Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

Discoidal complexes containing apolipoprotein E and their transformation by lecithin-cholesterol acyltransferase

The primary objectives of this study were to determine whether analogs to native discoidal apolipoprotein (apo)E-containing high-density lipoproteins (HDL) could be prepared in vitro, and if so, whether their conversion by lecithin-cholesterol acyltransferase (LCAT; EC 2.3.1.43) produced particles with properties comparable to those of core-containing, spherical, apoE-containing HDL in human plasma. Complexes composed of apoE and POPC, without and with incorporated unesterified cholesterol, were prepared by the cholate-dialysis technique. Gradient gel electrophoresis showed that these preparations contain discrete species both within (14-40 nm) and outside (10.8-14 nm) the size range of discoidal apoE-containing HDL reported in LCAT deficiency. The isolated complexes were discoidal particles whose size directly correlated with their POPC:apoE molar ratio: increasing this ratio resulted in an increase in larger complexes and a reduction in smaller ones. At all POPC:apoE molar ratios, size profiles included a major peak corresponding to a discoidal complex 14.4 nm long. Preparations with POPC:apoE molar ratios greater than 150:1 contained two distinct groups of complexes, also in the size range of discoidal apoE-containing HDL from patients with LCAT deficiency. Incorporation of unesterified cholesterol into preparations (molar ratio of 0.5:1, unesterified cholesterol:POPC) resulted in component profiles exhibiting a major peak corresponding to a discoidal complex 10.9 nm long. An increase of unesterified cholesterol and POPC (at the 0.5:1 molar ratio) in the initial mixture, increased the proportion of larger complexes in the profile. Incubation of isolated POPC-apoE discoidal complexes (mean sizes, 14.4 and 23.9 nm) with purified LCAT and a source of unesterified cholesterol converted the complexes to spherical, cholesteryl ester-containing products with mean diameters of 11.1 nm and 14.0 nm, corresponding to apoE-containing HDL found in normal plasma. Conversion of smaller cholesterol-containing discoidal complexes (mean size, 10.9 nm) under identical conditions resulted in spherical products 11.3, 13.3, and 14.7 nm across. The mean sizes of these conversion products compared favorably with those (mean diameter, 12.3 nm) of apoE-containing HDL of human plasma. This conversion of cholesterol-containing complexes is accompanied by a shift of some apoE to the LDL particle size interval. Our study indicates that apoE-containing complexes formed by the cholate-dialysis method include species similar to discoidal apoE-containing HDL and that incubation with LCAT converts most of them to spherical core-containing particles in the size range of plasma apoE-containing HDL. Plasma HDL particles containing apoE may arise in part from direct conversion of discoidal apoE-containing HDL by LCAT.     

Deletions of helices 2 and 3 of human apoA-I are associated with severe dyslipidemia following adenovirus-mediated gene transfer in apoA-I-deficient mice

The objective of this study was to determine the effect of two amino-terminal apolipoprotein A-I (apoA-I) deletions on high-density lipoprotein (HDL) biosynthesis and lipid homeostasis. Adenovirus-mediated gene transfer showed that the apoA-I[Delta(89-99)] deletion mutant caused hypercholesterolemia, characterized by increased plasma cholesterol and phospholipids, that were distributed in the very low density/intermediate density/low-density lipoprotein (VLDL/IDL/LDL) region, and normal triglycerides. The capacity of the mutant protein to promote ATP-binding cassette transporter A1- (ABCA1-) mediated cholesterol efflux and to activate lecithin:cholesterol acyltranserase (LCAT) was approximately 70-80% of the wild-type (WT) control. The phospholipid transfer protein (PLTP) activity of plasma containing the apoA-I[Delta(89-99)] mutant was decreased to 32% of the WT control. Similar analysis showed that the apoA-I[Delta(62-78)] deletion mutant in apoA-I-deficient mice caused combined hyperlipidemia characterized by increased triglycerides, cholesterol, and phospholipids in the VLDL/IDL region. There was enrichment of the VLDL/IDL with mutant apoA-I that resulted in reduction of in vitro lipolysis. The capacity of this mutant to promote ABCA1-mediated cholesterol efflux was normal, and the capacity to activate LCAT in vitro was reduced by 53%. The WT apoA-I and the apoA-I[Delta(62-78)] mutant formed spherical HDL particles, whereas the apoA-I[Delta(89-99)] mutant formed discoidal HDL particles. We conclude that alterations in apoA-I not only may have adverse effects on HDL biosynthesis but also may promote dyslipidemia due to interference of the apoA-I mutants on the overall cholesterol and triglycerides homeostasis.     

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

Lecithin:cholesterol acyltransferase-induced modifications of liver perfusate discoidal high density lipoproteins from African green monkeys

The role of lecithin:cholesterol acyltransferase (LCAT) in the formation of plasma high density lipoproteins (HDL) was studied in a series of in vitro incubations in which perfusates from isolated African green monkey livers were incubated at 37 degrees C with partially purified LCAT for between 1 and 13 hr. The HDL particles isolated from monkey liver perfusate stored at 4 degrees C and not exposed to added LCAT contained apoA-I and apoE, were deficient in neutral lipids, and were observed by electron microscopy as discoidal particles. Particle sizes, measured as Stokes' diameters by gradient gel electrophoresis (GGE), ranged between 7.8 nm and 15.0 nm. The properties of perfusate HDL were unchanged following incubation at 37 degrees C in the presence of an LCAT inhibitor. However, HDL subfractions derived from incubations at 37 degrees C with active LCAT contained apoA-I as the major apoprotein, appeared round by electron microscopy, and possessed chemical compositions similar to plasma HDL. The HDL isolated from perfusate incubations at 37 degrees C with low amounts of LCAT had a particle size and chemical composition similar to plasma HDL3a. In three of four perfusates incubated with higher levels of LCAT activity, the HDL products consisted of two distinct HDL subpopulations when examined by GGE. The major subpopulation was similar in size and composition to plasma HDL2a, while the minor subpopulation demonstrated the characteristics of plasma HDL2b. The data indicate that the discoidal HDL particles secreted by perfused monkey livers can serve as precursors to three of the major HDL subpopulations observed in plasma.     

Nascent high density lipoproteins from liver perfusates of orotic acid-fed rats

Uniformly fatty livers from orotic acid-fed rats secreted almost no very low density lipoproteins (VLDL) but normal amounts of nascent high density lipoproteins (HDL) accumulated in perfusates. When lecithin:cholesterol acyltransferase (LCAT) was inhibited, nascent HDL were uniformly discoidal and lacked cholesteryl esters. Lipid and apoprotein compositions of nascent HDL from normal and fatty livers were similar whether LCAT was inhibited or not. Apolipoprotein B-100 was not detected in perfusates of uniformly fatty livers, but small amounts of apolipoprotein B-48 were present in HDL2 fractions. Nascent lipoproteins were not seen in Golgi compartments, but lipid-rich particles were clearly evident in endoplasmic reticulum cisternae adjacent to the cis face of the Golgi complex, suggesting that orotic acid blocks VLDL secretion by preventing translocation of nascent particles from the endoplasmic reticulum to the cis Golgi compartment. The accumulation of normal amounts of discoidal HDL in liver perfusates despite virtual absence of triglyceride-rich lipoproteins in Golgi secretory compartments, the space of Disse, and the perfusate is inconsistent with the concept that nascent HDL are exclusively a product of surface remnants cast off during lipolysis of chylomicrons and VLDL.     

Pathways in the formation of human plasma high density lipoprotein subpopulations containing apolipoprotein A-I without apolipoprotein A-II

The lecithin:cholesterol acyltransferase (LCAT)-induced transformation of two discrete species of model complexes that differ in number of apolipoprotein A-I (apoA-I) molecules per particle was investigated. One complex species (designated 3A-I(UC)-complexes) contained 3 apoA-I per particle, was discoidal (13.5 X 4.4 nm), and had a molar composition of 22:78:1 (unesterified cholesterol (UC):egg yolk phosphatidylcholine (egg yolk PC):apoA-I). The other complex species (designated 2A-I(UC)complexes) containing 2 apoA-I per particle was also discoidal (8.4 X 4.1 nm) and had a molar composition of 6:40:1. Transformation of 3A-I(UC)complexes by partially purified LCAT yielded a product (24 hr, 37 degrees C) with a cholesteryl ester (CE) core, 3 apoA-I, and a mean diameter of 9.2 nm. The 2A-I(UC)complexes were only partially transformed to a core-containing product (24 hr, 37 degrees C) which also had 3 apoA-I; this product, however, was smaller (diameter of 8.5 nm) than the product from 3A-I(UC)complexes. Transformation of 3A-I(UC)complexes appeared to result from build-up of core CE directly within the precursor complex. Transformation of 2A-I(UC)complexes, however, followed a stepwise pathway to the product with 3 apoA-I, apparently involving fusion of transforming precursors and release of one apoA-I from the fusion product. In the presence of low density lipoprotein (LDL), used as a source of additional cholesterol, conversion of 2A-I(UC)complexes to the product with 3 apoA-I was more extensive. The transformation product of 3A-I(UC)complexes in the presence of LDL also had 3 apoA-I but was considerably smaller in size (8.6 vs. 9.2 nm, diameter) and had a twofold lower molar content of PC compared with the product formed without LDL. LDL appeared to act both as a donor of UC and an acceptor of PC. Transformation products with 3 apoA-I obtained under the various experimental conditions in the present studies appear to be constrained in core CE content (between 13 to 22 CE per apoA-I; range of 9 CE molecules) but relatively flexible in content of surface PC molecules they can accommodate (between 24 to 49 PC per apoA-I; range of 25 PC molecules). The properties of the core-containing products with 3 apoA-I compare closely with those of the major subpopulation of human plasma HDL in the size range of 8.2-8.8 nm that contains the molecular weight equivalent of 3 apoA-I molecules.     

Size transformations of intermediate and low density lipoproteins induced by unesterified fatty acids

Plasma from individual human subjects is known to contain multiple discrete subpopulations of low (LDL) and intermediate (IDL) density lipoproteins that differ in particle size and density. The metabolic origins of these subpopulations are unknown. Transformation of IDL and larger LDL to smaller, denser LDL particles had been postulated to occur as a result of the combined effects of triglyceride hydrolysis and lipid transfer. However, the presence of multiple small LDL subspecies has been described in patients lacking cholesteryl ester transfer protein. We have characterized an alternative pathway in which size decrements in IDL or LDL are produced in the presence of unesterified fatty acids and a source of apolipoprotein (apo) A-I. Incubation of IDL or LDL subfractions with palmitic acid and either high density lipoproteins (HDL), apoHDL, or purified apoA-I gives rise to apoA-I, apoB-containing complexes that can dissociate into two particles, an apoB-containing lipoprotein with particle diameter 10-30 A smaller than the starting material, and a still smaller species (apparent peak particle diameter 140-190 A) containing lipid and apoA-I but no apoB. The newly formed IDL or LDL are depleted in phospholipid and free cholesterol with no change in apoB-100 as assessed by SDS gel electrophoresis. We hypothesize that this reaction may contribute to the formation of discrete IDL and LDL subpopulations of varying size during the course of hydrolysis of triglyceride-rich lipoproteins in plasma.     

Interconversion between apolipoprotein A-I-containing lipoproteins of pre-beta and alpha electrophoretic mobilities.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.     

Molecular etiology of a dominant form of type III hyperlipoproteinemia caused by R142C substitution in apoE4

We have used adenovirus-mediated gene transfer in apolipoprotein (apo)E^−/− mice to elucidate the molecular etiology of a dominant form of type III hyperlipoproteinemia (HLP) caused by the R142C substitution in apoE4. It was found that low doses of adenovirus expressing apoE4 cleared cholesterol, whereas comparable doses of apoE4[R142C] greatly increased plasma cholesterol, triglyceride, and apoE levels, caused accumulation of apoE in VLDL/IDL/LDL region, and promoted the formation of discoidal HDL. Co-expression of apoE4[R142C] with lecithin cholesterol acyltransferase (LCAT) or lipoprotein lipase (LPL) in apoE^−/− mice partially corrected the apoE4[R142C]-induced dyslipidemia. High doses of C-terminally truncated apoE4[R142C]-202 partially cleared cholesterol in apoE^−/− mice and promoted formation of discoidal HDL. The findings establish that apoE4[R142C] causes accumulation of apoE in VLDL/IDL/LDL region and affects in vivo the activity of LCAT and LPL, the maturation of HDL, and the clearance of triglyceride-rich lipoproteins. The prevention of apoE4[R142C]-induced dyslipidemia by deletion of the 203-299 residues suggests that, in the full-length protein, the R142C substitution may have altered the conformation of apoE bound to VLDL/IDL/LDL in ways that prevent triglyceride hydrolysis, cholesterol esterification, and receptor-mediated clearance in vivo.     

Lipoprotein abnormalities associated with lipopolysaccharide-induced lecithin: cholesterol acyltransferase and lipase deficiency

Density gradient ultracentrifugation was used to isolate and characterize the plasma lipoproteins from African green monkeys before and 24 and 48 h after subcutaneous injection of 300 micrograms/kg lipopolysaccharide (LPS) to induce an acute phase response. Compared with 0 h values, reductions occurred in plasma cholesterol (39%), high density lipoprotein (HDL) cholesterol (54%), lecithin:cholesterol acyltransferase (LCAT) activity (55%), and post-heparin plasma lipase activity (68%) 48 h after LPS injection while plasma triglyceride concentrations increased 700%. Cholesterol distribution among lipoproteins shifted from 7 to 41% in very low density lipoproteins (VLDL), 65 to 38% in low density lipoproteins (LDL), and 28 to 21% in HDL after LPS injection. At 48 h after LPS injection, all lipoprotein classes were relatively enriched in phospholipid and triglyceride and depleted of cholesteryl ester. The plasma concentration of all chemical constituents in VLDL was increased 3-9-fold within 48 h after LPS injection. By negative stain electron microscopy, HDL were discoidal in shape while VLDL and LDL appeared to have excess surface material present. Even though total HDL protein concentration in plasma was unaffected, the plasma mass of the smallest HDL subfractions (HDL3b,c) doubled while the mass of intermediate-sized subfractions (HDL3a) was dramatically decreased within 24 h after treatment. HDL became enriched in apoE, acquired apoSAA, and became depleted of apoA-I, A-II, and Cs by 48 h after LPS injection while apoB-100 remained the major apoprotein of VLDL and LDL. We conclude that administration of LPS to monkeys prevents normal intravascular metabolism of lipoproteins and results in the accumulation of relatively nascent forms of lipoproteins in plasma. These immature lipoproteins resemble those isolated from the recirculating perfusion of African green monkey livers, which are relatively deficient of LCAT activity and those isolated from the plasma of patients with familial LCAT deficiency.     

Apolipoprotein E is the major physiological activator of lecithin-cholesterol acyltransferase (LCAT) on apolipoprotein B lipoproteins

Our previous studies have indicated that lecithin-cholesterol acyltransferase (LCAT) contributes significantly to the apoB lipoprotein cholesteryl ester (CE) pool. Cholesterol esterification rate (CER) in apoA-I(-)(/)(-) apoE(-)(/)(-) mouse plasma was <7% that of C57Bl/6 (B6) mouse plasma, even though apoA-I(-)(/)(-) apoE(-)(/)(-) plasma retained (1)/(3) the amount of B6 LCAT activity. This suggested that lack of LCAT enzyme did not explain the low CER in apoA-I(-)(/)(-) apoE(-)(/)(-) mice and indicated that apoE and apoA-I are the only major activators of LCAT in mouse plasma. Deleting apoE on low-density lipoprotein (LDL) reduced CER (1% free cholesterol (FC) esterified/h) compared to B6 (6% FC esterified/h) and apoA-I(-)(/)(-) (11% FC esterified/h) LDL. Similar sized LDL particles from all four genotypes were isolated by fast protein liquid chromatography (FPLC) after radiolabeling with [(3)H]-free cholesterol (FC). LDLs (1 microg FC) from each genotype were incubated with purified recombinant mouse LCAT; LDL particles from B6 and apoA-I(-)(/)(-) plasma were much better substrates for CE formation (5.7% and 6.3% CE formed/30 min, respectively) than those from apoE(-)(/)(-) and apoE(-)(/)(-) apoA-I(-)(/)(-) plasma (1.2% and 1.1% CE formed/30 min). Western blot analysis showed that the amount of apoA-I on apoE(-)(/)(-) LDLs was higher compared to B6 LDL. Adding apoE to incubations of apoA-I(-)(/)(-) apoE(-)(/)(-) very low density lipoprotein (VLDL) resulted in a 3-fold increase in LCAT CER, whereas addition of apoA-I resulted in a more modest 80% increase. We conclude that apoE is a more significant activator of LCAT than apoA-I on mouse apoB lipoproteins.     

ABCA1 promotes the de novo biogenesis of apolipoprotein CIII-containing HDL particles in vivo and modulates the severity of apolipoprotein CIII-induced hypertriglyceridemia

In this study, the ability of the lipid transporter ABCA1 and apolipoprotein CIII (apoCIII) to promote the de novo biogenesis of apoCIII-containing HDL in vivo and the role of this HDL in apoCIII-induced hypertriglyceridemia were investigated, using adenovirus-mediated gene transfer in apoE (-/-) x apoA-I (-/-) mice or ABCA1 (-/-) mice. Injection of apoE (-/-) x apoA-I (-/-) mice with 8 x 10 (8) pfu of an adenovirus expressing the wild-type human apoCIII (AdGFP-CIII g) generated HDL-like particles and triggered only a modest increase in plasma cholesterol and triglyceride levels of these mice, 3-5 days postinfection. Plasma human apoCIII was distributed among HDL, VLDL/IDL, and LDL in these mice. In contrast, ABCA1 (-/-) mice treated similarly failed to form HDL particles and developed severe hypertriglyceridemia which could be alleviated by coinfection with an adenovirus expressing human LpL, while their plasma cholesterol levels remained unchanged 3-5 days postinfection with AdGFP-CIII g. Human apoCIII in these mice accumulated exclusively on VLDL. Control experiments confirmed that the differences between apoE (-/-) x apoA-I (-/-) and ABCA1 (-/-) mice expressing human apoCIII were not due to differences in apoCIII expression. Overall, these data show that ABCA1 and human apoCIII promote the formation of apoCIII-containing HDL-like particles that are distinct from classical apoE- or apoA-I-containing HDL. Formation of apoCIII-containing HDL prevents excess accumulation of plasma apoCIII on VLDL and allows for the efficient lipolysis of VLDL triglycerides by LpL. Furthermore, the data establish that ABCA1 and apoCIII-containing HDL play key roles in the prevention of apoCIII-induced hypertriglyceridemia in mice.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

Lipid transfers between reconstituted high density lipoprotein complexes and low density lipoproteins: effects of plasma protein factors

In this study we examined the transfer of lipids between reconstituted high density lipoprotein discs (r-HDL) and human low density lipoproteins (LDL) in the presence and absence of lecithin:cholesterol acyltransferase (LCAT) or of plasma phospholipid transfer protein (PLTP). We found that spontaneous transfer of phospholipids from r-HDL to LDL occurred by an apparent first order reaction with a half-time of 5.8 to 6.9 hr depending on the phospholipid. During the time of incubation of r-HDL with LDL (from 0 to 25 hr), the phospholipid content of r-HDL decreased more than 30%, the free cholesterol content increased 2.5-fold, and low levels of cholesteryl esters appeared in r-HDL. These compositional changes gave rise to small discoidal particles with a limiting diameter of 77 A and two molecules of apoA-I per particle. When LCAT was included in the reaction mixture, the r-HDL lost even more phospholipid, lost some free cholesterol, and gained cholesteryl esters relative to the apolipoprotein content, due to the enzymatic reaction. The products of the LCAT reaction had a diameter of 93 A and three, rather than two, apoA-I molecules per particle. Inclusion of PLTP into the reaction mixture accelerated the transfer of phospholipids (half-time of 1.7 hr) and the formation of the 77 A product. In addition to these compositional and morphological changes, which may be important in the interconversions of native HDL subspecies, the prolonged incubations revealed some slow reactions, such as the esterification of LDL cholesterol by LCAT, a background formation of cholesteryl esters in r-HDL, and an apparent hydrolysis of cholesteryl esters in LDL in the presence of r-HDL.     

LCAT facilitates transacylation of 17 beta-estradiol in the presence of HDL3 subfraction

It has been shown that estrogens need to be metabolized to their hydrophobic estrogen ester derivatives to act as antioxidants in lipoproteins. Data suggest that 17beta-estradiol (E(2)) becomes esterified in LCAT-induced reactions and the esters are transported from HDL particles to LDL and VLDL particles by a CETP-dependent mechanism. In the present study we have further investigated the regulation of E(2) esterification by LCAT and focused on the importance of HDL structure and composition in the esterification process. Isolated LDL, HDL(2), HDL(3), and reconstituted discoidal HDL (rHDL) were incubated with labeled E(2), with and without purified LCAT, at 37 degrees C for 24 h. After purification of the lipoprotein fractions, there was a significant peak of radioactivity representing esterified estradiol attached to HDL(3) and rHDL, but HDL(2) and LDL contained only trace amounts of labeled estradiol ester. TLC analysis confirmed that the radioactivity migrated in a position corresponding to that of 17beta-E(2) 17-monoester standard. The amount of radioactivity associated with HDL(3) and rHDL representing esterified E(2) was significantly increased by addition of purified LCAT. However, only limited increases of radioactivity were observed in HDL(2) and LDL. In conclusion, HDL subfractions differ in their potential to regulate estradiol esterification by LCAT.     

Regulation of reconstituted high density lipoprotein structure and remodeling by apolipoprotein E

Apolipoprotein E (apoE) enters the plasma as a component of discoidal HDL and is subsequently incorporated into spherical HDL, most of which contain apoE as the sole apolipoprotein. This study investigates the regulation, origins, and structure of spherical, apoE-containing HDLs and their remodeling by cholesteryl ester transfer protein (CETP). When the ability of discoidal reconstituted high density lipoprotein (rHDL) containing apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein to act as substrates for LCAT were compared with that of discoidal rHDL containing apoA-I [(A-I)rHDL], the rate of cholesterol esterification was (A-I)rHDL > (E2)rHDL approximately (E3)rHDL > (E4)rHDL. LCAT also had a higher affinity for discoidal (A-I)rHDL than for the apoE-containing rHDL. When the discoidal rHDLs were incubated with LCAT and LDL, the resulting spherical (E2)rHDL, (E3)rHDL, and (E4)rHDL were larger than, and structurally distinct from, spherical (A-I)rHDL. Incubation of the apoE-containing spherical rHDL with CETP and Intralipid(R) generated large fusion products without the dissociation of apoE, whereas the spherical (A-I)rHDLs were remodeled into small particles with the formation of lipid-poor apoA-I. In conclusion, i) apoE activates LCAT less efficiently than apoA-I; ii) apoE-containing spherical rHDLs are structurally distinct from spherical (A-I)rHDL; and iii) the CETP-mediated remodeling of apoE-containing spherical rHDL differs from that of spherical (A-I)rHDL.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Point mutations in apolipoprotein A-I mimic the phenotype observed in patients with classical lecithin:cholesterol acyltransferase deficiency

We have analyzed the effect of charged to neutral amino acid substitutions around the kinks flanking helices 4 and 6 of apoA-I and of the deletion of helix 6 on the in vivo activity of LCAT and the biogenesis of HDL. The LCAT activation capacity of apoA-I in vitro was nearly abolished by the helix 6 point (helix 6P-apoA-I[R160V/H162A]) and deletion {helix 6Delta-apoA-I[Delta(144-165)]} mutants, but was reduced to 50% in the helix 4 point mutant (helix 4P-apoA-I[D102A/D103A]). Following adenovirus-mediated gene transfer in apoA-I deficient mice, the level of plasma HDL cholesterol was greatly reduced in helix 6P and helix 6Delta mutants. Electron microscopy and two-dimensional gel electrophoresis showed that the helix 6P mutant formed predominantly high levels of apoA-I containing discoidal particles and had an increased prebeta1-HDL/alpha-HDL ratio. The helix 6Delta mutant formed few spherical particles and had an increased prebeta1-HDL/alpha-HDL ratio. Mice infected with adenovirus expressing the helix 4P mutant or wild-type apoA-I had normal HDL cholesterol and formed spherical alpha-HDL particles. Coinfection of mice with adenoviruses expressing human LCAT and the helix 6P mutant dramatically increased plasma HDL and apoA-I levels and converted the discoidal into spherical HDL, indicating that the LCAT activity was rate-limiting for the biogenesis of HDL. The LCAT treatment caused only a small increase in HDL cholesterol and apoA-I levels and in alpha-HDL particle numbers in the helix 6Delta mutant. The findings indicate a critical contribution of residue 160 of apoA-I to the in vivo activity of LCAT and the subsequent maturation of HDL and explain the low HDL levels in heterozygous subjects carrying this mutation.