O.V. Rajaram

The interaction of cholesteryl ester transfer protein and unesterified fatty acids promotes a reduction in the particle size of high-density lipoproteins.

Purified human cholesteryl ester transfer protein (CETP) has been found, under certain conditions, to promote changes to the particle size distribution of high-density lipoproteins (HDL) which are comparable to those attributed to a putative HDL conversion factor. When preparations of either the conversion factor or CETP are incubated with HDL3 in the presence of very-low-density lipoproteins (VLDL) or low-density lipoproteins (LDL), the HDL3 are converted to very small particles. The possibility that the conversion factor may be identical to CETP was supported by two observations: (1) CETP was found to be the main protein constituent of preparations of the conversion factor and (2) an antibody to CETP not only abolished the cholesteryl ester transfer activity of the conversion factor preparations but also inhibited changes to HDL particle size. In additional studies, the changes to HDL particle size promoted by purified CETP were inhibited by the presence of fatty-acid-free bovine serum albumin; by contrast, albumin had no effect on the cholesteryl ester transfer activity of the CETP. The possibility that albumin may inhibit changes to HDL particle size by removing unesterified fatty acids from either the lipoproteins or CETP was tested by adding exogenous unesterified fatty acids to the incubations. In incubations of HDL with either VLDL or LDL, sodium oleate had no effect on HDL particle size. However, when CETP was also present in the incubation mixtures the capacity of CETP to reduce the particle size of HDL was greatly enhanced by the addition of sodium oleate. It is concluded that the changes in HDL particle size which were previously attributed to an HDL conversion factor can be explained in terms of the interacting effects of CETP and unesterified fatty acids.

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

Cholesteryl esters readily exchanges 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.

Partial purification and characterization of a triacylglycerol-transfer protein from rabbit serum

Rabbit lipoprotein-free serum was found to contain a protein fraction that mediates the transfer of [3H]triacylglycerol from low-density lipoprotein to high-density lipoprotein. Fractionation of rabbit lipoprotein-free serum by DEAE-Sephadex chromatography, ammonium sulphate precipitation, concanavalin-A-Sepharose chromatography, Sephadex G-200 gel filtration, phenyl-Sepharose chromatography and Sephadex G-100 gel filtration, yielded a preparation that had a 500-fold increase in transfer activity compared to that of the starting sample. The transfer activity appeared to reside in a glycoprotein of molecular weight in the range 100 000-155 000 and an isoelectric point at pH 9.

Species differences in the activity of a serum triglyceride transferring factor.

Low density lipoproteins (LDL), endogenously labelled with 3H in the triglyceride moiety, were isolated from rabbit serum and subsequently incubated in vitro at 37 degrees C with unlabelled preparations of rabbit high density lipoproteins (HDL) or very low density lipoproteins (VLDL). In incubations performed in the presence of phosphate buffer, there was no significant transfer of [3H]triglyceride from LDL to either HDL or VLDL; but when rabbit lipoprotein-free serum (the dialysed 1.21 g/ml infranatant) was added, transfer was apparent to both HDL and VLDL. The triglyceride transferring activity of the lipoprotein-free serum was abolished by heating at 85 degrees C for 10 min; all the transferring activity was found in the fraction which precipitated with ammonium sulphate at a concentration of less than 50% saturation. In direct contrast to the rabbit studies, rat serum failed to show a comparable process of triglyceride transfer. In subsequent experiments, mixtures of labelled LDL and unlabelled VLDL isolated either from rabbits or from rats were incubated with lipoprotein-free rabbit, rat or human serum. The lipoprotein-free serum of both the rabbit and man was effective in promoting transfer of 30--50% of LDL [3H]triglyceride into VLDL, regardless of the species origin of the lipoproteins. By contrast the lipoprotein-free serum of rats was only slightly more effective than buffer alone in promoting such transfers. It has been concluded that rabbit and human serum contains a triglyceride transferring factor of far greater activity than that in rat serum.

Increases in the particle size of high-density lipoproteins induced by purified lecithin: cholesterol acyltransferase: effect of low-density lipoproteins

Homogeneous subpopulations of human high-density lipoproteins subfraction-3 (HDL3) have been incubated at 37 degrees C with purified lecithin: cholesterol acyltransferase, human serum albumin and varying concentrations of human low-density lipoproteins (LDL). Changes in HDL particle size and composition during these incubations were monitored. Incubation of HDL3a (particle radius 4.3 nm) in the absence of LDL resulted in an esterification of more than 70% of the HDL free cholesterol after 24 h of incubation. This, however, was sufficient to increase the HDL cholesteryl ester by less than 10% and was not accompanied by any change in particle size. When this mixture was incubated in the presence of progressively increasing concentrations of LDL, which donated free cholesterol to the HDL, the molar rate of production of cholesteryl ester was much greater; at the highest LDL concentration HDL cholesteryl ester content was almost doubled after 24 h and there was an increase in the HDL particle size up to the HDL2 range. In the case of HDL3b (radius 3.9 nm), there were again only minimal changes in particle size in incubations not containing LDL. In the presence of the highest concentration of LDL tested, however, the particles were again enlarged into the HDL2 size range after 24 h incubation. These HDL2-like particles were markedly enriched with cholesteryl ester but depleted of phospholipid and free cholesterol when compared with native HDL2. Furthermore, the ratio of apolipoprotein A-I to apolipoprotein A-II resembled that in the parent-HDL3 and was very much lower than that in native HDL2. It has been concluded that purified lecithin: cholesterol acyltransferase is capable of increasing the size of HDL3 towards that of HDL2 but that other factors must operate in vivo to modulate the chemical composition of the enlarged particles.

Reactivity of human lipoproteins with purified lecithin: cholesterol acyltransferase during incubations in vitro

Studies have been performed to determine the proportion of the esterified cholesterol in high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) that is attributable to a direct action of lecithin: cholesterol acyltransferase on each lipoprotein fraction. Esterification of [3H]cholesterol was examined in 37 degrees C incubations of either: (a) unseparated whole plasma, (b) plasma reconstituted after prior ultracentrifugation to separate the 1.21 g/ml supernatant, (c) a mixture comprising the 1.21 g/ml supernatant of plasma and purified lecithin: cholesterol acyltransferase or (d) the same mixture as (c) after supplementation with a preparation of partially purified lipid transfer protein. Each of these incubations was performed using samples collected from four different subjects, two of whom had normal and two of whom had elevated concentrations of plasma triacylglycerol. At the completion of 3-h incubations, the lipoproteins were separated into multiple fractions by gel filtration to obtain a continuous profile of esterified [3H]cholesterol across the whole spectrum of lipoproteins. There was an appearance of esterified [3H]cholesterol in each of the major lipoprotein fractions in all incubations. In unseparated plasma, 56% of the total (mean of four experiments) was in HDL, 33% in LDL and 11% in VLDL. A comparable distribution was observed in the incubations of reconstituted plasma and in the samples to which partially purified lipid transfer protein had been added. In the absence of lipid transfer protein activity in incubations containing purified lecithin: cholesterol acyltransferase, 73% of the esterified [3H]cholesterol was in HDL, 25% in LDL and only 1% in VLDL. It has been concluded that at physiological concentrations of lipoproteins, 70-80% of the cholesterol esterifying action of lecithin: cholesterol acyltransferase is confined to the HDL fraction, with most of the remainder involving the LDL fraction. Of the newly formed esterified cholesterol incorporated into LDL during incubations of unseparated plasma, it was apparent that more than 70% was independent of activity of the lipid transfer protein. Of that incorporated into VLDL in unseparated plasma, in contrast, almost 90% was derived as a transfer from other fractions as a consequence of activity of the lipid transfer protein.

Sodium oleate dissociates the heteroexchange of cholesteryl esters and triacylglycerol between HDL and triacylglycerol-rich lipoproteins.

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 ester 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 less than 0.05) and a 14% increase in the transfer of triacylglycerol into HDL (P less than 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 less than 0.001) and the transfer of triacylglycerol into HDL was decreased by 25% (P less than 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.

6 Enzymes involved in plasma cholesterol transport

Summary Regulation of plasma cholesterol transport is to large extent a function of factors that regulate plasma cholesterol esterification and the transfers of cholesteryl esters between plasma lipoprotein fractions. Plasma cholesterol esterification is catalysed by the action of lecithin: cholesterol acyltransferase on lipids on the surface of HDL, while the transfers of cholesteryl esters require activity of a specific lipid transfer protein. Esterification of the cholesterol on the surface of HDL generates a concentration gradient down which unesterified cholesterol moves from tissues into the plasma. Once within the plasma and esterified, the newly formed cholesteryl esters are incorporated initially into the core of HDL particles before being redistributed to other classes of lipoproteins. The end result of these processes of esterification and transfer is that most of the cholesterol in human plasma is accommodated within the core of LDL, where its transport is a function of the highly regulated uptake by tissues of intact LDL particles. The capacity of HDL to act as substrates for lecithin: cholesterol acyltransferase varies inversely with HDL particle size. Thus, factors such as the concentration of triglyceride-rich lipoproteins and activities of the lipid transfer protein, hepatic lipase, lipoprotein lipase and the HDL conversion protein, which are known to influence HDL particle size, may also be important as regulators of plasma cholesterol esterification.

Influence of lipoprotein concentration on the exchanges of triacylglycerol between rabbit plasma low density and high density lipoproteins.

Molecular exchanges of triacylglycerol between rabbit serum low density lipoproteins (LDL) and high density lipoproteins (HDL) have been studied in 37 degrees C incubations performed in the presence of rabbit lipoprotein-free serum as a source of the triacylglycerol transfer protein. The molar rate of exchange of triacylglycerol between the two fractions increased with increasing incubation concentrations of LDL but was decreased as the HDL concentration was increased. When the concentration of both LDL and HDL was increased in parallel there was an increase in the molar rate of triacylglycerol exchange between the two fractions which flattened at higher concentrations, suggesting that the process was saturable. Fractionation of rabbit lipoprotein-free serum on a column of Sephadex G-200 resulted in the elution of the triacylglycerol transfer activity in a single peak. Addition of LDL to the lipoprotein-free serum had no effect on the position of elution of the triacylglycerol transfer activity. Addition of HDL, however, resulted in an elution of the transfer activity as two peaks, one in the original position and the other in the same position as HDL. The results of the kinetic studies have been interpreted in terms of a binding of the triacylglycerol transfer protein to HDL, but not to LDL.

Factors Regulating the Distribution of Cholesterol Between LDL and HDL

The importance of understanding factors which influence the partitioning of cholesterol between 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)1 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 lipoprotein fraction5 (Fig.l), the cholesteryl esters in these two lipoproteins must be close to equilibrium in vivo. 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 have recently reported, however, that the CETP-mediated equilibrium between LDL and HDL can be disrupted by Na oleate which, as a consequence, promotes a shift in the partitioning of cholesteryl esters from the non-atherogenic HDL to the atherogenic LDL6.