Garry J. Hopkins

Transfers of esterified cholesterol and triglyceride between high density and very low density lipoproteins: In vitro studies of rabbits and humans

Abstract Mixtures of rabbit very low density lipoproteins (VLDL) and high density lipoproteins (HDL), one of which was endogenously labeled with 3 H in the free and esterified cholesterol moieties and with 14 C in the triglyceride (TG) moiety, were incubated at 37°C in vitro in the presence of rabbit lipoprotein-free serum. There was a net mass transfer of esterified cholesterol from HDL to VLDL and of TG from VLDL to HDL. Both esterified 3 H-cholesterol and 14 C-TG, however, transferred in each direction between the two fractions. In the presence of parachlormercuryphenyl sulfonate (PCMPS), a thiol group blocker that inhibits lecithin:cholesterol acyltransferase (LCAT), the net mass transfers of esterified cholesterol and TG were reduced, as were the bidirectional transfers of 14 C-TG, but the bidirectional transfers of esterified 3 H-cholesterol were much less affected. Qualitatively similar results were obtained with incubations of human VLDL and HDL containing tracer amounts of either VLDL or HDL labeled with 3 H in the esterified cholesterol moiety. In incubations of unlabeled human VLDL, HDL, and lipoprotein-free plasma, the molar ratio of the net mass transfers of TG:esterified cholesterol was 0.55 ± 0.10 (mean ± SE, n=8) in the absence of PCMPS, and 0.25 ± 0.04 in the presence of PCMPS. The difference between these ratios was statistically significant ( p

The rabbit as an animal model of hepatic lipase deficiency

A natural deficiency of hepatic lipase in rabbits has been exploited to gain insights into the physiological role of this enzyme in the metabolism of plasma lipoproteins. A comparison of human and rabbit lipoproteins revealed obvious species differences in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL), with the rabbit lipoproteins being relatively enlarged, enriched in triacylglycerol and depleted of cholesteryl ester. To test whether these differences related to the low level of hepatic lipase in rabbits, whole plasma or the total lipoprotein fraction from rabbits was either kept at 4 degrees C or incubated at 37 degrees C for 7 h in (i) the absence of lipase, (ii) the presence of hepatic lipase and (iii) the presence of lipoprotein lipase. Following incubation, the lipoproteins were recovered and subjected to gel permeation chromatography to determine the distribution of lipoprotein components across the entire lipoprotein spectrum. An aliquot of the lipoproteins was subjected also to gradient gel electrophoresis to determine the particle size distribution of the LDL and HDL. Both hepatic lipase and lipoprotein lipase hydrolysed lipoprotein triacylglycerol and to a much lesser extent, also phospholipid. There were, however, obvious differences between the enzymes in terms of substrate specificity. In incubations containing hepatic lipase, there was a preferential hydrolysis of HDL triacylglycerol and a lesser hydrolysis of VLDL triacylglycerol. By contrast, lipoprotein lipase acted primarily on VLDL triacylglycerol. When more enzyme was added, both lipases also acted on LDL triacylglycerol, but in no experiment did lipoprotein lipase hydrolyse the triacylglycerol in HDL. Coincident with the hepatic lipase-induced hydrolysis of LDL and HDL triacylglycerol, there were marked reductions in the particle size of both lipoprotein fractions, which were now comparable to those of human LDL and HDL3, respectively.

Lipoprotein substrates for plasma cholesterol esterification: Influence of particle size and composition of the high density lipoprotein subfraction 3☆

Subpopulations of lipoproteins within the high density lipoprotein subfraction 3 (HDL3) have been isolated from human plasma and characterized in terms of their chemical composition and particle size. Since HDL3 are the major substrates for the esterification of plasma cholesterol, and thus play a central role in the transport of cholesterol through the plasma, these studies also examined the relative capacities of different HDL3 subpopulations to interact with lecithin: cholesterol acyltransferase. Substrate reactivity of a given preparation of lipoproteins was defined in terms of the Vmax of the cholesterol esterification reaction in incubations containing a fixed amount of human lipoprotein-free plasma as a source of the enzyme. Substrate reactivity was found to correlate inversely with the radius of HDL3 particles. This inverse relationship between particle size and substrate reactivity was independent of the particle content of cholesteryl ester.

Particle size distribution of high density lipoproteins as a function of plasma triglyceride concentration in human subjects

Polyacrylamide gradient gel electrophoresis has been used to examine the particle size distribution of high density lipoproteins (HDL) in human subjects with a wide range of plasma triglyceride concentrations. In studies of groups of both male and female subjects, it was confirmed that the concentration of HDL cholesterol decreases with increasing plasma triglyceride concentration. The HDL fraction from subjects with elevated concentrations of plasma triglyceride was depleted of cholesteryl ester and enriched in triglyceride. It was also confirmed that the proportion of HDL subfraction 2 (HDL2) declines as the plasma triglyceride increases. A new finding was that there were also significant changes in the size of particles in HDL subfraction 3 (HDL3). At low concentrations of plasma triglyceride the predominant subpopulation of HDL3 comprised particles of mean radius 4.3 nm. As the triglyceride concentration increased, however, there was a progressive appearance of HDL3 particles of radius 3.9 nm; in plasma samples with the highest concentrations of triglyceride there was an almost complete disappearance of the 4.3-nm particles, with the population of 3.9-nm particles now predominant.

Competitive inhibition of plasma cholesterol esterification by human high-density lipoprotein-subfraction 2.

Mixtures containing subfractions of human plasma high-density lipoproteins (HDL) and human lipoprotein-free plasma were incubated in vitro at 37 degrees C. Esterification of cholesterol was observed both in incubations containing HDL-subfraction 3 (HDL3) and in those containing HDL-subfraction 2 (HDL2). The implication that the lecithin: cholesterol acyltransferase in lipoprotein-free plasma may therefore interact with lipoproteins in both HDL subfractions was developed further by proposing a simple model in which the two HDL subfractions may compete for interactions with the enzyme. This model was described mathematically and tested in experiments in which a constant amount of the enzyme was incubated with a wide range of concentrations of HDL2 and HDL3 present either alone or in combination. The model was able to predict experimentally observed rates of cholesterol esterification with great accuracy. The best fit was obtained with a Vmax for HDL3 that was 2.4-4-times greater than that for HDL2 and values of the apparent Km for HDL3 free cholesterol and HDL2 free cholesterol of 43-60 nmol/ml and 167-391 nmol/ml, respectively. The model thus predicts that, at physiological concentrations of lipoproteins, HDL2 will function as a competitive inhibitor of the cholesterol esterification reaction by displacing lecithin: cholesterol acyltransferase from a more effective substrate, HDL3, to a less effective substrate, HDL2.

The role of lipid transfer proteins in plasma lipoprotein metabolism

Human plasma contains a number of proteins that promote movement of lipids between lipoprotein fractions. One of these proteins, designated lipid transfer protein, is known to promote bidirectional transfers of cholesteryl esters, triglyceride, and phospholipids between all plasma lipoprotein fractions. This report briefly reviews the role of lipid transfer protein in plasma cholesterol transport and in the regulation of the particle size distribution of high-density lipoproteins. Studies are described that show that the small particle size of high-density lipoproteins in human subjects with hypertriglyceridemia is a result of the combined actions of lipid transfer protein and hepatic lipase.

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: Cholesterol acyltransferase

A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

Pathways for the incorporation of esterified cholesterol into very low density and low density lipoproteins in plasma incubated in vitro

In vitro incubations of human or pig plasma containing a tracer amount of [3H]cholesterol have been performed to determine which lipoprotein fractions are the immediate recipients of the esterified cholesterol formed in the reaction catalysed by lecithin: cholesterol acyltransferase. In pig plasma, which is deficient in activity of the protein which promotes transfer of esterified cholesterol between different lipoprotein fractions, 87-90% of the lecithin: cholesterol acyltransferase-derived esterified cholesterol was incorporated into the high density lipoprotein (HDL) fraction. In human plasma there was an initial recovery of more than 80% in HDL, although the proportion recovered in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) became progressively greater with increasing duration of incubation, consistent with a transfer from an HDL -esterified cholesterol pool of increasing specific activity. Nevertheless, as in the pig plasma incubations, there was evidence that some 10-15% of the esterified cholesterol formed in the lecithin: cholesterol acyltransferase reaction was incorporated directly into human VLDL and LDL. In quantitative terms, however, it was found that most of the esterified cholesterol delivered to human VLDL and LDL was the result of transfers from HDL rather than as a direct incorporation from its site of synthesis.

Relative rates of incorporation of esterified cholesterol into human very low density lipoproteins and low density lipoproteins. In vitro studies of two separate pathways.

It has been shown previously that there are two pathways by which the esterified cholesterol formed in human plasma in the reaction catalysed by lecithin: cholesterol acyltransferase may be delivered to very low density lipoproteins (VLDL) and low density lipoproteins (LDL): (a) an indirect pathway in which esterified cholesterol which was incorporated initially into high density lipoproteins (HDL) is transferred subsequently to VLDL and LDL in a process mediated by an esterified cholesterol transfer/exchange protein and (b) a direct pathway in which a small proportion of the esterified cholesterol formed in the lecithin:cholesterol acyltransferase reaction is delivered to VLDL and LDL directly from its site of synthesis via a pathway which bypasses the bulk HDL fraction. These present studies have been designed to examine the incorporation of esterified cholesterol into VLDL relative to that into LDL via each of these two pathways. It has been found that a delivery of esterified cholesterol from HDL to VLDL and LDL via the indirect pathway has a marked preference for VLDL over LDL; equating the concentrations of esterified cholesterol in the two fractions revealed an incorporation into VLDL which was 7-11 times greater than that into LDL. By contrast, delivery via the direct pathway showed a marginal preference for LDL over VLDL.

Dissociation of the in vitro transfers of esterified cholesterol and triglyceride between human lipoproteins

This report describes the detailed time-course of in vitro net mass transfers of esterified cholesterol (EC) and triglyceride (TG) between human plasma lipoproteins incubated in the presence of EC and TG transfer proteins. In incubations containing high density lipoproteins (HDL), very low density lipoproteins (VLDL) and the thiol-group blocker parachloromercuriphenyl sulfonate (PCMPS) (which was added to inhibit EC production), the rate of EC transfer to VLDL was considerably more rapid than that of TG to HDL and equilibrium between the pools of EC was achieved much sooner than was the case for the pools of TG. When VLDL and low density lipoproteins (LDL) were incubated, the presence of PCMPS in the incubation mixture resulted in a marked reduction in the TG transfer to LDL, even though the EC transfer to VLDL was apparently unaffected. The molar ratio of the transfers of EC and TG was also examined in incubations of lipoproteins from a variety of human subjects. In incubations of HDL and VLDL (performed in the absence of PCMPS) there was a large individual variation (sixfold difference) in the molar ratio of the transfers of TG:EC. In incubations of LDL and VLDL from different subjects there was a twofold individual variation in the molar ratio of the transfers of TG:EC. It has been concluded that the net mass transfers of EC and TG between human lipoproteins do not involve a simple molecular exchange of one moiety for the other, as has been suggested previously, but are achieved by processes which are at least partially independent.