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.     

ApoA-IV metabolism in the rat: role of lipoprotein lipase and apolipoprotein transfer

Factors influencing the association of apoA-IV with high density lipoproteins (HDL) were investigated by employing a crossed immunoelectrophoresis assay to estimate the distribution of rat plasma apoA-IV between the lipoprotein-free and HDL fractions. Incubation of rat plasma at 37 degrees C resulted in the complete transfer of lipoprotein-free apoA-IV to HDL within 45 min. When plasma obtained from fat-fed rats was incubated at 37 degrees C in the presence of postheparin plasma as a source of lipolytic activity, there was a complete transfer of HDL apoA-IV to the lipoprotein-free fraction within 30 min. With extended incubation (120 min), lipoprotein-free apoA-IV began to transfer back to HDL. Similar patterns of apoA-IV redistribution were seen when plasma from fat-fed rats was incubated with postheparin heart perfusate or was perfused through a beating heart. Incubations conducted with plasma obtained from fasted rats showed similar but markedly attenuated apoA-IV responses. Similar observations were found in vivo following intravenous heparin administration. To determine whether the transfer of apolipoproteins from triglyceride-rich lipoproteins to HDL was partially responsible for the lipolysis-induced redistribution of apoA-IV, purified apoA-I, apoE, and C apolipoproteins were added to plasma from fasted rats. When added to plasma, all of the apolipoproteins tested displaced apoA-IV from HDL in a dose-dependent manner. Conversely, apolipoproteins were removed from HDL by adding Intralipid to plasma from fasted rats. With increasing concentrations of Intralipid, there was a progressive loss of HDL apoC-III and a progressive increase in HDL apoA-IV. Intravenous injection of a bolus of Intralipid to fasted rats resulted in a transient decrease of HDL apoC-III and concomitant increase in HDL apoA-IV. From these studies, we conclude that the binding of apoA-IV to HDL is favored under conditions that result in a relative deficit of HDL surface components, such as following cholesterol esterification by LCAT or transfer of apolipoproteins to nascent triglyceride-rich lipoproteins.     

Enzymatically induced alterations in the structure of rat serum lipoproteins

Incubation of freshly isolated rat serum induces a large number of changes in the properties of the serum lipoproteins, especially the high density lipoproteins (HDL). The particle diameter of the HDL increases from about 10.4 nm to 12.3 nm and the protein content appears to increase by about 60,000 Daltons. Reactions catalyzed by lecithin:cholesterol acyltransferase (LCAT) lead to a marked decrease in cholesterol and phospholipid content, and an even greater increase in cholesteryl ester content. Especially noteworthy are the marked increases in apoE and apoA-IV which are found associated with HDL as a result of this process. Data indicate that the affinity of apoE and apoA-IV for the HDL particles may be influenced by the proportion of surface to core lipid and by the presence of products of the LCAT reaction. Changes in the apoprotein content of very low density lipoproteins are also observed, with A-I and A-IV appearing in this density interval. All of the above changes can be prevented by the inclusion of 5,5'dithiobis(2-nitrobenzoate) or p-chloromercuriphenylsulfonate during the incubation, or by heat treatment of serum at 56 degrees C for 30 min; these treatments are known to inhibit LCAT activity. It is concluded that LCAT action is the major cause of the various changes in HDL structure that are observed and that alterations in apoprotein content occur to correct the resultant imbalance between core lipid and coverage of this core by amphiphilic components. Increased apoE association with cholesteryl ester-rich HDL may provide an efficient means for receptor-mediated removal of cholesterol from the circulation.     

Characterization and functional studies of lipoproteins, lipid transfer proteins, and lecithin:cholesterol acyltransferase in CSF of normal individuals and patients with Alzheimer's disease

We investigated the lipoprotein distribution and composition in cerebrospinal fluid (CSF) in a group of patients with Alzheimer's disease (AD) or affected by other types of dementia in comparison to non-demented controls. We found slightly decreased apolipoprotein (apo)E and cholesterol concentrations in CSF of AD patients and moderately increased apoA-I concentrations, while in patients suffering from other types of dementia the apoA-I CSF concentration was increased. ApoA-IV concentrations varied widely in human CSF, but were not associated with any clinical condition. HDL(2)-like apoE-containing lipoproteins represent the major lipoprotein fraction. In CSF of normal controls, only a minor HDL(3)-like apoA-I-containing lipoprotein fraction was observed; this fraction was more prevalent in AD patients. ApoA-II was recovered mostly in the HDL(3) density range, while apoA-IV was not associated with lipoproteins but appeared in a lipid-free form, co-localizing with LCAT immunoreactivity. Bi-dimensional analysis demonstrated pre-beta and alpha apoA-I-containing particles; apoE and apoA-II were detected only in alpha-migrating particles. ApoA-IV distributed both to pre-beta and gamma-migrating particles; the LCAT signal was co-localized in this gamma-migrating fraction. Enzymatically active LCAT was present in human CSF as well as PLTP activity and mass; no CETP mass was detected. In CSF from AD patients, LCAT activity was 50% lower than in CSF from normal controls. CSF lipoproteins induced a significant cholesterol efflux from cultured rat astrocytes, suggesting that they play an active role in maintaining the cholesterol homeostasis in brain cells.     

Acute and chronic effects of a 24-hour intravenous triglyceride emulsion challenge on plasma lecithin: cholesterol acyltransferase, phospholipid transfer protein, and cholesteryl ester transfer protein activities.

Lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and cholesteryl ester transfer protein (CETP) are key factors in remodeling of high density lipoproteins (HDL) and triglyceride-rich lipoproteins. We examined the effect of a large, 24 h intravenous fat load on plasma lipids and free fatty acids (FFA) as well as on plasma LCAT, PLTP, and CETP activity levels in 8 healthy men. The effect of concomitant insulin infusion was also studied, with 1 week between the study days. During Lipofundin(R) infusion, plasma triglycerides and FFA strongly increased after 8 and 24 h (P < 0.001), whereas HDL cholesterol decreased (P < 0.01). The increase in triglycerides was mitigated with concomitant insulin infusion (P < 0.05 from without insulin). Plasma LCAT activity increased by 17.7 +/- 7.7% after 8 h (P < 0.001) and by 26.1 +/- 11. 1% after 24 h (P < 0.001), PLTP activity increased by 19.7 +/- 15.6% after 24 h (P < 0.001), but CETP activity remained unchanged. Concomitant insulin infusion blunted the increase in plasma LCAT activity (P < 0.05 from without insulin), but not that in PLTP activity. One week after the first fat load, plasma non-HDL cholesterol (P < 0.02), and triglycerides (P = 0.05) were increased, whereas HDL cholesterol was decreased (P < 0.02). Plasma CETP and PLTP activity levels were increased by 34.8 +/- 30.4% (P < 0.02) and by 15.9 +/- 6.4% (P < 0.02), respectively, but LCAT activity was then unaltered. In summary, plasma LCAT, PLTP, and CETP activity levels are stimulated by a large intravenous fat load, but the time course of their responses and the effects of insulin coadministration are different. Changes in plasma LCAT and PLTP activities may be implicated in HDL and triglyceride-rich lipoprotein remodeling under the present experimental conditions.     

Association Between Lipids, Lipoproteins Composition of HDL Particles and Triglyceride-Rich Lipoproteins, and LCAT and CETP Activity in Post-renal Transplant Patients

High-density lipoprotein (HDL) remodeling within the plasma compartment and the association between lecithin-cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) activity, and lipid, lipoprotein concentrations and composition were investigated. The aim was to examine the high sensitivity of C-reactive protein (hsCRP), lipid, apolipoprotein B (apoB), apoAI, total apoAII, apoAIInonB, apoB-containing apoAII (apoB:AII), total apoCIII, apoCIIInonB, apoB-containing apoCIII (apoB:CIII) concentration and LCAT and CETP activity to gain an insight into the association between them and LCAT and CETP, 57 post-renal transplant (Tx) patients with and without statin therapy and in 15 healthy subjects. Tx patients had moderate hypertriglyceridemia, hypercholesterolemia, and dyslipoproteinemia, disturbed triglyceride-rich lipoproteins (TRLs) and HDL composition, decreased LCAT, and slightly increased hsCRP but no CETP activity. Spearman’s correlation test showed the association between lipids and lipoproteins and LCAT or CETP, and multiple ridge stepwise forward regression showed that immunosuppressive therapy in Tx patients can disturb HDL and TRLs composition. The results suggest that inhibition or activation of LCAT is due, in part, to HDL-associated lipoprotein. Lipoprotein composition of apoAI, apoAIInonB, and apoCIIInonB in HDL particle and apoB:AII TRLs can contribute to decrease LCAT mass in Tx patients. Tx patients without statin and with lower triglycerides but higher HDL cholesterol concentration and disturbed lipoprotein composition of ApoAI and apoAII in HDL particle can decrease LCAT, increase LDL cholesterol, aggravate renal graft, and accelerate atherosclerosis and chronic heart diseases.     

Lipoprotein ApoC-II activation of lipoprotein lipase. Modulation by apolipoprotein A-IV

Lipoprotein lipase (LPL)-mediated hydrolysis of triglycerides (TG) contained in chylomicrons requires the presence of a cofactor, apolipoprotein (apo) C-II. The physiological mechanism by which chylomicrons gain apoC-II necessary for LPL activation in whole plasma is not known. Using a gum arabic stabilized TG emulsion, activation of LPL by lipoprotein apoC-II was studied. Hydrolysis of TG by LPL was greater in the presence of serum than with addition of either high density lipoproteins (HDL) or very low density lipoproteins (VLDL). LPL activation by either VLDL or HDL increased with addition of the lipoprotein-free fraction of plasma. A similar increase in LPL activity by addition of the lipoprotein-free fraction together with HDL or VLDL was observed when another TG emulsion (Intralipid) or TG-rich lipoproteins from an apoC-II deficient subject were used as a substrate. Human apoA-IV, apoA-I, apoE, and cholesteryl ester transfer protein were assessed for their ability to increase LPL activity in the presence of VLDL. At and below physiological concentrations, only apoA-IV increased LPL activity. One hundred percent of LPL activity measured in the presence of serum was achieved using VLDL plus apoA-IV. In the absence of an apoC-II source, apoA-IV had no effect on LPL activity. Removal of greater than 80% of the apoA-IV from the nonlipoprotein-containing fraction of plasma by incubation with Intralipid markedly reduced its ability to activate LPL in the presence of VLDL or HDL. Gel filtration chromatography demonstrated that incubation of the nonlipoprotein-containing fraction of plasma with HDL and the TG emulsion caused increased transfer of apoC-II to the emulsion and association of apoA-IV with HDL. Our studies demonstrate that apoA-IV increases LPL activation in the presence of lipoproteins. We hypothesize that apoA-IV is required for efficient release of apoC-II from either HDL or VLDL, which then allows for LPL-mediated hydrolysis of TG in nascent chylomicrons.     

Plasma metabolism of apolipoprotein A-IV in humans

As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day. The results are consistent with the conclusions that: apoA-IV is present in human plasma in three distinct metabolic pools; apoA-IV associated with the triglyceride-rich lipoproteins is a precursor to the apoA-IV HDL and LFF pools; apoA-IV in LFF is not a free protein and its turnover rate is faster than that of apoA-IV in HDL; since no transfer of apoA-IV from the HDL or the LFF occurs, these pools may represent a terminal pathway for the catabolism of apoA-IV; and the catabolism of apoA-IV in HDL is dissociated from that of apoA-I although both apoproteins may reside on the same lipoprotein particles.     

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.     

The in vitro formation of HDL2 during the action of LCAT: the role of triglyceride-rich lipoproteins

We examined the effects of lecithin:cholesterol acyl transferase (LCAT) and of lipoprotein lipase (LPL) on the conversion of high density lipoproteins (HDL) towards fractions of lower densities using the analytical ultracentrifuge. Freshly isolated whole plasma was incubated for 24 h at 37 degrees C in the presence or absence of active enzyme systems. In some cases, lipoproteins were removed by selective precipitations; alternatively, we added triglyceride-rich lipoproteins (TGRLP) or Intralipid to the incubations. The results are as follows. 1) The incubation of whole plasma containing active LCAT leads to a conversion of HDL3 to a fraction of lower density, notably HDL2a. If LCAT is inhibited, the conversion is far less pronounced. 2) If very low and low density lipoproteins are removed by phosphotungstate precipitation and the supernatant is incubated with LCAT, HDL3 shifts towards higher densities. 3) The presence of phosphatidylcholine/cholesterol liposomes or the presence of blood cells as a source of additional LCAT substrate had only little influence on the HDL conversion in our system. 4) The addition of TGRLP or of Intralipid at minimal ratios of 2.5:1 caused an almost complete conversion of HDL3 to HDL2b. This conversion was dependent on active LCAT. 5) LPL also caused a shift of HDL3 to HDL2a if TGRLP was present. HDL2b, however, was not formed by LPL unless LCAT was active.     

Distribution of apolipoprotein A-IV among lipoprotein subclasses in rat serum

The distribution of apolipoproteins (apo) A-I, A-IV, and E in sera of fed and fasted rats was studied using various methods for the isolation of lipoproteins. Serum concentrations of apoA-I and apoA-IV decreased significantly during fasting (16 and 31%, respectively), while apoE concentrations remained essentially the same. Chromatography of sera on 6% agarose columns showed that apoA-IV is present on HDL and as so-called "free" apoA-IV. The concentration of "free" apoA-IV decreased six- to seven-fold during fasting, explaining the decrease in total serum apoA-IV. Serum apoA-I and apoE are almost exclusively associated with HDL-sized particles. When sera are centrifuged at a density of 1.21 g/ml, marked quantities of apoA-I (8-9%) and apoE (11-22%) are recovered in the "lipoprotein-deficient" infranatant, suggesting that ultracentrifugation affects the integrity of serum HDL. The nature of the chromatographically separated carriers of serum apoA-IV was investigated by quantitative immunoprecipitation. From these studies, it is concluded that apoA-IV in rat serum is present in at least three fractions: 1) particles with the size and composition of HDL, containing both apoA-I and apoA-IV and possibly minor quantities of apoE; 2) HDL-sized particles containing apoA-IV, but no apoA-I or apoE; 3) "free" apoA-IV probably containing small amounts of bound cholesterol and phospholipid.     

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI.These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.     

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.     

Distribution of apolipoprotein A-IV in human plasma

Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles.     

Human apolipoprotein A-IV: displacement from the surface of triglyceride-rich particles by HDL2-associated C-apoproteins

Human apolipoprotein A-IV rapidly dissociates from the surface of lymph chylomicrons following their entry into circulation by an unknown mechanism. We have therefore investigated the binding of human apoA-IV to triglyceride-rich particles and the interaction of these apoA-IV/lipid complexes with human HDL2. Human apoA-IV was purified from lipoprotein depleted serum (J. Lipid Res. 1983. 24:52-59). Triglyceride-rich particles of well-defined properties were isolated from Intralipid, a commercially available phospholipid-triglyceride emulsion. Various concentrations of radiolabeled human apoA-IV were incubated at 24 degrees C with a fixed quantity of lipid particles; the particles were reisolated by centrifugation, and bound and free apoA-IV were quantitated. In 50 mM Tris, pH 7.4, apoA-IV bound to the triglyceride-rich particles in a non-cooperative manner, with a Kd of 2.0 microM. The calculated maximal binding was 4.96 X 10(-4) mol of apoA-IV bound per mol of phospholipid. The addition of increasing amounts of human HDL2 to the incubations caused the progressive dissociation of apoA-IV from the triglyceride-rich particles. Analysis of the reisolated particles by isoelectric focusing demonstrated the presence of C-apoproteins, suggesting their transfer from HDL2. Addition of purified apoC-III-1 to the incubations at concentrations equivalent to those present in HDL2 caused a similar dissociation of apoA-IV. HDL2 was modified to selectively remove C-apoproteins, without alteration of other physical characteristics. This modified HDL2 was four times less effective in causing apoA-IV dissociation. These results demonstrate that the lipid binding properties of human apoA-IV may be quantitatively examined using triglyceride-rich particles as model chylomicrons. This approach reproduces in vitro the dissociation of apoA-IV that occurs in vivo when mesenteric lymph chylomicrons enter the circulation, and suggests that the primary mechanism for this phenomenon is the transfer of C-apoproteins from high density lipoproteins to the triglyceride-rich particle surface. We hypothesize that this mechanism may play an important role in the modulation of chylomicron apoA-IV content in man.     

Role of LCAT in HDL remodeling: investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (-77% and -13%), and LCAT activities (-99% and -39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated prebeta-1 and alpha-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with alpha-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, prebeta-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with prebeta-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of prebeta-1 into alpha-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.     

Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency

Our purpose was to compare HDL subpopulations, as determined by nondenaturing two-dimensional gel electrophoresis followed by immunoblotting for apolipoprotein A-I (apoA-I), apoA-II, apoA-IV, apoCs, and apoE in heterozygous, compound heterozygous, and homozygous subjects for cholesteryl ester transfer protein (CETP) deficiency and controls. Heterozygotes, compound heterozygotes, and homozygotes had CETP masses that were 30, 63, and more than 90% lower and HDL-cholesterol values that were 64, 168, and 203% higher than those in controls, respectively. Heterozygotes had approximately 50% lower pre-beta-1 and more than 2-fold higher levels of alpha-1 and pre-alpha-1 particles than controls. Three of the five heterozygotes' alpha-1 particles also contained apoA-II, which was not seen in controls. Compound heterozygotes and homozygotes had very large particles not observed in controls and heterozygotes. These particles contained apoA-I, apoA-II, apoCs, and apoE. However, these subjects did not have decreased pre-beta-1 levels. Our data indicate that CETP deficiency results in the formation of very large HDL particles containing all of the major HDL apolipoproteins except for apoA-IV. We hypothesize that the HDL subpopulation profile of heterozygous CETP-deficient patients, especially those with high levels of alpha-1 containing apoA-I but no apoA-II, represent an improved anti-atherogenic state, although this might not be the case for compound heterozygotes and homozygotes with very large, undifferentiated HDL particles.     

Phospholipid transfer protein gene knock-out mice have low high density lipoprotein levels, due to hypercatabolism, and accumulate apoA-IV-rich lamellar lipoproteins

Phospholipid transfer protein gene knock-out (Pltp KO) mice have defective transfer of very low density lipoprotein (VLDL) phospholipids into high density lipoprotein (HDL) and markedly decreased HDL levels (Jiang et al. 1999. J. Clin. Invest. 103: 907-914). These animals also accumulated VLDL- and LDL-sized lipoproteins on a high saturated fat diet. The goals of this study were to further characterize the abnormal lipoproteins of Pltp KO mice and to determine the mechanisms responsible for low HDL levels. A lipoprotein fraction enriched in lamellar structures was isolated from the low density lipoprotein (LDL) region and was shown to be phospholipid- and free cholesterol-rich and to have apoA-IV (55%) and apoE (25%) as major apolipoproteins. The lamellar lipoproteins accumulating in these mice probably represent surface material derived from triglyceride-rich lipoproteins (TRL). The HDL was found to be protein-rich (primarily apoA-I) and specifically depleted in phosphatidylcholine (PC) (28% in wild-type mice (WT) vs. 15% in Pltp KO mice, P < 0.001). Unexpectedly, turnover studies using autologous HDL revealed a profound 4-fold increase in the catabolism of HDL protein and cholesteryl ester in Pltp KO mice compared to wild-type, with minor differences in synthesis rates. In contrast, injection of WT mouse HDL into Pltp KO mice showed only a 2-fold increase in fractional catabolism. Reminiscent of the defect in Tangier disease, the failure of transfer of PC from TRL into the HDL fraction results in dramatic hypercatabolism of HDL. These results suggest that defective phospholipid transfer from TRL into HDL, arising from decreased lipolysis or decreased PLTP activity, could lead to hypoalphalipoproteinemia characterized by hypercatabolism of HDL protein. lipoprotein levels, due to hypercatabolism, and accumulate apoA-IV-rich lamellar lipoproteins.     

ApoA-IV promotes the biogenesis of apoA-IV-containing HDL particles with the participation of ABCA1 and LCAT

The objective of this study was to establish the role of apoA-IV, ABCA1, and LCAT in the biogenesis of apoA-IV-containing HDL (HDL-A-IV) using different mouse models. Adenovirus-mediated gene transfer of apoA-IV in apoA-I^−/− mice did not change plasma lipid levels. ApoA-IV floated in the HDL2/HDL3 region, promoted the formation of spherical HDL particles as determined by electron microscopy, and generated mostly α- and a few pre-β-like HDL subpopulations. Gene transfer of apoA-IV in apoA-I^−/− × apoE^−/− mice increased plasma cholesterol and triglyceride levels, and 80% of the protein was distributed in the VLDL/IDL/LDL region. This treatment likewise generated α- and pre-β-like HDL subpopulations. Spherical and α-migrating HDL particles were not detectable following gene transfer of apoA-IV in ABCA1^−/− or LCAT^−/− mice. Coexpression of apoA-IV and LCAT in apoA-I^−/− mice restored the formation of HDL-A-IV. Lipid-free apoA-IV and reconstituted HDL-A-IV promoted ABCA1 and scavenger receptor BI (SR-BI)-mediated cholesterol efflux, respectively, as efficiently as apoA-I and apoE. Our findings are consistent with a novel function of apoA-IV in the biogenesis of discrete HDL-A-IV particles with the participation of ABCA1 and LCAT, and may explain previously reported anti-inflammatory and atheroprotective properties of apoA-IV.     

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.