Multiple substrates for paraoxonase-1 during oxidation of phosphatidylcholine by peroxynitrite.

Paraoxonase (PON-1) is a high-density lipoprotein (HDL)-bound enzyme with activity toward multiple substrates. It hydrolyzes organic phosphate and aromatic carboxylic acid esters. It also inhibits accumulation of oxidized phospholipids in plasma lipoproteins by a mechanism yet to be determined. Therefore, we subjected apolipoprotein A-I proteoliposomes containing either 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine to oxidation by a peroxynitrite generator, SIN-1, in the presence and absence of purified PON-1. PON-1 modified the proportion of oxidation products without affecting the overall extent of PC oxidation. However, in the presence of PON-1, phosphatidylcholine isoprostanes were hydrolyzed to lysophosphatidylcholine. In addition, PON-1 hydrolyzed the phosphatidylcholine core aldehydes 1-palmitoyl-2-(9-oxo)nonanoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(5-oxo)valeroyl-sn-glycero-3-phosphocholine to lysophosphatidylcholine. This hydrolysis was not affected by pefabloc, a serine esterase inhibitor. There was no detectable release of linoleate, arachidonate, or their hydroperoxy or hydroxy derivatives in the presence of PON-1. We conclude that PON-1 minimizes the accumulation of phosphatidylcholine oxidation products by the hydrolysis of phosphatidylcholine isoprostanes and core aldehydes to lysophosphatidylcholine with a serine esterase-independent mechanism.     

Phosphatidylcholine transfer protein promotes apolipoprotein A-I-mediated lipid efflux in Chinese hamster ovary cells.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein of unknown function that catalyzes intermembrane transfer of phosphatidylcholines in vitro. Using stably transfected CHO cells, we explored the influence of PC-TP on apolipoprotein A-I- and high density lipoprotein 3 (HDL(3))-mediated lipid efflux. In proportion to its cellular level of expression, PC-TP accelerated apolipoprotein A-I-mediated phospholipid and cholesterol efflux as pre-beta-HDL particles. PC-TP increased rates of efflux of both lipids by >2-fold but did not affect mRNA levels or the activity of ATP-binding cassette A1, a plasma membrane protein that regulates apolipoprotein A-I-mediated lipid efflux. Overexpression of PC-TP was associated with only slight increases in HDL(3)-mediated phospholipid efflux and no changes in cholesterol efflux. In scavenger receptor BI-overexpressing cells, PC-TP expression minimally influenced apolipoprotein A-I- or HDL(3)-mediated lipid efflux. PC-TP did not affect cellular phospholipid compositions, phosphatidylcholine contents, or phosphatidylcholine synthetic rates. These findings suggest that a physiological function of PC-TP is to replenish the plasma membrane with phosphatidylcholines that are removed during pre-beta-HDL particle formation due to the activity of ATP-binding cassette A1.     

Transformation of large discoidal complexes of apolipoprotein A-I and phosphatidylcholine by lecithin-cholesterol acyltransferase

Using a cholate-dialysis recombination procedure, complexes of apolipoprotein A-I and synthetic phosphatidylcholine (1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC] were prepared in mixtures at a relatively high molar ratio of 150:1 phosphatidylcholine/apolipoprotein A-I. Particle size distribution analysis by gradient gel electrophoresis of the recombinant mixtures indicated the presence of a series of discrete complexes that included species migrating at RF values observed for discoidal particles in nascent high-density lipoproteins (HDL) in plasma of lecithin-cholesterol acyltransferase-deficient subjects. One of these complex species, designated complex class 6, formed with either phosphatidylcholine, was isolated by gel filtration and characterized at follows: discoidal shape (mean diameter 20.8 nm (POPC) and 19.0 nm (DOPC]; molar ratio, phosphatidylcholine/apolipoprotein A-I, 155:1 (POPC) and 130:1 (DOPC); and both containing 4 molecules of apolipoprotein A-I per particle. Incubation of class 6 complexes with lecithin-cholesterol acyltransferase (EC 2.3.1.43) and a source of unesterified cholesterol (low-density lipoprotein (LDL] was shown by electron microscopy to result in a progressive transformation of the discoidal particles (0 h) to deformable (2.5 h) and to spherical particles (24 h). The spherical particles (diameter 13.6 nm (POPC) and 12.5 nm (DOPC) exhibit sizes at the upper boundary of the interval defining the human plasma (HDL2b)gge (12.9-9.8 nm). The spherical particles contain a cholesteryl ester core that reaches a limiting molar ratio of approx. 50-55:1 cholesteryl ester/apolipoprotein A-I. The deformable particles assume a rectangular shape under negative staining and, relative to the 24-h spherical product, are enriched in phosphatidylcholine. Chemical crosslinking (by dimethyl suberimidate) of the isolated transformation products shows the 24-h spherical particle to contain predominantly 4 apolipoprotein A-I molecules; products produced after intermediate periods of time appear to contain species with 3 and 4 apolipoproteins per particle. Our in vitro studies indicate a potential pathway in the origins of large, apolipoprotein A-I-containing plasma HDL particles. The deformable species observed during transformation were similar in size and shape to particles observed in interstitial fluid.     

Paraoxonase-1 does not reduce or modify oxidation of phospholipids by peroxynitrite

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated esterase/lactonase implicated to play a role in protection against atherosclerosis. However, the exact mechanism(s) and substrates for PON1 are still uncertain. In this article, we review some of the evidence for PON1's antioxidant activity, as well as our efforts to identify the actual substrates and products for this activity. We originally reported that PON1 had phospholipase activity toward oxidized phosphatidylcholine (J. Biol. Chem. 276:24473-24481; 2001). Subsequently, Marathe et al. (J. Biol. Chem. 278:3937-3947; 2003) reported that this activity was due to a contaminating lipase. However, that article did not replicate the conditions used in our previous study. To address this controversy, we purified serum PON1 by a modified method that separates the paraoxonase activity from an activity detectable as platelet-activating factor acetyl hydrolase (PAF-AH) (Teiber et al., J. Lipid. Res. 2004; Epub ahead of print, PMID 15342686) and reexamined the oxidation of phosphatidylcholine by peroxynitrite using 3-morpholinosydnonimine as a peroxynitrite generator and apolipoprotein AI-phosphatidylcholine- PON1 complexes. The phosphatidylcholines were studied by electrospray ionization tandem mass spectrometry. PON1 preparations free of PAF-AH activity showed no phospholipase activity when reconstituted into apolipoprotein AI-phosphatidylcholine complexes. We conclude that PON1 does not affect the accumulation of phosphatidylcholine oxidation products. Further, we have no evidence that PON1 has an intrinsic phospholipase A2 activity toward oxidized phospholipids.     

The conformation of apolipoprotein A-I in high-density lipoproteins is influenced by core lipid composition and particle size: a surface plasmon resonance study

Plasma high-density lipoproteins (HDL) are a heterogeneous group of particles that vary in size as well as lipid and apoprotein composition. The effect of HDL core lipid composition and particle size on apolipoprotein (apo) A-I structure was studied using surface plasmon resonance (SPR) analysis of the binding of epitope-defined monoclonal antibodies. The association and dissociation rate constants of 12 unique apo A-I-specific monoclonal antibodies for isolated plasma HDL were calculated. In addition, the association rate constants of the antibodies were determined for homogeneous preparations of spherical reconstituted HDL (rHDL) that contained apo A-I as the sole apolipoprotein and differed either in their size or in their core lipid composition. This analysis showed that lipoprotein size affected the conformation of domains dispersed throughout the apo A-I molecule, but the conformation of the central domain between residues 121 and 165 was most consistently modified. In contrast, replacement of core cholesteryl esters with triglyceride in small HDL modified almost the entire molecule, with only two key N-terminal domains of apo A-I being unaffected. This finding suggested that the central and C-terminal domains of apo A-I are in direct contact with rHDL core lipids. This immunochemical analysis has provided valuable insight into how core lipid composition and particle size affect the structure of specific domains of apo A-I on HDL.     

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.     

Human high density lipoprotein (HDL3) binding to rat liver plasma membranes

The binding of human 125I-labeled HDL3 to purified rat liver plasma membranes was studied. 125I-labeled HDL3 bound to the membranes with a dissociation constant of 10.5 micrograms protein/ml and a maximum binding of 3.45 micrograms protein/mg membrane protein. The 125I-labeled HDL3-binding activity was primarily associated with the plasma membrane fraction of the rat liver membranes. The amount of 125I-labeled HDL3 bound to the membranes was dependent on the temperature of incubation. The binding of 125I-labeled HDL3 to the rat liver plasma membranes was competitively inhibited by unlabeled human HDL3, rat HDL, HDL from nephrotic rats enriched in apolipoprotein A-I and phosphatidylcholine complexes of human apolipoprotein A-I, but not by human or rat LDL, free human apolipoprotein A-I or phosphatidylcholine vesicles. Human 125I-labeled apolipoprotein A-I complexed with egg phosphatidylcholine bound to rat liver plasma membranes with high affinity and saturability, and the binding constants were similar to those of human 125I-labeled HDL3. The 125I-labeled HDL3-binding activity of the membranes was not sensitive to pronase or phospholipase A2; however, prior treatment of the membranes with phospholipase A2 followed by pronase digestion resulted in loss of the binding activity. Heating the membranes at 100 degrees C for 30 min also resulted in an almost complete loss of the 125I-labeled HDL3-binding activity.     

Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low density lipoprotein

Endothelial cells are known to play an important role in the regulation of vascular tone. Here we demonstrate that modified low density lipoprotein (LDL) with copper oxidation or phospholipase A2 treatment elicits a potent inhibitory action on endothelium-dependent relaxations evoked by acetylcholine, although native LDL does not affect endothelium-dependent relaxations. Phosphatidylcholine of native LDL is converted to lysophosphatidylcholine during these modifications. Furthermore, lysophosphatidylcholine fraction separated from oxidized LDL (0.5mg.protein/ml) by thin layer chromatography abolished endothelium-dependent relaxations, although the remaining lipid fraction had little effects on endothelium-dependent relaxations. These results indicate that lysophosphatidylcholine is the principal substance for the impairment of endothelium-dependent relaxations by oxidized LDL and phospholipase A2 treated LDL.     

Evidence in vitro that hepatic lipase reduces the concentration of apolipoprotein A-I in rabbit high-density lipoproteins

Incubation of rabbit plasma in vitro with hepatic lipase resulted in the hydrolysis of triacylglycerol in high-density lipoproteins (HDL) and a reduction in HDL particle size. These changes were accompanied by a decrease in the concentration of apolipoprotein A-I (apo A-I) in the HDL. The loss of apo A-I was demonstrated independently by ultracentrifugation, size exclusion chromatography and gradient gel-immunoblot analysis. It was unrelated to hydrolysis of HDL phospholipids but did correlate with the reduction in HDL particle size. These studies suggest that the concentration of apo A-I in HDL may be influenced by factors which regulate the metabolism of HDL core lipid constituents.     

Isolation and specificity of rat lecithin: cholesterol acyltransferase: comparison with the human enzyme using reassembled high-density lipoproteins containing ether analogs of phosphatidylcholine

Rat plasma lecithin: cholesterol acyltransferase, a 68 kDa glycoprotein, has been purified 14 000-fold by a modification of a procedure used for the human enzyme. The activity of lecithin: cholesteryl acyltransferase in human and rat plasma are the same, although activation of both enzymes by human apolipoprotein A-I is greater than that produced by rat apolipoprotein A-I. Using reassembled high-density lipoproteins composed of human apolipoprotein A-I, phosphatidylcholine ethers and a series of different phosphatidylcholines, the separate effects of molecular species specificity and microenvironment on the rate of cholesteryl ester formation was determined. Substitution of a fluid lipid, 1-palmityl-2-oleyl-sn-glycero-3-phosphorylcholine, for a solid lipid, 1,2-dipalmityl-sn-glycero-3-phosphorylcholine, produced an 8-fold increase in the activity of all molecular species of phosphatidylcholine. With either solid or fluid lipid environments, the activity decreased as a function of increasing chain length of saturated acyl groups. Addition of one or more double bonds greatly increased the activity of a given saturated homologue. One major difference between the molecular specificity of rat and human lecithin: cholesteryl acyltransferase was that the latter had a two-fold preference for phosphatidylcholines containing arachidonate at the sn-2-position.     

Isolation and specificity of rat lecithin : Cholesterol acyltransferase: Comparison with the human enzyme using reassembled high-density lipoproteins containing ether analogs of phosphatidylcholine

Rat plasma lecithin:cholesterol acyltransferase, a 68 kDa glycoprotein, has been purified 14000-fold by a modification of a procedure used for the human enzyme. The activity of lecithin : cholesteryl acyltransferase in human and rat plasma are the same, although activation of both enzymes by human apolipoprotein A-I is greater than that produced by rat apolipoprotein A-I. Using reassembled high-density lipoproteins composed of human apolipoprotein A-I, phosphatidylcholine ethers and a series of different phosphatidylcholines, the separate effects of molecular species specificity and microenvironment on the rate of cholesteryl ester formation was determined. Substitution of a fluid lipid, 1-palmityl-2-oleyl-sn-glvcero-3-phosphonlcholine. for a solid lipid, 1,2-dipalmityl-sn-glycero-3-phosphorylcholine, produced an 8-fold increase in the activity of all molecular species of phosphatidylcholine. With either solid or fluid lipid environments, the activity decreased as a function of increasing chain length of saturated acyl groups. Addition of one or more double bonds greatly increased the activity of a given saturated homologue. One major difference between the molecular specificity of rat and human lecithin:cholesteryl acyltransferase was that the latter had a two-fold preference for phosphatidylcholines containing arachidonate at the sn-2-position.     

Characterization of discoidal complexes of phosphatidylcholine, apolipoprotein A-I and cholesterol by gradient gel electrophoresis

Complexes of egg yolk phosphatidylcholine and apolipoprotein A-I were prepared by a detergent (sodium cholate)-dialysis method and characterized by gradient gel electrophoresis, gel filtration, electron microscopy and chemical analysis. Multicomponent electrophoretic patterns were obtained indicating formation of at least eight classes of discoidal complexes. The relative contribution of the different classes to the electrophoretic pattern was a function of the molar ratio of phosphatidylcholine:apolipoprotein A-I in the interaction mixture. Molar ratios of phosphatidylcholine:apolipoprotein A-I in isolated complexes were strongly and positively correlated with disc diameter obtained by electron microscopy. Incorporation of unesterified cholesterol into phosphatidylcholine/apolipoprotein A-I interaction mixtures also resulted in formation of unique complexes but with considerably different particle size distributions relative to those observed in the absence of cholesterol. One common consequence of cholesterol incorporation into interaction mixtures of 87.5:1 and 150:1 molar ratio of phosphatidylcholine:apolipoprotein A-I was the disappearance of a major complex class with diameter of 10.8 nm and the appearance of a major component with diameter of approximately 8.8 nm. Electrophoretic patterns of cholesterol-containing complexes showed a strong similarity to patterns recently published for high density lipoproteins from plasma of lecithin:cholesterol acyltransferase-deficient subjects, suggesting that the complexes formed in vitro by the detergent-dialysis method may serve as appropriate models for investigation of the origins of the HDL particle size distribution.     

Comparative analysis of lipid composition of normal and acute-phase high density lipoproteins

In the acute-phase response and in diseases with prolonged acute phases, normal HDL (NHDL) is converted into acute-phase HDL (APHDL) and becomes proinflammatory and unable to protect LDL against oxidative modification. Earlier work had demonstrated that these changes are associated with alterations in apolipoprotein composition and enzymatic activity of APHDL, but the effect of the acute-phase condition on the lipid composition of APHDL had remained obscure. The present study shows marked quantitative differences in lipid composition between NHDL and APHDL. Specifically, APHDL contained 25% less total lipid per milligram of protein. Up to 50% of cholesteryl ester in the lipid core of APHDL was replaced by triacylglycerol; however, the total phospholipid/total neutral lipid ratios were the same as in NHDL, both lipoproteins giving similar calculated lipid core radii. Furthermore, the phosphatidylcholine/sphingomyelin ratio in APHDL was nearly double that in NHDL, indicating a relative loss of sphingomyelin. A decrease was also seen in diacyl and alkenylacyl glycerophosphatidylethanolamine as well as in phosphatidylinositol of APHDL when compared with NHDL. APHDL contained proportionally more saturated and less polyunsaturated and isoprostane-containing species of phosphatidylcholine, as well as more saturated than unsaturated cholesteryl esters. APHDL also contained significantly more free fatty acids, lysophosphatidylcholine, and free cholesterol. These changes in the lipid composition of HDL are consistent with the alterations in the apoprotein composition and enzymatic activity of APHDL and indicate proinflammatory and proatherogenic roles for APHDL.     

Differential uptake and metabolism of free and esterified cholesterol from high-density lipoproteins in the ovary

Rat luteal cells utilize high-density lipoproteins (HDL) as a source of cholesterol for steroid synthesis. Both the free and esterified cholesterol of HDL are utilized by these cells. In this report, we have examined the relative uptake of free and esterified cholesterol of HDL by cultured rat luteal cells. Incubation of the cells with HDL labeled with [3H]cholesterol or [3H]cholesteryl linoleate resulted in 4-6-fold greater uptake of the free cholesterol compared to esterified cholesterol. The increased uptake of free cholesterol correlated with its utilization for progestin synthesis: utilization of HDL-derived free cholesterol was 3-6-fold higher than would be expected from its concentration in HDL. The differential uptake and utilization of free and esterified cholesterol was further examined using egg phosphatidylcholine liposomes containing cholesterol or cholesteryl linoleate as a probe. Liposomes containing free cholesterol were able to deliver cholesterol to luteal cells and support steroid synthesis in the absence of apolipoproteins, and the addition of apolipoprotein A-I (apo A-I) moderately increased the uptake and steroidogenesis. Similar experiments using cholesteryl linoleate/egg phosphatidylcholine liposomes showed that inclusion of apo A-I resulted in a pronounced increase in the uptake of cholesteryl linoleate and progestin synthesis. These experiments suggest that free cholesterol from HDL may be taken up by receptor-dependent and receptor-independent processes, whereas esterified cholesterol uptake requires a receptor-dependent process mediated by apolipoproteins.     

Effects of hydrophobicity on turnover of plasma high density lipoproteins labeled with phosphatidylcholine ethers in the rat

Rat high density lipoproteins (HDL) were labeled with a series of phosphatidylcholines and ether analogs of phosphatidylcholine. The rates of turnover of the phosphatidylcholine ethers in the rat decreased as a function of increasing hydrophobicity and were more than five times faster than those of apolipoprotein A-I turnover and spontaneous lipid transfer. The major tissue sites for uptake were the liver, adrenals, and ovaries. The rate of turnover of a phosphatidylcholine was faster than that of the corresponding ether analog due to the action of lecithin:cholesterol acyltransferase, although this activity was slow compared to the turnover of high density lipoprotein-phosphatidylcholine. Injection of a purified human phosphatidylcholine transfer protein increased the turnover rate of a phosphatidylcholine and its ether analog. We conclude that a major route for the turnover of plasma high density lipoprotein-phosphatidylcholine in the rat is independent of spontaneous lipid transfer, hydrolysis, and HDL particle uptake, and that it involves the activity of a plasma phosphatidylcholine transfer protein.     

Phosphatidylcholine substrate specificity of lecithin: cholestrol acyltransferase-in high density lipoproteins and in lipids dispersions.

Lecithin: cholesterol acyltransferase (LCAT) was more highly activated by apolipoprotein A-I (apoA-I) with dimyristoyl phosphatidylcholine (DMPC) than with dilinoleoyl phosphatidylcholine (DLPC) when lipid dispersion of cholesterol and each phosphatidylcholine was used as a substrate. When the enzyme reactions were activated by whole apolipoproteins of high density lipoproteins (HDL), DLPC was more available to the LCAT reaction than DMPC with high concentrations of apoHDL in an incubation mixture. However, no detectable enzyme reaction was observed with dipalmitoyl phosphatidylcholine (DPPC) under both conditions. On the other hand, all of these phosphatidylcholines acted as substrates of LCAT when they were incorporated into HDL coupled to Sepharose. The order of their relative reactivities to cholesterol was DMPC, DPPC, AND DLPC under the conditions used.     

Interplay of oxygen, vitamin E, and carotenoids in radical reactions following oxidation of Trp and Tyr residues in native HDL3 apolipoproteins. Comparison with LDL. A time-resolved spectroscopic analysis

It has been recently shown that the inhibition of apolipoprotein A-I (apoAI) reverse cholesterol transport activity during oxidation of HDL by myeloperoxidase may involve myeloperoxidase electron transfer pathways other than those leading to tyrosine chlorination. To better understand how such mechanisms might be initiated, the role of semioxidized Tyr and Trp residues in loss of apoAI and apolipoprotein A-II (apoAII) integrity has been assessed using selective Trp and Tyr one-electron oxidation by *Br2(-) radical-anions in HDL3 as well as in unbound apoAI and apoAII. Behavior of these radicals in apolipoprotein B of LDL has also been assessed. Formation of semioxidized Tyr in HDL3 is followed by partial repair during several milliseconds via reaction with endogenous alpha-tocopherol to form the alpha-tocopheroxyl radical. Subsequently, 2% of alpha-tocopheroxyl radical is repaired by HDL3 carotenoids. With LDL, a faster repair of semioxidized Tyr by alpha-tocopherol is observed, but carotenoid repair of alpha-tocopheroxyl radical is not. Only a small fraction of HDL3 particles contains alpha-tocopherol and carotenoids, which explains limited repair of semioxidized Tyr by alpha-tocopherol. All LDL particles normally contain multiple alpha-tocopherol and carotenoid molecules, and the lack of repair of alpha-tocopheroxyl radical by carotenoids probably results from hindered mobility of carotenoids in the lipid core. Western blots of gamma-irradiated HDL3 comparable to those reported for apoAI myeloperoxidase oxidation show that the incomplete repair of semioxidized Tyr and Trp induces apoAI and apoAII permanent damage including formation of a heterodimer of one apoAI with a monomeric apoAII at about 36 kDa.     

Apolipoprotein A-II: beyond genetic associations with lipid disorders and insulin resistance

PURPOSE OF REVIEW: Apolipoprotein A-II, the second major HDL apolipoprotein, was often considered of minor importance relatively to apolipoprotein A-I and its role was controversial. This picture is now rapidly changing, due to novel polymorphisms and mutations, to the outcome of clinical trials, and to studies with transgenic mice. RECENT FINDINGS: The -265 T/C polymorphism supports a role for apolipoprotein A-II in postprandial very-low-density lipoprotein metabolism. Fibrates, which increase apolipoprotein A-II synthesis, significantly decrease the incidence of major coronary artery disease events, particularly in subjects with low HDL cholesterol, high plasma triglyceride, and high body weight. The comparison of transgenic mice overexpressing human or murine apolipoprotein A-II has highlighted major structural differences between the two proteins; they have opposite effects on HDL size, apolipoprotein A-I content, plasma concentration, and protection from oxidation. Human apolipoprotein A-II is more hydrophobic, displaces apolipoprotein A-I from HDL, accelerates apolipoprotein A-I catabolism, and its plasma concentration is decreased by fasting. Apolipoprotein A-II stimulates ATP binding cassette transporter 1-mediated cholesterol efflux. Human and murine apolipoprotein A-II differently affect glucose metabolism and insulin resistance. A novel beneficial role for apolipoprotein A-II in the pathogenesis of hepatitis C virus has been shown. SUMMARY: The hydrophobicity of human apolipoprotein A-II is a key regulatory factor of HDL metabolism. Due to the lower plasma apolipoprotein A-II concentration during fasting, measurements of apolipoprotein A-II in fed subjects are more relevant. More clinical studies are necessary to clarify the role of apolipoprotein A-II in well-characterized subsets of patients and in the insulin resistance syndrome.     

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as 'free' apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and 'free' apolipoprotein A-IV. The increased level of 'free' A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of 'free' apolipoprotein A-IV present in serum obtained from fed animals.     

Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.