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J Biol Chem, Vol. 273, Issue 42, 27191-27198, October 16, 1998


The Influence of Apolipoproteins on the Hepatic Lipase-mediated Hydrolysis of High Density Lipoprotein Phospholipid and Triacylglycerol*

Neil J. HimeDagger §, Philip J. BarterDagger §, and Kerry-Anne Rye§parallel **

From the Dagger  Department of Medicine, University of Adelaide and parallel  Division of Cardiovascular Services, Royal Adelaide Hospital, Adelaide, South Australia, Australia 5000 and the § Lipid Research Laboratory, The Hanson Centre for Cancer Research, Frome Road, Adelaide, South Australia, Australia 5000

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

This study describes the influence of apolipoproteins on the hepatic lipase (HL)-mediated hydrolysis of phospholipids and triacylglycerol in high density lipoproteins (HDL). HL-mediated hydrolysis was assessed in well characterized, homogeneous preparations of spherical reconstituted high density lipoproteins (rHDL). The rHDL were comparable in size and lipid composition and contained either apoA-I ((A-I)rHDL) or apoA-II ((A-II)rHDL) as their sole apolipoprotein constituent. Preparations of rHDL containing only cholesteryl esters (CE) in their core, (A-I/CE)rHDL and (A-II/CE)rHDL, were used to assess phospholipid hydrolysis. Preparations of rHDL that contained triacylglycerol as their predominant core lipid, (A-I/TG)rHDL and (A-II/TG)rHDL, were used to assess both triacylglycerol and phospholipid hydrolysis. The rHDL contained trace amounts of either radiolabeled phospholipid or radiolabeled triacylglycerol. Hydrolysis was measured as the release of radiolabeled nonesterified fatty acids (NEFA) from the rHDL. Kinetic analysis showed that HL had a greater affinity for the phospholipids in (A-II/CE)rHDL (Km(app) = 0.2 mM) than in (A-I/CE)rHDL (Km(app) = 3.1 mM). This was also evident when hydrolysis was measured directly by quantitating NEFA mass. HL also had a greater affinity for the phospholipids and triacylglycerol in (A-II/TG)rHDL than in (A-I/TG)rHDL. The Vmax for phospholipid hydrolysis was, by contrast, greater for (A-I/CE)rHDL than for (A-II/CE)rHDL: 309.3 versus 49.1 nmol of NEFA formed/ml of HL/h. Comparable Vmax values were obtained for the hydrolysis of the phospholipids in (A-II/TG)rHDL and (A-I/TG)rHDL. In the case of triacylglycerol hydrolysis, the respective Vmax values for (A-I/TG)rHDL and (A-II/TG)rHDL were 1154.8 and 240.2 nmol of NEFA formed/ml of HL/h. These results show that apolipoproteins have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Hepatic lipase (HL)1 is a 476-amino acid glycoprotein of molecular weight 64,000-69,000 (1) that is bound to liver sinusoidal endothelial cells (2). HL hydrolyzes acyl ester bonds of triacylglycerol and the sn-1 acyl ester bond of phospholipids. The main plasma substrates for HL are very low density lipoproteins and high density lipoproteins (HDL). The role of HL in HDL metabolism is of considerable importance, as shown by strong negative associations between HL activity and plasma HDL2 levels (3-5) and the dramatic reduction in the HDL levels of rabbits that have been made transgenic for human HL (6).

Unlike lipoprotein lipase (LPL), which requires apolipoprotein C-II (apoC-II) for maximal activity, there is no known protein cofactor for HL. However, there is some conflicting evidence to suggest that the apoA-II in HDL may influence the HL-mediated hydrolysis of triacylglycerol in HDL (7-10). Some investigators have reported that apoA-II enhances (7, 8), while others have concluded that it inhibits, the HL-mediated hydrolysis of triacylglycerol in HDL (9, 10).

The present study was carried out in order to determine whether there are significant differences in the HL-mediated hydrolysis of phospholipids and triacylglycerol in HDL that differ in their apolipoprotein composition. This has been achieved by using well defined, homogeneous preparations of spherical reconstituted HDL (rHDL) as substrates for HL. The rHDL were comparable in size and lipid composition and contained either apoA-I or apoA-II as their sole apolipoprotein constituent. The results show that apolipoproteins not only have a major influence on the HL-mediated hydrolysis of the triacylglycerol and phospholipids in rHDL but also regulate the affinity of HL for the rHDL surface.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Purification of ApoA-I and ApoA-II-- ApoA-I and apoA-II were prepared from pooled human plasma donated by the Transfusion Service, Royal Adelaide Hospital. HDL were isolated from the plasma by sequential ultracentrifugation in the 1.07 < d < 1.21 g/ml density range (11). The isolated HDL were delipidated (12), and the resulting apoHDL was subjected to anion exchange chromatography on Q Sepharose Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) (13). The purified apoA-I and apoA-II appeared as single bands following electrophoresis on a homogeneous 20% SDS-polyacrylamide PhastGel (Amersham Pharmacia Biotech) and Coomassie staining.

Purification of Lecithin:Cholesterol Acyltransferase (LCAT)-- LCAT was purified from pooled human plasma (Transfusion Service, Royal Adelaide Hospital) as described previously (14). The purified LCAT appeared as a single band following electrophoresis on a homogeneous 20% SDS-gel and silver staining. LCAT activity was assessed as described by Piran and Morin (15) using as the substrate 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/unesterified cholesterol (UC)/apoA-I discoidal rHDL labeled with [1alpha ,2alpha -3H]cholesterol ([3H]UC). The POPC and [3H]UC were obtained, respectively, from Sigma and Amersham Pharmacia Biotech (Buckinghamshire, UK). The assay was linear as long as less than 30% of the [3H]UC was esterified. The preparation used in this study generated 562 nmol of cholesteryl esters (CE)/ml of LCAT/h.

Purification of Cholesteryl Ester Transfer Protein (CETP)-- CETP was isolated from pooled human plasma (Transfusion Service, Royal Adelaide Hospital) as described previously (16, 17). Transfer activity was quantitated as the transfer of [3H]CE from [3H]CE-HDL3 to low density lipoproteins (LDL) (18, 19). The assay was linear when less than 30% of the total counts were transferred from HDL3 to LDL during a 3-h incubation at 37 °C. The CETP preparation used in this study had 12.6 units of activity/ml, where 1 unit is the transfer activity of 1 ml of pooled, lipoprotein-deficient human plasma.

Purification of Phospholipid Transfer Protein (PLTP)-- PLTP was purified from pooled human plasma (Transfusion Service, Royal Adelaide Hospital) as described elsewhere (20). PLTP activity was quantitated as the transfer of L-3-1,2-di[1-14C]palmitoylphosphatidylcholine ([14C]DPPC) (112 mCi/mmol) (Amersham Pharmacia Biotech) from [14C]DPPC-labeled phospholipid vesicles to ultracentrifugally isolated HDL during a 2-h incubation at 37 °C (21). The PLTP preparation used in this study transferred 2700 nmol of phospholipid/ml of PLTP/h.

Preparation of Spherical rHDL (Fig. 1)-- Three pairs of substrates (a and b, c and d, and e and f) were used to study the HL-mediated hydrolysis of phospholipids and triacylglycerol in rHDL (Fig. 1). Each pair of substrates was comparable in size and lipid composition and contained either apoA-I or apoA-II as its sole apolipoprotein.


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  		Fig. 1.   Radiolabeled substrates for studying the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis. Three pairs of radiolabeled rHDL containing either apoA-I or apoA-II as their sole apolipoprotein constituent were used to study phospholipid and triacylglycerol hydrolysis. The first pair of substrates, a and b, designated (A-I/CE)rHDL and (A-II/CE)rHDL, contained CE as their sole core lipid and either apoA-I (open ovals) or apoA-II (shaded ovals) as their sole apolipoprotein and were labeled with [14C]DPPC (black circles). This pair of substrates was used to study HL-mediated phospholipid hydrolysis in the absence of triacylglycerol. The second pair of radiolabeled substrates, c and d, contained triacylglycerol as the predominant core lipid and either apoA-I or apoA-II as the sole apolipoprotein, and these substrates were labeled with [14C]DPPC. They were used to study phospholipid hydrolysis in the presence of triacylglycerol. The final two radiolabeled substrates, e and f, were identical to c and d, except they contained [3H]triolein (star TG) in their core. They were used to study triacylglycerol hydrolysis.

For most of the experiments, the substrates were labeled either with [14C]DPPC, for assessing phospholipid hydrolysis, or [9,10-3H]triolein (28 Ci/mmol) (NEN Life Science Products) ([3H]triolein), for monitoring triacylglycerol hydrolysis. The two substrates (a and b), which were labeled with [14C]DPPC and contained CE in their core, were used to monitor phospholipid hydrolysis in the absence of triacylglycerol hydrolysis. These substrates are designated (A-I/CE)rHDL and (A-II/CE)rHDL. Substrates c and d were also labeled with [14C]DPPC and contained triacylglycerol as their predominant core lipid. They are designated (A-I/TG)rHDL and (A-II/TG)rHDL and were used to study phospholipid hydrolysis against a background of triacylglycerol hydrolysis. The final two substrates, e and f, contained triacylglycerol in their core and were labeled with [3H]triolein. They were used to monitor triacylglycerol hydrolysis. Since the rate of triacylglycerol hydrolysis was much greater than that of phospholipid hydrolysis for both (A-I/TG)rHDL and (A-II/TG)rHDL, we were not able to study the kinetics of phospholipid and triacylglycerol hydrolysis in rHDL, which were doubly labeled with [14C]DPPC and [3H]triolein.

Preparation of (A-I/CE)rHDL and (A-II/CE)rHDL Labeled with [14C]DPPC: Substrates a and b-- Discoidal rHDL containing POPC, UC, and apoA-I were prepared by the cholate dialysis method (22). The discs were incubated with LDL and LCAT as described previously (23) to generate spherical rHDL with CE in their core and apoA-I as the sole apolipoprotein constituent, (A-I/CE)rHDL. The (A-I/CE)rHDL were dialyzed extensively against 0.01 M Tris-buffered saline (TBS) (pH 7.4) containing 0.15 M NaCl, 0.005% (w/v) EDTA-Na2, and 0.006% (w/v) NaN3 before use.

The (A-I/CE)rHDL were labeled with [14C]DPPC as follows. [14C] DPPC-labeled phospholipid vesicles were prepared by adding to a clean, dry test tube 0.35 mg POPC in chloroform/methanol (200 µl, 2:1 (v/v)), 1.25 µCi of [14C]DPPC, and 5 µl of 0.5 mM butylated hydroxytoluene in ethanol. The lipids were dispersed as a thin film on the walls of the tube and dried under N2 for 2 h at 40 °C. The phospholipids were resuspended in 0.5 ml of TBS and sonicated for 3 × 5 min, using a Sonifier B-12 (Branson Sonic Power Company, Danbury, CT) equipped with a microtip. The mixture was then centrifuged at 15,000 rpm for 10 min, and the supernatant, which contained the [14C]DPPC-labeled phospholipid vesicles, was collected.

Spherical (A-I/CE)rHDL (6.6 µmol phospholipid) were added to the [14C]DPPC-labeled phospholipid vesicles (0.66 µmol of phospholipid) and incubated for 3 h at 37 °C in the presence of purified PLTP (final transfer activity = 247 nmol of PL transferred/ml of PLTP/h) and bovine serum albumin (BSA) (final concentration = 20 mg/ml). The final volume of the incubation mixture was 5.5 ml. When the incubation was complete, the radiolabeled (A-I/CE)rHDL were isolated by ultracentrifugation at 100,000 rpm in the density range 1.063 < d < 1.21 g/ml using a TLA-100.4 rotor (Beckman Instruments, Fullarton, CA) with one 6-h spin at the lower density and another 16-h spin at the higher density. These procedures were carried out at 4 °C in a Beckman TL-100 tabletop ultracentrifuge (Beckman Instruments). The (A-I/CE)rHDL were dialyzed extensively against TBS prior to use. The specific activity of the (A-I/CE)rHDL was 4.8 × 105 dpm/mg of phospholipid.

Spherical (A-II/CE)rHDL labeled with [14C]DPPC were prepared by displacing all of the apoA-I from spherical [14C]DPPC-labeled (A-I/CE)rHDL with lipid-free apoA-II as described previously (23). The specific activity of the spherical [14C]DPPC-labeled (A-II/CE)rHDL was 5.1 × 105 dpm/mg of phospholipid.

Preparation of Unlabeled (A-I/CE)rHDL and (A-II/CE)rHDL-- These rHDL were prepared by displacing all the apoA-I from unlabeled spherical (A-I/CE)rHDL with lipid-free apoA-II. These unlabeled rHDL preparations were used in experiments where phospholipid hydrolysis was determined directly by measuring nonesterified fatty acid (NEFA) mass.

Preparation of (A-I/TG)rHDL and (A-II/TG)rHDL Labeled with [14C]DPPC: Substrates c and d-- Spherical (A-I/TG)rHDL containing triacylglycerol (and a small amount of CE) in their core were prepared as described by Rye et al. (17). Briefly, spherical (A-I/CE)rHDL (final CE concentration = 0.1 mmol/liter) were mixed with Intralipid (Kabi Pharmacia AB; final triacylglycerol concentration = 4 mmol/liter) and CETP (final concentration = 2.7 units/ml) and then incubated under N2 for 1.25 h at 37 °C. The final volume of the incubation mixture was 51.2 ml. The resulting (A-I/TG)rHDL were isolated by sequential ultracentrifugation in the density range 1.063 < d < 1.21 g/ml using a TLA-100.4 rotor (Beckman Instruments) as described previously (17). [14C]DPPC was incorporated into the (A-I/TG)rHDL as described for (A-I/CE)rHDL. Spherical (A-II/TG)rHDL labeled with [14C]DPPC were prepared by displacing all of the apoA-I from [14C]DPPC-labeled (A-I/TG)rHDL with lipid-free apoA-II (23). The specific activities of the [14C]DPPC-labeled (A-I/TG)rHDL and (A-II/TG)rHDL were 4.6 × 105 and 5.0 × 105 dpm/mg of phospholipid, respectively.

Preparation of (A-I/TG)rHDL and (A-II/TG)rHDL Labeled with [3H]Triolein: Substrates e and f-- Spherical (A-I/TG)rHDL were prepared as described by Rye et al. (17) with a slight modification to label the Intralipid with [3H]triolein. Briefly, 50 µCi of [3H]triolein were dried down under N2 and then taken up in 50 µl of ethanol. The [3H]triolein was added to Intralipid (200 mg of triacylglycerol) in a final volume of 1.05 ml, and the mixture was incubated for 3 h at 37 °C under N2. The [3H]triolein-labeled Intralipid (final triacylglycerol concentration = 4 mmol/liter) was then incubated for 1.5 h at 37 °C with (A-I/CE)rHDL (final CE concentration = 0.1 mmol/liter) and CETP (final concentration = 2.7 units/ml) in a final volume of 44.8 ml. The resulting [3H]triolein-labeled (A-I/TG)rHDL were isolated by sequential ultracentrifugation in the density range 1.063 < d < 1.21 g/ml using a TLA-100.4 rotor (Beckman Instruments) as described elsewhere (17). (A-II/TG)rHDL labeled with [3H]triolein were prepared by displacing apoA-I from [3H]triolein-labeled (A-I/TG)rHDL with lipid-free apoA-II (23). The specific activities of the [3H]triolein-labeled (A-I/TG)rHDL and (A-II/TG)rHDL were 11.0 × 105 and 10.7 × 105 dpm/mg of triacylglycerol, respectively.

Preparation of Native (A-I)HDL2 and (A-II)HDL2 Labeled with [14C]DPPC-- HDL2 were isolated from fresh human plasma by sequential ultracentrifugation in the 1.07 < d < 1.12 g/ml density range with two 24-h spins (50,000 rpm) at the lower density followed by a single 40-h spin (50,000 rpm) and one 16-h spin (100,000 rpm) at the higher density. The 50,000 rpm spins were carried out at 4 °C using a Ti-50 rotor in a Beckman L8-70M ultracentrifuge (Beckman Instruments). The 100,000 rpm spin was carried out at 4 °C using a TLA-100.4 rotor in a Beckman TL-100 tabletop ultracentrifuge. The isolated HDL2, which contained apoA-I as the predominant apolipoprotein, is designated (A-I)HDL2. [14C]DPPC was incorporated into the (A-I)HDL2 as described for (A-I/CE)rHDL. (A-II)HDL2 labeled with [14C]DPPC were prepared by displacing all of the apoA-I from [14C]DPPC-labeled (A-I)HDL2 with lipid-free apoA-II (23). The specific activities of the [14C] DPPC-labeled (A-I)HDL2 and (A-II)HDL2 were 3.65 × 105 and 3.53 × 105 dpm/mg of phospholipid, respectively.

Purification of HL-- HL was purified from the blood of patients injected with a bolus of 25,000 IU of heparin prior to undergoing angioplasty (Cardiovascular Investigational Unit, Royal Adelaide Hospital). Postheparin plasma was isolated by centrifugation at 3,000 rpm for 10 min at 4 °C and stored at -70 °C. The pooled postheparin plasma was thawed and added to an equal volume of 0.005 M sodium barbitone buffer, 0.45 M NaCl (pH 7.4). The postheparin plasma was applied to an HR 10/30 column containing Heparin Sepharose Fast Flow (Amersham Pharmacia Biotech) preequilibrated with 0.005 M sodium barbitone, 0.15 M NaCl (pH 7.4). HL was eluted from the column at a flow rate of 5 ml/min with a linear 0.8-1.3 M NaCl gradient. Fifty IU of heparin was added to the eluted fractions (8 ml) before dialysis against TBS containing 6.3 IU/ml heparin. Active fractions were pooled and concentrated approximately 20-fold in a Centriprep-10 concentrator (Amicon Inc., Beverly, MA). Heparin was added to the pooled fractions to give a final concentration of 500 IU/ml. The purified HL appeared as a single band following SDS-polyacrylamide gel electrophoresis on a 20% homogeneous PhastGel (Amersham Pharmacia Biotech) and staining with Coomassie Blue. The HL was stored at -70 °C.

HL activity was assessed as the nmol of triacylglycerol hydrolyzed/ml of HL/h using (A-I/TG)rHDL as a substrate. Purified HL (24 µl) was incubated at 37 °C for 1 h with (A-I/TG)rHDL (final triacylglycerol concentration = 0.15 mmol/liter) and heparin (final concentration = 500 IU/ml) in the presence or absence of 1 M NaCl. The final incubation volume was 120 µl. Triacylglycerol hydrolysis was measured as the decrease in triacylglycerol mass compared with a control incubation that did not contain HL. Since the amount of triacylglycerol hydrolysis was identical in the presence and absence of 1 M NaCl, the HL was judged to be free of LPL activity. It should also be noted that the rHDL preparations were deficient in apoC-II, the cofactor for LPL-mediated triacylglycerol hydrolysis. Triacylglycerol mass was measured by enzymatic assay (24) using Boehringer Mannheim standards (Mannheim, Germany). The amount of HL activity in individual experiments is presented in the figure legends.

Determination of HL-mediated Hydrolysis in Radiolabeled Substrates-- All incubations were carried out in stoppered plastic tubes in a shaking water bath at 37 °C. Details of individual incubations are described in the legends to the figures. Chloroform/methanol (1 ml, 2:1 (v/v)), was added to stop the hydrolysis reactions. The lipids were extracted by the method of Folch et al. (25). NEFA were separated from the other rHDL (or native HDL2) lipids by thin layer chromatography on 20 × 20-cm Silica gel 60 plastic sheets (Merck, Darmstadt, Germany). The sheets were developed in chloroform/methanol/water (65:25:4, v/v/v) until the solvent front was 8 cm from the origin. The sheets were dried and then run in hexane/diethyl ether/acetic acid (70:30:1, v/v/v) until the solvent front was 17 cm from the origin. A mixture of triolein (Sigma), POPC, and sodium oleate (Sigma) (0.4 mg/ml of each dissolved in chloroform/methanol (2:1, v/v)) was used as a standard. The NEFA and the other lipids were visualized with I2. The spots corresponding to phosphatidylcholine, triacylglycerol, and NEFA were cut from the sheets and placed directly into 10 ml of Ready SafeTM liquid scintillation mixture (Beckman Instruments). Radioactivity was determined using a Beckman LS 6000TA liquid scintillation counter with automatic quenching correction (Beckman Instruments). The silica gel had a negligible effect on the counting.

Determination of HL-mediated Hydrolysis in Unlabeled (A-I/CE)rHDL and (A-II/CE)rHDL-- These incubations were carried out exactly as described above for the radiolabeled substrates. Phospholipid hydrolysis was determined directly by assaying the mass of NEFA released from the rHDL. At the end of the incubation period, the tubes containing the incubation mixtures were placed on ice prior to assaying for NEFA. The NEFA concentration was determined using an enzymatic colorimetric assay kit (Wako Pure Chemical Industries, Osaka, Japan).

Calculations-- HL-mediated hydrolysis in the radiolabeled substrates was determined as the amount of radiolabel in the NEFA relative to the total radiolabel in the substrate. HL-mediated hydrolysis in the unlabeled (A-I/CE)rHDL and (A-II/CE)rHDL was determined by direct mass assay of the NEFA formed. The kinetic parameters Km(app) and Vmax were estimated from the line of best fit by linear regression analysis of a Lineweaver-Burk double-reciprocal plot of the rate of hydrolysis versus the concentration of substrate. In all cases, the regression coefficients (r) were >0.98. Vmax was determined as the reciprocal of the intercept on the y axis. The Km(app) was calculated as the product of the slope and Vmax.

Other Techniques-- All chemical analyses were carried out on a Cobas Fara centrifugal analyzer (Roche Diagnostics, Zurich, Switzerland). Boehringer Mannheim kits were used for phospholipid, UC, and total cholesterol assays. CE concentrations were calculated as the difference between the total and UC concentrations. The concentrations of apoA-I and apoA-II were determined by an immunoturbidometric assay (26). The size of the rHDL and native HDL2 was determined by electrophoresis on 3-35% nondenaturing polyacrylamide gradient gels (Gradipore, Sydney, Australia) (27).

Statistical Methods-- The one-tailed, Student's t test for two samples with equal variance was used to determine whether differences between values were significant.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Physical Properties of rHDL and Native HDL2 (Table I)-- The respective molar ratios of phospholipid/UC/CE/A-I/A-II in the (A-I/CE)rHDL and (A-II/CE)rHDL labeled with [14C]DPPC were 89.2/9.4/71.4/3.0/0.0 and 79.4/8.1/63.6/0.1/6.0. Since cross-linking studies have shown that spherical (A-I)rHDL and (A-II)rHDL of comparable size and composition contain three and six apolipoprotein molecules/particle, respectively (16), the molar ratios in Table I are expressed relative to the number of apolipoprotein molecules. The composition of the native HDL2 is expressed as percentage of mass. The data in Table I confirm that displacement of apoA-I from (A-I/CE)rHDL by lipid-free apoA-II does not promote displacement of rHDL lipid constituents (23). As such, the (A-I/CE)rHDL and (A-II/CE)rHDL differed only in their apolipoprotein composition. This was also the case for both the (A-I/TG)rHDL and (A-II/TG)rHDL and the (A-I)HDL2 and (A-II)HDL2. The slight increase in the diameter of the (A-II/CE)rHDL and (A-II/TG)rHDL relative to their apoA-I-containing precursors is consistent with what has been reported earlier by this laboratory (23). The diameter of the (A-II)HDL2 was also slightly larger than that of the (A-I)HDL2.

                              
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Table I
Physical properties of spherical rHDL and HDL2
(A-I/CE)rHDL were prepared by incubating discoidal rHDL with LDL and LCAT. Radiolabeled (A-I/CE)rHDL were labeled with [14C]DPPC as described under "Experimental Procedures." (A-I/TG)rHDL were prepared and labeled with [14C]DPPC or with [3H]triolein-labeled Intralipid as described under "Experimental Procedures." (A-II/CE)rHDL, (A-II/TG)rHDL, and (A-II)HDL2 were prepared by displacing all of the apoA-I from (A-I/CE)rHDL, (A-I/TG)rHDL, and (A-I)HDL2 with lipid-free apoA-II.

Kinetics of the HL-mediated Hydrolysis of Phospholipids and Triacylglycerol in rHDL-- The aim of these studies was to determine how apolipoproteins influence the HL-mediated hydrolysis of phospholipids and triacylglycerol in rHDL. Preliminary experiments established that the hydrolysis of phospholipids and triacylglycerol in both (A-I)rHDL and (A-II)rHDL was linear up to 30% (results not shown). Consequently, the kinetic studies described below were all conducted under conditions that gave less than 30% phospholipid or triacylglycerol hydrolysis.

Kinetics of HL-mediated Phospholipid Hydrolysis in (A-I/CE)rHDL and (A-II/CE)rHDL (Figs. 2 and 3 and Table II)-- (A-I/CE)rHDL and (A-II/CE)rHDL labeled with [14C]DPPC were used to monitor the kinetics of the HL-mediated hydrolysis of phospholipids in the absence of triacylglycerol (Fig. 2). In this experiment, the substrate concentration was increased progressively in incubations that contained a constant amount of HL. The duration of the incubation was 3 h. Fig. 2A shows that the rate of phospholipid hydrolysis increased as the concentration of the (A-I/CE)rHDL and (A-II/CE)rHDL increased from 0.05 to 0.4 mM phospholipid. For phospholipid concentrations from 0.05 to 0.2 mM, the rate of hydrolysis was greater in (A-II/CE)rHDL (open symbols) than in (A-I/CE)rHDL (closed symbols) (p < 0.01). At concentrations of 0.3 and 0.4 mM, the (A-I/CE)rHDL and (A-II/CE)rHDL exhibited comparable rates of phospholipid hydrolysis. A Lineweaver-Burk double-reciprocal plot of the data in Fig. 2A is shown in Fig. 2B. The kinetic parameters derived from the double reciprocal plot are shown in Table II. The Vmax was greater for (A-I/CE)rHDL than for (A-II/CE)rHDL (309.3 versus 49.1 nmol of NEFA formed/ml of HL/h). However, HL had a greater affinity for the phospholipids in (A-II/CE)rHDL (Km(app) = 0.2 mM) than in (A-I/CE)rHDL (Km(app) = 3.1 mM). The catalytic efficiency of the HL-mediated phospholipid hydrolysis (Vmax/Km(app)) was greater for (A-II/CE)rHDL than for (A-I/CE)rHDL.


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  		Fig. 2.   Kinetics of the hydrolysis of phospholipids in (A-I/CE)rHDL and (A-II/CE)rHDL. (A-I/CE)rHDL (bullet ) and (A-II/CE)rHDL (open circle ) were radiolabeled with [14C]DPPC, and aliquots in which the phospholipid concentration varied from 0.05 to 0.4 mM were incubated at 37 °C for 3 h with HL (29 µl of a preparation that hydrolyzed 62 nmol of triacylglycerol/ml of HL/h). The incubation mixtures also contained BSA (final concentration 20 mg/ml) and heparin (final concentration 500 IU/ml) in a final incubation volume of 50 µl. The rate of phospholipid hydrolysis as a function of substrate concentration is shown in A. The values are the means of triplicate determinations (*, p < 0.01). A double reciprocal plot of the kinetic data in A is shown in B.


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  		Fig. 3.   Kinetics of the hydrolysis of phospholipids in (A-I/CE)rHDL and (A-II/CE)rHDL as deterimined by mass assay of NEFA. (A-I/CE)rHDL (bullet ) and (A-II/CE)rHDL (open circle ) without radiolabel were incubated at 37 °C for 3 h with HL. The rHDL phospholipid concentration varied from 0.08 to 0.8 mM, and the incubation mixtures contained BSA at the same concentration as in Fig. 2. The rate of phospholipid hydrolysis as a function of substrate concentration is shown. The values are the means of quaduplicate determinations (*, p < 0.01; #, p < 0.05). A double reciprocal plot of the kinetic data in A is shown in B.

                              
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Table II
Kinetic parameters of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL
A range of concentrations of spherical rHDL were incubated with a constant amount of HL as described in the legends to Figs. 2-6. For the radiolabeled substrates, the resulting NEFA were separated from the other rHDL lipids by thin layer chromatography as described under "Experimental Procedures." Kinetic parameters were estimated from double-reciprocal plots of the rate of hydrolysis versus the concentration of rHDL phospholipid or triacylglycerol. All values are means of triplicate determinations. Since different preparations of HL were used for each study, the Vmax values cannot be compared for the different experiments.

To ensure that the hydrolysis of [14C]DPPC in the radiolabeled rHDL reflected the hydrolysis of the rHDL bulk phospholipid, NEFA formation was measured directly by mass assay in unlabeled (A-I/CE)rHDL and (A-II/CE)rHDL (Fig. 3A). As the NEFA mass assay is much less sensitive than the measurement of radiolabel, the lowest concentration of substrate used in these experiments was 0.08 mM phospholipid (compared with 0.05 mM phospholipid for the radiolabeled substrate). These results were comparable with what was obtained using [14C]DPPC-labeled (A-I/CE)rHDL and (A-II/CE)rHDL. When these results were transformed into a Lineweaver-Burk double reciprocal plot (Fig. 3B), it was apparent that, as with the radiolabeled substrate, HL has a greater affinity for the phospholipid in (A-II/CE)rHDL than in (A-I/CE)rHDL, with the Vmax being greater for (A-I/CE)rHDL than for (A-II/CE)rHDL. These results show that the hydrolysis of [14C]DPPC, when present in trace amounts in rHDL, reflects the hydrolysis of the bulk phospholipid.

Kinetics of HL-mediated Phospholipid Hydrolysis in Native (A-I)HDL2 and (A-II)HDL2 (Fig. 4)-- To ensure that the above results are an accurate reflection of phospholipid hydrolysis in native HDL, an additional experiment was carried out with HDL2 that had been isolated from human plasma and radiolabeled with [14C]DPPC. ApoA-I constituted more than 90% of the apolipoproteins in this preparation (Table I). The HDL2 also contained a minimal amount of triacylglycerol. Native HDL2, in which apoA-II comprised more than 90% of the apolipoproteins, was prepared as described under "Experimental Procedures." The relationship between HL-mediated phospholipid hydrolysis in native (A-I)HDL2 and (A-II)HDL2 (Fig. 4) was comparable with what was observed in (A-I/CE)rHDL and (A-II/CE)rHDL. At HDL2 phospholipid concentrations of 0.05 and 0.1 mM, the rate of phospholipid hydrolysis was greater in (A-II)HDL2 (open symbols) than in (A-I)HDL2 (closed symbols) (p < 0.01). At HDL2 phospholipid concentrations of 0.7 and 1.0 mM, the rate of phospholipid hydrolysis was greater in (A-I)HDL2 than in (A-II)HDL2 (p < 0.01). Since transformation of this data into a Lineweaver-Burk double reciprocal plot did not yield a straight line, the kinetic parameters were not determined. The nonlinearity of the transformed data highlights the problems associated with using substrates that are heterogeneous in size and composition.


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  		Fig. 4.   Kinetics of the hydrolysis of phospholipids in native (A-I)HDL2 and (A-II)HDL2. (A-I)HDL2 (bullet ) and (A-II)HDL2 (open circle ) were radiolabeled with [14C]DPPC, and aliquots in which the phospholipid concentration varied from 0.05 to 1.0 mM were incubated at 37 °C for 3 h with HL (5 µl of a preparation that hydrolyzed 247 nmol of triacylglycerol/ml of HL/h). The incubation mixtures also contained BSA and heparin at the same concentrations as in Fig. 2. The rate of phospholipid hydrolysis as a function of substrate concentration is shown. The values are the means of triplicate determinations (*p < 0.01).

Kinetics of HL-mediated Phospholipid Hydrolysis in (A-I/TG)rHDL and (A-II/TG)rHDL (Fig. 5, Table II)-- (A-I/TG)rHDL and (A-II/TG)rHDL labeled with [14C]DPPC were used to assess the HL-mediated hydrolysis of phospholipids in the presence of triacylglycerol. The design of this study was identical to that described above for (A-I/CE)rHDL and (A-II/CE)rHDL. The rate of phospholipid hydrolysis as a function of rHDL phospholipid concentration is shown in Fig. 5A. At all concentrations of phospholipid, the rate of hydrolysis was greater in (A-I/TG)rHDL (closed symbols) than in (A-II/TG)rHDL (open symbols) (p < 0.01). The Vmax was greater for (A-I/TG)rHDL than for (A-II/TG)rHDL (625.3 versus 254.4 nmol of NEFA formed/ml of HL/h) (Table II). The HL had a greater affinity for the phospholipids in (A-II/TG)rHDL (Km(app) = 0.4 mM) than in (A-I/TG)rHDL (Km(app) = 0.9 mM). The Vmax/Km(app) values for the two substrates were comparable.


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  		Fig. 5.   Kinetics of the hydrolysis of phospholipids in (A-I/TG)rHDL and (A-II/TG)rHDL. (A-I/TG)rHDL (bullet ) and (A-II/TG)rHDL (open circle ) were radiolabeled with [14C]DPPC, and aliquots in which the phospholipid concentration varied from 0.05 to 0.4 mM were incubated at 37 °C for 3 h with HL (10 µl of a preparation that hydrolyzed 102 nmol of triacylglycerol/ml of HL/h). The incubation mixtures also contained BSA and heparin at the same concentrations as in Fig. 2. The rate of phospholipid hydrolysis as a function of substrate concentration is shown in A. The values are the means of triplicate determinations (*, p < 0.01). A double reciprocal plot of the kinetic data in A is shown in B.

Kinetics of HL-mediated Triacylglycerol Hydrolysis in (A-I/TG)rHDL and (A-II/TG)rHDL (Fig. 6, Table II)-- The kinetics of triacylglycerol hydrolysis in (A-I/TG)rHDL and (A-II/TG)rHDL radiolabeled with [3H]triolein is shown in Fig. 6A. In this study, increasing concentrations of substrate were incubated for 1 h with a constant amount of HL. At rHDL triacylglycerol concentrations of 0.02 and 0.03 mM, the rate of hydrolysis was greater in (A-II/TG)rHDL (open symbols) than in (A-I/TG)rHDL (closed symbols) (see inset). The (A-I/TG)rHDL and (A-II/TG)rHDL exhibited comparable rates of hydrolysis at a triacylglycerol concentration of 0.1 mM. At rHDL triacylglycerol concentrations of 0.24 and 0.33 mM, the rate of triacylglycerol hydrolysis was greater in (A-I/TG)rHDL than in (A-II/TG)rHDL (p < 0.01). The Vmax for (A-I/TG)rHDL was greater than for (A-II/TG)rHDL (1154.8 versus 240.2 nmol of NEFA formed/ml of HL/h) (Table II). As with the phospholipid hydrolysis, HL had a greater affinity for the triacylglycerol in (A-II/TG)rHDL (Km(app) = 0.1 mM) than in (A-I/TG)rHDL (Km(app) = 1.0 mM). The Vmax/Km(app) for triacylglycerol hydrolysis in (A-II/TG)rHDL was approximately double that for (A-I/TG)rHDL.


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  		Fig. 6.   Kinetics of the hydrolysis of triacylglycerol in (A-I/TG)rHDL and (A-II/TG)rHDL. (A-I/TG)rHDL (bullet ) and (A-II/TG)rHDL (open circle ) were radiolabeled with [3H]triolein, and aliquots in which the triacylglycerol concentration varied from 0.02 to 0.33 mM were incubated at 37 °C for 1 h with HL (9 µl of a preparation that hydrolyzed 47 nmol of triacylglycerol/ml of HL/h). The incubation mixtures also contained BSA and heparin at the same concentrations as in Fig. 2. The rate of triacylglycerol hydrolysis as a function of substrate concentration is shown in A. The inset shows an expanded view of the area of the graph below 0.075 mM triacylglycerol. Each value is the mean of triplicate determinations (*, p < 0.01; #, p < 0.05). A double reciprocal plot of the kinetic data in A is shown in B.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, we have used well characterized, homogeneous, apolipoprotein-specific preparations of spherical rHDL and preparations of native HDL2 to investigate the influence of apoA-I and apoA-II on the HL-mediated hydrolysis of HDL phospholipids and triacylglycerol. The (A-I)rHDL and (A-II)rHDL used in this study were comparable in size and lipid composition and differed only in their apolipoprotein content. The results show unequivocally that, although HL has a higher affinity for the phospholipids and triacylglycerol in (A-II)rHDL than in (A-I)rHDL, the maximal rate of hydrolysis for both constituents is greater in (A-I)rHDL than in (A-II)rHDL.

The HDL in human plasma consist of two major apolipoprotein-specific subpopulations of particles: those containing apoA-I without apoA-II, (A-I)HDL, and those with both apoA-I and apoA-II, (A-I/A-II)HDL (28). There is also a minor HDL subpopulation that contains apoA-II without apoA-I, (A-II)HDL (29). The present studies conducted in vitro with rHDL and native HDL2 raise the possibility that there may be major differences in the interaction of HL with (A-I)HDL and (A-II)HDL in vivo. Although it is not known whether (A-I/A-II)HDL resemble (A-I)HDL or (A-II)HDL in terms of their interactions with HL in plasma, it would be surprising if the presence of apoA-II in HDL did not have an impact on the HL-mediated hydrolysis of phospholipids and triacylglycerol in vivo.

There are several reasons why apolipoproteins may influence the interaction of HL with HDL. For instance, the different affinity of HL for (A-I)rHDL relative to (A-II)rHDL may reflect differences in particle charge. Rye and Barter (23) have reported that the surface of (A-II)rHDL is less negatively charged than that of (A-I)rHDL. Since HL has a net negative charge (30), it is likely to have a greater affinity for the surface of (A-II)rHDL compared with (A-I)rHDL. Consistent with this, Laboda et al. (30) showed that the HL-mediated hydrolysis of triolein is reduced when a negative charge is incorporated into a lipid monolayer. Another explanation for the different affinities of HL for (A-I)rHDL and (A-II)rHDL relates to lipid-water interfacial hydration. It has been shown in earlier work from this laboratory that the lipid-water interface of (A-I)rHDL is more hydrated than that of (A-II)rHDL (23). Since the catalytic sites of most lipases are hydrophobic (31, 32), it is conceivable that such enzymes would associate preferentially with the less hydrated lipid-water interface of (A-II)rHDL.

The present data show that when the substrate concentration is not rate-limiting, HL hydrolyzes both phospholipids and triacylglycerol more rapidly in (A-I)rHDL than in (A-II)rHDL. This is consistent with the phospholipid and triacylglycerol acyl chains in (A-I)rHDL being more accessible to the active site of HL than those in (A-II)rHDL. One explanation for this observation relates to the less ordered phospholipid head group packing in (A-I)rHDL compared with (A-II)rHDL (16). Such a difference may lead to significant structural changes in the microenvironment of the rHDL interface, which could influence access of the enzyme to its substrates in such a way as to enhance the hydrolysis of both phospholipids and triacylglycerol in (A-I)rHDL (33).

Tansey et al. reported recently on the HL-mediated hydrolysis of phospholipids in discoidal and spherical rHDL that had been prepared with a range of phospholipids, including DPPC (34). In that study, minimal phospholipid hydrolysis was observed in either the discoidal or spherical rHDL that contained DPPC. This is an interesting finding given the results of the present study, where [14C]DPPC was used as a tracer to monitor POPC hydrolysis. It should be noted that other investigators have also used [14C]DPPC successfully as a tracer to monitor phospholipid hydrolysis (35, 36). These discrepancies suggest that HL is sensitive to the phase state of substrate lipids. Indeed, Thuren et al. have found this to be the case in their monolayer studies (37). Thus, although DPPC is a poor substrate for HL when it is present as the bulk lipid in an interface, it is hydrolyzed readily by HL when it is present in trace amounts in a more physiological substrate. The current results show clearly that this is the case, since comparable results were obtained when phospholipid hydrolysis was determined using rHDL containing a trace amount of [14C]DPPC (Fig. 2) as well as in experiments where NEFA formation was measured directly with a mass assay (Fig. 3).

There are several conflicting reports as to the effects of apolipoproteins on the HL-mediated hydrolysis of phospholipids and triacylglycerol in HDL. On the one hand, it has been suggested that apoA-II inhibits (9, 10), while others have found that it enhances, HL-mediated hydrolysis of HDL triacylglycerol (7, 8, 38, 39). Mowri et al. (38) reported that hydrolysis of phospholipids and triacylglycerol in HDL is greater in (A-I/A-II)HDL2 compared with (A-I)HDL2. However, those observations cannot be compared directly with what was observed in the present study, since the HDL2 used by Mowri et al. (38) were heterogeneous in size and composition. They also contained apolipoproteins other than apoA-I and apoA-II that may have influenced the hydrolysis (40). Finally, Mowri et al. (38) measured total fatty acid liberation (i.e. phospholipid and triacylglycerol hydrolysis combined) rather than the hydrolysis of individual lipids.

The results of Zhong et al. (9) are, to a large extent, consistent with the present findings. Those investigators found that the HL-mediated hydrolysis of triacylglycerol in a lipid emulsion was inhibited by the addition of HDL from mice that had been made transgenic for either human apoA-I or human apoA-II or for both human apoA-I and apoA-II. Since the HDL from the apoA-II and apoA-I/apoA-II transgenic mice mediated a greater reduction in the rate of HL-mediated triacylglycerol hydrolysis than the HDL from the apoA-I transgenic mice, it was concluded that apoA-II inhibits HL activity more than apoA-I. Given the findings in the present study, which show that HL has a greater affinity for the less reactive apoA-II-containing rHDL, it would be predicted that (A-II)HDL would be more effective than (A-I)HDL as inhibitors of triacylglycerol hydrolysis in lipid emulsions.

In another study, Jahn et al. (39) found that reconstituted particles containing apoA-II and HDL lipids enhanced the HL-mediated hydrolysis of triacylglycerol in microemulsions. These investigators obtained similar results when the reconstituted particles were substituted with ultracentrifugally isolated HDL. Jahn et al. (39) explained their observations in terms of apoA-II partitioning from the reconstituted particles (or isolated HDL) to the triacylglycerol emulsion and thus increasing the affinity of HL for the triacylglycerol emulsion. However, these investigators also found that the triacylglycerol hydrolysis in the microemulsions decreased at high concentrations of reconstituted particles (or HDL). This may have been caused by the high concentrations of reconstituted particles or HDL competing with the triacylglycerol emulsion for HL.

The results of the present study may explain some of the conflicting conclusions that have been drawn about the relative effects of apoA-I and apoA-II on HL-mediated phospholipid and triacylglycerol hydrolysis (9, 38). The lower Km(app) values for both the phospholipids and triacylglycerol in (A-II)rHDL relative to (A-I)rHDL suggest that HL has a greater affinity for HDL that contain apoA-II than for HDL that contain apoA-I. In other words, at a low concentration of substrate, the amount of HL interacting with HDL (and hydrolyzing HDL lipids) may be much greater in HDL that contain apoA-II than in (A-I)HDL. Therefore, when a study is conducted at a low substrate concentration, it may be concluded that apoA-II-containing HDL are superior to (A-I)HDL as substrates for HL. By contrast, if experiments are conducted at high substrate concentrations, under conditions where substrate availability is no longer a limiting factor, and the Vmax for phospholipid and triacylglycerol hydrolysis is greater in (A-I)rHDL than in (A-II)rHDL, it may be concluded that (A-I)HDL are superior substrates compared with HDL that contain apoA-II.

In conclusion, these studies provide the first description of the kinetics of the HL-mediated hydrolysis of phospholipids and triacylglycerol in spherical, apolipoprotein-specific rHDL. In addition, they show that apolipoproteins have a major impact on these processes. It remains to be determined whether the interaction of (A-I/A-II)HDL with HL resembles that of either the (A-I)rHDL or (A-II)rHDL or whether it is distinct from each.

    ACKNOWLEDGEMENTS

The technical assistance of Richard Bright is gratefully acknowledged. The staff of the Cardiovascular Investigational Unit, Royal Adelaide Hospital, are acknowledged for assistance in the collection of postheparin blood samples from angioplasty patients.

    FOOTNOTES

* This work was supported by the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Postgraduate Science Research Scholarship from the National Heart Foundation of Australia.

** A Royal Adelaide Hospital Florey Fellow. To whom correspondence should be addressed: Lipid Research Laboratory, Level 1, Hanson Centre, Frome Road, Adelaide, South Australia, Australia 5000. Tel.: 61-8-8222-3448; Fax: 61-8-8222-3154; E-mail: karye{at}camtech.net.au.

The abbreviations used are: HL, hepatic lipase; HDL, high density lipoprotein(s); HDL2, high density lipoprotein(s), subfraction 2LPL, lipoprotein lipaseapo, apolipoproteinrHDL, reconstituted high density lipoproteinsLCAT, lecithin:cholesterol acyltransferasePOPC, 1-palmitoyl-2-oleoylphosphatidylcholineUC, unesterified cholesterolCE, cholesteryl ester(s)CETP, cholesteryl ester transfer proteinLDL, low density lipoprotein(s)PLTP, phospholipid transfer proteinDPPC, 1,2-dipalmitoylphosphatidylcholineTBS, Tris-buffered salineNEFA, nonesterified fatty acid(s)BSA, bovine serum albumin.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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Role of apoA-II in lipid metabolism and atherosclerosis: advances in the study of an enigmatic protein
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In vivo interactions of apoA-II, apoA-I, and hepatic lipase contributing to HDL structure and antiatherogenic functions
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Influence of phospholipid depletion on the size, structure, and remodeling of reconstituted high density lipoproteins
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ApoA-II maintains HDL levels in part by inhibition of hepatic lipase: studies in apoA-II and hepatic lipase double knockout mice
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T. A. Ramsamy, T. A.-M. Neville, B. M. Chauhan, D. Aggarwal, and D. L. Sparks
Apolipoprotein A-I Regulates Lipid Hydrolysis by Hepatic Lipase
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[Abstract] [Full Text] [PDF]

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