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Originally published In Press as doi:10.1074/jbc.M306580200 on August 7, 2003

J. Biol. Chem., Vol. 278, Issue 42, 40859-40866, October 17, 2003
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Lipid Transfer Inhibitor Protein Defines the Participation of High Density Lipoprotein Subfractions in Lipid Transfer Reactions Mediated by Cholesterol Ester Transfer Protein (CETP)*

Viktor M. Paromov and Richard E. Morton {ddagger}

From the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Received for publication, June 20, 2003 , and in revised form, July 28, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cholesterol ester transfer protein (CETP) moves triglyceride (TG) and cholesteryl ester (CE) between lipoproteins. CETP has no apparent preference for high (HDL) or low (LDL) density lipoprotein as lipid donor to very low density lipoprotein (VLDL), and the preference for HDL observed in plasma is due to suppression of LDL transfers by lipid transfer inhibitor protein (LTIP). Given the heterogeneity of HDL, and a demonstrated ability of HDL subfractions to bind LTIP, we examined whether LTIP might also control CETP-facilitated lipid flux among HDL subfractions. CETP-mediated CE transfers from [3H]CE VLDL to various lipoproteins, combined on an equal phospholipid basis, ranged 2-fold and followed the order: HDL3 > LDL > HDL2. LTIP inhibited VLDL to HDL2 transfer at one-half the rate of VLDL to LDL. In contrast, VLDL to HDL3 transfer was stimulated, resulting in a CETP preference for HDL3 that was 3-fold greater than that for LDL or HDL2. Long-term mass transfer experiments confirmed these findings and further established that the previously observed stimulation of CETP activity on HDL by LTIP is due solely to its stimulation of transfer activity on HDL3. TG enrichment of HDL2, which occurs during the HDL cycle, inhibited CETP activity by ~2-fold and LTIP activity was blocked almost completely. This suggests that LTIP keeps lipid transfer activity on HDL2 low and constant regardless of its TG enrichment status. Overall, these results show that LTIP tailors CETP-mediated remodeling of HDL3 and HDL2 particles in subclass-specific ways, strongly implicating LTIP as a regulator of HDL metabolism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cholesteryl ester transfer protein (CETP)1 promotes the net transfer of lipids among lipoproteins (1, 2). Because of its unique capacity to mediate net exchange of triglyceride (TG) and cholesteryl ester (CE) between TG-rich VLDL and CE-rich lipoproteins, such as LDL and HDL, CETP plays an essential role in regulating plasma cholesterol levels. By altering lipoprotein core lipid composition, CETP participates in the catabolism of VLDL to LDL (1, 3, 4) and affects the level of LDL subfractions (5–7). Additionally, CETP facilitates HDL metabolism by promoting the formation of larger TG-rich HDL2 from smaller HDL3 subspecies (8, 9), stimulating LCAT activity (10, 11), and enhancing the formation of pre{beta}-HDL (12). These processes influence the reverse transport of peripheral tissue cholesterol to the liver (2, 13, 14).

In a previous study (15), we demonstrated that CETP possesses no functional preference for HDL over LDL. Consequently, in the absence of other factors, LDL is the most active donor of CE to VLDL due to the relative abundance of CE in this lipoprotein in normal plasma. However, CETP activity is controlled by several apolipoproteins including lipid transfer inhibitor protein (LTIP) (15–19). LTIP has been purified and characterized as an acidic glycoprotein with a molecular mass of 33 kDa (17, 20). Molecular cloning of LTIP had determined its identity with apolipoprotein F (20). LTIP prevents CETP activity by binding to lipoproteins and disrupting their interaction with CETP (17, 21). LTIP appears to alter the relative participation of HDL versus LDL in the lipid transfer processes by selectively interacting with the latter lipoprotein and reducing its capacity to bind CETP. Thus, in plasma-like lipoprotein mixtures, HDL and LDL are equal donors of CE to VLDL in the absence of LTIP, but HDL becomes a preferred CETP substrate and donates more CE in the presence of LTIP (15). However, the interaction of LTIP with LDL may be not exclusive. For instance, LTIP effectively binds to HDL and displaces CETP from its surface (21). Also, in two-component assays with CE-labeled lipoproteins, LTIP inhibits CETP-induced lipid transfer between VLDL and HDL, although to a lower extent than transfer between VLDL and LDL (17).

It is well known that HDL is heterogeneous in composition and function. The two most abundant HDL subpopulations, HDL2 and HDL3, differ not only in terms of particle size, but also in lipid content, lipid/protein ratio, apoprotein A-I/apoprotein A-II ratio, and, presumably, in apoprotein conformation (22–25). These structural differences of HDL subspecies lead to functional diversity, such as in their interaction with lecithin cholesterol acyltransferase (LCAT) (26, 27), hepatic lipase (28–30), CETP (27, 31, 32), and the scavenger receptor-BI receptor (33).

Given that HDL2 and HDL3 vary significantly in their interaction with plasma enzymes and transfer factors, and previous observations that LTIP can bind HDL (21, 34), we have investigated the capacity of LTIP to modify CETP-mediated lipid flux from these HDL subfractions. We have analyzed lipid transfers in 2-, 3-, and 4-component, short-term radiolabeled lipid transfer experiments with native and modified lipoproteins, and in long-term mass transfer assays to determine how LTIP alters the pattern of lipid movement among HDL fractions. We have observed marked differences in the substrate properties of HDL2 and HDL3 for both CETP and LTIP. The data suggest that LTIP may be important in controlling the maturation of HDL species.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Lipoprotein Isolation and Labeling—VLDL, LDL, HDL2, and HDL3 were isolated by sequential ultracentrifugation from fresh human plasma obtained from the Blood Bank of the Cleveland Clinic Foundation as described (35). Lipoproteins were extensively dialyzed against 0.9% NaCl, 0.01% EDTA, 0.02% NaN3, pH 7.4, and stored at 4 °C. Radiolabeled lipoproteins were prepared by CETP-induced transfer (15) of [1{alpha},2{alpha}(n)-3H]cholesteryl oleate (48.0 Ci/mmol, Amersham Biosciences) from phosphatidylcholine cholesterol-[3H]CE liposomes prepared as previously described (36). After the labeling procedure, VLDL and LDL were isolated by affinity chromatography on heparin-Sepharose (15), whereas HDL2 and HDL3 were re-isolated within their original density intervals by ultracentrifugation. Radiolabeled lipoproteins contained 400–1200 cpm 3H per µg of total cholesterol. Biotin-LDL and avidin-Sepharose were prepared as previously described (37). Lipoproteins were coupled to CNBr-activated Sepharose 4B (Amersham Biosciences) as previously detailed (21) at the lipoprotein to Sepharose ratios given under "Results."

Purification of CETP and LTIP—Partially purified CETP (~1 mg of total protein/ml, ~4 µg of CETP/ml) was isolated from lipoprotein-deficient human plasma using preparative chromatography as described earlier (38) and stored in 0.27 mM EDTA, pH 7.4 at 4 °C. Partially purified LTIP (~0.5 mg of total protein/ml) was also isolated from human plasma according to the procedure described earlier (17, 19). Based on the comparison of the activity of several preparations with the LTIP activity of lipoprotein-deficient human plasma, 3 µg of total protein of the partially purified LTIP was equivalent to the LTIP activity contained in 1 µl of human lipoprotein-deficient plasma (17). Neither LTIP nor CETP preparations contained phospholipid transfer protein (17), which has been previously shown to stimulate CETP activity in vitro (39).

Radiolabeled Lipid Transfer Assays—[3H]CE-labeled lipoproteins were prepared as described above. VLDL, LDL (or biotin-LDL), HDL2 and HDL3 were added to assays on an equal phospholipid basis (4.6 µg phospholipid each). Depending on the design of the experiment, assays contained a radiolabeled lipoprotein (donor) and one to three unlabeled lipoproteins (acceptors). The lipoprotein mixture was incubated in the presence of 1% bovine serum albumin, 21 mM Tris-HCl (pH 7.4), 0.5% NaCl, 0.011% NaN3, and 0.006% EDTA (total volume, 1.4 ml) ±CETP and ±LTIP (as indicated) for 1.5 h at 37 °C. After incubation, samples were cooled on ice and lipoprotein fractions were separated at 4 °C. For assays containing biotin-LDL, LDL was removed by addition of 100 µl of avidin-Sepharose (37). Otherwise, LDL or VLDL was precipitated by manganese-phosphorus reagent (17). Finally, HDL2 and HDL3 were separated by a single ultracentrifugation step (d = 1.125 g/ml), or at d = 1.21 g/ml when assays contained only one of these HDL subfractions. In parallel studies not shown, we have confirmed data derived from this hybrid separation approach with assays using native lipoproteins that were separated by sequential ultracentrifugation (35) alone. The radioactivity (cpm) of each fraction was measured by scintillation counting. Lipid transfer rates, inhibition values, and mass of the lipid transferred were calculated as previously described (15, 17). Blank samples (no CETP added) were incubated under the same conditions and subtracted from the values shown. Samples were assayed in duplicate.

In general, the presence of CETP or LTIP did not influence the separation of lipoproteins at the end of transfer assays. However, we observed in samples containing LTIP that HDL2 was partially recovered in the d > 1.125 g/ml density fraction, presumably because LTIP or proteins in this fraction bind to HDL2 and increase its density. Thus, for each HDL2 and LTIP preparation we determined, under assay conditions, the dose-relationship between the fraction of HDL2 recovered in the d > 1.125 g/ml fraction and LTIP concentration. This correction was then applied to the values reported for all assays containing both HDL2 and HDL3. Without this correction, the reported differences in LTIP activity between HDL2 and HDL3 would be larger.

Lipid Mass Transfer Assays—VLDL, LDL, HDL2, and HDL3 (225, 500, 120, and 120 µg of total cholesterol, respectively) were incubated in the presence of 1% bovine serum albumin, 50 mM Tris-HCl (pH 7.4), 0.9% NaCl, 0.02% NaN3, and 0.01% EDTA (total volume 3.0 ml) ±CETP and ±LTIP (as indicated) for 20 h at 37 °C. Also, 1 mM (final concentration) diethyl p-nitrophenyl phosphate was added to inhibit residual LCAT activity co-isolated with HDL subfractions. After incubation, samples were cooled on ice and separated by sequential ultracentrifugation at 4 °C (35). Dialyzed samples were assayed for total and unesterified cholesterol, TG, and protein content. Changes in CE and TG content were calculated from the recovery corrected differences in the lipid content of CETP-containing samples (±LTIP) incubated at 37 °C and blank (no CETP added) samples (±LTIP) incubated at 4 °C. Preference values were calculated as previously described (15). These values reflect the ratio between the amount of lipid transferred to a given lipoprotein acceptor expressed as a fraction of all transferred lipid, and the amount of transferable lipid in that acceptor expressed as a fraction of whole transferable lipid in the acceptor pool. Protein content of the lipoprotein fractions was quantitated according to the Lowry method as modified by Peterson (40) using bovine serum albumin as a standard. Total cholesterol and TG were assayed by InfinityTM Cholesterol and InfinityTM Triglyceride kits (Sigma). Unesterified cholesterol was determined by the Free Cholesterol C kit from Wako Diagnostics (Richmond, VA). Lipid phosphorus was assayed by the method of Bartlett (41). For calculations, 653, 885, and 800 were used as average molecular weights of CE, TG, and phospholipid, respectively. All assays were performed in duplicate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LTIP Binding to LDL, HDL2, and HDL3—LTIP binds HDL and displaces CETP from the HDL surface (21). To compare the LTIP binding capacity of HDL subspecies with LDL, isolated LDL, HDL2 and HDL3 were immobilized on Sepharose CL-4B. Since LTIP is a surface active apoprotein that interacts primarily with the phospholipid monolayer of lipoproteins (42), the capacity of solid-phase lipoproteins to bind LTIP were compared on an equal phospholipid basis. LTIP was applied to columns prepared with these matrices, the columns extensively washed, and bound LTIP was quantified. HDL2 and HDL3 bound similar amounts of LTIP, and this binding exceeded that for LDL by 2-fold under these conditions (Fig. 1). Thus, HDL2 and HDL3 are comparable in their ability to form stable complexes with LTIP.



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  		FIG. 1.
Binding of LTIP to plasma lipoproteins. 500 µg of partially purified LTIP was applied to columns (2 ml) containing Sepharose CL 4B alone, or Sepharose-bound LDL (0.69 mg of phospholipid, 1.5 mg of total cholesterol), HDL2 (0.69 mg of phospholipid, 0.53 mg of total cholesterol) or HDL3 (0.69 mg of phospholipid, 0.45 mg of total cholesterol) and eluted with 50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN3, 0.01% EDTA, pH 7.4 buffer at ~8 ml/h. Fractions (1 ml) were collected and LTIP content was determined by a competitive, semi-quantitative immunoassay using solid phase-bound LTIP and recombinant LTIP antibody (20). LTIP bound to each resin was calculated as the difference between applied LTIP and that eluted for each resin. Bound LTIP was not displaced by extensive (6 column-volume) washing of the matrix. Values are the mean ± S.D. (n = 2) and are representative of two experiments.

 

Effect of LTIP on CETP-mediated CE Transfer to HDL2 and HDL3—In our previous studies of LTIP, where lipoproteins were added to transfer assays on an equal cholesterol basis, we observed that LTIP preferentially inhibited CETP activity with LDL (15, 17). However, the binding studies above suggest that this preference toward LDL may not be observed when lipoproteins are compared on a phospholipid basis. To address this issue, we initially assessed the capacity of LTIP to affect CETP-mediated CE transfer between HDL2 and HDL3 in the absence of other lipoproteins to determine if the binding of LTIP to HDL2 and HDL3 noted above resulted in CETP suppression (Fig. 2A). Without LTIP, the CETP-mediated transfer of CE between these lipoproteins was the same (inset). LTIP did not disrupt the balance of CE flux between these two lipoproteins but equally suppressed CE transfer from both HDL2 and HDL3 in the dose-dependent manner.



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  		FIG. 2.
LTIP effects on [3H]CE transfers in 2-component assays. Panel A, [3H]CE-labeled HDL2 or HDL3 were incubated with native HDL3, or HDL2 (respectively) in the presence of CETP (10 µg) ± LTIP (as indicated) for 1.5 h at 37 °C. See "Experimental Procedures" for details. Inset, CE transfer activity in the absence of LTIP. Panel B, [3H]CE-labeled VLDL was incubated with whole HDL, HDL2, or HDL3 in the presence of CETP (10 µg) ± LTIP (as indicated) for 1.5 h at 37 °C. Inset, CE transfer activity in the absence of LTIP. Values are the mean ± S.D. (n = 2) and are representative of four similar experiments.

 

Since CETP transfer occurs largely through an exchange mechanism (1, 2), an equivalent effect of LTIP on bidirectional flux between two lipoproteins is expected and consistent with our previous findings (17). To further investigate the capacity of LTIP to inhibit lipid transfers to HDL2 and HDL3, we measured the effect of this protein on CETP-mediated CE transfer from VLDL to these lipoprotein fractions. Although TG transfer from VLDL to HDL is more physiologically relevant, the very small pool of TG in HDL, when added to assays at the same phospholipid concentration as VLDL, prevented the transfer of this lipid from being accurately measured. CE transfer to each of these lipoproteins in the absence of LTIP was similar, with transfer to HDL2 being modestly but not significantly higher than that to HDL3 (Fig. 2B, inset). LTIP caused a dose-dependent suppression of CE transfer from VLDL regardless of the acceptor lipoprotein (Fig. 2B). However, CETP activity from VLDL to HDL2 was much more sensitive to LTIP. At higher LTIP levels, the inhibition of VLDL to HDL2 was 1.7-fold more than that for VLDL to HDL3. As expected, unfractionated HDL gave an intermediate response. These results suggest that LTIP may influence CETP activity on HDL subfractions differently.

Having observed that LTIP suppresses lipid transfers to HDL2 more than HDL3 in simple 2-component assays, we subsequently investigated how CE transfer from VLDL was affected by LTIP when both HDL2 and HDL3 were present as acceptors. In the absence of LTIP, CETP-mediated CE transfer from VLDL to HDL2 was lower than that from VLDL to HDL3 (Fig. 3A, inset). Among four similar experiments, VLDL to HDL3 transfers averaged ~30% higher than VLDL to HDL2 transfers. The influence of LTIP on these CE transfers was unique. In contrast to that seen when HDL2 and HDL3 were added separately to transfer reactions, here we observed that VLDL to HDL2 CE transfer was inhibited in a dose-dependent manner by LTIP, but CE transfer from VLDL to HDL3 was stimulated by almost 40% at the highest LTIP level (Fig. 3A). Similar LTIP effects were observed in lipid transfer assays measuring lipid flux in the reverse direction from HDL2 or HDL3 to VLDL (i.e. HDL2 to VLDL + HDL3 and HDL3 to VLDL + HDL2, data not shown). Total CE flux from VLDL to HDL2 + HDL3 was unaffected by LTIP (Fig. 3B). This indicates that LTIP alters the destination of CE transferred from VLDL but not the overall rate of CE efflux from this lipoprotein. This shift in lipid transfer pathways is illustrated by the calculated preference values of CETP for each acceptor in the absence and presence of LTIP (Fig. 3C). In the absence of LTIP, lipid flux from VLDL to HDL3 was favored modestly over flux to HDL2. However, at the highest LTIP dose tested the preference of CETP for VLDL to HDL3 transfers was 3-fold greater than that involving HDL2. Thus in this experimental design, LTIP did not inhibit CETP-mediated lipid transfer from VLDL per se, but influenced which lipoprotein was the recipient of that lipid.



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  		FIG. 3.
LTIP effects on [3H]CE transfers from VLDL to HDL2 plus HDL3. Panel A, [3H]CE-labeled VLDL was incubated with native HDL2 + HDL3 in the presence of CETP (10 µg) ± LTIP (as indicated) for 1.5 h at 37 °C. Inset, CE transfer activity in the absence of LTIP. Panel B, total [3H]CE transferred from VLDL in the absence or presence of LTIP (as indicated). Panel C, CETP preference for HDL2 and HDL3 in the absence or presence of LTIP (as indicated). Preference values were calculated as described under "Experimental Procedures." Values are the mean ± S.D. (n = 2) and are representative of four similar experiments.

 

We previously reported that LTIP preferentially blocks CETP activity on LDL (15, 17, 20). Thus in transfer assays containing LDL, the influence of LTIP on lipid flux pathways involving HDL2 and HDL3 may be different. In fact, we have observed that although LTIP effectively inhibited CETP-mediated transfers between LDL and HDL2 as well as between LDL and HDL3, no statistically significant difference was evident (data not shown). Also, no significant difference in LTIP effect was detected for the VLDL -> HDL2 and VLDL -> HDL3 transfers in assays that also contained LDL (i.e. VLDL to LDL + HDL2 versus VLDL to LDL + HDL3) (data not shown). Together, these findings suggest that the LTIP-LDL interaction may modulate the interaction of LTIP with HDL subfractions. However, in each of these experimental settings the transfer assays contained HDL2 or HDL3, but not both.

Since we observed in Fig. 3 that the differential affects of LTIP on HDL subfractions are most evident when these lipoproteins are together, we examined the influence of LTIP on lipid flux pathways in four-component assays containing VLDL donor, and LDL, HDL2, and HDL3 as acceptors. In the absence of LTIP, lipid fluxes from VLDL to LDL and HDL3 were similar and almost 2-fold higher than the transfer to HDL2 (Fig. 4A, inset). Although the magnitude of the LTIP effects on HDL2 and HDL3 transfers was quantitatively less than that observed in the absence of LDL (Fig. 3), the unique influence of LTIP on these lipoproteins observed above persisted when LDL was present. As expected, VLDL to LDL transfers were the most sensitive to LTIP inhibition, but lipid transfer to HDL2 was also inhibited in a dose-dependent fashion by LTIP (Fig. 4A). In contrast, lipid transfer from VLDL to HDL3 was mildly stimulated. These changes in lipid flux among LDL, HDL2, and HDL3 revealed marked changes in the preference of CETP for these lipoproteins. Among multiple assays without (n = 5) and with (n = 4) LTIP (15 µg) the preference values for LDL decreased and HDL3 increased significantly in the presence of LTIP (Fig. 4B). The relative preference of CETP for HDL2 was unchanged by LTIP. Consistent with our previous findings (15), here we observe that in the absence of LTIP the preference values for LDL and total HDL are near one and these values decrease for LDL and increase for HDL in the presence of LTIP (Fig. 4B). These data also clearly demonstrate, however, that CETP has a marked preference for HDL3 as a substrate and that HDL2 is the least preferred substrate among the three acceptors studied. This preference is accentuated by LTIP. Thus, HDL2 and HDL3, when compared on an equal phospholipid basis, are very different in their functional response to both CETP and LTIP.



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  		FIG. 4.
LTIP effects on [3H]CE transfers from VLDL to LDL, HDL2, and HDL3. Panel A, [3H]CE-labeled VLDL was incubated with native LDL + HDL2 + HDL3 in the presence of CETP (10 µg) ± LTIP (as indicated) for 1.5 h at 37 °C. Inset, transfer rates in the absence of LTIP. B, CETP preferences for the lipoprotein acceptors in the absence or presence of LTIP. Values are the mean ± S.D. (n = 2) and are representative of four similar experiments.

 

CETP has been reported to have small but measurably different affinities for LDL and HDL, or HDL2 and HDL3, when measured with isolated lipoproteins under steady-state and non steady-state conditions (21, 32). How these differences in CETP binding affinity influence lipid flux among a mixture of lipoproteins has not been rigorously examined under conditions where these lipoproteins are added to transfer assays on an equal phospholipid, and therefore equal CETP interaction site, basis. To investigate this, we measured the transfer of CE from VLDL to LDL, HDL2 and HDL3 as a function of CETP concentration. We were surprised to observe that the transfers of CE to LDL and HDL2 are strongly and inversely affected by CETP concentration (Fig. 5A). This resulted in a linear relationship between the ratio of CE transfer to LDL versus HDL2 and CETP concentration (Fig. 5B). On the other hand, the transfer of CE from VLDL to HDL3 remained rather constant on a fractional basis (Fig. 5A) and on an absolute basis (20.4 versus 20.5% transfer for 10 versus 50 µg CETP, respectively). The marked sensitivity of LDL and HDL2 transfer activities to CETP concentration was also seen in 3-component assays lacking HDL3 (Fig. 5B). Overall, these data show that lipid transfer to HDL3 is unaffected by a severalfold change in CETP concentration, whereas HDL2 and LDL are highly sensitive to CETP concentration and appear to compete for the transfer factor. By comparison with our published data (15), we believe that the highest CETP level tested represents a physiologically relevant ratio of CETP to lipoprotein. Thus these data suggest that subnormal levels of CETP will disrupt the normal pattern of lipid flux between lipoproteins.



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  		FIG. 5.
Influence of CETP level on [3H]CE transfer to various lipoprotein acceptors in multicomponent assays. Panel A, percent distribution of CE transferred from VLDL to various lipoprotein acceptors as a function of CETP concentration. Panel B, influence of CETP concentration on CE transfer to LDL versus HDL2 in 3-component assays without HDL3 (i.e. VLDL to LDL + HDL2) and in 4-component assays with HDL3 (i.e. VLDL to LDL + HDL2 + HDL3).

 

Effect of LTIP on Net Lipid Transfer from HDL2 and HDL3— The net movement of neutral lipids between lipoproteins by CETP is mediated by a heteroexchange mechanism where TG is transferred from VLDL to LDL and HDL with the return of CE (15, 43, 44). This remodeling of lipoprotein composition is probably the most important function of CETP. In the foregoing studies we measured the transfer of radiolabeled CE from VLDL, which primarily reflects CE-CE homoexchange since this reaction is heavily favored over heteroexchange (15). To verify the unique responses of HDL2 and HDL3 to CETP and LTIP observed above, we performed long term incubations of native lipoproteins followed by quantification of net changes in lipoprotein CE content. Also, in contrast to above, for these experiments lipoproteins were combined at physiologically relevant ratios instead of an equal phospholipid basis. As previously reported, in the absence of LTIP, LDL was the major donor of CE (to VLDL) (Fig. 6). As suggested by radiolabel experiments, the addition of LTIP significantly reduced the loss of CE mass from LDL and modestly inhibited the CE loss in HDL2. Further, LTIP caused a 3-fold stimulation of CE net efflux from HDL3. LTIP did not reduce the total loss of CE mass from LDL and HDL fractions (280 nmol of CE (–LTIP) versus 312 nmol of CE (+LTIP)), but greatly altered the source of that CE. In the presence of LTIP, HDL3 became the predominate donor of CE to VLDL.



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  		FIG. 6.
Modulation of CE mass transfer by LTIP. Native VLDL, LDL, HDL2, and HDL3 (225, 500, 120, and 120 µg of total cholesterol, respectively) were incubated in the presence of CETP (150 µg) and/or LTIP (750 µg) for 24 h at 37 °C. Changes in CE content for each lipoprotein fraction were determined by comparing CETP containing samples (± LTIP) with control samples (± LTIP) where CETP was not active (incubated at 4 °C). Values are the mean ± S.D. (n = 2) and are representative of four similar experiments.

 

Since we and others have reported tight coupling of the transfer process (43), we were surprised to find in these studies that the loss of CE from LDL, HDL2, and HDL3 was not balanced by an equimolar gain of TG in these lipoproteins (Table I). Here, the mole loss of CE typically exceeded TG gain by 2–3-fold. This imbalance was not influenced by LTIP. While similar imbalances have been occasionally reported (45, 46), the mechanism of this unequal transfer is unclear. As seen by its TG/CE ratio, LTIP caused a mark change in HDL3 composition beyond that achieved by CETP alone (Table I).


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  		TABLE I
Influence of CETP and LTIP activities on lipoprotein composition

VLDL, LDL, HDL2 and HDL3 (225, 500, 120, 120 µg of cholesterol, respectively, which corresponds to 185, 197.5, 215, and 540 µg of protein), were incubated with CETP (150 µg) and LTIP (750 µg), as indicated, for 24 h at 37 °C, or at 4 °C in the absence of these factors. After separation by ultracentrifugation, lipoprotein lipid composition was determined as described under "Experimental Procedures." FC, free cholesterol.

 

Influence of TG Enrichment on CETP and LTIP Activities with HDL2—During the HDL cycle, HDL2 becomes progressively enriched in TG through the TG/CE heteroexchange mechanism of CETP. Following hepatic lipase action, small HDL3 like particles are formed, thus permitting HDL particles to cycle between the HDL3 and HDL2 compartments (9, 47). Control of this process is important in HDL homeostasis. Here we have examined how changes in TG content affect CETP and LTIP activities on HDL2. TG-enriched HDL2 (CE/TG ratio 0.53) was prepared in vitro by incubation of HDL2 (CE/TG ratio 3.38) with VLDL in the presence of CETP followed by ultracentrifugal reisolation (see "Experimental Procedures"). In a three-component transfer assay with VLDL as a donor, and LDL, and HDL2 (native or TG-enriched) as acceptors, CE transfer from VLDL to TG-enriched HDL2 was ~2-fold lower than for native HDL2 (Fig. 7, A and B). There was a modest compensatory increase in CE flux from VLDL to LDL in the presence of TG-HDL2 compared with native HDL2. LTIP strongly inhibited (76–78%) CE transfer from VLDL to LDL and this was not affected by the TG status of HDL2. Compared with that with LDL, LTIP suppressed CE transfer from VLDL to native HDL2 to a lesser degree (50% inhibition), consistent with that observed above. In contrast, LTIP had almost no effect on lipid transfer to TG-HDL2 (11% inhibition). Notably, CE transfer from VLDL to HDL2, regardless of TG status, was the same in the presence of both CETP and LTIP. Thus, the reduced CETP activity toward TG-HDL2 combined with a much lower LTIP activity on this HDL combine to maintain CETP activity constant. This suggests that LTIP functions to govern CETP activity on HDL2 and maintain the rate of remodeling regardless of the particle's TG content.



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  		FIG. 7.
Influence of TG enrichment of HDL2 on CETP and LTIP activities. TG-enriched HDL2 (TG-HDL2) (CE/TG ratio 0.53) was obtained after an overnight incubation of freshly purified HDL2 (CE/TG ratio 3.38) with VLDL in the presence of CETP followed by re-isolation by ultracentrifugation. [3H]CE labeled VLDL was incubated with LDL and HDL2 (native (panel A) or TG-rich (panel B)) in the presence of CETP (10 µg) ± LTIP (as indicated) for 1.5 h at 37 °C. CE transfer activity (CETA) values in the absence and presence of LTIP are shown. Percent CE transfer rates in the absence of LTIP were 17.7% for LDL and 25.1% for native HDL2 (panel A), and 22.5% for LDL and 14.1% for TG-rich HDL2 (panel B). Values are the mean ± S.D. (n = 2) and are representative of four similar experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We previously observed that LTIP, through its suppression of lipid transfer to LDL, stimulates the net efflux of CE from HDL to VLDL (15, 17). We have proposed that this shift in lipid movement among lipoproteins enhances the function of HDL in its role as the central mediator of reverse cholesterol transport (48). However, HDL is heterogeneous and its two main subfractions, HDL2 and HDL3, have diverse functions (26–33,49). Information on the substrate properties of these two subfractions for CETP is limited, and the role of LTIP in regulating CETP activity on these distinct particles is unknown.

Comparison of lipoproteins of different composition and size is complicated because these particles can be added to assays based on their protein content or the level of a lipid component. For CETP and LTIP, sufficient evidence exist to suggest that these proteins interact with the surface phospholipid monolayer (21, 42, 50, 51), providing a strong rationale for comparing the functional properties of these lipoproteins by adding them to assays on an equal phospholipid basis. In [3H]CE transfer experiments we evaluated the interaction of CETP and LTIP with HDL2 and HDL3 in a series of progressively more complex studies. We observed that LTIP inhibits CETP activity with HDL2 to a greater extent than with HDL3. This differential effect is observed primarily when HDL2 and HDL3 are both present in the transfer assay. In assays containing all four major lipoprotein fractions, LTIP activity followed the order of LDL > HDL2 >> HDL3. Although LTIP is still observed to preferentially suppress lipid transfer with LDL, its inhibition of lipid transfers to HDL2 is highly significant, averaging about one-half that observed with LDL.

Notably, in all assay configurations where HDL2 and HDL3 were both present as acceptors in lipid transfer assays, LTIP consistently inhibited CETP activity on HDL2 but stimulated CETP transfer to HDL3. Since CETP concentrations are rate limiting in these assays, we suggest that this occurs because LTIP interacts with HDL2 (and LDL) where it displaces CETP. This CETP is then available to bind to HDL3. However, binding studies show that LTIP binds to HDL3 and HDL2 to a similar extent. This suggests that LTIP can bind to both HDL2 and HDL3 but is able to dissociate CETP only from HDL2. Further, these data show that the extent of LTIP binding does not directly correlate with CETP suppression. We have made similar observations with chemically modified LDL where LTIP binding is unchanged but LTIP activity is markedly altered (52). These findings suggest that the ability of LTIP, once bound, to displace CETP from the lipoprotein surface varies among lipoproteins and is much higher for LDL and HDL2 than HDL3. This possibility will be investigated in more detail in future studies.

Multiple, radiolabeled CE transfer assays, which principally measure CE homoexchange, demonstrated that LTIP affects lipid transfers to and from HDL2 and HDL3 differently. In mass transfer assays under more plasma-like conditions, we recapitulated these observations and clearly demonstrated that LTIP augments the net efflux of CE from HDL3 at the expense of LDL and HDL2. Together, these radiolabel and mass transfer assays show that our previous observation that lipid transfer to whole HDL is stimulated by LTIP (15, 17) is solely due to increased transfer activity with HDL3.

CETP in plasma (53) or in in vitro lipoprotein mixtures (32) has been shown to be preferentially associated with HDL3 compared with HDL2. The consequences of this interaction on CETP activity are not well understood since it has been reported that these two HDL fractions support equivalent CETP activity when added to assays on a CE basis (54). However, here we observed that not only are HDL2 and HDL3 unique in their response to LTIP, these lipoproteins, when compared on an equivalent phospholipid bases, also supported very different CETP activities. In the presence of all plasma lipoproteins, HDL2 was the least preferred acceptor of lipid from VLDL, with transfer rates ~60% of that for LDL and HDL3. Calculated preference values (15) indicate that HDL3 is the most active CETP substrate, followed by LDL and HDL2. The preference of CETP for HDL3 was almost 2-fold higher than that for HDL2. As discussed above, the addition of LTIP further favored HDL3 as the acceptor of choice, resulting in preference values for HDL3 more than 3-fold higher than that for HDL2. These findings are contrary to non steady-state binding kinetic measurements, which show that the affinity of HDL2 for CETP exceeds that for HDL3 (32). Our finding that HDL3 is a superior CETP transfer substrate suggests that kinetic values obtained under non steady-state conditions may not be applicable to the steady-state conditions of a transfer assay. Furthermore, these new findings qualify our previous conclusion that CETP has no preference for LDL versus HDL (15) since, clearly, preferences exists among HDL subfractions.

The preference of CETP for HDL3 was further demonstrated in assays where CETP concentration was varied. Over a wide range of CETP levels we observed that lipid transfer to HDL3 remained rather constant whereas CE transfer to LDL and HDL2 was strongly influenced by CETP levels. We were surprised to find that lipid transfer to HDL2 decreased and that to LDL increased as CETP concentration increased. The mechanism for this unusual response is not known. Based on comparison with our published data (15), we concluded that the highest CETP level tested reflects a physiologically relevant CETP/lipoprotein value. Direct calculation of this ratio is not feasible since, at the very low lipoprotein concentrations used in transfer assays, a large portion of CETP is likely to be free (non-lipoprotein bound) in solution (21, 32). Given this assumption, these data suggest that as plasma CETP concentrations are decreased the relative rates of lipid transfer to LDL, HDL2, and HDL3 change whereas the absolute rate of transfer to HDL3 is rather constant. This observation is potentially important given current interests in providing atherogenic protection through pharmacological suppression of CETP. While preliminary studies of CETP inhibitors in animals and humans have been encouraging (55–57), our observations suggest that decreasing CETP activity will have a more complex effect that simply proportionally suppressing all transfer reactions. Whether this is correct, and whether the lipid transfer profile between plasma lipoproteins that result when CETP is suppressed is beneficial remains to be determined.

Maturation of HDL2 involves its enrichment with TG through CETP mediated heteroexchange. Here we investigated how TG enrichment of HDL2 influences its substrate properties toward CETP and LTIP. Our data show that TG-rich HDL2 is an inferior substrate for CETP compared with native HDL2. Interestingly, concomitantly there is a nearly complete loss of reactivity with LTIP. The combined effect of these changes in reactivity, however, is neutral since the decline in CETP reactivity as HDL2 became TG-enriched was matched by a lost of the suppressive action by LTIP. We suggest that a role for LTIP may be to maintain CETP-mediated remodeling of HDL2 low and constant as it matures and becomes TG enriched.

In summary, we report here that HDL3 is the preferred CETP substrate when lipoproteins are compared on an equal phospholipid (i.e. CETP "binding site") basis. In a plasma-like mixture of lipoproteins, this preference is maintained. Further, we show that the previously recognized stimulation of VLDL-HDL transfer events by LTIP is due exclusively to enhanced lipid transfer activity with HDL3. Overall, LTIP accentuates the capacity of CETP to remodel HDL3 while maintaining lipid transfer reactions to LDL and HDL2 well below that expected based on their CE content. We suggest that there may be several metabolic advantages to the altered lipid flux among HDL subfractions that occurs in the presence of LTIP. LCAT prefers HDL3 as a substrate (26) and its activity is stimulated by CETP (10, 11), presumably because CETP removes CE and minimizes product inhibition of the enzyme (58). Our studies show that LTIP stimulates this function of CETP, and thus would improve the reactivity of these particles with LCAT. Subsequently, HDL3, primarily due to the action of LCAT (59), progressively enlarges and becomes HDL2. LTIP is more active and CETP is less active on HDL2 particles. We suggest that this combined effect is to slow further remodeling of HDL2 and prolong the plasma lifetime of these particles. As these particles are slowly remodeled into TG-enriched HDL2 through the action of CETP, a reduction in sensitivity to LTIP offsets a natural decline in the capacity of these TG-rich particles to function as CETP substrates. Thus, variations in LTIP activity sustain the rate of HDL2 remodeling regardless of TG status. By retarding the enrichment of HDL2 with TG, LTIP would stabilize these particles and prevent their premature clearance from plasma (60, 61). Overall, our data show that LTIP regulates CETP-mediated remodeling of HDL in a subclass-specific manner. We suggest that the effect of LTIP on HDL metabolism will be to augment specific functions of HDL2 and HDL3 involved in reverse cholesterol transport. We propose that inhibiting CETP by elevating LTIP activity may promote a more beneficial lipoprotein metabolic profile than approaches that globally suppress CETP activity.


   FOOTNOTES
 
* This research was supported in part by Grant HL60934 from the NHLBI, National Institutes of Health and Grant 0050075N from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Cell Biology, NC 10, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-5850; Fax: 216-444-9404; E-mail: mortonr{at}ccf.org.

1 The abbreviations used are: CETP, cholesteryl ester transfer protein; LTIP, lipid transfer inhibitor protein; CE, cholesteryl ester; TG, triglyceride; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; HDL2, 1.063 < d < 1.125 g/ml fraction of HDL; HDL3, 1.125 < d < 1.21 g/ml fraction of HDL; LCAT, lecithin cholesterol acyltransferase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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