressure pump. The lipoproteins were eluted with TBS at a flow rate of 30 ml/h and fractions of 0.6 ml each were collected for assaying cholesteryl esters (panel A) and triacylglycerol (panel B) . The vertical broken lines indicate the positions of elution of LDL and HDL as defined by the elution of the peaks of apo B and apo A-I respectively, in non-incubated samples.

20 TABLE 2 INCUBATION OF A MIXTURE OF HDL AND LDL IN THE PRESENCE OF CETP AND SODIUM OLEATE: EFFECTS ON THE COMPOSITION OF HDL Incubation (h)

HDL: percent composition by mass

0 3 6 12 24

Apo A-I

Apo A-II

PL*

UC *

CE *

TG *

29.4 25.3 41.1 51.4 53.2

8.6 10.3 12.0 15.0 15.6

24.9 25.3 24.1 23.8 23.1

3.8 4.3 4.3 2.2 1.9

26.3 18.3 12.3 3.8 3.2

7.9 6.5 6.3 3.8 2.9

These results were obtained from the same experiment as presented in Figs. 4-7. The individual column fractions which contained HDL as defined by apo A-I and apo A-II were pooled and assayed for chemical composition. * PL, phospholipids; UC, unesterified cholesterol; CE, cholesteryl esters; TG, triacylglycerol.

particles of radius 3.7 nm. The changes in composition of HDL which accompanied the changes in particle size are shown in Table 2.

(c) Distribution of apolipoproteins within the HDL fraction (Fig. 7). There was a quantitative recovery of both apo A-I and apo A-II in the HDL 0

A Unestenfwd

a0

cholesterol

LDL

Phosphollpids Oh

HDL

40

0 a0

3h

z . aJ 40 a E c

0 00

6h

5 kl ;; Y

40

e ” D a pz ; a0

i

I

aJ ‘: 5

40

C 0C

40

C 15

20

25

30

35 Fraction

15

20

25

30

35

number

Fig. 5. Changes to the distribution of unesterified cholesterol and phospholipids between LDL and HDL during incubation with CETP and sodium oleate. The incubation conditions and processing of samples were as described in the legend to Fig 4. The distribution of unesterified cholesterol (panel A) and phospholipids (panel B) are shown.

21 fraction after incubation in the presence and in the absence of CETP and sodium oleate. There was no evidence of a transfer of either apolipoprotein to the LDL fraction in any case. Thus, only the size exclusion chromatography fractions containing HDL are shown in Fig. 7. In non-incubated samples (0 h) and in samples incubated for 24 h in the absence of CETP and sodium oleate (result not shown), apo A-I and apo A-II coeluted, each peaking between fractions 27 and 28. There was relatively little change after 3 h

Apohpoprotems

25

10

0

3

50

20

25

IO

0

3 20

10

0 22

24

Zb

28 Fracl~on

30

32

34

36

number

Fig. 7. Changes to the distribution of apo A-I and apo A-II during incubation of LDL and HDL with CETP and sodium oleate. The incubation conditions were as described in the legend to Fig. 4. Fractions recovered from the size exclusion and apo A-II column were assayed for apo A-I (0 -0) 0). Since each apolipoprotein was recovered quanti(Otatively in fractions 25-34, only these fractions are shown.

\ 5/2

4’3

Stokes’

;9 rodlus

37 (nm)

Fig. 6. Changes to the particle size distribution of HDL during incubation of LDL and HDL with CETP and sodium oleate. The incubation conditions were as described in the legend to Figure 4. Following incubation, samples were subjected to ultracentrifugation at d =1.25 g/ml; the supematant fraction was then further separated by non-denaturing gradient gel electrophoresis on 4-30% gradient gels to define the particle size distribution of HDL. The profiles represent the densitometric scans of gels stained with Coomassie blue. Stoke’s radii (nm) were calculated from known high molecular weight standards.

of incubation. By 6 h, however, and even more so after 12 h, the elution of apo A-I was retarded relative to apo A-II, consistent with the newly formed very small particles (Fig. 6) being enriched with apo A-I. By 24 h, the elution of apo A-II was also retarded with the two apolipoproteins again coeluting, although in this case the peaks were in fraction 30.

Discussion

It has been well documented that CETP catalyses bidirectional transfers of cholesteryl esters between HDL and LDL [3,4,16,17] at a rate which

22 is rapid relative to that of lipoprotein catabolism [5]. It would be predicted, therefore, that the pools of cholesteryl esters in LDL and HDL are close to equilibrium in vivo. Indeed, when HDL and LDL are incubated in vitro in the presence of lipoprotern-deficient plasma as a crude source of CETP, there is a rapid transfer of isotopically labelled cholesteryl esters between the 2 fractions but in incubations lasting for up to 6 h, there is minimal net mass transfer of cholesteryl esters in either direction [5,16]. In the present studies the incubations were prolonged for up to 24 h and contained purified rather than crude preparations of CETP. Under these conditions there were obvious net mass transfers of cholesteryl esters from HDL to LDL, indicating either that the pools of cholesteryl esters in HDL and LDL had not yet come into equilibrium at the commencement of the incubation or, if already equilibrated, that the equilibrium had in some way been disrupted during the incubation. This issue was addressed by supplementing incubation mixtures of HDL, LDL and CETP with a tracer amount of HDL in which the cholesteryl ester moiety had been labelled isotopically during prior incubations with [3H]cholesterol and LCAT. It should be noted that this method of labelling modifies the HDL in such a way that there is a reduction in the rate at which CETP promotes the transfer of cholesteryl esters out of the modified particles [18]. Thus, the rate at which the labelled cholesteryl esters equilibrated between the HDL and LDL in the present studies would have represented an underestimate of the true rate of equilibration. Yet, within 3 h of adding the labelled HDL to the incubation, the specific activity of cholesteryl esters was identical in HDL and LDL, indicating that the added [3H]cholesteryl esters had equilibrated throughout the initially unlabelled pools in both fractions. Despite this, over the next 21 h there was a time-dependent, CETPmediated net mass transfer of cholesteryl esters from HDL to LDL. Furthermore, although neither fatty acid-poor albumin nor sodium oleate had any apparent effect on the rate of equilibration of the [3H]cholesteryl esters, the CETP-mediated net mass transfer from HDL to LDL were inhibited by the albumin but enhanced by sodium oleate. Thus, we have concluded: (i) that the net mass

transfer of cholesteryl esters from HDL to LDL promoted by CETP cannot be explained in the simple terms of two non-equilibrated pools coming into equilibrium and (ii) that the process responsible for the net mass transfer is inhibited by fatty acid-poor albumin and stimulated by sodium oleate. The mechanism by which CETP and sodium oleate interact to promote a net mass transfer of cholesteryl esters from HDL to LDL is not addressed by these studies. However, some insights may be obtained by reviewing the postulated mechanism of action of CETP. Two general models have been proposed: (i) a shuttle model [19] and (ii) a ternary collision complex model [20]. According to the shuttle model, CETP picks up molecules of cholesteryl ester and triacylglycerol and circulates as a CETP-lipid complex [21]. It has been postulated that this complex binds transiently to lipoprotein particles during which there is an exchange of lipids between CETP and the lipoprotein. The CETP-lipid complex then dissociates from the lipoprotein and circulates in plasma until again it binds to a lipoprotein particle and again exchanges lipids. In this way, CETP acts as a shuttle which promotes an exchange and thus an equilibration of cholesteryl esters and triacylglycerol between lipoprotein particles; this includes an exchange between particles within a given lipoprotein class as well as between particles in different classes [19]. The net effect of the process is an exchange of lipid molecules between lipoprotein particles, whether cholesteryl ester for cholesteryl ester, cholesteryl ester for triacylglycerol or triacylglycerol for triacylglycerol; it does not, however, result in a net change in the total core lipid content of lipoprotein particles. Thus, while the shuttle model can explain the heteroexchange of cholesteryl ester for triacylglycerol promoted by CETP in incubations of HDL and VLDL [22], it cannot account for the net mass transfer of cholesteryl esters from HDL to LDL observed in the present studies. CETP has also been suggested to act by mediating a ternary collision complex with HDL and LDL during which lipid constituents redistribute between the lipoprotein particles [20]. It is possible that the formation of a ternary complex of HDL, CETP and LDL may lead to a remodelling

23 of the lipoprotein particles which results in a net movement of constituents from HDL to LDL. In these terms, one consequence of the formation of a ternary complex may be the conversion of HDL into the small, lipid-poor, protein-rich particles observed in the present studies. Furthermore, since it is known that non-esterified fatty acids enhance the binding of CETP to lipoproteins [8], it is not unreasonable to speculate that non-esterified fatty acids may also enhance the formation of ternary complexes. We postulate that both the shuttle and the ternary complex mechanisms operate and that CETP may act both to promote the shuttling of lipids between HDL and LDL and the formation of collision complexes between the lipoprotein particles. In the absence of non-esterified fatty acids on the lipoprotein surface, we postulate that the shuttle mechanism predominates and that the major lipid transfer process is one of exchange. As the non-esterified fatty acid concentration on the surface of lipoproteins is increased, and there is a consequent enhancement in the binding of CETP to the particles, we postulate that the formation of ternary complexes of HDL-CETP-LDL is favoured, resulting in a net mass transfer of core lipids from HDL to LDL. There is strong circumstantial evidence from other studies that non-esterified fatty acids on the surface of plasma lipoproteins interact with and modify the function of CETP. For example, when a mixture of HDL and CETP is supplemented by the addition of very low density lipoproteins (VLDL) which have been pretreated with lipoprotein lipase, there is an enhanced formation of small HDL particles [23]. It has also been reported that lipolytic products, specifically non-esterified fatty acids, result in both an increase in the binding of CETP to lipoproteins and an increase in the rate of cholesteryl ester transfer from HDL to VLDL [8]. The synergistic effects of hepatic lipase and CETP in promoting the reduction in size of HDL [24] may also reflect an involvement of non-esterified fatty acids. In the present studies, CETP promoted a net mass transfer of cholesteryl esters from HDL to LDL even in incubations which had not been supplemented with exogenous sodium oleate. However. the observation that this CETP-media-

ted net mass transfer was markedly inhibited by fatty acid-poor but not by fatty acid-rich albumin suggested that albumin and CETP may compete for the endogenous non-esterified fatty acids which exist as normal components of the surface of plasma lipoproteins [8]. It will be of interest to investigate whether the capacity of albumin to inhibit the CETP-mediated net mass transfer of cholesteryl esters from HDL to LDL correlates with its content of non-esterified fatty acids over the range of concentrations encountered physiologically. The interaction of CETP and sodium oleate produced changes which extended beyond a simple redistribution of cholesteryl esters between HDL and LDL (Figs. 4-7). Initially, there was a transfer of core constituents (both cholesteryl esters and triacylglycerol) from HDL to LDL with minimal change to the distribution of surface components. However, as the process progressed there was also a loss of surface lipids from the HDL fraction, leaving HDL particles which were relatively enriched in protein (Table 2) and markedly reduced in size. Ultimately, the original populations of HDL, and HDL, were replaced completely by a single population of very small particles of Stoke’s radius 3.7 nm. Coinciding with the loss of lipids and the reduction in size of HDL particles, there were redistributions of the HDL apolipoproteins. The observation that there was an initial delay in the elution of apo A-I but not apo A-II suggested a preferential modification to apo A-I-containing particles. It was only when the HDL had been converted quantitatively into particles of radius 3.7 nm that the apo A-II was retarded to the same extent as that of apo A-I and the two apolipoproteins again coeluted. The implication that apo A-II-containing HDL may therefore be relatively resistant to reduction in particle size is supported by previous studies in which human whole plasma was incubated in vitro [25]. In conclusion, these studies performed in vitro do not address issues of physiological significance. Further investigation is required to determine whether variations in plasma non-esterified fatty acid metabolism play a role, either physiologically or pathologically, in regulating the distribution of cholesterol between the non-atherogenic HDL and the atherogenic LDL fractions.

24 Acknowledgements

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