Evidence that phospholipids play a key role in pre-beta apoA-I formation and high-density lipoprotein remodeling

The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid-poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high-density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation.     

Apolipoproteins regulate the kinetics of endothelial lipase-mediated hydrolysis of phospholipids in reconstituted high-density lipoproteins

Endothelial lipase (EL) is a newly identified member of the triglyceride lipase gene family that hydrolyzes high-density lipoprotein (HDL) phospholipids. This study investigates the ability of the major apolipoproteins of rHDL to regulate the kinetics of EL-mediated phospholipid hydrolysis in well-characterized, homogeneous preparations of spherical rHDL. The rHDL contained either apoA-I as the only apolipoprotein, (A-I)rHDL, apoA-II as the only apolipoprotein, (A-II)rHDL, or apoA-I as well as apoA-II, (A-I/A-II)rHDL. The rHDL were comparable in terms of size and lipid composition and contained cholesteryl esters (CE) as their sole core lipid. Phospholipid hydrolysis was quantitated as the mass of nonesterified fatty acids (NEFA) released from the rHDL during incubation with EL. The V(max) of phospholipid hydrolysis for (A-I/A-II)rHDL [391.9 +/- 12.9 nmol of NEFA formed (mL of EL)(-1) h(-1)] was greater than (A-I)rHDL [152.8 +/- 4.7 nmol of NEFA formed (mL of EL)(-1) h(-1)]. The energy of activation (E(a)) for the hydrolysis reactions was calculated to be 52.1 and 34.8 kJ mol(-1) for (A-I)rHDL and (A-I/A-II)rHDL, respectively. Minimal phospholipid hydrolysis was observed for the (A-II)rHDL. Kinetic analysis showed that EL has a higher affinity for the phospholipids in (A-I)rHDL [K(m)(app) = 0.10 +/- 0.01 mM] than in (A-I/A-II)rHDL [K(m)(app) = 0.27 +/- 0.03 mM]. Furthermore, (A-I)rHDL is a competitive inhibitor of the EL-mediated phospholipid hydrolysis of (A-I/A-II)rHDL. These results establish that apolipoproteins are major determinants of the kinetics of EL-mediated phospholipid hydrolysis in rHDL.     

Inhibition of lecithin:cholesterol acyltransferase activity by synthetic phosphatidylcholine species containing eicosapentaenoic acid or docosahexaenoic acid in the sn-2 position.

Phospholipids isolated from the plasma of monkeys fed a diet enriched in fish oil were poor substrates for cholesteryl ester (CE) synthesis by the lecithin:cholesterol acyltransferase (LCAT) reaction relative to those from animals fed a lard containing diet when the phospholipids were used for the preparation of recombinant particles by cholate dialysis (Parks, J. S., B. C. Bullock, and L. L. Rudel. 1989. J. Biol. Chem. 264: 2545-2551). The purpose of the present study was to directly test the influence of eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) in the sn-2 position of phosphatidylcholine (PC) on the activity of LCAT. PC species containing 1-palmitoyl-2-oleoyl PC (POPC), 1-palmitoyl-2-linoleoyl PC (PLPC), 1-palmitoyl-2-arachidonoyl PC (PAPC), 1-palmitoyl-2-eicosapentaenoyl PC (PEPC), or 1-palmitoyl-2-docosahexaenoyl PC (PDPC) were purchased or synthesized and made into recombinant particles of uniform size and composition with [14C]cholesterol and apoA-I using the cholate dialysis procedure. The recombinant particles (PC:cholesterol:apoA-I molar ratio = 42:1.9:1) exhibited the following order of reactivity towards purified human LCAT in vitro: POPC greater than PLPC greater than PEPC = PAPC greater than PDPC. The apparent Vmax/Km for recombinant particles containing PEPC and PDPC was 17% and 7% that of particles containing POPC, respectively. There was a linear decrease in CE formation when the percentage of PEPC or PDPC was increased from 0 to 100% relative to POPC in recombinant particles with a constant PC:cholesterol:apoA-I molar ratio, suggesting that the PEPC and PDPC were competitive inhibitors of the LCAT reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of highly unsaturated lipids were examined by Raman spectroscopy. Bilayer assemblies were composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), PAPC (1-palmitoyl-2-arachidonylphosphatidylcholine), and PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine), lipid systems possessing saturated sn-1 chains and unsaturated sn-2 chains with one, four, and six double bonds, respectively. Raman spectra were recorded in the acyl chain 2800-3100-cm-1 carbon-hydrogen (C-H) stretching and 1100-1200-cm-1 carbon-carbon (C-C) stretching mode regions, spectral intervals reflecting both the inter- and intrachain order/disorder properties of the various lipid dispersions. In order to obtain C-H stretching mode spectra relevant solely to the sn-1 chains of PAPC and PDPC, liquid-phase spectra of arachidonic and docosahexaenoic acid, respectively, were subtracted from the observed phospholipid spectra. The unsaturated sn-2 chains of PAPC and PDPC undergo minimal conformational reorganizations as the bilayers pass from the gel to liquid-crystalline phases. Phase transition temperatures, Tm, derived from statistically fitting the temperature-dependent Raman spectral data are approximately -2.5, -22.5, and -3 degrees C for POPC, PAPC, and PDPC, respectively. As the degree of unsaturation increases from POPC to PAPC and PDPC, the cooperativity of the phase transition, as measured by its breadth, decreases. Estimates of the transition widths from the temperature profiles are approximately 15 degrees C for PAPC and 20 degrees C for PDPC. The behavior of various Raman spectral parameters for the lipid gel phase reflects the formation of lateral microdomains, or clusters, whose packing properties maximize the van der Waals interactions between sn-1 chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-Glycosylation regulates endothelial lipase-mediated phospholipid hydrolysis in apoE- and apoA-I-containing high density lipoproteins

Endothelial lipase (EL) is a member of the triglyceride lipase gene family with high phospholipase and low triacylglycerol lipase activities and a distinct preference for hydrolyzing phospholipids in HDL. EL has five potential N-glycosylation sites, four of which are glycosylated. The aim of this study was to determine how glycosylation affects the phospholipase activity of EL in physiologically relevant substrates. Site-directed mutants of EL were generated by replacing asparagine (N) 62, 118, 375, and 473 with alanine (A). These glycan-deficient mutants were used to investigate the kinetics of phospholipid hydrolysis in fully characterized preparations of spherical reconstituted high density lipoprotein (rHDL) containing apolipoprotein E2 (apoE2) [(E2)rHDL], apoE3 [(E3)rHDL], apoE4 [(E4)rHDL], or apoA-I [(A-I)rHDL] as the sole apolipoprotein. Wild-type EL hydrolyzed the phospholipids in (A-I)rHDL, (E2)rHDL, (E3)rHDL, and (E4)rHDL to similar extents. The phospholipase activities of EL N118A, EL N375A, and EL N473A were significantly diminished relative to that of wild-type EL, with the greatest reduction being apparent for (E3)rHDL. The phospholipase activity of EL N62A was increased up to 6-fold relative to that of wild-type EL, with the greatest enhancement of activity being observed for (E2)rHDL. These data show that individual N-linked glycans have unique and important effects on the phospholipase activity and substrate specificity of EL.     

Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I

Endothelial lipase (EL) is a triglyceride lipase gene family member that has high phospholipase and low triglyceride lipase activity. The aim of this study was to determine whether the phospholipase activity of EL is sufficient to remodel HDLs into small particles and mediate the dissociation of apolipoprotein A-I (apoA-I). Spherical, reconstituted HDLs (rHDLs) containing apoA-I only [(A-I)rHDLs], apoA-II only [(A-II)rHDLs], or both apoA-I and apoA-II [(A-I/A-II) rHDLs] were prepared. The rHDLs, which contained only cholesteryl esters in their core and POPC on the surface, were incubated with EL. As the rHDLs did not contain triacylglycerol, only the POPC was hydrolyzed. Hydrolysis was greater in the (A-I/A-II)rHDLs than in the (A-I)rHDLs. The (A-II)rHDL phospholipids were not hydrolyzed by EL. EL remodeled the (A-I)rHDLs and (A-I/A-II)rHDLs, but not the (A-II)rHDLs, into smaller particles. The reduction in particle size was related to the amount of phospholipid hydrolysis, with the diameter of the (A-I/A-II)rHDLs decreasing more than that of the (A-I)rHDLs. These changes did not affect the conformation of apoA-I, and neither apoA-I nor apoA-II dissociated from the rHDLs. Comparable results were obtained when human plasma HDLs were incubated with EL. These results establish that the phospholipase activity of EL remodels plasma HDLs and rHDLs into smaller particles without mediating the dissociation of apolipoproteins.     

Lipoprotein electrostatic properties regulate hepatic lipase association and activity.

The effect of lipoprotein electrostatic properties on the catalytic regulation of hepatic lipase (HL) was investigated. Enrichment of serum or very low density lipoprotein (VLDL) with oleic acid increased lipoprotein negative charge and stimulated lipid hydrolysis by HL. Similarly, enrichment of serum or isolated lipoproteins with the anionic phospholipids phosphatidylinositol (PI), phosphatidic acid, or phosphatidylserine also increased lipoprotein negative charge and stimulated hydrolysis by HL. Anionic lipids had a small effect on phospholipid hydrolysis, but significantly stimulated triacylglyceride (TG) hydrolysis. High density lipoprotein (HDL) charge appears to have a specific effect on lipolysis. Enrichment of HDL with PI significantly stimulated VLDL-TG hydrolysis by HL. To determine whether HDL charge affects the association of HL with HDL and VLDL, HL-lipoprotein interactions were probed immunochemically. Under normal circumstances, HL associates with HDL particles, and only small amounts bind to VLDL. PI enrichment of HDL blocked the binding of HL with HDL. These data indicate that increasing the negative charge of HDL stimulates VLDL-TG hydrolysis by reducing the association of HL with HDL. Therefore, HDL controls the hydrolysis of VLDL by affecting the interlipoprotein association of HL. Lipoprotein electrostatic properties regulate lipase association and are an important regulator of the binding and activity of lipolytic enzymes.     

Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.     

Metabolism of high density lipoprotein lipids by the rat liver: evidence for participation of hepatic lipase in the uptake of cholesteryl ester.

In order to determine the role of hepatic lipase in the hepatic uptake and metabolism of high density lipoprotein (HDL) triglycerides, cholesteryl esters, and phospholipids, isolated rat livers were perfused with a reconstituted HDL (rHDL) radiolabeled with [3H]triolein and [14C]cholesteryl oleate or palmitoyl-[14C]linoleoyl phosphatidylcholine. A bolus of radiolabeled rHDL was injected into the portal vein and livers were perfused for 5 min using a nonrecirculating perfusion system. Recovery of rHDL triolein in the liver as intact triolein was used to determine the amount of unmetabolized rHDL remaining in the liver. After correcting for the amount of unmetabolized rHDL remaining in the liver, about 30% of the rHDL triolein was hydrolyzed of which 19% was recovered in the liver and 11% in the perfusate. Moreover, about 7% of the rHDL phosphatidylcholine was hydrolyzed to lysophosphatidylcholine, all of which was recovered in the perfusate. Although there was no hydrolysis of rHDL cholesteryl oleate, about 30% of the cholesteryl oleate was taken up by the liver. Preperfusion of the liver with heparin to deplete the liver of hepatic lipase resulted in about a 70% reduction in rHDL triolein hydrolysis and about a 75% reduction in rHDL cholesteryl oleate uptake. Although hepatic lipase hydrolyzes both triglycerides and phosphatidylcholines, elimination of the triolein from rHDL had no effect on the uptake of rHDL cholesteryl oleate, but replacement of the rHDL phosphatidylcholine with a nonhydrolyzable phosphatidylcholine diether resulted in an 87% reduction in cholesteryl oleate uptake. These results indicate that hepatic lipase is necessary for the hepatic uptake of both HDL triglycerides and cholesteryl esters and that the uptake of cholesteryl esters is not dependent on the hydrolysis of HDL triglycerides but is dependent on the hydrolysis of HDL phospholipids.     

Role of apolipoprotein E in hepatic lipase catalyzed hydrolysis of phospholipid in high-density lipoproteins.

We reported earlier that hepatic lipase (HL)-catalyzed hydrolysis of phospholipid monolayers is activated by apolipoprotein (apo) E [Thuren et al. (1991b) J. Biol. Chem. 266, 4853-4861]. On the basis of these studies, it was postulated that apoE-rich high-density lipoproteins (HDL) were preferred substrates for HL. In the present study, we tested this hypothesis, as well as further characterizing the activation of HL hydrolysis of phospholipid by apoE. The apoE-rich HDL, referred to as HDL-I, were isolated by heparin-Sepharose chromatography, and the phospholipid hydrolysis by HL was compared to an apoE-poor HDL, designated HDL-II. The hydrolysis of HDL-I phosphatidylcholine was approximately 3-fold higher than HDL-II, supporting the hypothesis that HL preferably hydrolyzes the phospholipids in apoE-rich HDL. In order to gain additional insight into the nature of the activation, we used phospholipid monolayers as model systems. Comparison of the ability of the two thrombolytic fragments of apoE (22 kDa, residues 1-191; 12 kDa, residues 192-299) revealed that only the 12-kDa fragment was capable of activating the hydrolysis of phospholipid by HL (1.75-fold). However, activation was less than with the intact protein (2.8-fold for apoE3), suggesting that the intact protein was required for full activation. The fact that the 12-kDa fragment, which represents a major lipid region of the protein, did activate HL suggests that activation occurs at the lipid-water interface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorogenic substrates for high-throughput measurements of endothelial lipase activity

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum.     

Measurement of the phospholipase activity of endothelial lipase in mouse plasma

Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL''s hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface.     

Endothelial lipase: a new lipase on the block

Endothelial lipase (EL) is a newly described member of the triglyceride lipase gene family. It has a considerable molecular homology with lipoprotein lipase (LPL) (44%) and hepatic lipase (HL) (41%). Unlike LPL and HL, this enzyme is synthesized by endothelial cells and functions at the site where it is synthesized. Furthermore, its tissue distribution is different from that of LPL and HL. As a lipase, EL has primarily phospholipase A1 activity. Animals that overexpress EL showed reduced HDL cholesterol levels. Conversely, animals that are deficient in EL showed a marked elevation in HDL cholesterol levels, suggesting that it plays a physiologic role in HDL metabolism. Unlike LPL and HL, EL is located in the vascular endothelial cells and its expression is highly regulated by cytokines and physical forces, suggesting that it may play a role in the development of atherosclerosis. However, there is only a limited amount of information available about this enzyme. Some of our unpublished data in addition to previously published data support the possibility that the enzyme plays a role in the formation of atherosclerotic lesion.     

Apolipoprotein E enhances hepatic lipase-mediated hydrolysis of reconstituted high-density lipoprotein phospholipid and triacylglycerol in an isoform-dependent manner

This study compares the kinetics of hepatic lipase (HL)-mediated phospholipid and triacylglycerol hydrolysis in spherical, reconstituted high-density lipoproteins (rHDL) that contain either apolipoprotein E2 (apoE2), apoE3, apoE4, or apoA-I as the sole apolipoprotein. HL-mediated phospholipid hydrolysis was assessed by incubating various concentrations of rHDL that contained only cholesteryl esters (CE) in their core, (E2/CE)rHDL, (E3/CE)rHDL, (E4/CE)rHDL, and (A-I/CE)rHDL, with a constant amount of HL. The rate of phospholipid hydrolysis was determined as the formation of nonesterified fatty acid mass. HL-mediated triacylglycerol hydrolysis was assessed in rHDL containing CE, unlabeled triacylglycerol, and [(3)H]triacylglycerol in their core, (E2/TG)rHDL, (E3/TG)rHDL, (E4/TG)rHDL, and (A-I/TG)rHDL. Triacylglycerol hydrolysis was determined as the ratio of (3)H-labeled hydrolysis products to (3)H-labeled unhydrolyzed triacylglycerol. The rates of phospholipid hydrolysis in the (E2/CE)rHDL, (E3/CE)rHDL, and (E4/CE)rHDL were significantly greater than that in the (A-I/CE)rHDL. The rates of triacylglycerol hydrolysis were also greater in the (E2/TG)rHDL, (E3/TG)rHDL, and (E4/TG)rHDL compared to the (A-I/TG)rHDL, although to a lesser degree than observed with phospholipid hydrolysis. Furthermore, the rates of both phospholipid and triacylglycerol hydrolyses were greater in the (E2)rHDL than in either the (E3)rHDL or the (E4)rHDL. These results show that apoE increases the rate of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL and that this influence is isoform dependent.     

Glycosylation of endothelial lipase at asparagine-116 reduces activity and the hydrolysis of native lipoproteins in vitro and in vivo

We previously identified that four of five putative N-linked glycosylation sites of human endothelial lipase (EL) are utilized and suggested that the substitution of asparagine-116 (Asn-116) with alanine (Ala) (N116A) increased the hydrolytic activity of EL. The current study demonstrates that mutagenesis of either Asn-116 to threonine (Thr) or Thr-118 to Ala also disrupted the glycosylation of EL and enhanced catalytic activity toward synthetic substrates by 3-fold versus wild-type EL. Furthermore, we assessed the hydrolysis of native lipoprotein lipids by EL-N116A. EL-N116A exhibited a 5-fold increase in LDL hydrolysis and a 1.8-fold increase in HDL2 hydrolysis. Consistent with these observations, adenovirus-mediated expression of EL-N116A in mice significantly reduced the levels of both LDL and HDL cholesterol beyond the reductions observed by the expression of wild-type EL alone. Finally, we introduced Asn-116 of EL into the analogous positions within LPL and HL, resulting in N-linked glycosylation at this site. Glycosylation at this site suppressed the LPL hydrolysis of synthetic substrates, LDL, HDL2, and HDL3 but had little effect on HL activity. These data suggest that N-linked glycosylation at Asn-116 reduces the ability of EL to hydrolyze lipids in LDL and HDL2.     

Relationship between serum HDL-C levels and common genetic variants of the endothelial lipase gene in Japanese school-aged children

Endothelial lipase (EL) is a new member of the triglyceride lipase family, the genes of which play a central role in dietary fat absorption, energy homeostasis, and plasma lipoprotein metabolism. One physiologic role of EL is thought to be hydrolysis of high-density lipoprotein (HDL) phospholipid, although the precise function of endothelial lipase has yet to be fully clarified. Furthermore, genetic variation in EL has been suggested to influence serum HDL-C levels. In the present study, we detected two common single nucleotide polymorphisms in the EL gene associated with serum HDL cholesterol levels in healthy school-aged children. Our data support the hypothesis that variations in the EL gene are one of the genetic determinants of serum HDL-C levels.     

Identification and analysis of products formed from phospholipids in the free radical oxidation of human low density lipoproteins

Phospholipids reside in the surface layer of LDLs and constitute approximately 20-25% of the particle by weight. We report a study of the primary products generated from the most abundant molecular species of phosphatidylcholines present in LDL during in vitro free radical oxidations. The 13-hydroperoxides of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) and 1-stearoyl-2-linoleoyl-sn-glycero-phosphocholine (SLPC) and the 15-hydroperoxides of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) and 1-stearoyl-2-arachidonoyl-sn-glycero-phosphocholine (SAPC) were found to increase in a time-dependent manner and in significant amounts even in the presence of alpha-tocopherol. Phospholipid alcohols also formed during the course of the oxidations. Early in the LDL oxidations, while alpha-tocopherol was still present, the thermodynamically favored trans,trans products of PLPC and SLPC were found to form in significantly larger quantities than those formed from cholesteryl linoleate. Additionally, quantities of PAPC 11-hydroperoxide (11-OOH) decreased over time relative to PAPC 15-OOH, even while alpha-tocopherol was still present in the oxidation, presumably as a result of further oxidation of PAPC 11-OOH to form cyclic peroxide oxidation products. These results suggest that alpha-tocopherol is more closely associated with the inner cholesteryl ester-rich hydrophobic core of an LDL particle and is not as effective as an antioxidant in the outer phospholipid layer as it is in the lipid core.     

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.     

Evidence that apolipoprotein A-I facilitates hepatic lipase-mediated phospholipid hydrolysis in reconstituted HDL containing apolipoprotein A-II

This study examines hepatic lipase (HL) mediated phospholipid hydrolysis in mixtures of apolipoprotein-specific, spherical reconstituted high-density lipoproteins (rHDL). We have shown previously that apolipoprotein A-I (apoA-I) and apoA-II have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in well-characterized, homogeneous preparations of spherical rHDL [Hime, N. J., Barter, P. J., and Rye, K.-A. (1998) J. Biol. Chem. 273, 27191-27198]. In the present study, phospholipid hydrolysis was assessed in mixtures of rHDL containing either apoA-I only, (A-I)rHDL, apoA-II only, (A-II)rHDL, or both apoA-I and apoA-II, (A-I/A-II)rHDL. The rHDL contained trace amounts of radiolabeled phospholipid, and hydrolysis was measured as the formation of radiolabeled nonesterified fatty acids (NEFA). As predicted from our previous kinetic studies, the (A-II)rHDL acted as competitive inhibitors of HL-mediated phospholipid hydrolysis in (A-I)rHDL. Less expected was the observation that the rate of phospholipid hydrolysis in (A-II)rHDL was enhanced when (A-I)rHDL were also present in the incubation mixture. The rate of phospholipid hydrolysis in (A-I/A-II)rHDL was also greater than in (A-II)rHDL, indicating that apoA-I enhances phospholipid hydrolysis when it is present as a component of (A-I/A-II)rHDL. It is concluded that apoA-I enhances HL-mediated phospholipid hydrolysis in apoA-II containing rHDL, irrespective of whether the apoA-I is present in the same particle as the apoA-II [as in (A-I/A-II)rHDL] or whether it is present as a component of a different particle, such as when (A-I)rHDL are added to incubations of (A-II)rHDL.     

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.