© 1999 American Heart Association, Inc.
Original Contributions |
Lipid Transfer Inhibitor Protein Defines the Participation of Lipoproteins in Lipid Transfer Reactions
CETP Has No Preference for Cholesteryl Esters in HDL Versus LDL
From the Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH (A.P.S., R.E.M.); and Department of Biochemistry, National Research Center for Preventive Medicine, Moscow, Russia (A.P.S.).
Correspondence to Richard E. Morton, PhD, Dept. of Cell Biology, NC 10, The Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. E-mail mortonr{at}cesmtp.ccf.org
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Abstract |
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Abstract—Cholesteryl ester transfer protein (CETP) catalyzes the net transfer of cholesteryl ester (CE) between lipoproteins in exchange for triglyceride (heteroexchange). It is generally held that CETP primarily associates with HDL and preferentially transfers lipids from this lipoprotein fraction. This is illustrated in normal plasma where HDL is the primary donor of the CE transferred to VLDL by CETP. However, in plasma deficient in lipid transfer inhibitor protein (LTIP) activity, HDL and LDL are equivalent donors of CE to VLDL (Arterioscler Thromb Vasc Biol. 1997;17:1716–1724). Thus, we have hypothesized that the preferential transfer of CE from HDL in normal plasma is a consequence of LTIP activity and not caused by a preferential CETP-HDL interaction. We have tested this hypothesis in lipid mass transfer assays with partially purified CETP and LTIP, and isolated lipoproteins. With a physiological mixture of lipoproteins, the preference ratio (PR, ratio of CE mass transferred from a lipoprotein to VLDL versus its CE content) for HDL and LDL in the presence of CETP alone was
1 (ie, no preference).
Fourfold variations in the LDL/HDL ratio or in the levels of HDL in the
assay did not result in significant preferential transfer from any
lipoprotein. On addition of LTIP, the PR for HDL was increased up to
2-fold and that for LDL decreased in a concentration-dependent manner.
Under all conditions where LDL and HDL levels were varied, LTIP
consistently resulted in a PR >1 for CE transfer from HDL.
Short-term experiments with radiolabeled lipoproteins and either
partially purified or homogenous CETP confirmed these observations and
further demonstrated that CETP has a strong predilection to mediate
homoexchange (bidirectional transfer of the same lipid) rather than
heteroexchange (CE for TG); LTIP had no effect on the selection of CE
or TG by CETP or its mechanism of action. We conclude, in contrast to
current opinion, that CETP has no preference for CE in HDL versus LDL,
suggesting that the previously reported stable binding of CETP to HDL
does not result in selective transfer from this lipoprotein. These data
suggest that LTIP is responsible for the preferential transfer of CE
from HDL that occurs in plasma. CETP and LTIP cooperatively determine
the extent of CETP-mediated remodeling of individual lipoprotein
fractions.
Key Words: cholesteryl ester transfer protein • lipid transfer inhibitor protein • lipoprotein preference • cholesteryl ester • heteroexchange
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Introduction |
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Cholesteryl ester transfer protein (CETP) is a plasma protein that mediates the exchange and net transfer of cholesteryl ester (CE) and triglyceride (TG) molecules between lipoprotein particles during their circulatory lifetime.1 2 The ability of CETP to facilitate the remodeling of lipoprotein composition endows an important role to this protein in the intravascular metabolism of lipoproteins. CETP participates in determining the ratio of HDL2 to HDL3,3 4 5 facilitates the loss of apo A-I from
-migrating HDL resulting in an
increase of preß HDL,6 and enhances reverse
cholesterol transport.2 7 8 CETP also affects
the level of LDL subfractions and their interaction with the LDL
receptor,9 10 11 12 13 and facilitates the conversion of VLDL to
LDL.1 14 Many of these observations have been verified and
extended through studies of humans who are genetically deficient in
CETP15 16 and in transgenic mice bearing the CETP
transgene.17 18 Overall, CETP activity significantly
influences the structure and function of the lipoproteins with which it
interacts. We and others have suggested that the function of CETP, that is, its capacity to affect changes in the composition of individual lipoprotein fractions, is modulated by a second plasma protein, lipid transfer inhibitor protein (LTIP).19 20 21 22 23 24 LTIP is a LDL-associated protein of ~30 kd that preferentially reduces the action of CETP on LDL, probably through its capacity to prevent CETP binding to lipoproteins.23 25 In whole plasma, CETP mediates the net flux of CE from LDL and HDL to VLDL with the return of TG.1 26 In normolipidemic individuals, VLDL TG, not CETP concentration, is rate limiting for these net lipid transfers.27 28 As a result, LDL and HDL compete for a limited supply of TG in VLDL. When whole plasma is supplemented with exogenous LTIP, net transfer of TG to LDL is diminished, but, because TG is rate-limiting, net transfer with HDL increases in a LTIP-dependent manner.23 It is likely that the overall affect of CETP activity on atherogenesis is modulated by LTIP activity because this protein, at least in vitro, can alter the CETP-mediated flux of lipids through individual lipoprotein fractions.
Although LTIP affects the extent to which CETP modifies lipoproteins, it is widely held that CETP itself has a strong preference for interaction with HDL. CETP has been shown to be almost exclusively associated with HDL in plasma by gel filtration and affinity chromatography, and by electrophoretic methods.29 30 31 32 33 In vitro, CETP forms stable complexes with HDL under nonequilibrium conditions, but it stably binds to VLDL or LDL only after the negative charge of these lipoproteins has been enhanced.34 35 36 In simple 2-lipoprotein assay systems, kinetic analyses of CETP activity have consistently suggested that HDL is a preferred substrate compared with the same amount of VLDL and LDL on a protein or cholesterol basis.37 Furthermore, in whole plasma, HDL is the major donor of CE to VLDL compared with LDL when transfers are expressed relative to the CE content of these lipoproteins.38 39
In contrast to these observations, some studies suggest that HDL is not a preferential substrate for CETP. Under equilibrium conditions, steady-state binding studies indicate that VLDL, LDL, and HDL have very similar affinities for CETP, and the number of CETP "binding sites" is nearly the same for these lipoproteins when expressed relative to their phospholipid surface area.25 We have shown that there is no preferential transfer of CE from HDL in plasma from patients undergoing continuous peritoneal dialysis, in contrast to control plasma.39 A possible explanation for these findings is that CETP has no inherent preference for HDL as a substrate, but rather, the preferential transfer of CE from HDL seen in control subjects is due to the action of LTIP, whose activity is greatly diminished in these patients.
Considering the foregoing, we have proposed an alternative hypothesis that CETP itself has no inherent preference for HDL as a substrate. To investigate the substrate specificity of CETP for LDL and HDL, and the effect of LTIP on this specificity, we have performed detailed analyses of lipid fluxes among VLDL, LDL, and HDL in short-term, radiolabeled tracer studies and in long-term studies where CETP affects mass changes in the TG and CE content of lipoproteins. These studies also provide further insight into the transfer process and the selection of CE and TG for transfer. The results suggest that LTIP activity, not CETP lipoprotein specificity, is responsible for the higher rate of lipid transfer from HDL.
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Methods |
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Materials
Glycerol tri[9,10-3H]oleate (26.8 Ci/mmol) was obtained from New England Nuclear, and [1
,2(n)-3H]cholesterol (45.6
to 48.4 Ci/mmol) and cholesteryl [1-14C]oleate
(56 mCi/mmol) were purchased from Amersham Corp. BSA (fraction V),
diethyl p-nitrophenyl phosphate, egg phosphatidylcholine,
butylated hydroxytoluene, dithiothreitol, and all reagents for salt and
buffer solutions were obtained from Sigma Chemical Co.
Cholesterol was purchased from NuChek and solutions were
prepared in chloroform and stored at -20°C. Phenyl Sepharose CL-4B,
heparin-Sepharose and dextran sulfate
(Mr=500 000) were from Pharmacia Biotech, Inc.,
and CM52-cellulose was from Whatman Chemical Separations, Inc.
Isolation of CETP and LTIP
Partially purified CETP was isolated from lipoprotein-deficient
human plasma by hydrophobic and ion-exchange
chromatography as previously described40
and stored in 0.27 mmol/L EDTA, pH 7.4. These preparations
typically contained 4.1 µg CETP protein/mL based on immunoassay of
CETP mass with TP2 monoclonal antibody.30 During the
purification of CETP, lipid transfer activity was routinely assayed by
determining the extent of radiolabel transferred from
[3H]CE-labeled LDL to unlabeled HDL (10 µg
cholesterol each) in the presence of 0.5% BSA in a total
volume of 0.7 mL.41 42 LTIP was partially purified as
previously described.19 23 Based on the specific activity
of these preparations, and the LTIP activity contained in
lipoprotein-deficient plasma,23 1 µg (protein) of
partially purified LTIP was equivalent to the LTIP activity contained
in 1 µL of lipoprotein-deficient plasma. The characteristics of LTIP
and CETP activities in these preparations are not different from those
determined in highly purified preparations.25 33
Additionally, based on the ineffectiveness of 56°C heating, neither
LTIP nor CETP preparations contained phospholipid transfer
protein,23 43 which has been previously shown to stimulate
CETP activity in vitro.44 For selected experiments,
homogeneous CETP was isolated from the partially purified
fraction by chromatofocusing and gel filtration as previously
described.40 LTIP was isolated in greater purify by
fractionation of LDL-associated proteins as described in the text.
Lipoprotein Isolation and Radiolabeling
Fresh human plasma from the Blood Bank of the Cleveland Clinic
Foundation was the source of VLDL, LDL, and HDL. Lipoproteins were
isolated at 4°C by sequential
ultracentrifugation,45 extensively
dialyzed against 0.9% NaCl, 0.01% EDTA, 0.02%
NaN3, pH 8.5, and stored at 4°C. Lipoproteins
were quantitated based on their total cholesterol content.
In some instances, before use in transfer assays, isolated lipoproteins
were radiolabeled by CETP-mediated transfer of radiolabeled lipids from
liposomes. Briefly, phosphatidylcholine –cholesterol
liposomes (4:1 mole ratio) containing tracer quantities of
3H-TG and 14C-CE were
prepared by cholate dialysis.46 Liposomes (
0.4 ng
phospholipid per µg lipoprotein cholesterol) were
incubated with VLDL, LDL or HDL, CETP, and 1% BSA in 50 mmol/L
tris-HCl, 150 mmol/L NaCl, 0.01% EDTA, 0.02%
NaN3, pH 7.4 (tris/NaCl buffer). After 6 hours at
37°C, HDL was isolated by sequential centrifugation
as above; VLDL and LDL were isolated by affinity
chromatography. In the latter case, samples were
applied to a column of heparin-Sepharose equilibrated in 50 mmol/L
NaCl, 0.01% EDTA, pH 7.4, extensively washed with the same buffer, and
the lipoproteins were eluted with 300 mmol/L NaCl, 0.01% EDTA, pH
7.4. Under these labeling conditions, lipoproteins contained 1.2 to
5.2x103 cpm 3H and 2.2 to
10x102 cpm 14C/µg
cholesterol. Approximately 90% of each isotope was
transferred from the liposomes to the lipoprotein under these
conditions. This method permitted radiolabeling of lipoproteins without
measurable changes in lipoprotein composition.
Lipid Transfer Assays
Mass Transfer Assays
The CETP-mediated net transfer of CE among lipoproteins was
measured in assays containing isolated VLDL, LDL, and HDL (isolated
from the same plasma donor pool) at levels indicated in the figure
legends. Lipoproteins were combined with CETP±LTIP as noted in the
legends in the presence of 0.2 mmol/L diethyl
p-nitrophenyl phosphate, 0.4% BSA and tris/NaCl buffer in a
final volume of 3 to 4 mL. Diethyl p-nitrophenyl phosphate
was added to inhibit residual LCAT contained in isolated HDL; this
compound had no effect on LTIP activity (unpublished observation) and
did not alter the selectivity of CETP for TG and
CE.40 After incubation at 37°C for 24 hours,
samples were cooled and fractionated by
ultracentrifugation.45 Dialyzed samples
were assayed for total and free cholesterol, and protein
content. Lipoprotein recovery was assessed by the recovery of protein
in each fraction. Changes in CE content were calculated from the
difference in CE content, after correction for recovery, between
samples incubated at 37°C and 4°C (control). Preference
ratios were determined as the fraction of total CE transferred to
VLDL derived from a given lipoprotein divided by the fraction of
transferable CE (LDL+HDL CE) in that lipoprotein fraction. A preference
ratio of 1 indicates that there is no selective interaction of CETP
with that lipoprotein.
Radiolabeled Lipid Transfer Assays
Doubly labeled lipoproteins were prepared as described above.
VLDL, LDL, and HDL (10, 40, and 16.8 µg cholesterol,
respectively) were incubated in 1% BSA and tris-NaCl buffer (total
volume=0.7 mL) ±CETP and ±LTIP as indicated in the figure legends.
Within a mixture of these 3 lipoproteins, the lipoprotein that was
initially radiolabeled was systematically varied to permit quantitation
of the 6 unidirectional lipid transfer reactions for each lipid. After
incubation for 2 hours at 37°C, the samples were cooled, 0.5 mL
unlabeled plasma was added as a carrier, and lipoprotein fractions were
isolated by sequential
ultracentrifugation.45 The radiolabel
content of each fraction was determined by scintillation counting. The
mass of CE or TG transferred was calculated as previously
described41 from the fraction of radiolabeled lipid
transferred from the labeled donor lipoprotein to an acceptor
lipoprotein and the initial specific activity of the lipid. Blank
transfer in the absence of CETP was subtracted before calculating the
fractional transfer.
Analytical Procedures
Protein was quantitated by the method of Lowry et
al47 as modified by Peterson,48 with BSA as a
standard. Total cholesterol of lipoproteins was assayed by
a colorimetric, enzymatic method using a
Cholesterol 100 reagent kit (Sigma). Free
cholesterol was determined by a kit from Wako
Diagnostics, and CE was calculated from the difference
between total and free cholesterol values times 1.69 to
correct for the fatty acid content of CE. The cholesterol
content of organic solutions of lipids was assayed by the method of Zak
et al.49 TG was measured by the glycerol phosphate
oxidase-Trinder enzymatic method (Sigma). For mole calculations, 653
and 885 were used as average molecular weights of CE and TG,
respectively. Lipid phosphorus was quantitated by the method of
Bartlett,50 and a conversion factor of 25 was used to
determine phospholipid mass. All chemical assays were performed in
triplicate.
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Results |
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Participation of LDL and HDL in CE Mass Transfer
To evaluate the capacity of LDL and HDL to participate in net CE transfer reactions with VLDL, these lipoproteins were isolated, recombined at their plasma equivalent levels (15% VLDL, 60% LDL, 25% HDL cholesterol), and incubated with CETP±LTIP for 24 hours to affect net lipid flux between lipoproteins (Figure 1
). In the absence of LTIP, LDL was the
major donor of CE to VLDL, accounting for more than 70% of the CE
transfer to the TG-rich lipoprotein fraction (Figure 1A).
Addition of LTIP led to a dose-dependent decrease in CE loss from LDL
and an increase in the donation of CE from HDL to VLDL. When these
transfers were compared with the relative amounts of CE in LDL and HDL
(preference ratios, see Methods), LDL and HDL were equivalent donors of
CE to VLDL (preference value of 1) in the absence of LTIP, whereas
the participation of LDL decreased and that of HDL increased as LTIP
concentration was increased (Figure 1B). At the highest LTIP
level studied, HDL contributed twice as much CE to VLDL, as would be
expected based on its content of CE. At the lipoprotein concentrations
used in these assays, which strongly determines LTIP
activity,19 20 51 this level of LTIP is equivalent to that
in plasma.23 A preference value of 2 is similar to that
observed in intact, control plasma.39
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The lack of preferential CE net transfer from HDL seen in Figure 1
was not unique to the ratio of lipoproteins present in
normal plasma. When the LDL to HDL ratio in the assay was varied
>4-fold while holding the sum of these lipoproteins constant, there
was minimal preferential transfer of CE from HDL in the absence of LTIP
(Figure 2A). As noted above, when LTIP is
present, HDL became the preferred donor of CE to VLDL, although
this preference tended to diminish as the assay content of LDL
increased (Figure 2B). Likewise, when the assay content of LDL
and VLDL was constant but the amount of HDL varied 7-fold, there was
little preferential transfer of CE from HDL under any condition (Figure 3A). As before, the addition of LTIP
altered the participation of LDL and HDL in CE net transfer such that
HDL was consistently the preferred donor (Figure 3B). In
these assays (Figure 3), the percentage inhibition of CE
transfer from LDL to VLDL by LTIP was constant at 30.0±2.7%
(mean±SE) even though the assay concentration of HDL was varied
7-fold. This result is consistent with our earlier report that
LTIP selectively interacts with LDL,23 which was held
constant in this experiment.
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The cumulative results from multiple studies with differing amounts of
LDL and HDL in the assay demonstrate that in the absence of LTIP, the
preference ratios for net CE transfer from LDL and HDL to VLDL are near
unity (0.90±0.03 and 1.16±0.05, respectively, mean±SE, n=14). As
shown below, this lack of marked lipoprotein preference was also seen
with pure CETP. We suggest that the small departure of these values
from unity, where no preferential transfer exists, may be due to the
presence of residual LTIP in LDL preparations. We estimate, based on
the LTIP activity recovered in the lipoprotein-free fraction of plasma
(d>1.21 g/mL), which is 75% of that in plasma, that about 25% of
the LTIP activity originally associated with LDL is retained in the
lipoprotein fraction after standard ultracentrifugal isolation.
Role of LDL and HDL in Lipid Exchange Among Lipoproteins
Radiolabeled lipid transfer experiments were performed to verify
the conclusion reached in the mass transfer studies discussed above and
to further assess the CETP-mediated lipid flux in the presence and
absence of LTIP. Short-term, radioisotopic studies also obviate
concerns that arise from long-term incubations where the resultant
modification of lipoprotein composition may cause changes in
lipoprotein ultracentrifugation properties. Three
assays of identical design were performed, differing only in the
lipoprotein fraction prelabeled with 3H-TG and
14C-CE. This permitted the concurrent
determination of bidirectional CE and TG fluxes among all assay
lipoproteins (VLDL, LDL, and HDL). As was observed in mass transfer
studies, the total lipid flux (CE+TG) from LDL and HDL to VLDL was
balanced by an equivalent flux of lipids returning from VLDL (Figure 4). Although LTIP reduced lipid transfer
between lipoproteins, this balance was not affected by LTIP.
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Although total lipid flux was the same between a given lipoprotein pair
(Figure 4), the transfer of CE and TG was not the same,
resulting in a net transfer of CE to VLDL and an efflux of TG from this
lipoprotein (see below). The net balance of lipid transfers in LDL and
HDL (CE loss versus TG gain) was well matched, with the imbalance
accounting for only 2.2% and 4.0% of the total CE+TG flux between
VLDL-LDL and VLDL-HDL, respectively. This small, apparent imbalance may
reflect the fact that the 14C-CE label
(cholesteryl oleate) may not accurately measure total CE flux since
cholesteryl oleate is transferred slightly faster than other
cholesteryl ester species.52 The tight coupling of CE loss
and TG gain in LDL and HDL was not dissociated by LTIP.
As observed in long-term mass transfer studies, LTIP did lead to a
progressive decrease in net radiolabel transfer from LDL (Figure 5A). Net transfer from HDL was not
increased by LTIP as was noted in long-term studies. This is probably
because CETP activity is rate-limiting in these short-term isotope
studies, whereas in the long-term studies, VLDL TG is rate limiting.
Despite this difference, LTIP did lead to a progressive increase in the
preferential transfer of CE from HDL and decreased relative transfer
from LDL (Figure 5B) as seen before. In the absence of LTIP, the
preference ratios for transfers from LDL and HDL were near unity and
similar to the average values noted above from mass transfer studies.
Therefore, the lack of a strong preferential transfer from HDL is
confirmed in these short-term, radiolabeled lipid studies.
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The greater suppression of lipid transfers involving LDL by LTIP, as
observed above in assays containing mixtures of VLDL, LDL, and HDL, is
also measurable with assays containing a single donor and acceptor
lipoprotein,19 and is especially evident with assays where
LDL serves as both donor and acceptor.51 To demonstrate
that this LDL preference not unique to the CM-cellulose fraction of
LTIP, we isolated LTIP from LDL and studied its inhibitor
properties. LDL-derived LTIP had a 4-fold higher specific activity than
the CM-cellulose fraction and contained only 2 protein components: a
protein of 33 kd, consistent with the molecular weight of
LTIP,19 22 and contaminating proteins of <10 kd, which
were absent from the CM-cellulose fraction of LTIP. This preparation
lacked apolipoprotein D, which accounts for >90% of the protein in
the CM-cellulose fraction.23 Thus, these LTIP preparations
do not contain common contaminates. Although only selected comparisons
were possible because of limiting amounts of LDL-derived LTIP, these
assays clearly demonstrated that both LTIP fractions preferentially
suppress lipid transfers involving LDL (Table 1). This was observed when LTIP activity
in lipid transfer assays containing VLDL and LDL was compared with
those containing VLDL and HDL, and when transfer assays containing only
LDL were contrasted with those containing LDL and HDL. Although
LDL-derived LTIP tended to show slightly lower LDL specificity, this
difference was not statistically significant.
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Heteroexchange Versus Homoexchange
Further analysis of radiolabeled-lipid flux rates among
VLDL, LDL, and HDL (Figures 4 and 5) provides additional
insight into the lipid transfer process. The total efflux of lipids
from LDL (CE+TG) to all other lipoproteins was 10.46 nmol, compared
with 11.93 nmol for HDL. It is notable that the lipoprotein surface
areas (expressed as phospholipid content) of LDL and HDL in the assay
were very similar (31.2 and 37.1 µg phospholipid, respectively).
Thus, the rates of CETP-mediated lipid flux from LDL and HDL are
equivalent relative to the phospholipid surface available for
interaction with CETP on each lipoprotein. The importance of the
phospholipid surface for CETP binding has been previously
reported.25 36 This further suggests that LDL and HDL are
equivalent in their capacity to serve as substrates for CETP when
equated based on surface phospholipid. Under similar assay conditions,
homogenous CETP also showed little preference for lipid transfers from
HDL compared with LDL, but marked preference for transfer from HDL
versus LDL was noted with pure CETP when LTIP was present (Table 2). As was observed in mass transfer
assays, doubling the HDL content of these assays changed the absolute
lipid flux values but did not alter the calculated preference values
(data not shown).
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Lipid flux between lipoproteins is composed of exchanges of like lipids
(CE for CE and TG for TG; ie, homoexchange) and of dissimilar lipids
(CE for TG; ie, heteroexchange). Among VLDL, LDL, and HDL, which were
present in physiologically relevant ratios
(Figures 4 and 5), the sum of homoexchange reactions
exceeded heteroexchange. Compared with the rates of CE-TG
heteroexchange (relative rate=1) CE-CE and TG-TG homoexchanges were
6.1- and 1.3-fold faster, respectively. A large portion of the CE-CE
homoexchange occurred between LDL and HDL, where no net lipid transfer
occurs because of the similarity of the TG/CE ratio in the core of
these 2 lipoproteins. However, among those pathways where net transfer
is possible (HDL-VLDL and LDL-VLDL), the homoexchange of CE still was
the predominant reaction, with relative rates of CE-CE, TG-TG, and
CE-TG exchange reactions of 3.6:1.3:1.
For CE transfer from LDL and HDL to VLDL, CE homoexchange exceeded
CE-TG heteroexchange by 3- to 4-fold (Figure 6). It is notable that CE homoexchange
between LDL or HDL and VLDL occurs at a high rate given the paucity of
CE in VLDL compared with TG. That is, even though CE is the predominant
lipid in LDL or HDL, and thus is likely to be transferred at a greater
absolute rate than TG, it is remarkable that CE was the preferred
exchange partner since it is much less abundant in the VLDL core than
TG. As shown in Table 3, when the
composition of the exchange partner is considered, both CE and TG
homoexchange reactions were 7- to 10-fold more probable than
heteroexchange. LTIP did not effect the relative rates of homoexchange
and heteroexchange (Figure 6). Thus, LTIP alters CETP activities
without changing the mechanism of transfer as assessed by the selection
of CE and TG for transfer.
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Discussion |
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In our studies, in contrast to the general tenor of published data, we demonstrate that CETP has little if any functional preference for HDL over LDL as a lipid donor to other lipoproteins. This is illustrated by several lines of evidence. In long-term mass transfer assays we demonstrate that CETP alone has no preference for facilitating net CE transfer from HDL versus LDL to VLDL when these lipoproteins are compared relative to their content of transferable substrate (CE). This observation was unchanged under a variety of assay conditions that mimic plasma HDL/LDL ratios seen in hyperbeta- and hyperalphalipoproteinemia. Likewise, in short-term assays with radiolabeled lipids, the capacity of LDL and HDL to support net CE transfer (heteroexchange) to VLDL was the same. These results indicate that when the experimental endpoint is CE mass transfer to VLDL, LDL and HDL are equivalent contributors when equated on CE content. In a similar manner, in radiolabel transfer experiments, LDL and HDL were equally active donors of neutral lipid (CE+TG) for transfer to other lipoproteins. This similarity in total lipid transfer activity was evident when lipoproteins were equated by their phospholipid surface area. Thus when total, unidirectional lipid flux is the experimental endpoint, which reflects the number of transfer events that occur on the lipoprotein surface, LDL and HDL are equivalent if their concentrations are equated by the number of potential CETP interaction sites (ie, phospholipid, see References 25 and 3625 36 ). Collectively these data indicate that LDL and HDL are comparable substrates for CETP.
A necessary corollary to these findings is that the often-reported stable binding of CETP to HDL does not result in selective transfer from this lipoprotein fraction. It is evident, based on published studies, that the stable binding of CETP to lipoprotein surfaces is not essential for CETP activity. This is clearly evidenced by numerous observations that CETP readily mediates lipid transfer between VLDL and LDL, lipoproteins that do not form isolatable complexes with CETP.34 35 36 However, it is generally accepted that the stable association of CETP with a lipoprotein results in increased transfer from that particle. This is supported by several studies where native lipoprotein composition has been modified in vitro.29 34 35 36 Our data indicate that the capacity of CETP and HDL to form stable complexes does not lead to enhanced transfer. Thus, although Nishida et al34 have shown that HDL has the highest affinity for CETP under nonequilibrium conditions, our experiments, which used CETP to lipoprotein ratios that varied from 0.26- to 1.8-fold of that present physiologically, were unable to detect preferential transfer from this lipoprotein. It is notable that no preference was observed when CETP concentrations were low since this condition should magnify any differences in lipoprotein affinity for CETP. This lack of lipoprotein specificity by CETP is not an in vitro artifact due to lipoprotein isolation since no preference for HDL is seen in whole plasma from patients deficient in LTIP activity.39 We have recently observed that increased negative charge on lipoproteins results in increased stable CETP-lipoprotein binding, but that the effects of these modification on CETP activity are highly dependent on the nature of the derivatization itself (Morton R.E. and Greene D.J., unpublished observations). The role of stable binding in the lipid transfer process, if any, is presently under investigation.
A preference for transfer from HDL could be induced by the addition of LTIP to transfer assays. LTIP mediated a dose-dependent decrease in CE transfer from LDL and enhanced transfer from HDL in long-term mass transfer assays. CETP plus near-physiological levels of LTIP replicated the preferential transfer of CE from HDL typically seen in plasma, whereas CETP alone could not. These findings are consistent with our recent observations in an LTIP activity-deficient patient population,39 where CETP-mediated CE fluxes from LDL and HDL to VLDL were equivalent when the lipoproteins are compared on the basis of their CE content. We suggest that the apparent preferential transfer of HDL lipids by CETP in plasma38 39 53 54 reflects the activity of LTIP, not the lipoprotein selectivity of CETP.
Detailed analysis of the simultaneous, bidirectional flux of radiolabeled CE and TG among VLDL, LDL, and HDL affords further insights into the transfer process between lipoproteins. Under these assay conditions, lipid transfer is a tightly coupled process with gain and loss of lipid from a given lipoprotein being closely matched. While the coupling of these events is supported by some long-term mass studies where CE loss and TG gain are nearly equimolar,1 39 55 56 this is not always observed.38 57 58 59 We suggest that these short-term radiolabeled lipid studies provide an accuracy not afforded by mass transfer assays since the extent of CE mass lost from LDL or HDL in these latter studies is deduced from small decrements in a comparatively large CE pool. Measurements of all the lipid fluxes among VLDL, LDL, and HDL by isotope demonstrate that CETP-mediated lipid transfer is an exchange process with no detectable net, unidirectional flux of lipids. LTIP did not alter this transfer mechanism. Whether this coupling is disrupted by free fatty acids as previously suggested60 remains to be determined.
While it has been inferred from previous studies that the homoexchange of CE and TG proceed at faster rates than heteroexchange, these radioisotope studies provide the first quantitation of these processes. On an absolute basis, the exchange of CE for CE was the most prevalent reaction in a plasma-like mixture of lipoproteins, exceeding TG-TG and CE-TG transfers by almost 5-fold. When the lipid composition of the transfer partner is considered, both CE-CE and TG-TG homoexchange occurred at a disproportionately high frequency compared with heteroexchange. These results indicate that once a lipid is bound to CETP, there is a 7- to 10-fold higher probability that the same lipid class will be used in the return reaction when TG and CE are present at equal concentrations in the "acceptor." The high preference of CETP for facilitating homoexchange reactions even when the participating lipoproteins contain markedly different TG/CE content, suggests that once CETP has adopted a conformation to accommodate a CE or TG molecule, it is easier to exchange that lipid for the same lipid species on transfer at the acceptor surface. Again, it was observed that LTIP did not alter the relative rates of homoexchange and heteroexchange indicating that LTIP does not affect the way CETP interacts with its lipid substrates.
In summary, when present at the same CE concentration, LDL and HDL are nearly equivalent in their capacity to increase VLDL CE mass via CETP. Thus, CETP itself has no apparent specificity for HDL as a substrate. The addition of LTIP causes a preferential net transfer of CE from HDL to VLDL under all conditions tested, and mimics the preferential transfer that occurs in plasma. We suggest, based on published studies,23 25 that LTIP alters the relative participation of lipoproteins in CETP-mediated transfers by selectively interacting with LDL, displacing CETP from its surface, and reducing its capacity to serve as a CETP substrate. LTIP decreases lipid transfer without altering the selection of TG versus CE by CETP (homoexchange versus heteroexchange) and without influencing the balance of TG and CE exchange between lipoproteins. If the properties of LTIP determined in vitro can be directly applied to lipid transfer processes in vivo, we hypothesize that LTIP directly affects the function and catabolism of lipoproteins through its capacity to influence the flow of lipids among lipoproteins and defines the extent to which LDL and HDL are remodeled during their circulatory lifetime by CETP. The influence of other factors, such as lipases, LCAT, and phospholipid transfer protein on the in vivo expression of LTIP activity remains to be determined.
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Acknowledgments |
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This research was supported by grant no. HL29582 from the National Heart, Lung and Blood Institute, National Institutes of Health.
Received February 26, 1998; accepted September 28, 1998.
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