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Originally published In Press as doi:10.1074/jbc.M005436200 on August 15, 2000

J. Biol. Chem., Vol. 275, Issue 43, 33480-33486, October 27, 2000
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Apolipoprotein A-I Regulates Lipid Hydrolysis by Hepatic Lipase*

Tanya A. RamsamyDagger, Tracey A.-M. Neville, Bobby M. Chauhan, Dhiraj Aggarwal, and Daniel L. Sparks§

From the Lipoprotein and Atherosclerosis Research Group and the Departments of Pathology & Laboratory Medicine and Biochemistry, Microbiology & Immunology, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7, Canada

Received for publication, June 21, 2000, and in revised form, July 25, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Association of hepatic lipase (HL) with pure heparan sulfate proteoglycans (HSPG) has little effect on hydrolysis of high density lipoprotein (HDL) particles, but significantly inhibits (>80%) the hydrolysis of low (LDL) and very low density lipoproteins (VLDL). Lipolytic inhibition is associated with a differential ability of the lipoproteins to remove HL from the HSPG. LDL and VLDL are unable to displace HL, whereas HDL readily displaces HL from the HSPG. These data show that HSPG-bound HL is inactive. Purified apolipoprotein (apo) A-I is more efficient than HDL at liberating HL from HSPG, and HL displacement is associated with the direct binding of apoA-I to HSPG. However, displacement of HL by apoA-I does not enhance hydrolysis of VLDL particles. This appears due to the direct inhibition of HL by apoA-I. Both apoA-I and HDL are able to inhibit VLDL lipid hydrolysis by up to 60%. Inhibition of VLDL hydrolysis is associated with the binding of apoA-I to the surface of the VLDL particle and a concomitant decreased affinity for HL. These data show that apoA-I can regulate lipid hydrolysis by HL by liberating/activating the enzyme from cell surface proteoglycans and by directly modulating lipoprotein binding and hydrolysis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human hepatic lipase (HL)1 is a 64-kDa, 476-amino acid glycoprotein that is synthesized and secreted primarily by the liver (1). The enzyme is found anchored by heparan sulfate proteoglycans (HSPG) to the surface of endothelial cells and hepatocytes (2) and is postulated to function as a lipolytic enzyme and as a cell surface ligand for lipoprotein uptake. As a ligand, HL is thought to interact with cell surface HSPG (3) and specific receptors including the low density lipoprotein (LDL) receptor and LDL receptor-related protein (4, 5) in order to enhance the clearance of lipoprotein particles (3, 4, 6, 7). The catalytic function of HL involves the hydrolysis of sn-1 fatty acyl ester bonds of phospholipids (PL) and mono-, di-, and triglycerides (TG) found in all classes of lipoproteins (8-11). It is believed that HL is involved in the remodeling of high density lipoprotein (HDL) and in the hydrolysis of the intermediate density lipoprotein to form LDL (12).

HSPG are composed of two main components: a protein core and sulfated polysaccharide side chains known as glycosaminoglycans. The protein core functions to anchor the molecule to the cell surface and to covalently bind glycosaminoglycans. HSPG are present in most tissues and can be located within the cell as well as on the pericellular and extracellular matrices (13). Studies have suggested that interactions with HSPG may affect the catalytic activity of lipolytic enzymes. Waite et al. (8) identified changes in the substrate specificity of HL after it was released from the liver by heparin. In addition, studies with lipoprotein lipase (LPL) have suggested that the binding of this enzyme to HSPG may inhibit its catalytic activity (14, 15).

In humans, most HL is thought to be associated with cell surface HSPG, therefore experiments were undertaken to determine how the association of HL with HSPG may affect the function of this enzyme. We show that lipid hydrolysis by HL is inhibited when the enzyme is associated with HSPG. This study further shows that apolipoprotein (apo) A-I has the ability to displace HL from HSPG and also to inhibit the hydrolysis of very low density lipoprotein (VLDL) lipids by this enzyme.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HSPG were purchased from Sigma. The free fatty acid diagnostic kits were purchased from Roche Diagnostics (Laval, Quebec (PQ), Canada). The anti-mouse IgG, horseradish peroxidase (HRP)-linked whole antibody (isolated from sheep) and the broad range molecular weight markers were obtained from Amersham Pharmacia Biotech (Baie d'Urfé, PQ, Canada). The SuperSignal West Pico Chemiluminescent Substrate was purchased from Pierce. The anti-HL monoclonal antibody (mAb) (3-6) was obtained from Dr. A. Bensadoun. The anti- apoA-I mAbs (5F6 and 4H1) and the anti-apoB mAb (1D1) were obtained from Drs. Y. Marcel and R. Milne. All other reagents were of analytical grade.

Isolation of Lipoproteins-- Plasma from fasting, normolipidemic subjects was collected and VLDL, LDL, and HDL were isolated by sequential ultracentrifugation within the density ranges d < 1.006, d = 1.019-1.063, and d = 1.063-1.21 g/ml, respectively. The protein content of the lipoprotein fractions was determined by the Lowry method as modified by Markwell et al. (16). PL, cholesterol, and TG contents were determined enzymatically using diagnostic kits (Roche Diagnostics, Laval, PQ, Canada).

Hepatic Lipase Purification-- HL was purified from post-heparin human plasma by heparin affinity chromatography as described by Ehnholm et al. (17). Post-heparin human plasma was collected from normolipidemic subjects and a 20% TG emulsion (Intralipid 20%, Pharmacia & Upjohn Inc., Missassauga, Ontario, Canada) was added to the plasma. Lipid cakes were harvested centrifugally and delipidated. The resuspended, aqueous solution of crude HL was loaded onto a heparin-Sepharose CL-6B column and eluted with a linear salt gradient of 0.15-1.5 M NaCl in 5 mM sodium barbital, pH 7.4, 20% glycerol and the pooled fractions stored at -80 °C until use.

HL activity was characterized using a TG emulsion and quantitated into units of enzyme activity (where 1 unit = 1 µmol of fatty acid hydrolyzed/h). The protein concentration of the purified HL was 0.114 mg/ml, and the specific activity of the isolated HL was determined to be 19,455 units/mg of protein. The isolated HL was further characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE) and immunoblotting using the anti-HL mAb. A single band with an apparent molecular mass of 66 kDa was evident.

Binding of Hepatic Lipase to Proteoglycans-- The binding of human HL to HSPG was investigated in assays performed in 96-well Removawell plates. Removawells were incubated with 5 µg of pure HSPG for 2 h at room temperature, washed three times with 50 mM sodium phosphate, pH 7.2, 150 mM NaCl (PBS) and blocked with PBS containing 1% essentially fatty acid-free bovine serum albumin (FAF-BSA) overnight at 4 °C. The Removawells were again washed three times with PBS and incubated for 2 h at room temperature with HL (120 units) in PBS (final volume, 125 µl). After incubation, Removawells were washed once with PBS to remove any unbound HL.

Substrate Specificity of Bound Versus Free Hepatic Lipase-- Native lipoproteins were characterized as substrates for purified HL bound to HSPG (as described above) or free in solution. Each Removawell contained the lipoprotein substrate, 26 units of purified HL, 75 µl of incubation buffer (0.33 M Tris-HCl, pH 8.3, 1% FAF-BSA, 5 mM CaCl2), and PBS (final volume, 250 µl) in the presence or absence of HSPG. Incubations were carried out for 30 min (inhibition studies) or 3 h at 37 °C and terminated by placing the plate on ice. The total amount of fatty acid released during the incubation was determined using a free fatty acid diagnostic kit.

Determination of Hepatic Lipase Binding to Proteoglycans-- HSPG-bound HL was incubated for 3 h with the lipoprotein substrates, and then Removawells were rinsed with PBS. 60 µl of an elution buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS, 0.5% (w/v) bromphenol blue) was added to the Removawells, to elute the HSPG-bound HL, and the mixture was incubated at 37 °C for 30 min. Samples were electrophoresed on a 12% acrylamide gel, under denaturing conditions, and then transferred to a nitrocellulose membrane. The membrane was cut at the 35-kDa marker, and the upper portion of the nitrocellulose was blocked overnight at 4 °C in blocking solution A (PBS containing 1% BSA and 0.2% Tween 20). After a 2-h incubation at room temperature with the anti-HL mAb (3:5000 dilution) in blocking solution A containing 0.02% sodium azide, the membranes were rinsed three times and washed four times for 15 min each with PBS containing 0.2% Tween 20 (PBS-Tween). Sheep anti-mouse IgG, HRP-linked whole antibody was used as a secondary antibody and diluted (1:5000) in blocking solution A. After a 1-h incubation, the membranes were rinsed three times and washed four times for 15 min each with PBS-Tween. A 10-min incubation with the Pierce Super Signal West Pico Chemiluminescent Substrate was used to visualize the HL. The membranes were exposed to film for various times and scanned, and the percentage of HL that remained bound to the HSPG under the various conditions was determined by densitometry using Bio-Rad Quantity One (version 4.03) software.

Determination of ApoA-I Binding to Proteoglycans-- After the samples were electrophoresed and transferred and the nitrocellulose membrane cut, the lower portion of the nitrocellulose was blocked overnight at 4 °C in blocking solution B (PBS containing 5% skim milk and 0.2% Tween 20). After a 1-h incubation at room temperature with the anti-apoA-I mAbs 5F6 and 4H1 (1:5000 dilution each) in blocking solution B containing 0.02% sodium azide, the membranes were rinsed and washed three times for 10 min with PBS-Tween. Sheep anti-mouse IgG, HRP-linked whole antibody was used as a secondary antibody and diluted (1:5000) in blocking solution B. After a 1-h incubation, the membranes were washed three times with PBS-Tween. The apoA-I that bound to the HSPG was visualized, scanned, and analyzed as described for HL. Similar protocols were used to evaluate the binding of apoB (mAb 1D1) and apoE (mAb 1D7) to HSPG.

Determination of ApoA-I Binding to VLDL-- Removawells were coated with an anti-apoB mAb (1D1) (at a predetermined dilution) overnight at 4 °C, washed with PBS, and saturated with 0.5% gelatin in PBS. Serial dilutions of VLDL ± HDL or apoA-I (in PBS with 0.1% gelatin), that had been incubated with HL for 3 h at 37 °C, were incubated with the previously coated Removawells for 2 h at room temperature. After 3 washes (PBS with 0.05% Tween 20), the Removawells were incubated for 1 h with 125I-labeled anti-apoA-I mAbs (4H1 and 5F6) in PBS with 0.1% gelatin (~200,000 cpm/0.015 µg/well). Wells were washed three times with PBS-Tween, and their radioactivity was measured.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activity of HSPG-bound Hepatic Lipase-- Removawells were coated with pure HSPG (5 µg/Removawell), incubated with FAF-BSA to block any nonspecific binding of HL, and incubated with excess purified human HL (120 units) for 2 h. Control incubations showed that negligible amounts of HL bound to albumin or albumin treated plates. Unbound HL was removed by washing, and 60 µl of elution buffer was added and incubated for 30 min at 37 °C to elute the HSPG-bound HL. SDS-PAGE followed by immunoblot analysis using an anti-HL mAb was performed to estimate the amount of HL bound. Maximal binding was shown to occur within 2 h, and ~26 units of HL was shown to associate with 5 µg of HSPG. All subsequent experiments were therefore carried out with a 2-h association of HL with HSPG, followed by a brief wash to remove excess unbound HL. To evaluate whether the association of HL to HSPG alters the catalytic activity of the enzyme, Removawells were coated with HSPG, blocked with FAF-BSA, incubated with excess HL for 2 h, and washed. VLDL, LDL, or HDL (350 µM TG) were added to the wells and incubated for 3 h at 37 °C. Extended incubation times (3 h) and high substrate (TG) concentrations were required to promote enough hydrolysis to allow for comparison of hydrolytic rates for the best (VLDL and LDL) and poorer (HDL) HL substrates (Fig. 1). Fig. 1 shows that the association of HL with HSPG has a significant inhibitory affect on VLDL and LDL lipid hydrolysis, as compared with the control (BSA-blocked) Removawell that contained 26 units of HL, but no HSPG. In contrast, when HDL was evaluated as a substrate for HL, in the presence and absence of HSPG, the difference in total hydrolytic activity of the enzyme was much less (Fig. 1). Association of HL with HSPG inhibited LDL and VLDL hydrolysis by >80%, but inhibited HDL hydrolysis by ~40%.


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  		Fig. 1.   Hydrolysis of VLDL, LDL, and HDL by HSPG-bound hepatic lipase. Removawells were incubated with 5 µg of HSPG for 2 h at 24 °C. HSPG-coated (solid bars) and deficient (open bars) wells were washed three times with PBS and blocked with 1% FAF-BSA in PBS overnight at 4 °C. The HSPG-coated wells were incubated with 120 units of purified HL in PBS for 2 h at 24 °C and washed to remove any unbound HL. HSPG-deficient wells were incubated with 26 units of HL for 2 h, and then all Removawells were incubated with ultracentrifugally isolated VLDL, LDL or HDL (350 µM TG) for 3 h at 37 °C. An aliquot was removed, and fatty acid release was measured enzymatically. Hydrolytic values are the mean ± S.D. of triplicate determinations and are representative of two different experiments.

Displacement of HL from Proteoglycans-- To investigate whether the various lipoproteins affected the binding/association of HL with the HSPG, Removawells were coated with HSPG, blocked with FAF-BSA, incubated with excess HL for 2 h, washed, and incubated with different lipoproteins. After a standard 3-h incubation, the supernatant was removed and the Removawells were washed with PBS. Elution buffer was added, and the eluants were analyzed by SDS-PAGE and immunoblotted with anti-HL mAb. The amount of HL that remained bound to HSPG after a 3-h incubation with VLDL, LDL, and HDL was estimated, relative to a control Removawell incubated with PBS. Fig. 2 (upper panel) shows that LDL and VLDL were almost unable to displace HL from pure HSPG. When either LDL or VLDL was incubated with HSPG-bound HL, >90% of the HL remained bound. In contrast, HDL readily displaced the HL; only ~40% of the HL remained bound after a 3-h incubation. Displacement of HL was time-dependent; minimal displacement occurred by 30 min, and maximal displacement occurred by 3 h (data not shown). Experiments were also undertaken with lipoproteins from other subjects, and a substantive variation in displacement was evident. Some HDL preparations almost completely dissociated HL from HSPG and some VLDL caused slightly more displacement (5-10%) than that illustrated in Fig. 2.


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  		Fig. 2.   Effect of lipoproteins on the association of hepatic lipase with HSPG. Removawells were coated with HSPG and HL (described in Fig. 1) and incubated with various plasma lipoproteins or PBS (control) for 3 h at 37 °C. Wells were washed, and the HSPG-bound proteins were eluted from the Removawells by incubating with 60 µl of elution buffer at 37 °C for 30 min. The eluants were electrophoresed on SDS-PAGE and electrotransferred to nitrocellulose. Samples were probed with an anti-HL mAb (upper panel) or with anti-apoA-I mAbs (lower panel) and with an anti-mouse IgG HRP-linked secondary antibody. Apparent molecular weight determinations were derived from broad range molecular weight markers. Images are representative of triplicate determinations of two different experiments.

To determine if some apoprotein component of the different lipoproteins was able to displace HL by binding to the proteoglycan, the HSPG eluant was probed with mAbs to specific apoproteins. Although some apoB retention to the HSPG was identified with LDL and VLDL, as others have reported (18-20), no detectable amounts of apoE were found associated with the HSPG after incubation with any of the lipoproteins (data not shown). In contrast, large amounts of apoA-I were retained on the HSPG when HDL was incubated with the HSPG-bound HL (Fig. 2, lower panel). Small amounts of apoA-I were also detectable in incubations with LDL and VLDL, suggesting that some apoA-I may have been endogenously associated with these lower density lipoproteins. Fig. 3 illustrates an experiment that was undertaken to determine if HDL and pure, lipid-free apoA-I were equally effective at displacing HL from HSPG. When equal amounts of protein were incubated with HSPG-bound HL, apoA-I was shown to be able to promote ~1.5 times the HL release, as compared with HDL (Fig. 3, upper panel). In addition, twice as much apoA-I was retained on the HSPG with incubations of the lipid free protein, as compared with HDL (Fig. 3, lower panel). ApoA-I titration experiments were then performed to assess the effect of apoA-I concentration on HL displacement from HSPG. The results showed that even very low concentrations of apoA-I (14 µg/ml) were able to promote a maximal displacement of HL and that a 10-fold increase in apoA-I concentration had no substantial effect on the liberation of HL from HSPG (data not shown).


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  		Fig. 3.   Effect of HDL and apoA-I on the association of hepatic lipase with HSPG. Removawells were coated with HSPG and HL as described and then incubated with HDL or apoA-I (164 µg of protein) for 3 h at 37 °C. Wells were washed, and the HSPG-bound proteins were eluted and electrophoresed by SDS-PAGE. Samples were transferred to nitrocellulose and probed with an anti-HL mAb (upper panel) or with anti-apoA-I mAbs (lower panel). Images are representative of triplicate determinations from three different experiments.

Effect of ApoA-I on VLDL Hydrolysis-- Since HL appeared inactive when bound to HSPG and apoA-I can liberate the enzyme, experiments were performed to determine if the liberation of HL from HSPG by apoA-I could stimulate the hydrolysis of VLDL. Incubations were performed with free and HSPG-bound HL as described in Fig. 1; however, in some wells, VLDL was co-incubated with either pure apoA-I or HDL. Although the displacement of HSPG-bound HL by heparin was shown to recover the activity of the enzyme, Fig. 4 shows that addition of apoA-I or HDL to incubations of HSPG-bound HL and VLDL had little effect on the rate of lipid hydrolysis, relative to that expected for a displaced enzyme. Fig. 5 further shows that this "retained inhibition" was partly due to an impaired displacement of HL by either apoA-I or HDL, in the presence of VLDL. In this experiment, apoA-I and HDL were able to displace between 40% and 50% of the HSPG-bound HL. In the presence of VLDL, HL displacement fell to less than 10%. However, it is evident that, even though some HL displacement had occurred in these incubations, hydrolysis rates remained unaffected. These data suggest that apoA-I may have a secondary affect on HL-mediated hydrolysis.


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  		Fig. 4.   Effect of apoA-I or HDL on the hydrolysis of VLDL by HSPG-bound hepatic lipase. Removawells were coated with HSPG and HL and then were incubated with ultracentrifugally isolated VLDL, in the presence or absence of HDL (130 µM TG) or apoA-I (equivalent HDL protein concentration) for 3 h at 37 °C. An aliquot was removed, and fatty acid release was measured enzymatically. Hydrolytic values are the mean ± S.D. of triplicate determinations and are representative of two different experiments.


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  		Fig. 5.   Effect of VLDL on the dissociation of hepatic lipase by HDL and apoA-I. Removawells were coated with HSPG and HL as described and incubated with VLDL (350 µM TG), HDL (130 µM TG), apoA-I (equivalent HDL protein concentration), or VLDL plus HDL or apoA-I for 3 h at 37 °C. Wells were washed, and the HSPG-bound proteins were eluted and subjected to SDS-PAGE. Samples were transferred to nitrocellulose and probed with an anti-HL mAb. The histograms show the percentage of HL displaced from the HSPG (relative to the control PBS incubation) and were estimated from the Western blot shown. Values are representative of two different experiments.

To determine whether apoA-I or HDL could directly affect the hydrolysis of VLDL by HL, standard hydrolytic assays, in the absence of HSPG, were performed. Fig. 6 shows that increasing the concentration of apoA-I or HDL significantly inhibited the hydrolysis of VLDL, up to a maximum of 60%. Similar observations were made with LDL; both apoA-I and HDL can also directly inhibit the HL-mediated hydrolysis of LDL (data not shown). In addition, at the lower concentrations, apoA-I was up to 5 times more inhibitory to HL than HDL. This is also evident in Fig. 7, which shows HL hydrolytic rates as a function of VLDL concentrations, but at constant inhibitor concentration. The figure shows that 5 times more HDL is required to accomplish the equivalent inhibition as a given amount of apoA-I. The inset in Fig. 7 further shows that inhibition of HL is associated with an increase in the apparent Michaelis-Menten constant (Km) values (inverse of x intercept) and concomitant decrease in the maximum velocity (Vmax) of the enzyme. We (21), and others (22), have previously shown that this apparent Km for an interfacial enzyme, when represented as a protein concentration, indicates the amount of VLDL particles required for a half-maximal velocity and therefore reflects a measure of binding affinity. The data therefore suggest that apoA-I may inhibit HL by decreasing its binding affinity to VLDL.


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  		Fig. 6.   Effect of inhibitor concentration on the hydrolysis of VLDL by hepatic lipase. VLDL (350 µM TG) was incubated with HL (26 units) and increasing amounts of HDL or apoA-I for 30 min at 37 °C. An aliquot was removed, and fatty acid release was measured enzymatically. Hydrolytic values are the mean of triplicate determinations and representative of two experiments.


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  		Fig. 7.   Effect of apoA-I or HDL on the hydrolysis of VLDL by hepatic lipase. Various amounts of VLDL were incubated with HL (26 units) and a constant amount of HDL or apoA-I (as indicated) for 30 min at 37 °C. An aliquot was removed and fatty acid release was measured enzymatically. Hydrolytic values are the mean ± S.D. of triplicate determinations. Inset, double-reciprocal plots are shown where reciprocals of hydrolytic rates (µM/h) are plotted against reciprocals of VLDL concentration (µg/ml protein).

The data shown in Fig. 5 suggested that, with co-incubations, VLDL interfered in the displacement capacity of apoA-I. One way this could have occurred is if apoA-I was prevented from interacting with HSPG by instead binding directly to the VLDL. To determine if inhibition of HL may also be associated with the binding of apoA-I to VLDL, several experiments were performed to show the direct binding of apoA-I to VLDL. Fig. 8 illustrates an experiment demonstrating VLDL-apoA-I association by a solid phase radio-immunometric assay. Plates coated with a mAb to apoB (1D1) were incubated with VLDL that had been incubated (3 h) with HL ± HDL or apoA-I. ApoA-I association with the VLDL was then measured with a mixture of two 125I-labeled mAbs to apoA-I (4H1 and 5F6). Significant apoA-I immunoreactivity is evident with the VLDL that had been incubated with apoA-I or HDL, but not with the native VLDL (Fig. 8). The omission of HL from these incubations had no affect on apoA-I association with VLDL (data not shown). Similar incubations with HL ± HDL or apoA-I were performed, and then VLDL was isolated by density gradient ultracentrifugation and probed for apoA-I. These studies also showed significant association of apoA-I with VLDL particles (data not shown) and confirmed the view that significant amounts of apoA-I can become associated with VLDL. In addition, VLDL incubated with apoA-I or HDL also exhibited a reduced electrophoretic mobility on agarose gels (data not shown), which suggests that association of apoA-I with VLDL affects its electrostatic properties and reduces the net negative charge on this lipoprotein.


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  		Fig. 8.   VLDL-apoA-I association by a solid phase radio-immunometric assay. Removawells coated with a mAb to apoB (1D1) were incubated with VLDL that had been pre-incubated (3 h) with HL ± HDL or apoA-I. ApoA-I association with the VLDL was then measured by addition of a mixture of two 125I-labeled mAbs to apoA-I (4H1 and 5F6), followed by washing and counting of radioactivity. Values are the mean ± S.D. of triplicate determinations and are representative of two experiments.

To determine the physiological relevance of HL inhibition by HDL, experiments were performed to estimate the effect of HDL concentration in plasma on lipid hydrolysis by HL. Plasma lipoproteins from a fasting normolipidemic subject were isolated within the density ranges d < 1.063 g/ml and d = 1.063-1.25 g/ml and then recombined in various ratios. Mixtures contained the equivalent amount of apoB-containing lipoproteins, to the original plasma concentrations, and progressively increasing amounts of HDL, representing from 0% to 100% of the original amount of HDL. Unmodified plasma and lipoprotein mixtures were then incubated with a constant amount of purified HL for various times. Fig. 9 shows that HL-mediated lipid hydrolysis in plasma is almost completely inhibited. Hydrolysis was maximal for the apoB-containing lipoproteins alone and was progressively inhibited by increasing amounts of HDL. The experiment shows that approximately 70% of the HL inhibition in plasma is likely due to HDL/apoA-I specific effects, but that some other components in plasma also have complementary inhibitory effects.


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  		Fig. 9.   Effect of HDL concentration on lipid hydrolysis in plasma. Plasma lipoproteins from a fasted normolipidemic subject were isolated within the density ranges, d < 1.063 g/ml and d = 1.063-1.25 g/ml. Mixtures of the plasma lipoproteins were prepared to contain the equivalent amount of apoB-containing lipoproteins, to the original plasma concentration, and progressively increasing amounts of HDL. HDL concentration is illustrated as 0-100% of the original amount of HDL in the plasma sample. Unmodified plasma and lipoprotein mixtures were then incubated with a constant amount of purified HL (26 units) for 0-2 h at 37 °C. An aliquot was removed, and fatty acid release was measured enzymatically. Hydrolytic values are the mean ± S.D. of triplicate determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The majority of plasma LDL is thought to be produced by HL through the hydrolysis of VLDL and intermediate density lipoprotein and the transfer/exchange of lipids and apoproteins with HDL (23, 24). The ability of HL to promote the production of potentially atherogenic lipoprotein particles has led to the view that HL may increase the risk of developing atherosclerosis (25, 26). This is consistent with observations from a number of laboratories that have identified an inverse relationship between HL activity and plasma HDL levels. Studies have shown that HDL2 levels in the plasma are inversely related to post-heparin plasma HL activity (27-29) and to the risk of developing coronary artery disease (30). Although several anti-atherogenic HDL functions have been proposed, some views have maintained that HDL levels are merely reflective of an efficient lipolytic system and, through this link, a marker for coronary artery disease risk (31). The present work, however, suggests quite the opposite and shows that HDL/apoA-I levels may play an active role in regulating TG metabolism.

HL is anchored by HSPG to the lumenal and sublumenal surfaces of liver sinusoidal endothelial cells, on the external surfaces of hepatocyte microvilli located in the space of Disse and in the interhepatocyte space (2). It is specifically bound to glycosaminoglycans, which are long, unbranched, highly charged carbohydrate side chains composed of repeating disaccharide subunits (32). Although human HL is primarily found anchored to cell surfaces, most of the studies to date have evaluated the hydrolytic activity of this enzyme when free in solution. Early studies (33-35) identified HL as a triglyceride lipase activity in post-heparin plasma, heparin perfusates of liver, and in heparin-treated rat liver plasma membranes. Waite et al. (8) later reported a change in the substrate specificity of HL after it was released by heparin and suggested that the function/activity of an HSPG-bound enzyme may differ. Investigations with a closely related enzyme, LPL, suggest that HSPG association may have inhibitory effects. Studies by both Posner (14) and Clark (15) have looked at the effect of immobilizing LPL onto heparin-Sepharose. They found significant reductions in Michaelis-Menten constants (Km) and maximum velocities (Vmax) for the heparin-bound enzyme, when compared with LPL in solution. More recently, de Man et al. (36) demonstrated that the binding of LPL to HSPG also partially inhibited the lipolysis of VLDL. This perspective has been indirectly confirmed in vivo. Numerous reports have demonstrated significantly increased rates of TG hydrolysis when HL and LPL are released from cell surface HSPG by heparin administration (37, 38). Our data show that VLDL hydrolysis by HL is also significantly inhibited when HL is bound to HSPG. These data suggest that lipolytic activities are impaired when LPL or HL is HSPG-bound. Efficient lipolysis may therefore require the displacement of these lipases from the cell surface matrix and the factors that regulate displacement may be critical to achieve efficient lipolysis.

In the study by Posner et al. (14), the authors concluded that the lower Vmax values obtained for heparin-Sepharose-bound LPL might have been partly due to a reduced accessibility of VLDL for the bound LPL. In the present study, we observed significantly decreased hydrolysis of both LDL and VLDL when HL was bound to HSPG. This reduced hydrolytic activity observed for the HSPG-bound HL supports the view that these lipoproteins may not be able to gain access to the HSPG-bound enzyme. This hypothesis is further confirmed by the observation that neither LDL nor VLDL could significantly displace HL from the proteoglycan. These data show that the HSPG-bound HL is inactive and suggest that hydrolysis of the larger TG-enriched lipoprotein substrates may require the dissociation of HL (and perhaps LPL) from cell surface HSPG. Again, this may explain the increased rates of lipid hydrolysis evident in post-heparin plasmas (37, 38).

On the other hand, this study shows that association of HL with HSPG has much less effect on the hydrolysis of HDL lipids by HL. This reduced inhibition of the catalytic activity of HL suggests that either the HSPG-bound enzyme is readily accessible to the smaller lipoproteins or that some component of HDL may act to liberate HL and catalyze its activity. All lines of evidence support this latter perspective. With all lipoproteins studied, the rate of hydrolysis in the presence of HSPG closely correlates to the capacity of the lipoprotein to displace HL from the HSPG. Minimal displacement by LDL and VLDL paralleled maximal inhibition, whereas 60% displacement by HDL was associated with a 40% inhibition of HL. HDL preparations from different subjects sometimes exhibited a greater ability to displace HL, and in these instances, less inhibition was observed. These data show that some component/constituent of HDL, i.e. apoA-I, can directly access the bound HL. ApoA-I appears to mediate HL liberation from the HSPG by binding to sites on the proteoglycan and dislodging the bound HL. Purified apoA-I is in fact more effective than HDL at HL displacement, which suggests that only a fraction of HDL apoA-I is functional in this respect. This may indicate that HL displacement from HSPG is facilitated by a more exchangeable/loosely bound fraction of apoA-I on HDL (Fig. 10, steps 1 and 2). Several earlier studies have shown that a loosely bound fraction of apoA-I does indeed exist on HDL particles (39, 40), and higher levels of this fraction have been identified in hypertriglyceridemic subjects (41). Since co-incubations with VLDL inhibit HL displacement by HDL, it appears that this loosely bound apoA-I fraction may also be transferable to VLDL.


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  		Fig. 10.   Proposed model showing the effect of apoA-I on the displacement and catalytic regulation of hepatic lipase. A fraction of the apoA-I molecules on HDL are less tightly bound and can become dissociated from the lipoprotein particle. These apoA-I can bind to cell surface proteoglycans (step 1) and cause the displacement of proteoglycan-bound HL (step 2). Displaced HL becomes catalytically active and binds to lipoprotein particles in the plasma (step 3). The activity of the lipoprotein-bound HL is then regulated by the amount of lipoprotein-associated apoA-I, which inhibits lipid hydrolysis by this enzyme (step 4).

Preliminary studies in this laboratory have shown that VLDL from different subjects also vary in their ability to displace HL from HSPG and be hydrolyzed by this enzyme. As shown in Fig. 2, a small amount of HL displacement by VLDL also coincided with the association of endogenous apoA-I on the HSPG. ApoA-I has been well described to be a constituent of VLDL (42-44), and these data may suggest that the small amount of apoA-I associated with VLDL particles may play a role in regulating HL displacement and lipid hydrolysis. Some data actually suggest that apoA-I may be a substantial component of VLDL (43), but that its contribution to VLDL protein may be underestimated by its highly exchangeable, ultracentrifugally dissociable nature (42). In unpublished work, we too have shown that significant amounts of apoA-I are pelleted when VLDL is ultracentrifugally washed. Therefore, substantial amounts of loosely bound apoA-I may also exist on VLDL (and LDL), and this component may control the displacement of HL from HSPG and indirectly affect the remodeling of these lipoproteins.

Although loosely bound apoA-I appears to indirectly affect the actions of HL, the protein also has a very direct effect on lipid hydrolysis by HL. Addition of purified apoA-I or HDL to incubations of HL and VLDL significantly inhibited lipid hydrolysis, by decreasing HL affinity for VLDL and catalytic activity (Fig. 10, steps 3 and 4). This is consistent with the work of Hime et al. (45), which also showed a reduced HL affinity for apoA-I enriched HDL, relative to apoA-II-containing particles. However, this report also showed that the apoA-II HDL were substantially poorer substrates for HL than apoA-I HDL and promoted significantly reduced rates of hydrolysis of both TG and PL. The data suggest that apoA-II may also be an inhibitor of HL, and this agrees with the view of Plump et al. (46) that the mild hypertriglyceridemia in apoA-I knock out mice may be partially due to the apoA-II-enriched HDL being poorer substrates for HL. However, it appears unlikely that apoA-II would have contributed significantly to the HL inhibitory capacity of HDL on VLDL hydrolysis, since this protein has a very high lipid affinity and is not readily transferred to other lipoproteins (47). In addition, it is notable that, at low inhibitor concentrations, apoA-I is more effective than HDL (containing both apoA-I and apoA-II) at inhibiting HL, but, at higher concentrations, both apoA-I and HDL are very similar in their ability to inhibit this enzyme.

HL inhibition by apoA-I appeared to parallel the association of apoA-I with VLDL surface lipids, and, as with HL displacement, apoA-I appeared more effective than HDL particles at inhibiting HL. These data suggest that it is only a fraction of loosely bound apoA-I on the HDL that can be transferred to VLDL. Since co-incubations with VLDL also impair the ability of HDL and apoA-I to displace HL from HSPG, it appears that it is the same loosely bound apoA-I that can either liberate HSPG-bound HL or bind to VLDL. VLDL has the capacity to bind substantial amounts of apoA-I, which again is suggestive of the view that much more apoA-I may be associated with this lipoprotein in vivo. The movement of apoA-I between HDL and VLDL appears to be a passive and spontaneous event, but may be affected by changes in the composition and structural properties of these lipoproteins. ApoA-I transfer from HDL to LDL and VLDL during lipolysis has been previously demonstrated and is thought to be promoted by the products of lipolysis, specifically free fatty acids (48). However, in the present work, the association of apoA-I with VLDL was unaffected by lipolysis and appeared similar in magnitude in the presence or absence of HL. The products of lipolysis do appear to affect the dissociation of HDL constituents from apoB-containing lipoproteins. Musliner et al. (49) have shown that free fatty acids can promote the dissociation of various lipid and protein components from LDL and VLDL and enhance the formation of HDL particles in vitro. Therefore, the amount of apoA-I exchange with VLDL and the factors that control its dissociation from the surface of lipoprotein particles may regulate both the liberation of HSPG-bound HL and the formation of LDL particles in vivo.

These studies show that the hydrolysis of TG-enriched lipoproteins may require the displacement of HL from cell surface HSPG. It appears that this phenomenon may have significant consequences on lipid metabolism in vivo. It is conceivable that if the ability of different lipoproteins to displace lipolytic enzymes affects the plasma clearance of TG, this pathway may play an important role in regulating the duration and magnitude of the postprandial response. The data suggest that the kind of VLDL produced in a particular patient may directly affect its hydrolysis and conversion to smaller LDL particles. Metabolic studies have shown that an increased production of large TG-enriched VLDL will promote the formation of the more atherogenic, small, dense LDL (50). If these larger VLDL have less surface (inhibitory) apoA-I or are more able to displace HL from cell surface HSPG, it is possible that they may be hydrolyzed to a greater extent and this may underlie their propensity to form the small LDL species. This relationship may also partly explain the inverse relationship between apoA-I levels and HL activity. High levels of apoA-I in the plasma may directly inhibit the formation of LDL particles by slowing down VLDL conversions and enhancing their clearance as remnant particles. Since this apoA-I directly affects the association of HL with cell surface HSPG, the amount of loosely bound apoA-I in plasma may also control the ligand functions of this enzyme and affect the uptake and clearance of remnant lipoprotein particles. This work therefore suggests that HDL and apoA-I play important roles in regulating the function and actions of HL.

    ACKNOWLEDGEMENTS

We thank Drs. Y. Marcel and R. Milne for valuable discussions and Dr. A. Bensadoun for providing the anti-HL monoclonal antibody.

    FOOTNOTES

* This work was supported by a grant from the Medical Research Council of Canada.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.

Dagger Supported by a postgraduate scholarship from the Ontario Graduate Scholarship Program and the Natural Sciences and Engineering Research Council of Canada.

§ To whom correspondence should be addressed: Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Inst., 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-4822; Fax: 613-761-5281; E-mail: dsparks@ottawaheart.ca.

Published, JBC Papers in Press, August 15, 2000, DOI 10.1074/jbc.M005436200

    ABBREVIATIONS

The abbreviations used are: HL, hepatic lipase; apoA-I, apolipoprotein A-I; BSA, bovine serum albumin; FAF-BSA, essentially fatty acid free-bovine serum albumin; HDL, high density lipoprotein; HRP, horseradish peroxidase; HSPG, heparan sulfate proteoglycan; LDL, low density lipoprotein; LPL, lipoprotein lipase; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PL, phospholipid; TG, triglyceride; VLDL, very low density lipoprotein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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