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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1912.)
© 2002 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Metabolism of ApoA-I as Lipid-Free Protein or as Component of Discoidal and Spherical Reconstituted HDLs

Studies in Wild-Type and Hepatic Lipase Transgenic Rabbits

Patrick Kee; Kerry-Anne Rye; John L. Taylor; P. Hugh R. Barrett; Philip J. Barter

From the University Department of Medicine (P.K., P.J.B.), Royal Adelaide Hospital, and the Lipid Research Laboratory (P.K., K.-A.R., P.J.B.), Hanson Institute, Adelaide, Australia; the Gladstone Institute of Cardiovascular Disease (J.L.T.), University of California, San Francisco; and the Department of Medicine (P.H.R.B.), University of Western Australia and the Western Australian Institute for Medical Research, Perth, Australia.

Correspondence to Prof Philip J. Barter, Cardiovascular Investigation Unit, Level 6, Royal Adelaide Hospital, North Terrace, Adelaide, SA 5000, Australia. E-mail philip.barter{at}adelaide.edu.au

	Abstract

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Objective— Apolipoprotein (apo)A-I exists in 3 forms in plasma: as lipid-free apoA-I, as a component of pre–ß-migrating discoidal high density lipoproteins (HDLs), and as a component of -migrating spherical HDLs. This study investigates (1) the in vivo metabolism of apoA-I in each of these forms and (2) the effects of hepatic lipase (HL) on apoA-I metabolism.

Methods and Results— Wild-type and HL transgenic rabbits were studied. When lipid-free ¹²⁵I-apoA-I and ¹²⁵I-apoA-I in pre–ß-migrating discoidal reconstituted HDLs (rHDLs) were injected into wild-type rabbits, the label rapidly appeared in -migrating particles and decayed with the same fractional catabolic rate (FCR) as when they were injected as a component of spherical rHDLs. Spherical rHDLs did not change in size when they were injected into wild-type rabbits but were reduced in size in HL transgenic rabbits. The FCR of apoA-I in HL transgenic rabbits was double that in wild-type rabbits.

Conclusions— In vivo, (1) lipid-free apoA-I rapidly incorporates into preexisting -migrating particles, (2) pre–ß-migrating discoidal HDLs are rapidly converted into -migrating HDLs, (3) the FCR of apoA-I is independent of the form in which it is introduced into plasma, and (4) HL reduces the size of -migrating HDLs and increases the rate of catabolism of apoA-I.

Key Words: apolipoprotein A-I • hepatic lipase • high density lipoproteins • metabolism • rabbits

	Introduction

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The discovery that HDLs protect against the development of atherosclerosis^1,2 has stimulated a major interest in the factors that regulate these lipoproteins. This interest has applied particularly to the regulation of apoA-I, the main protein constituent of HDL.

Most of the HDLs in normal plasma are spherical particles that exhibit -migration when subjected to agarose gel electrophoresis. They consist of a surface monolayer of apolipoproteins, phospholipids, and unesterified cholesterol (UC) surrounding a core of cholesteryl esters (CEs) and triglycerides. However, there is the potential for apoA-I also to circulate in a lipid-free (or lipid-poor) form and as a component of pre–ß-migrating discoidal complexes containing apoA-I, phospholipid, and UC.³

In vitro studies have shown that apoA-I cycles between the lipid-free and lipid-associated forms in processes mediated by lecithin:cholesterol acyltransferase (LCAT), CE transfer protein (CETP), phospholipid transfer protein, and hepatic lipase (HL).⁴ It has also been shown in vitro that pre–ß-migrating discoidal HDLs are converted into -migrating spherical particles by the action of LCAT.^5,6 In vitro studies have also shown that -migrating HDLs are increased in size by LCAT and that they decrease in size when they interact with CETP, HL, and triglyceride-rich lipoproteins.⁴ The reduction in size is accompanied by a dissociation of lipid-free apoA-I from the HDL.^7–9

Despite the abundant evidence that HDL remodeling occurs in vitro, its contribution to HDL metabolism in vivo remains unclear. Indeed, the fact that apoA-I exists predominantly in -migrating spherical HDLs in the plasma of most species, including humans, raises questions about the relevance of these in vitro findings to HDL metabolism in vivo.

The present study investigates how the different forms of apoA-I are related in vivo and whether the processes that operate in vitro also have an impact on the metabolism of HDL in vivo. Three specific questions have been addressed in the present study: (1) Does lipid-free apoA-I incorporate into preexisting -migrating HDL in vivo, and is the process sufficiently rapid to explain why lipid-free apoA-I is normally not detectable in plasma? (2) Are pre–ß-migrating discoidal HDLs converted to -migrating particles in vivo, and is the process sufficiently rapid to account for a virtual absence of such particles in normal plasma? (3) Is there evidence in vivo that -migrating spherical HDLs can increase and decrease in size, as has been demonstrated in vitro?

	Methods

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Rabbits
The present study was carried out in mature male wild-type New Zealand White (NZW) rabbits and human HL transgenic rabbits. The rabbits were fed a normal rabbit chow diet. Two sets of experiments were performed. The first was conducted at the Hanson Institute in Adelaide, Australia, and involved only wild-type NZW rabbits that were bred in Adelaide. The second set was conducted at the Gladstone Institute of Cardiovascular Disease in San Francisco, Calif, and involved wild-type and HL transgenic NZW rabbits of the Charles River strain. The transgenic rabbits were provided by Prof John Taylor of the Gladstone Institute. The generation of the HL transgenic rabbits and their lipoprotein characteristics have been described in detail.^10–12 They contain a liver-specific sequence from the human apoE gene, the hepatic control region of the apoE/C-I locus, and a full-length human HL cDNA. Rabbits containing the HL transgene were identified by Southern blot analysis. Activity of HL in the transgenic rabbits was measured in postheparin plasma by using a triolein-based substrate.¹³ Among the colony of 13 HL transgenic rabbits, the HL activity of the HL transgenic rabbits was 5.2±1.7 (mean±SE) µmol fatty acid (FA) released · mL^-1 · h^-1. Experiments were performed on 2 transgenic animals expressing high HL activity (21.6 and 7.8 µmol FA released · mL^-1 · h^-1) and 2 transgenic animals expressing moderate HL activity of (3.2 and 2.8 µmol FA released · mL^-1 · h^-1). Activity of HL in the wild-type rabbits was 0.6±0.3 µmol FA released · mL^-1 · h^-1. These values should be compared with activity of HL in normal human subjects of 6.6±2.7 µmol FA released · mL^-1 · h^-1.¹⁴

Purification and Radiolabeling of ApoA-I
HDLs were isolated from rabbit plasma by sequential ultracentrifugation in the following range: 1.07<density<1.21 g/mL. ApoA-I was obtained by delipidation of ultracentrifugally isolated HDL.¹⁵ The resulting apo-HDL was loaded onto a Q Sepharose Fast Flow column (Pharmacia Biotech AB) to isolate apoA-I.^16,17 The purity of apoA-I was confirmed by SDS-PAGE (Phast System, Pharmacia Biotech AB). The purified apoA-I was iodinated with Na¹²⁵I (NEN Life Sciences) and IODO-BEAD iodination reagent (Pierce), according to the manufacturers’ instructions. Free iodine was separated from ¹²⁵I-apoA-I on a PD-10 column (Pharmacia Biotech AB). ¹²⁵I-apoA-I was dialyzed against 5x1 L of 0.01 mol/L Tris-buffered saline (TBS, pH 7.4) containing 0.15 mol/L NaCl, 0.005% (wt/vol) EDTA Na₂, and 0.006% (wt/vol) NaN₃ before use. The specific activity of labeled apoA-I ranged from 60 to 120 cpm/ng protein.

Preparation of Discoidal and Spherical ApoA-I rHDLs
Discoidal reconstituted HDLs (rHDLs) containing ¹²⁵I-apoA-I, 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC, Sigma Chemical Co), and UC (Sigma) were prepared by the cholate dialysis method¹⁸ and dialyzed against 3x1 L of TBS before use. Spherical rHDLs were prepared by incubating the discoidal rHDLs with LDLs and LCAT at 37°C for 24 hours.¹⁹ The spherical rHDLs were isolated by ultracentrifugation in the range of 1.07<density<1.21 g/mL²⁰ and dialyzed against 3x1 L of TBS before use.

In Vitro Studies
Five micrograms of ¹²⁵I-apoA-I, as lipid-free protein or as a component of discoidal or spherical rHDLs, was added to 2 mL of rabbit plasma and incubated at 37°C for 0, 5, 10, 30, 90, 180, and 360 minutes. The size and electrophoretic mobility of the labeled apoA-I were determined by 3% to 40% nondenaturing polyacrylamide gradient gel electrophoresis and agarose gel electrophoresis, respectively.

In Vivo Studies
The experimental protocols were reviewed and approved by animal ethics committees at the Institute of Medical and Veterinary Science, Adelaide, Australia, and the University of California, San Francisco. Five hundred micrograms (20 µCi) of ¹²⁵I-apoA-I, either in the lipid-free form or as a component of discoidal or spherical rHDLs, was injected into the left marginal ear vein of the rabbit. Blood samples were drawn from the right marginal ear vein at 2, 10, 30, and 60 minutes and at 2, 4, 6, 24, 50, 76, and 100 hours. The samples were stored at 4°C in tubes containing 0.1 mg/mL EDTA Na₂ and 2 mmol/L DTNB. Plasma samples collected during the first 6 hours were subjected to nondenaturing polyacrylamide gradient gel electrophoresis and agarose gel electrophoresis. All of the samples were used for kinetic analysis.

Kinetic Analyses
The fractional catabolic rate (FCR) of ¹²⁵I-apoA-I when injected in the various forms was determined by fitting a 2-compartment model to the tracer data.²¹ The model assumes that labeled HDL was injected into a central compartment and subsequently exchanged with an extravascular compartment. Irreversible loss of HDL was assumed to have occurred from the central compartment.

Other Analysis
Nondenaturing polyacrylamide gradient gel electrophoresis and agarose gel electrophoresis were carried out as described.²² The electrophoretic migration of ¹²⁵I-apoA-I was determined by exposing the gels to a PhosphorImager plate that was read by a Molecular Dynamics PhosphorImager (Molecular Dynamics). Chemical assays were carried out on a Cobas Fara centrifugal analyzer (Roche Diagnostics). Enzymatic kits were used to measure the total cholesterol, UC, and phospholipid concentrations (Boehringer-Mannheim, GmbH). CE concentrations were calculated as the difference between the total and UC concentrations. The concentration of apoA-I was measured by an immunoturbidometric method using sheep anti-rabbit apoA-I.⁷

Statistical Analysis
Student t test and 1-way ANOVA were performed with JMP software (SAS Institute).

	Results

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Characterization of Lipid-Free ¹²⁵I-ApoA-I, Discoidal rHDLs, and Spherical rHDLs
Discoidal and spherical rHDLs containing PLPC, UC, and ¹²⁵I-apoA-I were prepared. The molar ratios of PLPC/CE/UC/apoA-I in the discoidal and spherical rHDLs were 56:0:6:1 and 41:10:2:1, respectively. The respective diameters of the discoidal and spherical rHDLs were 9.3 and 8.8 nm. The electrophoretic mobility of the discoidal rHDL was -0.49 µm s^-1/Vcm^-1 compared with -0.72 µm · s^-1/V · cm^-1 for the spherical rHDL. When compared with discoidal and spherical rHDLs containing unlabeled apoA-I, discoidal and spherical rHDLs containing radiolabeled apoA-I with ¹²⁵I had no effect on either electrophoretic mobility or particle size (not shown).

Metabolism of ApoA-I in Plasma of Wild-Type Rabbits
Lipid-Free ApoA-I
In vitro
When lipid-free ¹²⁵I-apoA-I was added to rabbit plasma, its size and electrophoretic mobility changed rapidly. Within 30 minutes of incubation, all of the pre–ß-migrating apoA-I was in particles with mobility (please see online Figure IA, available at http://www.ahajournals.org). This change in electrophoretic mobility was accompanied by a rapid appearance of ¹²⁵I-apoA-I in particles the size of HDLs (please see online Figure IB).

In vivo
When lipid-free ¹²⁵I-apoA-I was injected into wild-type rabbits, the changes in electrophoretic mobility (please see online Figure IC) and size (please see online Figure ID) of apoA-I were complete in 2 minutes (versus 30 minutes in vitro). All of the ¹²⁵I was incorporated into large -migrating particles.

ApoA-I in Discoidal rHDLs
In vitro
When ¹²⁵I-apoA-I was added to rabbit plasma as a component of discoidal rHDL, its electrophoretic mobility changed from pre-ß to within 10 minutes (please see online Figure IIA, available at http://www.ahajournals.org). This is consistent with a rapid conversion of the discoidal particles into spheres. There were also changes in the size distribution of the ¹²⁵I-apoA-I–containing particles. At the earliest time points, ¹²⁵I-apoA-I appeared in 2 populations of particles: 7.8- and 8.8-nm-diameter particles (please see online Figure IIB). Lipid-free apoA-I was also observed at 0 and 5 minutes (please see online Figure IIB). By 1 hour, the smaller particles had disappeared, and all of the ¹²⁵I-apoA-I had been incorporated into larger particles.

In vivo
When ¹²⁵I-apoA-I was injected into wild-type rabbits as a component of discoidal rHDL, the changes in size and electrophoretic mobility were similar to those observed in vitro. The electrophoretic mobility of the ¹²⁵I-apoA-I changed from pre-ß to at 2 minutes after injection (please see online Figure IIC). This is consistent with a rapid conversion of the pre–ß-migrating discoidal particles into -migrating spherical HDLs. The injected ¹²⁵I-apoA-I initially appeared in 2 populations of particles: 7.6- and 8.5-nm-diameter particles (please see online Figure IID). As in the in vitro studies, there was a progressive disappearance of the smaller particles, with all of the ¹²⁵I-apoA-I residing in the larger particles 60 minutes after injection.

ApoA-I in Spherical rHDLs
In vitro
When ¹²⁵I-apoA-I was added to rabbit plasma as a component of -migrating spherical rHDLs, its electrophoretic mobility did not change (please see online Figure IIIA, available at http://www.ahajournals.org). By 6 hours, larger particles (9.8 nm in diameter) were apparent (please see online Figure IIIB).

In vivo
When ¹²⁵I-apoA-I was injected intravenously into wild-type rabbits as a component of spherical rHDLs, its electrophoretic mobility (please see online Figure IIIC) and size distribution (please see online Figure IIID) did not change.

Kinetics of the Disappearance of ¹²⁵I-ApoA-I in Wild-Type Rabbits
Wild-type rabbits were divided into 3 groups (6 animals per group) and injected intravenously with tracer amounts of ¹²⁵I-apoA-I either in the lipid-free form or as a component of discoidal rHDLs or spherical rHDLs. Regardless of the form in which it was injected, the ¹²⁵I-apoA-I displayed virtually identical biphasic decay curves (Figure 1A). The FCRs of the apoA-I introduced in the lipid-free form or as a component of discoidal or spherical rHDLs were, respectively, 0.78±0.10, 0.89±0.14, and 0.74±0.11 pools per day (mean±SD). The differences between these values were not statistically significant.

View larger version (21K): [in this window] [in a new window]   Figure 1. In vivo metabolism of 125I-apoA-I in wild-type and HL transgenic rabbits. A, Studies were conducted with wild-type rabbits at the Hanson Institute in Adelaide, Australia. Animals were divided into 3 groups (6 animals per group) and injected intravenously with tracer amounts of 125I-apoA-I either in the lipid-free form (n=6) or as a component of discoidal rHDLs (n=6) or spherical rHDLs (n=6). The decay of label is presented as a percentage of the injected dose. The FCRs of the apoA-I introduced in the lipid-free form or as a component of discoidal or spherical rHDLs (calculated as described in Methods) were, respectively, 0.78±0.10, 0.89±0.14, and 0.74±0.11 pools per day (mean±SD). B, Studies were conducted in wild-type and HL transgenic rabbits at the Gladstone Institute in San Fran-cisco, Calif. Tracer amounts of 125I-apoA-I as a component of spherical rHDL were injected into wild-type rabbits (n=6) and HL transgenic rabbits (n=4). The FCRs of the injected apoA-I in the wild-type and HL transgenic rabbits were, respectively, 0.42±0.09 and 0.98±0.03 (mean±SEM) pools per day (P<0.05).

Metabolism of ApoA-I in Plasma of HL Transgenic Rabbits
The studies of wild-type rabbits described above were conducted at the Hanson Institute in Adelaide, Australia, whereas the studies of HL transgenic rabbits were conducted at the Gladstone Institute in San Francisco, Calif. Although NZW rabbits were used at both sites, it is known that there is a degree of heterogeneity within the strain. Thus, rather than comparing the HL transgenic animals from the Gladstone Institute with wild-type animals from Australia, the studies at the Gladstone Institute included wild-type NZW rabbits (Charles River strain) with the same genetic background as the transgenic rabbits.

Compared with wild-type rabbits, the HL transgenic rabbits had lower levels of plasma total cholesterol, HDL cholesterol, and apoA-I (Table). When ¹²⁵I-apoA-I was injected into the wild-type and transgenic rabbits as a component of spherical rHDLs, its electrophoretic mobility did not change (Figure 2A and 2C). In the wild-type rabbits, the size of the ¹²⁵I-apoA-I–containing particles did not change (Figure 2B). In the case of the transgenic rabbits, the ¹²⁵I-apoA-I–containing particles progressively decreased in size from 8.8 to 7.4 nm (Figure 2D).

View this table: [in this window] [in a new window]   Table 1. Lipid Profile of Wild-Type and Hepatic Lipase Transgenic Rabbits*

View larger version (97K): [in this window] [in a new window]   Figure 2. Changes in the electrophoretic mobility (A and C) and size distribution (B and D) of spherical rHDLs in wild-type rabbits and in HL transgenic rabbits. 125I-ApoA-I as a component of spherical rHDL was injected into wild-type (A and B) and HL transgenic(C and D) rabbits. These studies were conducted at the Gladstone Institute and used NZW rabbits of the Charles River strain. Serial blood samples were taken at the indicated intervals. Analyses of the samples were identical to those described in Figure 1. The samples from individual studies are representative of 6 such studies conducted in wild-type rabbits and 4 such studies conducted in HL transgenic rabbits.

Kinetics of the Disappearance of ¹²⁵I-ApoA-I When Injected Into HL Transgenic Rabbits
Tracer amounts of ¹²⁵I-apoA-I as a component of spherical rHDLs were injected into 6 of the wild-type and 4 of the HL transgenic rabbits from the Gladstone Institute (Figure 1B). The decay of ¹²⁵I-apoA-I after injection of labeled spherical rHDLs into the transgenic rabbits was substantially faster than the decay in the nontransgenic rabbits (Figure 1B). The corresponding FCRs of ¹²⁵I-apoA-I in spherical rHDLs in the transgenic and nontransgenic rabbits were 0.98±0.03 and 0.42±0.09 (mean±SD) pools per day, respectively (P<0.05). Note that the FCR of apoA-I in the wild-type rabbits from the Gladstone Institute was substantially slower than the FCR of apoA-I in the equivalent wild-type animals studied in Australia, emphasizing the importance of comparing animals from the same genetic background.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study describes the in vitro and in vivo metabolism of 3 forms of apoA-I in plasma: lipid-free apoA-I, pre–ß-migrating discoidal HDLs, and -migrating spherical HDLs. The in vivo experiments were carried out in wild-type and HL transgenic rabbits. In wild-type rabbits, there was (1) a rapid appearance of lipid-free apoA-I in preexisting -migrating HDL and (2) a rapid appearance of the apoA-I injected as a component of pre–ß-migrating discoidal HDLs in first smaller and then larger -migrating particles. These results are consistent with the fact that most of the apoA-I in rabbit plasma circulates in -migrating particles, with very little existing in either the lipid-free or pre–ß-migrating forms.

By using apoA-I in 3 different forms, the present study has given a unique insight into the metabolism of HDL. Previous investigations of the in vivo metabolism of apoA-I in humans and other species have used 2 main techniques. Most commonly, preparations of HDL have been isolated from plasma, labeled exogenously with ¹²⁵I-apoA-I, and then reinjected intravenously into the study subject.^23,24 More recently, experiments have been conducted with the use of an endogenous labeling technique, whereby the kinetics of the incorporation of amino acids (labeled with stable isotopes) into HDL apolipoproteins are analyzed.^25,26 Although the kinetic parameters generated by the 2 approaches are similar,²⁷ neither of these techniques can answer the questions being asked in the present study. The exogenous labeling of native HDLs is appropriate for studying -migrating HDLs. However, this approach cannot be used to study pre–ß-migrating HDLs, which are not normally present in significant quantities in plasma. The endogenous labeling technique, on the other hand, cannot be used to investigate the remodeling of HDL subpopulations because of the rapidity of these processes relative to the overall rate of HDL catabolism.

The in vivo relationship between the different forms of apoA-I can be answered only by tracing the metabolism of apoA-I when introduced directly into the plasma in each of its forms. To this end, we assembled rHDLs of defined shape, size, and electrophoretic mobility^9,19,20,28 containing isotopically labeled apoA-I in each of its forms. The IODO-BEAD iodination technique used in the present study exposes the apoA-I to relatively low levels of radiation and thus minimizes damage to the protein.²⁹ However, the possibility that the process alters apoA-I in some way cannot be excluded, and the results must be interpreted with this reservation. Another reservation in studies of this type relates to possible modification of the injected HDLs by prior ultracentrifugation. However, the fact that the FCR was the same when apoA-I was injected as a component of spherical rHDLs (that were subject to ultracentrifugation) and when it was lipid free or was a component of discoidal rHDLs (that had not been ultracentrifuged) argues strongly against a major effect of the prior centrifugation.

Rabbits were used to study the in vivo metabolism of each form of apoA-I. This species is well suited for studies of this type because their plasma lipoprotein profiles resemble those of humans. Rabbits also have high CETP levels, a protein known to play a major role in HDL metabolism in humans but which is deficient in the plasma of many other species.³⁰ The fact that rabbits are deficient in activity of HL,³¹ another protein that has an impact on human HDL metabolism, has been turned into an advantage by using transgenic rabbits expressing human HL.

The studies conducted in the wild-type rabbits have provided the first demonstration in vivo that lipid-free apoA-I is incorporated rapidly and quantitatively into -migrating HDLs. It was also apparent that when it is injected as a component of pre–ß-migrating discoidal rHDLs, the labeled apoA-I appeared sequentially in small and then in larger -migrating particles. Although this is consistent with a conversion of the disks into first small and then larger spheres, the possibility that apoA-I may have exchanged between discoidal and spherical particles cannot be excluded. The incorporation of lipid-free apoA-I and discoidal apoA-I into -migrating particles was so rapid in vivo that the fate of apoA-I injected into the wild-type rabbits was independent of the form in which it was delivered. Within minutes of injection, each form of the apoA-I was found exclusively in -migrating HDL particles. The rapidity of these processes also provides an explanation for the virtual absence of the pre–ß-migrating apoA-I in normal plasma.

The observation of an increase in size of -migrating HDLs in vivo has been reported previously.²³ The fact that the size of injected HDL particles may also be decreased in vivo has not previously been reported. The absence of such a decrease in HDL size in vivo in wild-type rabbits is predictable from their low activity of HL, inasmuch as it has been shown in vitro that a decrease in HDL particle size depends on the joint activities of CETP and HL.^8,32 In this process, CETP transfers CEs from HDL to the triglyceride-rich lipoproteins in exchange for a transfer of triglycerides into the HDLs to generate triglyceride-enriched HDLs. When HL subsequently hydrolyzes the HDL triglyceride, the core lipid content and size of the HDL decreases in a process that is accompanied by dissociation of apoA-I from the particle.^8,33 Thus, given the relative deficiency of HL in wild-type rabbits, it was quite predictable that HDL particle size would not be reduced in vivo in these animals. It was, perhaps, also predictable that HDL particle size would be reduced in the transgenic rabbits in which HL was expressed.

Kinetic analysis showed that the FCR of ¹²⁵I-apoA-I in HDLs was enhanced in the HL transgenic rabbits, although the FCR did not correlate with the level of activity of HL in the individual transgenic rabbits. HL activities of 21.6, 7.8, 3.2, and 2.8 µmol FA released · mL^-1 · h^-1 in the 4 transgenic rabbits were associated with FCRs of 1.05, 0.91, 0.95, and 1.01 pools per day, respectively. This suggests that once the HL activity is above a certain threshold level, it may no longer be rate limiting.

The explanation for the increased FCR in the HL transgenic rabbits is not known, although it has been reported that HL participates in the direct uptake of HDL CEs by the liver.³⁴ Whether this explains the increased FCR of apoA-I is not known. The increased FCR may also relate to the ability of HL to promote the dissociation of apoA-I from the particle.³³ Any dissociated lipid-poor apoA-I would not remain in this form for long, as evidenced by the rapid disappearance of pre–ß-migrating apoA-I after addition of lipid-free apolipoprotein to rabbit plasma in vitro or after its injection into intact rabbits. Lipid-poor apoA-I has several potential fates. It may be relipidated by ATP-binding cassette transporter A1,³⁵ it may be incorporated into preexisting HDL, or it may be filtered by the glomerulus and be lost in the urine.³⁶ Given the likelihood that urinary loss will be a function of the amount filtered, any increase in the dissociation of lipid-poor apoA-I from HDL would be predicted to increase the urinary excretion and thus contribute to an increased rate of catabolism of apoA-I in the HL transgenic rabbits.

In conclusion, these results show that lipid-free apoA-I and the apoA-I in discoidal HDLs rapidly appear in -migrating HDLs in vitro and in vivo. The results also show for the first time that HL has the capacity to decrease the size of -migrating HDL and also to enhance their rate of clearance in vivo. The pathophysiological implications of these findings remain to be determined.

	Acknowledgments

 
This work was supported by a Pfizer Cardiovascular Lipid Research Grant. P.K. was supported by a fellowship from the Helpman Bequest. K.-A.R. is a Principal Research Fellow of the Heart Foundation of Australia. P.H.R.B. is a Career Development Fellow of the Heart Foundation of Australia and is supported in part by National Institutes of Health grant RR-12609.

Received August 14, 2002; accepted September 4, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Gordon D, Probstfield J, Garrison R, Neaton J, Castelli WP, Knoke J, Jacobs D, Bangdiwala S, Tyroler HA. High-density lipoprotein cholesterol and cardiovascular disease: four prospective American studies. Circulation. 1989; 79: 8–15.[Abstract/Free Full Text]
2. Miller NE. Associations of high-density lipoprotein subclasses and apolipoproteins with ischemic heart disease and coronary atherosclerosis. Am Heart J. 1987; 113: 589–597.[CrossRef][Medline] [Order article via Infotrieve]
3. Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein. Biochemistry. 1988; 27: 25–29.[CrossRef][Medline] [Order article via Infotrieve]
4. Rye KA, Clay MA, Barter PJ. Remodelling of high density lipoproteins by plasma factors. Atherosclerosis. 1999; 145: 227–238.[CrossRef][Medline] [Order article via Infotrieve]
5. Nichols AV, Blanche PJ, Gong EL, Shore VG, Forte TM. Molecular pathways in the transformation of model discoidal lipoprotein complexes induced by lecithin: cholesterol acyltransferase. Biochim Biophys Acta. 1985; 834: 285–300.[Medline] [Order article via Infotrieve]
6. Liang HQ, Rye KA, Barter PJ. Remodelling of reconstituted high density lipoproteins by lecithin: cholesterol acyltransferase. J Lipid Res. 1996; 37: 1962–1970.[Abstract]
7. Clay MA, Rye KA, Barter PJ. Evidence in vitro that hepatic lipase reduces the concentration of apolipoprotein A-I in rabbit high-density lipoproteins. Biochim Biophys Acta. 1990; 1044: 50–56.[Medline] [Order article via Infotrieve]
8. Clay MA, Newnham HH, Forte TM, Barter PJ. Cholesteryl ester transfer protein and hepatic lipase activity promote shedding of apo A-I from HDL and subsequent formation of discoidal HDL. Biochim Biophys Acta. 1992; 1124: 52–58.[Medline] [Order article via Infotrieve]
9. Hime NJ, Barter PJ, Rye KA. The influence of apolipoproteins on the hepatic lipase-mediated hydrolysis of high density lipoprotein phospholipid and triacylglycerol. J Biol Chem. 1998; 273: 27191–27198.[Abstract/Free Full Text]
10. Barbagallo C, Fan J, Blanche P, Rizzo M, Taylor J, Krauss R. Overexpression of human hepatic lipase and apoE in transgenic rabbits attenuates response to dietary cholesterol and alters lipoprotein subclass distributions. Arterioscler Thromb Vasc Biol. 1999; 19: 625–632.[Abstract/Free Full Text]
11. Fan J, Wang J, Bensadoun A, Lauer SJ, Dang Q, Mahley RW, Taylor JM. Overexpression of hepatic lipase in transgenic rabbits leads to a marked reduction of plasma high density lipoproteins and intermediate density lipoproteins. Proc Natl Acad Sci U S A. 1994; 91: 8724–8728.[Abstract/Free Full Text]
12. Fan J, McCormick SP, Krauss RM, Taylor S, Quan R, Taylor JM, Young SG. Overexpression of human apolipoprotein B-100 in transgenic rabbits results in increased levels of LDL and decreased levels of HDL. Arterioscler Thromb Vasc Biol. 1995; 15: 1889–1899.[Abstract/Free Full Text]
13. Cheng CF, Bensadoun A, Bersot T, Hsu JS, Melford KH. Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase: reactivity with monoclonal antibodies to hepatic triglyceride lipase. J Biol Chem. 1985; 260: 10720–10727.[Abstract/Free Full Text]
14. Connelly PW, Maguire GF, Lee M, Little JA. Plasma lipoproteins in familial hepatic lipase deficiency. Arteriosclerosis. 1990; 10: 40–48.[Abstract/Free Full Text]
15. Osborne J. Delipidation of plasma lipoproteins. Methods Enzymol. 1986; 128: 213–222.[Medline] [Order article via Infotrieve]
16. Rye K-A. Interaction of apolipoprotein A-II with recombinant HDL containing egg phosphatidylcholine, unesterified cholesterol and apolipoprotein A-I. Biochim Biophys Acta. 1990; 1042: 227–236.[Medline] [Order article via Infotrieve]
17. Weisweiler P. Isolation and quantitation of apolipoproteins A-I and A-II from human high-density lipoproteins by fast-protein liquid chromatography. Clin Chim Acta. 1987; 169: 249–254.[CrossRef][Medline] [Order article via Infotrieve]
18. Matz C, Jonas A. Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions. J Biol Chem. 1982; 257: 4535–4540.[Abstract/Free Full Text]
19. Rye KA, Barter PJ. The influence of apolipoproteins on the structure and function of spheroidal, reconstituted high density lipoproteins. J Biol Chem. 1994; 269: 10298–10303.[Abstract/Free Full Text]
20. Rye KA, Hime NJ, Barter PJ. The influence of cholesteryl ester transfer protein on the composition, size, and structure of spherical, reconstituted high density lipoproteins. J Biol Chem. 1995; 270: 189–196.[Abstract/Free Full Text]
21. Lewis GF, Lamarche B, Uffelman KD, Heatherington C, Honing MA, Szeto LW, Barrett PHR. Clearance of post-prandial and lipolytically modified human HDL in rabbits and rats. J Lipid Res. 1997; 38: 1771–1781.[Abstract]
22. Rye KA. Interaction of the high density lipoprotein conversion factor with recombinant discoidal complexes of egg phosphatidylcholine, free cholesterol, and apolipoprotein A-I. J Lipid Res. 1989; 30: 335–346.[Abstract]
23. Colvin P, Moriguchi E, Barrett P, Parks J, Rudel L. Small HDL particles containing two apoA-I molecules are precursors in vivo to medium and large HDL particles containing three and four apoA-I molecules in nonhuman primates. J Lipid Res. 1999; 40: 1782–1792.[Abstract/Free Full Text]
24. Fidge N, Nestel P, Ishikawa T, Reardon M, Billington T. Turnover of apoproteins A-I and A-II of high density lipoprotein and the relationship to other lipoproteins in normal and hyperlipidemic individuals. Metabolism. 1980; 29: 643–653.[CrossRef][Medline] [Order article via Infotrieve]
25. Tilly-Kiesi M, Lichtenstein AH, Joven J, Vilella E, Cheung MC, Carrasco WV, Ordovas JM, Dolnikowski G, Schaefer EJ. Impact of gender on the metabolism of apolipoprotein A-I in HDL subclasses LpAI and LpAI: AII in older subjects. Arterioscler Thromb Vasc Biol. 1997; 17: 3513–3518.[Abstract/Free Full Text]
26. Velez-Carrasco W, Lichtenstein AH, Li Z, Dolnikowski GG, Lamon-Fava S, Welty FK, Schaefer EJ. Apolipoprotein A-I and A-II kinetic parameters as assessed by endogenous labeling with [(2)H(3)]leucine in middle-aged and elderly men and women. Arterioscler Thromb Vasc Biol. 2000; 20: 801–806.[Abstract/Free Full Text]
27. Ikewaki K, Rader DJ, Schaefer JR, Fairwell T, Zech LA, Brewer HB Jr. Evaluation of apoA-I kinetics in humans using simultaneous endogenous stable isotope and exogenous radiotracer methods. J Lipid Res. 1993; 34: 2 207–2215.[Abstract]
28. Rye KA, Hime NJ, Barter PJ. Evidence that cholesteryl ester transfer protein-mediated reductions in reconstituted high density lipoprotein size involve particle fusion. J Biol Chem. 1997; 272: 3953–3960.[Abstract/Free Full Text]
29. Markwell MAK. A new solid-state reagent to iodinate proteins: conditions for the efficient labeling of antiserum. Anal Biochem. 1982; 125: 427–432.[CrossRef][Medline] [Order article via Infotrieve]
30. Ha YC, Barter PJ. Differences in plasma cholesteryl ester transfer activity in sixteen vertebrate species. Comp Biochem Physiol B Biochem Mol Biol. 1982; 71: 265–269.[CrossRef][Medline] [Order article via Infotrieve]
31. Clay MA, Hopkins GJ, Ehnholm CP, Barter PJ. The rabbit as an animal model of hepatic lipase deficiency. Biochim Biophys Acta. 1989; 1002: 173–181.[Medline] [Order article via Infotrieve]
32. Newnham HH, Barter PJ. Synergistic effects of lipid transfers and hepatic lipase in the formation of very small high-density lipoproteins during incubation of human plasma. Biochim Biophys Acta. 1990; 1044: 57–64.[Medline] [Order article via Infotrieve]
33. Clay MA, Newnham HH, Barter PJ. Hepatic lipase promotes a loss of apolipoprotein A-I from triglyceride-enriched human high density lipoproteins during incubation in vitro. Arterioscler Thromb. 1991; 11: 415–422.[Abstract/Free Full Text]
34. Lambert G, Chase MB, Dugi K, Bensadoun A, Brewer HB Jr, Santamarina-Fojo S. Hepatic lipase promotes the selective uptake of high density lipoprotein-cholesteryl esters via the scavenger receptor B1. J Lipid Res. 1999; 40: 1294–1303.[Abstract/Free Full Text]
35. Oram JF, Vaughan AM. ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Curr Opin Lipidol. 2000; 11: 253–260.[CrossRef][Medline] [Order article via Infotrieve]
36. Horowitz B, Goldberg I, Merab J, Vanni T, Ramakrishnan R, Ginsberg H. Increased plasma and renal clearance of an exchangeable pool of apolipoprotein A-I in subjects with low levels of high density lipoprotein cholesterol. J Clin Invest. 1993; 91: 1743–1752.[Medline] [Order article via Infotrieve]

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