FACTORS REGULATING THE DISTRIBUTION OF CHOLESTEROL BETWEEN LDL AND HDL

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.1), 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•

Samples of the plasma fraction of density 1.019-1.21 gfml containing a mixture of HDL and LDL were incubated at 37oC for 24 hours in the presence of various additions 6• Incubations performed in the presence of buffer alone produced no change in the distribution of cholesteryl esters between the two lipoproteins. Incubation in the presence of CETP (purified 5000-fold relative to lipoprotein-free plasma and added at a concentration of 2.4 units/ml), on the other hand, resulted in a net mass transfer of 40% of the HDL cholesteryl esters to the LDL fraction. When, however, identical mixtures of HDL, LDL and CETP were supplemented with fatty acid-poor human serum albumin at a protein concentration of 40 mg/ml, there was a 50% inhibition of the net mass transfer, with only about 20% 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. The implication that non-esterified fatty acids may, therefore, have been involved in promoting the net mass transfers was supported by finding that 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 estgr mass now being transferred o LDL during the 24 hours of incubation • In other experiemnts it was found that the enhancement of the CETP-mediated net mass transfer by Na oleate was concentration dependent over the range 0.06 mM to 0.24 mM. In the absence of CETP, on the other hand, addition of Na oleate at concentrations of up to 0.24 mM had no effect on the distribution of cholesteryl esters between HDL and LDL.

Given that CETP promotes bidirectional transfers and thus an equilibration of cholesteryl esters between HDL and LDL (Fig.l), the observation of a net mass transfer from HDL to LDL in the presence of CETP in vitro indicates either that equilibration is still incomplete at the commencement of the incubation or, if complete, that the equilibrium is disrupted during the incubation. To differentiate between these two possibilities, time course studies were performed with mixtures of HDL and LDL supplemented with a tracer amount of HDL labelled isotopically

in the cholesteryl ester moiety. In the presence of CETP, the isotopically-labelled cholesteryl esgers equilibrated between HDL and LDL within three hours of incubation • The net mass transfers, by contrast, were progressive for up to 24 hours. Furthermore, despite the effects of fatty acid-poor albumin and Na oleate in respectively decreasing and increasing the magnitude of the net mass transfers of cholesteryl esters from HDL to LDL, neither albumin nor Na oleate had any discernable effect on the rate at which CETP promoted the equilibration of the isotopically-labelled cholesteryl esters between the two fractions. Thus, the redistribution of cholesteryl ester mass was quite distinct from the isotopic transfers and could therefore not be explained simply as the completion of a process of equilibration. Rather, the fact of a net mass transfer between already equilibrated pools indicated that the equilibrium was being disrupted. We postulate that this disruption is caused by non-esterified fatty acids which, in the presence of CETP, promote a redistribution of cholesteryl esters from HDL to LDL.

The mechanism by which CETP and Na oleate interact to promote a net mass transfer of cholesteryl esters from HDL to LDL is not known. However, some insights may be obtained by reviewing the postulated mechanism of action of CETP. Two general models have been Qroposed: (i) a shuttle model 7 and (ii) a ternary collision complex modelti. According to the shuttle model, CETP picks up molecules of cholesteryl ester and triacylglycerol and circulates as a CETP-lipid complex9. 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 7• 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 VLDL10, it cannot account for a net mass transfer of cholesteryl esters from HDL to 1016•

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 8• It is possible that the formation of a ternary complex of HDL, CETP and LDL may lead to a remodelling 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 as observed recently in incubations of HDL, LDL, CETP and Na oleate11 Furthermore, since it is known that non-esterified fatty acids enhance the binding of CETP to lipoproteins 12, 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 (Fig.2).

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 li oprotein lipase, there is an enhanced formation of small HDL particles 1 • 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 f increase in the rate of cholesteryl ester transfer from HDL to VLDL • The synergistic efects of hepatic lipase and CETP in promoting the reduction in size of HDL13 may also reflect an involvement of non­ esterified fatty acids.

It has been observed that CETP promotes a net mass transfer of cholesteryl esters from HDL to LDL even in incubations which have not been supplemented with exogenous Na oleate6• However, the fact that this CETP-mediated net mass transfer was markedly inhibited by fatty acid-poor but not by fatty acid-rich albumin 6 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 12• 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.

It is still uncertain whether an interaction of CETP and non­ esterified fatty acids is of physiological importance in modulating the partitioning of cholesterol between HDL and LDL. Nevertheless, it is tempting to speculate that variations in plasma non-esterified fatty acid metabolism may play a major role in regulating the distribution of cholesterol between plasma lipoproteins. For example, the low concentrations of HDL cholesterol in obese subjects and in smokers may reflect increased concentrations of plasma non-esterified fatty acids in such subjects. There are also obvious implications for subjects in whom the non-esterified fatty acid concentration is increased as a consequence of exposure to chronic stress. Clearly, therefore, an involvement of non-esterified fatty acids in modulating plasma cholesterol transport is a phenomenon of great potential importance.

Much more investigation is required to define the mechanism and both the physiological and pathological implications of the role played by non­ esterified fatty acids in determining the partitioning of cholesterol between the non-atherogenic HDL and the atherogenic LDL fractions.

REFERENCES

1. W. B. Kannel, W. P. Castelli, T. Gordon and P. H. McNamara, Serum cholesterol, lipoproteins and the risk of coronary heart disease, Ann. Int. Med. 74:1 (1971).

2. G. J. Miller and N. E. Miller, Plasma-high-density-lipoprotein concentration and the development of ischaemic heart disease, Lancet i:l6 (1975).

3. N. M. Pattnaik, A. Montes, L. B. Hughes and D. B. Zilversmit, Cholesteryl ester exchange protein in human plasma: isolation and characterisation, Biochim. Biophys. Acta 530:428 (1978).

4. P. J. Barter and J. I. Lally, In vitro exchanges of esterified cholesterol between serum lipoprotein fractions: studies of humans and rabbits, Metabolism 28:230 (1979).

5. P. J. Barter and M. E. Jones, Rate of exchange of esterified cholesterol between human low and high density lipoproteins, Atherosclerosis 34:67 (1979).

6. P. J. Barter, L. B. F. Chang, H. H. Newnham, K. A. Rye and 0. V.

Rajaram, Roles of lecithin: cholesterol acyltransferase, lipid transfer proteins and nonesterified fatty acids in plasma cholesterol transport, J. Drug Devel. (in press, 1990).

7. P. J. Barter and M. E. Jones, Kinetic studies of the transfer of esterified cholesterol between human plasma low and high density lipoproteins, J. Lipid Res. 21:238 (1980).

8. J. Ihm, D. M. Quinn, S. J. Busch, B. Chataing and J. A. K. Harmony, Kinetics of plasma protein-catalyzed exchange of phosphatidylcholine and cholesteryl ester between plasma lipoproteins, J. Lipid Res. 23:1328 (1982).

9. T. L. Swenson, R. W. Brocia and A. R. Tall, Plasma cholesteryl ester transfer protein has binding sites for neutral lipids and phospholipids, J. Biol. Chern. 263:5150 (1988).

10. G. J. Hopkins, L. B. F. Chang and P. J. Barter, Role of lipid transfers in the formation of a subpopulation of small high density lipoproteins, J. Lipid Res. 26:218 (1985).

11. P. J. Barter, L. B. F. Chang, H. H. Newnham, K. A. Rye and 0. V.

Rajaram, The interaction of cholesteryl ester transfer protein and unesterified fatty acids promotes a reduction in the particle size of high density lipoproteins, Biochim. Biophys. Acta (in press, 1990).

12. D. Sammett and A. R. Tall, Mechanisms of enhancement of cholesteryl ester transfer activity by lipolysis, J. Biol. Chern. 260:6687 (1985).

13. J. L. Ellsworth, M. L. Kashyap, R. L. Jackson and J. A. K. Harmony, Human plasma lipid transfer protein catalyses the speciation of high density lipoproteins, Biochim. Biophys. Acta 918:260 (1987).

14. G. J. Hopkins and P. J. Barter, Role of triglyceride-rich lipoproteins and hepatic lipase in determining the particle size and compositions of high density lipoproteins, J. Lipid Res. 27"1265 (1986).