Lipoprotein lipase prevents the hepatic lipase-induced reduction in particle size of high density lipoproteins during incubation of human plasma

Atherosclerosis, 82 (1990) 167-176 Elsevier Scientific Publishers Ireland,

ATHERO

167 Ltd.

04475

Lipoprotein lipase prevents the hepatic lipase-induced reduction in particle size of high density lipoproteins during incubation of human plasma Harvey H. Newnham,

Garry J. Hopkins,

Shona Devlin and Philip J. Barter

Baker Medical Research Institute, Melbourne (Australia) (Received 25 October 1989) (Revised, received 19 January 1990) (Accepted 29 January 1990)

Summary Human plasma lipoproteins or human whole plasma have been incubated in vitro with canine hepatic lipase (HL) and bovine milk lipoprotein lipase (LPL) to determine the effects of the lipases on the particle size distribution of HDL. Confirming previous reports, HL preferentially hydrolysed high density lipoprotein (HDL) triacylglycerol while LPL hydrolysed predominantly very low density lipoprotein (VLDL) triacylglycerol; however, neither lipase altered HDL particle size unless both VLDL and cholesteryl ester transfer protein (CETP) were present. Under these conditions HL promoted marked reduction in HDL particle size in a process dependent on the concentration of VLDL triacylglycerol while LPL was virtually without effect. When both LPL and HL were included in the same incubation, however, LPL prevented the effects of HL. These results are consistent with a proposition that HL has a direct effect on HDL particle size in a process which is dependent on concurrent lipid transfers between HDL and VLDL and that LPL reduces the effect of HL by reducing the concentration of VLDL triacylglycerol.

Key words:

Lipoprotein

lipase;

Hepatic

lipase;

HDL particle

size; Cholesteryl

ester transfer

protein

Introduction Correspondence to: Dr. Philip J. Barter, Baker Medical Research Institute, Commercial Road, P&an, Victoria 3181, Australia. Abbreviations: apo A-I, apohpoproteiu A-I; apo A-II, apolipoprotein A-II; apo B, apolipoprotein B; CETP, cholesteryl ester transfer protein; HL, hepatic lipase; LPL, lipoprotein lipase; LCAT, lecithin : cholesterol acyltransferase; HDL, high density lipoproteins; LDL, low density lipoproteins; VLDL, very low density lipoproteins.

0021-9150/90/$03.50

0 1990 Elsevier Scientific

Publishers

Ireland,

The observation that the concentration of high density lipoproteins (HDL) correlates negatively with the risk of developing premature coronary heart disease has stimulated intense interest in factors regulating HDL metabolism [l]. The HDL in human plasma are heterogeneous, consisting of several discrete subpopulations of particles varyLtd.

168 ing in size, density, composition and metabolic function [2-41. A number of plasma factors are implicated in changing the size, density and composition of these particles; these include lecithin : cholesterol acyltransferase (LCAT) [5], the cholesteryl ester transfer protein (CETP) [6], a putative HDL conversion factor [7] and the endothelial lipases, lipoprotein lipase (LPL) [8] and hepatic lipase (HL) [9]. In this report we focus on the regulation of HDL particle size by activity of LPL and HL. LPL and HL may have opposing effects on the particle size distribution of HDL. In vivo, for example, the concentration of the larger, less dense HDL, subfraction correlates positively with the postheparin activity of LPL [lo] but negatively with that of HL [lO,ll]. Some studies performed in vitro have shown that the increase in HDL particle size mediated by LPL is due to transfer of excess surface constituents from VLDL to HDL,, producing particles of a density similar to that of HDL, [8,12]. On the other hand, the reduction in HDL particle size which occurs when plasma is incubated with HL has been attributed to the hydrolysis of HDL core triacylglycerol by HL [9,13,14]. This process is markedly enhanced when HDL are first enriched with triacylglycerol, either by incubation in vitro with triacylglycerol-rich particles and a source of CETP [15] or in vivo after a fat meal [16]. In the present report we have directly compared the effects of LPL and HL on HDL particle size during incubation in vitro. We have confirmed that the combined effects of lipid transfer and HL markedly reduce HDL particle size [15], but have been unable to demonstrate any substantial direct effect of LPL on the particle size of HDL. LPL has, however, been found to abolish the reduction in HDL particle size promoted by HL. Materials and methods Isolation

made with solid KBr [17]. The total lipoprotein fraction was isolated from plasma as the supernatant after two 24-h spins at a density of 1.21 g/ml. VLDL were isolated from plasma as the supernatant after two 16-h spins at a density of 1.006 g/ml. The combined low density lipoprotein (LDL) + HDL fraction (density 1.006-1.21 g/ml) was isolated by a single 20-h spin at a density of 1.006 g/ml and two 24-h spins at a density of 1.21 g/ml. All isolated lipoproteins were dialyzed against 0.02 M phosphate buffer, pH 7.4, which contained 0.15 M NaCl, 0.01% (w/v) disodium EDTA and 0.02% (w/v) NaN, (PBS) prior to use. Isolation

of enzymes

LPL was purified from bovine milk as previously described [18]. HL was purified from dog post-heparin plasma by affinity chromatography on Sepharose 4B (Pharmacia LKB, Uppsala, Sweden) containing covalently bound heparin. The procedure was essentially as described by Boberg et al. [19] except that HL activity was eluted from the column with a linear gradient of 0.55-1.1 M NaCl in 0.005 M sodium barbitone, pH 7.4. The active fractions were pooled and concentrated by vacuum dialysis (Biomolecular Dynamics, Beaverton, OR, U.S.A.) against the PBS described above. Activity of the enzyme was not inhibited by 1 M NaCl, but more than 96% of the activity was removed following incubation in the presence of a specific antibody raised against dog HL (courtesy of Dr. C.P. Ehnholm) 1201. Phospholipase A, was obtained from Boehringer Mannheim (Mannheim, F.R.G.). Assay of lipolytic activity

HL and LPL activities were measured at high and low salt concentrations, respectively, using an artificial triolein emulsion as described by Huttunen et al. [20]. Activities are expressed as units (U)/ml, where 1 U = 1 pmol free fatty acid released per h.

of lipoproteins

Blood from healthy male subjects who had fasted for 12 h was collected into tubes containing disodium EDTA (1 mg/ml), placed on ice and the plasma separated by centrifugation at 4 o C. Lipoproteins were isolated at 4O C in a Beckman 50.3 Ti rotor at 173000 x g. Density adjustments were

Isolation

of cholesteryl

ester transfer protein

Partially pure CETP was isolated as described previously [21]. Briefly, the ultracentrifugally separated plasma fraction of d < 1.25 g/ml was subjected to chromatography on phenyl-Sepharose and CM-52 cellulose to produce CETP which was

169 TABLE

1

INCUBATION

OF TOTAL

LIPOPROTEINS

IN THE PRESENCE

OF HEPATIC

LIPASE

AND

LIPOPROTEIN

LIPASE

The plasma fraction of density i 1.21 g/ml was kept at 4“ C or incubated at 37 o C for 6 h. Incubations at 37 o C were performed in the absence or presence of either hepatic lipase, lipoprotein lipase or phospholipase A,. All incubations were supplemented with bovine serum albumin (2.5%. w/v) and heparin 100 U/ml. Following incubation the lipoproteins were reisolated as the fraction of d < 1.25 g/ml and analyzed as described in Materials and methods. Results are expressed per ml of incubation mixture. Concentration

Incubation “C

Addition

4O 37” 37”

HLh LPL c Phospholipase

37O 37”

d

Apo A-I

Apo A-II

Apo B (I.lg/mB

EC = (nmol/ml)

FC a (nmol/ml)

PL it (nmol/ml)

(I.Lg/ml)

@g/ml)

988 981 964 1008 985

364 327 366 345 322

782 736 835 814 795

4168 4218 4165 4291 4318

1099 1102 1092 1101 1097

2459 2593 2363 2337 1509

a EC, esterified cholesterol; FC, free cholesterol; PL, phospholipid; TG, triacylglycerol. h.c.d Incubations contained: b 30.2 U of HL per ml, 4.4 U of LPL per ml, and d 0.29 mg of phospholipase activities were measured in the presence of low and high salt concentrations as described in Materials

purified about lOOO-fold compared with the lipoprotein-free fraction of plasma. It did not contain activity of LCAT. CETP activity was determined by measuring the transfer of [3H]cholesteryl ester from LDL to HDL during incubation at 37” C [22]. Transfer activity was expressed as U/ml, the number of units being the rate constant, k, for the transfer of [ 3H]cholesteryl ester from LDL to HDL per 3 h using the formula of Pattnaik et al. [22]. Using this assay, the activity of CETP in the lipoprotein-free fraction of pooled human plasma is approximately 1 U/ml.

Incubations Whole plasma or the plasma fractions of d < 1.21 and 1.006-1.21 g/ml, supplemented with bovine serum albumin (l-2.58 (w/v) as indicated; BSA, fraction V fatty acid-free, Sigma, St. Louis, MO, U.S.A.) were kept at 4°C or incubated at 37°C in the absence of presence of added HL or LPL. All incubations contained heparin (100 U/ml) to stabilize lipase activity [23,24]. Following incubation, the samples were placed immediately on ice and then subjected to ultracentrifugation at 4 o C (173 000 x g for 24 h) to isolate the fraction of d < 1.25 g/ml. The results shown in Figs. l-5 and in Table 1 are generally representative of 2 or more experiments.

-_ TC; ’ (nmol/ml) 1198 1233 610 600 1257 --

A, per ml. HL and LPL and methods.

Size exclusion chromatography Lipoproteins of d < 1.25 g/ml were fractionated by size exclusion chromatography on a Superose 6 HR lo/30 column (Pharmacia-LKB, Uppsala, Sweden) connected to a high pressure P-500 pump (Pharmacia-LKB, Uppsala, Sweden). VLDL, LDL and HDL were eluted with 0.05 M Tris-HCl (pH 7.4) containing 0.15 M NaCl at a flow rate of 30 ml/h. Fractions of 0.6 ml were collected. Gradient gel electrophoresis The lipoproteins in the 1.25 g/ml supernatant were also separated by non-denaturing gradient gel electrophoresis on 4-30% polyacrylamide gels (Pharmacia LKB, Uppsala, Sweden) as previously described [25]. Chemical analyses All assays were performed on a Cobas-Bio centrifugal analyzer (Roche Diagnostics, Zurich, Switzerland). Concentrations of total cholesterol, free cholesterol and phospholipid were measured using enzymatic assays as previously described [15]. The concentration of esterified cholesterol was calculated as the difference between the concentrations of total (esterified plus free) cholesterol and free cholesterol. Triacylglycerol was measured using an assay which corrects for free glycerol

170 (Boehringer, Mannheim, F.R.G.). Protein concentrations were measured using the method of Lowry et al. [26] adapted for use on the Cobas-Bio. Concentrations of apolipoprotein (apo) A-I, apo A-II and apo B were measured immunoturbidimetrically as previously described [27] using specific antisera from Boehringer (Mannheim, F.R.G.).

higher concentrations of LPL, the triacylglycerol in the VLDL fraction was completely hydrolysed (Fig. lE-H). At the highest concentration of LPL there was a modest reduction of LDL triacylglycerol but no reduction of HDL triacylglycerol. Indeed, incubation in the presence of LPL was associated with a slight increase in the concentration of HDL triacylglycerol (panels EH).

Results Effects of HL and LPL on HDL particle size Substrate specificities of HL and LPL (a) Lipids

The lipoprotein rich fraction of human plasma (d < 1.21 g/ml) was incubated for 6 h at 37 o C in

the presence of HL or LPL (Table 1). In both cases the concentration of triacylglycerol in the incubation mixture was reduced by approx. 50%. There was a slight reduction in the concentration of phospholipid but no change in the concentration of apo A-I, apo A-II, apo B, esterified cholesterol or free cholesterol (Table 1). To ensure that this system was able to detect hydrolysis of phospholipid, the lipoproteins were also incubated in the presence of phospholipase A, which was shown to reduce the concentration of phospholipid by about 40% without affecting the other components (Table 1). (b) Lipoproteins

To determine which lipoprotein fraction of plasma triacylglycerol was hydrolysed by LPL and HL, aliquots of the plasma fraction of d< 1.21 g/ml were first incubated at 37” C for 6 h with increasing amounts of either HL or LPL and then resolved by size exclusion chromatography (Fig. 1). At the lowest concentrations of HL there was a substantial reduction in HDL triacylglycerol and proportionately, a smaller reduction of VLDL triacylglycerol (panel A). At higher concentrations of HL there was a progressive hydrolysis of VLDL triacylglycerol and, to a lesser extent, of LDL triacylglycerol, although at the highest concentration of HL, hydrolysis of VLDL and LDL triacylglycerol was still incomplete (Fig. 1, A-D). By contrast, incubation in the presence of the lowest concentration of LPL promoted significant hydrolysis of VLDL triacylglycerol (panel E). At

Aliquots of the incubation mixture from the experiments shown in Fig. 1 were loaded onto gradient gels to determine the HDL particle size (Fig. 2). Despite the extensive hydrolysis of triacylglycerol, neither HL nor LPL changed the particle size distribution of HDL. This result was not unexpected in view of the fact that triacylglycerol is only a minor constituent of HDL. In fact, previous studies have shown that lipases have a significant effect on HDL size only if the HDL have first been depleted of cholesteryl esters and enriched with triacylglycerol as a result of CETP-mediated lipid transfers [15]. The effects of HL and LPL on HDL particle size were therefore compared in incubations which contained activity of CETP. The plasma fraction of 1.006-1.21 g/ml (LDL + HDL) was supplemented with CETP and then incubated with HL or LPL in the presence or absence of added VLDL (Fig. 3). Incubation in the absence of HL, LPL or VLDL (profile B) had no effect on the particle size of HDL when compared to a sample kept at 4 o C (profile A). In the presence of HL alone there was a slight reduction in HDL particle size (profile C), but in the presence of both HL and VLDL there was a marked reduction in the particle size of HDL which appeared as a single population of particles of Stoke’s radius 3.7 nm (profile D). Incubation in the presence of LPL and CETP, on the other hand, had little effect on HDL particle size whether or not VLDL was present (profiles E and F), despite hydrolysis of approx. 40% of the triacylglycerol in the incubation mixture in each case. Studies were performed to determine the effects on HDL particle size distribution after adding HL and LPL to incubations of human whole plasma

171

300

r

Hepatic

lipase

Lipoprotein

V

A

200

200I.

100

100

0 z Z

E

.5 5 E e E m

E

0,

300I.

;"\

lipase

I; 1\

L H

b

A

0 4

"

300

E 2

200

L

200

100

5 E

100

300

E

9

F

9

G

e 0

$

0 -4

: 8

300

z Pk

200

0, F

100

.? :

0 -6 300 200

100

0

6

12

16

24

Fraction

0 +4 0

6

12

16

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Fraction

Fig. 1. Incubation of the total lipoprotein fraction of plasma in the presence of increasing amounts of HL or LPL: effects on the distribution and concentration of triacylglycerol. The human plasma fraction of density i 1.21 g/ml was incubated at 37O C for 6 h 0, final concentration in the absence of added lipase ( 0- - - - - - 0, all panels) or in the presence of increasing amounts of HL (0 0.6, 1.6, 2.7 and 18.2 U/ml, respectively, in 1.1, 3.2, 5.1 and 54 U/ml, respectively, in panels A, B, C and D) or LPL (0 -0, panels E, F, G and H). All incubations were supplemented with bovine serum albumin (2.5%. w/v) and heparin (100 U/ml). Following incubation the lipoproteins were isolated as the fraction of density cl.25 g/ml and subjected to size exclusion chromatography on a Superose 6 HR lo/30 column as described in Materials and Methods. Individual column fractions were assayed for triacylglycerol.

(Fig. 4). Plasma containing DTNB to inhibit activity of LCAT was supplemented with additional VLDL and CETP to ensure that the concentrations of these constituents were not rate limiting. In the absence of the added VLDL and CETP, incubation of plasma with HL resulted in a slight reduction in the size of the major HDL population from particles of Stoke’s radius of 4.3 to those of radius 4.1 nm, whereas incubation with LPL had no effect on the particle size of HDL (result not shown). In the plasma supplemented with VLDL and CETP, however, the 2 lipases had quite different effects on the size of HDL (Fig. 4). When the supplemented plasma was incubated at 37 o C for

12 h in the absence of added lipase there was relatively little change to the overall particle size distribution of HDL although a minor population of particles of 3.7 nm radius was formed (profile B). Incubation in the presence of an amount of HL sufficient to hydrolyse 55% of the triacylgiycerol in the supplemented plasma promoted an almost total conversion of HDL into very small particles of radius 3.7 nm (profile C). By contrast, when the supplemented plasma was incubated in the presence of an amount of LPL sufficient to hydrolyse 90% of the triacylglycerol in the mixture, there was little change in the HDL particle size (profile D). When both HL and LPL

172

+ HL

VLDL there was minimal change to the particle size distribution of HDL (profile B). When the incubation was supplemented with VLDL to achieve a final exogenous VLDL triacylglycerol concentration of 0.16 mM, there was a slight increase in the proportion of particles of radius 3.9 nm (profile C). When the exogenous VLDL triacylglycerol concentration was 0.4 or 0.8 mM (profiles D and E), there was a concomitant formation of smaller particles of Stoke’s radius 3.7 nm. Addition of sufficient VLDL to achieve a final concentration of exogenous VLDL triacylglycerol in the incubation of 1.6 mM resulted

+ LPL 5.3 4.3 3.9 3.7 Stokes’ radius (nm)

Fig. 2. The particle size distribution of HDL after incubating the d < 1.21 g/ml fraction of plasma in the presence of HL or LPL. Aliquots of the incubation mixtures from the experiments shown in Fig. 1 were subjected to gradient gel electrophoresis. Particle size distributions of HDL are shown for the samples stored at 4 o C (A), incubated for 6 h in the absence of lipase (B) or incubated for 6 h in the presence of HL (C) or LPL (D). The final concentrations of HL and LPL were 54 and 18.2 U/ml respectively. Stoke’s radii (mn) were calculated by reference to known high molecular weight standards.

were present there was no conversion to small HDL (profile E), suggesting that activity of LPL had inhibited the effects of HL. This inhibitory effect was only apparent if LPL was added prior to the HL (profile F); there was no evidence that LPL was able to reverse pre-existing changes promoted by HL (profile G). To determine whether this inhibitory effect of LPL may have been secondary to its capacity to reduce the concentration of VLDL triacylglycerol, an experiment was performed to examine the influence of VLDL concentration on the reduction of HDL particle size induced by HL. Aliquots of normal human plasma containing DTNB to inhibit LCAT were supplemented with CETP and HL and then incubated in the presence of increasing amounts of exogenous VLDL (Fig. 5). In a sample kept at 4 o C the HDL contained particles with radii of 5.3, 4.3 and 3.9 nm (profile A). After incubation for 6 h at 37OC without exogenous

Stokes’ radius

(nm)

Fig. 3. Incubation of the 1.006-1.21 g/ml fraction of plasma supplemented with CETP: effects of adding VLDL, HL and LPL on the particle size distribution of HDL. The 1.006-1.21 g/ml fraction of plasma (38 pl containing 550 umol of total cholesterol) was supplemented with CETP (final concentration 3.2 U/ml) and either stored at 4OC (A) or incubated for 12 h at 37O C in the absence of further addition (B) or in the presence of HL (44 U/ml) (C), HL (44 U/ml) and VLDL (VLDL triacylglycerol 2.0 mM) (D), LPL (32 U/ml) (E) or LPL (32 U/ml) and VLDL (VLDL triacylglycerol 2.0 mM) (F). Each incubation contained bovine serum albumin (l%, w/v) and heparin (100 U/ml) and was adjusted to a final volume of 0.25 ml with PBS. Following incubation, samples were subjected to ultracentrifugation to recover the fraction of d cl.25 g/ml which was further separated by non-denaturing gradient gel electrophoresis on 4-30% polyacrylamide gradient gels. The profiles represent laser densitometric scans of stained gels.

173

particle size of HDL. Consistent with previous reports [15], we have demonstrated that, so long as triacylglycerol-rich lipoproteins and a source of CETP were also present, HL promoted a marked reduction in HDL particle size. By contrast, the only demonstrable effect of LPL on HDL size was its ability to prevent the changes mediated by HL. In terms of their lipid substrate specificity, the preparations of HL and LPL used in the present studies were comparable to those described elsewhere, with each enzyme hydrolysing triacylglycerol in preference to other lipoprotein

5.3 4.3 3.9

3.7

Stokes’ radius (nm)

Fig. 4. Incubation of whole plasma with HL and LPL: effect on HDL particle size. Aliquots (0.1 ml) of a sample of plasma (cholesterol 5.2 mM and triacylglycerol 0.6 mM) were supplemented with VLDL (to provide a final concentration of exogenous VLDL triacylglycerol of 2.0 mM in the incubation mixture) and with CETP (final concentration 3.2 U/ml) and either stored at 4OC (A) or incubated for 12 h in the absence of lipase (B) or in the presence of HL (60 U/ml) (C), LPL (40 U/ml) (D) or both HL and LPL (E-G). In (E) both HL and LPL were present for the entire 12 h. In (F) LPL was present throughout the 12 h incubation with HL present for only the last 6 h while in (G) HL was present for 12 h and LPL present for only the last 6 h. Each incubation contained heparin (100 U/ml), DTNB (1.4 mM) and was adjusted to a final volume of 0.25 ml with PBS. Following incubation the samples were processed as described in the legend to Fig. 3. The profiles represent laser densitometric scans of stained 4-308 gradient gels.

in the complete conversion of the HDL to small particles of Stoke’s radius 3.7 nm (profile F). Discussion

These studies have revealed marked differences in the capacities of HL and LPL to alter the

I

5.3

I,#

4.3 3.9 3.7

Stokes’ radius

(nm)

Fig. 5. Reduction of HDL particle size induced by HL: dependence on the concentration of added VLDL. Aliquots (0.1 ml) of a sample of plasma (cholesterol 4.7 mM and triacylglycerol 0.4 mM) were supplemented with CETP (final concentration 3.6 U/ml) and HL (36 U/ml) and either stored at 4” C (A) or incubated for 6 h at 37OC in the absence (B) or presence (C-F) of increasing concentrations of added VLDL (final exogenous VLDL triacylglycerol concentrations of 0.16, 0.4, 0.8, 1.6 mM, respectively). Each incubation contained heparin (100 U/ml) and DTNB (1.4 mM) and was adjusted to a final volume of 0.25 ml with PBS. Following incubation the samples were processed as described in the legend to Fig. 3. The profiles represent laser densitometric scans of stained 4-30s gradient gels.

174 constituents [13,14,28,29]. As reported previously [9,13,30], however, the two enzymes showed different lipoprotein specificities, with HL preferring HDL triacylglycerol and LPL preferring that in VLDL (Fig. 1). In fact, in the case of LPL, a reduction in the concentration of VLDL triacylglycerol was accompanied by a slight increase in the triacylglycerol in HDL (Fig. 1). Neither lipase, however, promoted any change to the particle size distribution of HDL in the absence of CETP, presumably because triacylglycerol is only a minor constituent of the core of HDL which has not been modified by lipid transfers. The combined effects of lipid transfers and endothelial lipases on HDL particle size have been reported previously [15,31]. When HDL and triacylglycerol-rich lipoproteins are incubated in the presence of a source of CETP, reciprocal transfer of cholesteryl ester and triacylglycerol result in HDL becoming depleted of cholesteryl esters and enriched with triacylglycerol [15]. Subsequent incubation of these modified HDL with either HL [15] or LPL [31] results in hydrolysis of a proportion of the HDL triacylglycerol and the consequent formation of HDL particles which are reduced in size and increased in density. In the present studies in which the processes of lipid transfer and lipolysis were operating simultaneously rather than sequentially, small HDL were formed in incubations containing HL but not in those containing LPL. In other studies LPL has been reported to promote an increase in the size and a decrease in the density of HDL particles during incubation in vitro [8,12]. These changes have been attributed to the transfer to HDL of surface constituents made redundant by the action of LPL on triacylglycerol-rich lipoproteins [8,12]. In the present studies, however, there was no evidence that LPL promoted enlargement of HDL particles whether or not the incubation mixture contained triacylglycerol-rich lipoproteins. In fact, LPL did not substantially alter the particle size of HDL under any of the incubation conditions employed in the present studies. It did, however, prevent the changes promoted by HL. We postulate that this inhibitory effect of LPL is secondary to its effect in reducing the concentration of VLDL triacylglycerol, a suggestion supported by the observation

that the capacity of HL to reduce the particle size of HDL was dependent not only on the presence of a source of CETP but also on the concentration of VLDL triacylglycerol; the effect was markedly attenuated when VLDL was absent (Fig. 3) or present at very low concentrations (Fig. 5). Thus, an inhibitory effect of LPL is readily explicable in terms of the action of LPL in reducing the concentration of VLDL triacylglycerol. Relationships between the activities of HL and LPL in postheparin plasma and the HDL subpopulation distribution in vivo have been well documented. The inverse correlation between the concentration of HDL, (the larger HDL species) and both the activity of HL [lO,ll] and the concentration of triacylglycerol-rich lipoproteins [32] is consistent with the interaction between lipid transfers and HL observed in the present and other studies performed in vitro [15,31]. The positive correlation in vivo between the concentration of HDL, and activity of LPL [lo], on the other hand, has generally been attributed to an increase rate of transfer to HDL of surface constituents from VLDL or chylomicrons [8,12,33]. The present studies, however, suggest that the positive correlation with LPL may be no more than a reflection of the capacity of LPL to inhibit the changes promoted by HL. It has been postulated previously that the positive correlation between activity of LPL and the concentration of HDL, after a fat meal is the consequence of the role of LPL in regulating the amount of triacylglycerolrich lipoproteins which are available to interact with HDL [lo]. The particle size distribution of HDL in vivo is undoubtedly the reflection of a complex balance between factors such as LCAT which act to increase HDL particle size and factors such as HL which act in conjunction with lipid transfer to reduce the particle size of HDL. If the effect of HL were to be reduced because of a low concentration of triacylglycerol-rich lipoproteins secondary to a high level of activity of LPL, the balance would shift in favour of factors promoting particle enlargement. While this would readily explain a positive correlation between HDL particle size and activity of LPL, it should be emphasized that results obtained in vitro may have little bearing on the physiological function of enzymes

175

which normally act in situ on the surface of endothelial cells. Nevertheless, the results of the present study are consistent with previous reports and provide a logical explanation for important and well documented observations made in vivo.

12 13

Acknowledgements 14

This work was supported by a grant from the National Health & Medical Research Council of Australia. We are grateful for the technical assistance of Ms. Margaret Morey.

15

References 16 1 Miller, G.J. and Miller, N.E., Plasma-high-density-lipoprotein concentration and development of ischaemic heart disease, Lancet, i (1975) 16. 2 Kostner, G.M., Isolation, subfractionation, and characterization of human serum high-density lipoproteins. In: C.E. Day (Ed.), High Density Lipoprotein, Marcel-Dekker, New York, 1988, pp. 1. 3 Blanche, P.J., Gong, E.L., Forte, T.M. and Nichols, A.V., Characterization of human high-density lipoproteins by gradient gel electrophoresis, Biochim. Biophys. Acta, 665 (1981) 408. 4 Barter, P.J., Hopkins, G.J. and Gorjatschko, L., Lipoprotein substrates for plasma cholesterol esterification. Influence of particle size and composition of the high density lipoprotein subfraction 3, Atherosclerosis, 58 (1985) 97. 5 Daerr, W.H. and Greten, H., In vitro modulation of the distribution of normal human plasma high density lipoprotein subfractions through the lecithin : cholesterol acyltransferase reaction, Biochim. Biophys. Acta, 710 (1982) 128. 6 Hopkins, G.J., Chang, L.B.F. and Barter, P.J., Role of lipid transfer in the formation of a subpopulation of small high density lipoproteins, J. Lipid Res., 26 (1985) 218. 7 Rye, K.A. and Barter, P.J., Changes in the size and density of human high density lipoproteins promoted by a plasmaconversion factor, B&him. Biophys. Acta, 875 (1986) 429. 8 Patsch, J.R., Gotto, A.M., Jr., Olivecrona, T. and Eisenberg, S., Formation of HDL,-like particles during lipolysis of VLDL in vitro, Proc. Natl. Acad. Sci. USA, 75 (1978) 4519. 9 Shirai, K., Barnhart, R.L. and Jackson, R.L., Hydrolysis of human plasma high density lipoprotein,-phospholipids and triglycerides by hepatic lipase, B&hem. Biophys. Res. Commun., 100 (1981) 591. 10 Patsch, J.R., Prasad, S., Gotto, A.M. and Patsch, W., High density lipoprotein,. Relationship of the plasma levels of this lipoprotein species to its composition, to the magnitude of postprandial lipemia, and to the activities of lipoprotein lipase and hepatic lipase, J. Clin. Invest., 80 (1987) 341. 11 Kuusi, T.. Saarinen, P. and Nikkill, E.A., Evidence for the

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