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BBALIP 50311 BBA Report

Sodium oleate dissociates the heteroexchange of cholesteryl esters and triacylglycerol between HDL

and triacylglycerol-rich lipoproteins

P.J. Barter, L.B.F. Chang and O.V. Rajaram

(Received 17 September 1990)

Key words: Sodium oleate; Cholesteryl ester transfer protein: Triacylglycerol-rich lipoprotein

Mixtures of human high-density lipoproteins (HDL) and triacylglycerol-rich lipoproteins (TGRL) have been incubated in the presence of partially pure cholesteryl ester transfer protein (CETP). There were net mass transfers of cholesteryl esters from HDL to TGRL and of triacylglycerol from TGRL to HDL which were accompanied by the formation of minor subpopulations of small HDL particles. When the mixture of HDL, TGRL and CETP was supplemented with fatty acid-poor bovine serum albumin (40 mg/ml) there was a 7% reduction in the transfer of cholesteryl esters out of HDL (P < 0.05) and a 14% increase in the transfer of triacylglycerol into HDL ( P < 0.05); there was also a reduction in the formation of very small HDL particles. In contrast, when the mixture of HDL, TGRL and CETP was supplemented with 0.16 mM sodium oleate the transfer of cholesteryl esters out of HDL was increased by 31% (P -c 0.001) and the transfer of triacylglycerol into HDL was decreased by 25% (P < 0.01); under these conditions the formation of very small HDL particles was enhanced. It has been concluded that in the presence of sodium oleate, there is a dissociation of the CETP-mediated heteroexchange of cholesteryl esters and triacylglycerol between HDL and TGRL.

The cholesteryl ester transfer protein (CETP) plays a

fundamental role in plasma lipid transport by catalysing

transfers of both cholesteryl esters and triacylglycerol

between plasma lipoprotein fractions [l]. In the case of high-density lipoproteins (HDL) and triacylglycerol-rich

lipoproteins (TGRL), CETP promotes a net mass trans- fer of cholesteryl esters from HDL to TGRL in ex-

change for a reciprocal transfer of triacylglycerol in the reverse direction [2]. In some studies this heteroex- change has been found to be equimolar [3] such that each molecule of cholesteryl ester transferred out of HDL is replaced by a molecule of triacylglycerol. Since the molecular volume of triacylglycerol is greater than that of cholesteryl ester [4], such an exchange results in the formation of HDL which are both triacylglycerol-

enriched and enlarged [5]. In recent studies, however, it

has been reported that the formation of large HDL particles during incubations with TGRL and CETP is abolished when the mixture also contains sodium oleate

Abbreviations: CETP, cholesteryl ester transfer protein; HDL. high- density lipoprotein; LDL, low-density lipoprotein; TGRL, tri-

acylglycerol-rich lipoprotein.

Correspondence: P.J. Barter, Baker Medical Research Institute, Com-

mercial Road, Prahran. Melbourne, Victoria (Australia).

which interacts in some way with CETP so as to favour

the creation of very small HDL particles [6]. It was postulated that the small particles were formed as a

consequence of transfers of cholesteryl esters from HDL to TGRL which were not balanced by mole for mole reciprocal transfers of triacylglycerol and which there-

fore left the HDL depleted of total core lipids. This issue has been addressed in the present studies

which investigate the influence of sodium oleate on the

heteroexchange of cholesteryl esters for triacylglycerol in incubations of HDL, TGRL and CETP. It has been found that while, in confirmation of a previous report

[7], sodium oleate enhances the transfer of cholesteryl esters from HDL to TGRL, it also inhibits the recipro- cal transfer of triacylglycerol from TGRL to HDL; thus the HDL become depleted of total core lipid and

markedly reduced in particle size. Blood from healthy male and female subjects aged

23-44 was collected about I h after a light breakfast into tubes containing disodium EDTA (7 mg/ml) and placed immediately on ice. Lipoprotein fractions (TGRL, d < 1.006 g/ml and HDL, 1.070-1.21 g/ml) were isolated at 4°C by sequential ultracentrifugation as described previously [6]. Partial purification of CETP was also performed as described previously [6] to pro- duce a preparation which was purified about 5000-fold compared with lipoprotein-free plasma. Cholesteryl es-

0005-2760/90/$03.50 G 1990 Elsevier Science Publishers B.V. (Biomedical Division)

ter transfer activity in lo-100 ~1 of the test sample was measured in terms of its capacity to promote the trans- fer of [3H]cholesteryl ester from human LDL to human HDL [8], the number of transfer units being the fraction of the LDL [3H]cholesteryl ester transferred during a 3-h incubation. This assay was linear so long as the transfer of [3H]cholesteryl ester was less than 40%. Using this assay system, human lipoprotein-free plasma contains approx. 1 transfer unit/ml.

Incubations of mixtures of HDL, TGRL and CETP were carried out in stoppered tubes in a shaking water bath at 37” C. Non-incubated control samples were stored at 4” C. In some of the experiments the incuba- tion mixtures were supplemented with either sodium oleate (Sigma Chemical, St Louis, MO, U.S.A.) at a final concentration of 0.16 mM or with fatty acid-poor bovine serum albumin (Sigma) at a final concentration of 40 mg/ml. Following incubation, samples were placed on ice while awaiting further processing. The particle size distribution of HDL was determined by gradient gel electrophoresis on non-denaturing 4-30% polyacryl- amide gels (Pharmacia-LKB) [9] as performed previ- ously [6]. The TGRL and HDL fractions were separated by 5 h of ultracentrifugation at 1.019 g/ml in a Beck- man TL-100 table top ultracentrifuge using a Beckman TLA-100.2 rotor at a speed of 100000 rpm; complete- ness of separation was established by the consistent observation that apolipoprotein (apo)B was measurable only in the supernatant fraction while apo A-I was measurable only in the infranatant. All chemical assays were performed on a Cobas-Bio centrifugal analyser (Roche Diagnostics, Zurich, Switzerland) as described previously [6].

The effects of incubation on the particle size distri- bution of HDL are illustrated in the representative experiment (five such studies were performed) shown in Fig. 1. The non-incubated HDL consisted of two popu- lations of particles of Stokes’ radii 5.2 nm (HDL,,) and 4.3 nm (HDL,,) (Profile I). Following 24 h of incuba- tion of this HDL in a mixture containing TGRL and CETP there was an appearance of two additional popu- lations of smaller HDL particles of radii 3.9 nm and 3.7 nm (Profile II). When the mixture of HDL, TGRL and CETP was further supplemented with fatty acid-free bovine serum albumin, there was a marked inhibition in the formation of the population of smallest particles (Profile III). By contrast, when the incubation mixture of HDL, TGRL and CETP was supplemented with 0.16 mM sodium oleate, the formation of very small HDL particles was enhanced; under these conditions, the original HDL,, and HDL,, particles were converted almost completely into the very small particles of Stokes’s radius 3.7 nm (Profile IV). If, however, fatty acid-poor bovine serum albumin was included in the incubation of HDL, TGRL, CETP and sodium oleate, the effects of sodium oleate were abolished (Profile V).

/ /\\

5.2 4.3 3.9 3.7

STOKES’ RADIUS (nm)

I

II

III

IV

V

Fig. 1. Effects of albumin and sodium oleate on the changes to HDL

particle size during incubation with TGRL and CETP. Aliquots of

HDL (final concentration of HDL cholesterol 0.38 mM) were mixed

with TGRL (final TGRL triacylglycerol concentration 0.44 mM) and

CETP (final concentration 3.1 units per ml) and kept at 4O C (Profile

I) or incubated at 37’C for 24 h either without further addition

(Profile II) or after being supplemented with fatty acid-poor bovine

serum albumin (final concentration 40 mg/ml) (Profile III), sodium

oleate (final concentration 0.16 mM) (Profile IV) or both sodium

oleate (0.16 mM) and bovine serum albumin (40 mg/ml) (Profile V).

Following incubation, samples were subjected to ultracentrifugation

to recover the 1.25 g/ml supematant fraction which was further

separated by non-denaturing polyacrylamide gradient gel electro-

phoresis on 4-30s gradient gels to define the particle size distribution

of HDL. The profiles represent the densitometric scans of gels stained

with Coomassie blue. Stokes’ radii (nm) were calculated from known

high molecular weight standards.

Incubation of a mixture of HDL and TGRL in the presence of either bovine serum albumin or sodium oleate in the absence of CETP resulted in no changes to the particle size of HDL which remained identical to that in the non-incubated sample (result not shown).

To determine whether the enhanced formation of very small HDL in the presence of sodium oleate was the consequence, as suggested previously [6], of an inhibition of the transfer of triacylglycerol into HDL, studies were performed to define the effects of incuba- tion on the distribution of cholesteryl esters and tri- acylglycerol between HDL and TGRL (Table I). In- cubation of HDL and TGRL for 24 h in the presence of CETP as the sole addition resulted in a net mass trans-

296

fer of cholesteryl esters from HDL to TGRL of 201

nmol/ml (Table I). This was accompanied by a recipro- cal transfer of triacylglycerol from TGRL to HDL of

116 nmol/ml. The molar ratio of these transfers, tri-

acylglycerol/cholesteryl esters, was 0.58. When the mix-

tures of HDL, TGRL and CETP also contained fatty acid-poor bovine serum albumin, there was a 7% reduc-

tion in the transfer of cholesteryl esters from HDL to

TGRL (P < 0.05), while the transfer of triacylglycerol from TGRL to HDL was increased by 14% (P < 0.05); as a consequence, the molar ratio of the transfers, triacylglycerol/cholesteryl esters, increased to 0.71 (P < 0.01). In contrast to the effects of albumin, the ad- dition of sodium oleate to mixtures of HDL, TGRL and

CETP resulted in a 31% increase in the transfer of cholesteryl esters from HDL to TGRL and a 25%

decrease in the reciprocal transfer of triacylglycerol into

HDL (P -c 0.01). The resulting molar ratio of the trans-

fer, triacylglycerol/cholesteryl esters, under these con- ditions, fell to 0.33 (P < 0.001). When mixtures of HDL

and TGRL were incubated at 37 o C for 24 h with either

bovine serum albumin or sodium oleate in the absence of CETP, there was no redistribution of either cholesteryl esters or triacylglycerol between the lipopro-

tein fractions (result not shown).

net reduction in the total core lipid (cholesteryl esters plus triacylglycerol) content of HDL particles.

There is previous circumstantial evidence that non- esterified fatty acids on the surface of plasma lipopro-

teins interact with and modified the function of CETP.

It has been reported that when a mixture of HDL and

CETP is supplemented by the addition of TGRL which

have been pretreated with lipoprotein lipase, there is an enhanced formation of small HDL particles [lo]. It has

also been found that lipolytic products, specifically non- esterified fatty acids, promote an increase in the binding of CETP to lipoproteins and an increase in the rate of cholesteryl ester transfer from HDL to TGRL [7]. The synergism between CETP and hepatic lipase in reducing

the particle size of HDL [ll] may also reflect an in-

volvement of nonesterified fatty acids. In direct contrast with the effects of sodium oleate,

albumin was found in the present studies to reduce the

CETP-mediated transfer of cholesteryl esters from HDL

to TGRL and to increase that of triacylglycerol in the reverse direction. This result probably reflects the

capacity of albumin to bind nonesterified fatty acids and thus to deplete plasma lipoproteins of their endoge-

nous nonesterified fatty acids [7] which would otherwise have been available to interact with CETP.

These studies provide fundamental new information The mechanism by which sodium oleate dissociates

regarding the heteroexchange of cholesteryl esters and the heteroexchange of cholesteryl esters and tri- triacylglycerol between HDL and TRGL: while con- acylglycerol between HDL and TGRL is not addressed firming the capacity of sodium oleate to enhance the in these studies, although it is worth speculating briefly CETP-mediated transfer of cholesteryl esters from HDL on what may be occurring. While it is possible that

to TGRL [7], these studies provide the first indication sodium oleate has opposing effects on the capacity of that under the same conditions sodium oleate inhibits CETP to transport cholesteryl esters and triacylglycerol,

the CETP-mediated transfer of triacylglycerol in the it is also possible that sodium oleate exerts its primary

reverse direction from TGRL to HDL. Thus, sodium effect on the lipoproteins, For example, sodium oleate

oleate modulates the activity of CETP so as to favour a may modify HDL particles in such a way that their

TABLE1

Transfers of cholesteryl esters and triacylglycerol between HDL and TGRL

Mixtures of TGRL (d < 1.006 g/ml) and HDL (d 1.070-1.21 g/ml) obtained from each of seven subjects were either: (I) kept at 4°C or incubated

for 24 h at 37 o C in the presence of: (II) CETP (3.1 units/ml), (III) CETP (3.1 units/ml) plus fatty acid-poor bovine serum albumin (BSA, 40

mg/ml) or (IV) CETP (3.1 units/ml) plus sodium oleate (0.16 mM). Final incubation volumes were 300 ~1. After incubation the TGRL and HDL

were separated by ultracentrifugation at d =1.019 g/ml and assayed for cholesteryl esters and triacylglycerol. The transfer was calculated by

reference to the control sample, I, which was kept at 4O C. Values represent mean* S.D. of seven separate experiments, each of which was

performed in duplicate.

Differences between the transfers in incubations containing CETP alone and those supplemented with BSA or sodium oleate were determined by Student’s r-test for paired samples: * P c 0.05, * * P < 0.01, * * * P < 0.001.

Temp. Addition to Cholesteryl esters (nmol/ml) Triacylglycerol (nmol/ml) Ratio of

(04 incubation

mixture TGRL HDL transfer TGRL HDL transfer

transfers

(TG/CE)

I 4 Nil 90+57 443*83 _ 443+23 47k23 _ _

II 37 CETP 293 k 77 244*60 201+ 24 325 + 27 161*20 116510 0.58 f 0.06

III 37 CETP + BSA 276 f 70 255 + 68 187 * +20 310520 177+28 132 * +17 0.71 * * kO.03

IV 37 CETP + sodium oleate 355 f 88 18Ok61 264 *** t37 _ 355&33 132 + 26 87 ** k28 0.33 *** +0.10

capacity to donate core lipid to CETP is enhanced while their capacity to accept core lipid is reduced; this would translate into both an increase in the net efflux of cholesteryl esters out of HDL, and a decrease in the net influx of triacylglycerol. To test this and other possible affects, however, will require further investigation as will the issue of the potential physiolo~~al significance of the phenomenon.

Acknowledgements

This work was supported by grants from the Na- tional Health & Medical Research Council of Australia and from the National Heart Foundation of Australia. The technical assistance of MS Shona Devlin is acknowledged.

297

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