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
Objective— Apolipoprotein (apo)A-I exists in 3 forms in plasma: as lipid-free apoA-I, as a component of pre–β-migrating discoidal high density lipoproteins (HDLs), and as a component of α-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–β-migrating discoidal reconstituted HDLs (rHDLs) were injected into wild-type rabbits, the label rapidly appeared in α-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 α-migrating particles, (2) pre–β-migrating discoidal HDLs are rapidly converted into α-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 α-migrating HDLs and increases the rate of catabolism of apoA-I.
The discovery that HDLs protect against the development of atherosclerosis1,2⇓ 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 α-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–β-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–β-migrating discoidal HDLs are converted into α-migrating spherical particles by the action of LCAT.5,6⇓ In vitro studies have also shown that α-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 α-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 α-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–β-migrating discoidal HDLs converted to α-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 α-migrating spherical HDLs can increase and decrease in size, as has been demonstrated in vitro?
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 5×1 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 3×1 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 3×1 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.
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.
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
Student t test and 1-way ANOVA were performed with JMP software (SAS Institute).
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
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–β-migrating apoA-I was in particles with α 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).
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 α-migrating particles.
ApoA-I in Discoidal rHDLs
When 125I-apoA-I was added to rabbit plasma as a component of discoidal rHDL, its electrophoretic mobility changed from pre-β to α 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.
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-β to α at 2 minutes after injection (please see online Figure IIC). This is consistent with a rapid conversion of the pre–β-migrating discoidal particles into α-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
When 125I-apoA-I was added to rabbit plasma as a component of α-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).
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.
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).
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.
The present study describes the in vitro and in vivo metabolism of 3 forms of apoA-I in plasma: lipid-free apoA-I, pre–β-migrating discoidal HDLs, and α-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 α-migrating HDL and (2) a rapid appearance of the apoA-I injected as a component of pre–β-migrating discoidal HDLs in first smaller and then larger α-migrating particles. These results are consistent with the fact that most of the apoA-I in rabbit plasma circulates in α-migrating particles, with very little existing in either the lipid-free or pre–β-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 α-migrating HDLs. However, this approach cannot be used to study pre–β-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 mobility9,19,20,28⇓⇓⇓ 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 α-migrating HDLs. It was also apparent that when it is injected as a component of pre–β-migrating discoidal rHDLs, the labeled apoA-I appeared sequentially in small and then in larger α-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 α-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 α-migrating HDL particles. The rapidity of these processes also provides an explanation for the virtual absence of the pre–β-migrating apoA-I in normal plasma.
The observation of an increase in size of α-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–β-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 α-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 α-migrating HDL and also to enhance their rate of clearance in vivo. The pathophysiological implications of these findings remain to be determined.
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; revision accepted September 4, 2002.
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