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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3041-3048

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3041-3048.)
© 1997 American Heart Association, Inc.

Articles

Suppression of Lipid Transfer Inhibitor Protein Activity by Oleate

A Novel Mechanism of Cholesteryl Ester Transfer Protein Regulation by Plasma Free Fatty Acids

Richard E. Morton; ; Diane J. Greene

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

Correspondence to Richard E. Morton, PhD, Department of Cell Biology, NC10, Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail mortonr{at}cesmtp.ccf.org


*    Abstract
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 *Abstract
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Abstract Cholesteryl ester transfer protein (CETP) mediates the interlipoprotein exchange of cholesteryl ester (CE) and triglyceride. A second plasma protein, lipid transfer inhibitor protein (LTIP), binds to lipoproteins and inhibits CETP activity by displacing CETP from the lipoprotein surface. Since free fatty acids (FFAs) enhance the binding of CETP to lipoproteins, we have examined the possible role of FFAs in modulating LTIP activity. Partially purified CETP, LTIP, and lipoproteins were incubated with 0 to 30 µmol/L sodium oleate, and the transfer of CE between a labeled donor lipoprotein and a given acceptor lipoprotein was measured. Without LTIP, oleate stimulated CETP-mediated CE transfer between VLDL, LDL, and HDL up to threefold. This stimulation was unique in both magnitude and oleate concentration dependence for each donor-acceptor lipoprotein pair. In contrast to CETP activity, in transfer reactions involving LDL or VLDL as donor, LTIP activity was suppressed (>80%) by 10 to 15 µmol/L oleate. LTIP activity in transfer reactions with HDL as donor was less sensitive. Similar results to these were observed when lipid transfer reactions were measured in the total lipoprotein fraction isolated from FFA-enriched plasma. The FFA content of lipoproteins was strongly influenced by the concentration of FFA in plasma; lipoprotein FFA levels sufficient to suppress LTIP activity by 50% to 100% were achieved in plasma containing 0.8 to 1.0 mmol/L FFA. We conclude that LTIP may be functionally inactive during periods of transient elevations of plasma FFA levels, such as during postprandial lipemia or overnight fasting, or chronically suppressed in disease states in which plasma FFA levels are increased. The suppression of LTIP activity by FFA allows for maximum CETP-mediated lipid transfer between all lipoproteins, including lipid transfer reactions involving LDL that are normally preferentially suppressed by LTIP.


Key Words: cholesteryl ester transfer protein • lipid transfer inhibitor protein • free fatty acid


*    Introduction
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 *Introduction
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Plasma CETP mediates the remodeling of lipoprotein composition by promoting the net transfer of TG and CE between TG-rich lipoproteins and CE-rich lipoproteins.1 2 The activity of CETP is modulated by multiple factors, including CETP mass levels,3 the concentration of VLDL in plasma,4 and the chemical composition of the lipoprotein substrates.5 6 7 8 Additionally, LTIP modifies the pattern of lipid fluxes between lipoproteins mediated by CETP by preferentially suppressing lipid transfer reactions involving LDL.9 LTIP disrupts the binding of CETP to the lipoprotein surface;10 this binding is essential for CETP activity.10 Consistent with its preferential suppression of transfer pathways involving LDL, LTIP primarily associates with LDL in native plasma,9 although it may be recovered, in part, in the HDL fraction after ultracentrifugation.11 This preferential suppression of CETP reactions involving LDL results in enhanced mass transfer of CE from HDL to VLDL and increases plasma LCAT activity.9 Both of these events are expected to enhance the reverse cholesterol transport process.

Binding of CETP to the lipoprotein surface is necessary but not sufficient for lipid transfer to occur.10 12 The mechanisms of CETP binding remain unclear but likely involve specific residues in the carboxy-terminal portion of the molecule.12 CETP readily binds to phospholipid surfaces, and this binding is enhanced by the presence of negatively charged components.13 14 Binding is disrupted by positively charged groups or by the presence of divalent cations that can compete for interaction with phospholipid head groups.13

During postprandial lipolysis, CETP activity is increased.15 A portion of this increase is mediated by the accumulation of FFAs in lipoproteins, which results in both enhanced binding and redistribution of bound CETP among the plasma lipoproteins.15 16 The possible roles of FFAs in modifying CETP activity have been investigated in vitro by Barter and colleagues.17 Elevated FFAs enhance the "HDL conversion" activity of CETP, and augment both the CETP-mediated exchange of radiolabeled CE and the mass transfer of CE from HDL3 to LDL and VLDL.18 19 20 These investigators have proposed that elevated FFAs dissociate the normal heteroexchange process of CE and TG19 and convert the transfer process from a lipid shuttle mechanism,21 which is proposed to predominate at low FFA levels, to a ternary collision complex mechanism22 that may be important for the resizing of lipoprotein particles.23

LTIP appears to mediate the suppression of CETP activity by competing with the transfer protein for residence on the lipoprotein surface.10 Given the strong influence of FFAs on CETP binding to lipoproteins and the apparent competition of CETP and LTIP for similar sites on the lipoprotein surface10 we have investigated the potential modulation of LTIP activity by FFAs. The capacity of sodium oleate to modulate CETP and LTIP activities has been measured in CE transfer assays involving VLDL, LDL, and HDL. These results are compared with the FFA levels in plasma lipoproteins under normal and pathological conditions.


*    Methods
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 *Methods
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Materials
Glycerol tri[9,10-3H]oleate (26.8 Ci/mmol) and [9,10-3H(n)]-oleic acid (14 Ci/mmol) were obtained from New England Nuclear. [1{alpha},2{alpha}(n)-3H]Cholesteryl oleate (48 Ci/mmol) and cholesteryl [1-14C]oleate (53.9 mCi/mmol) were purchased from Amersham Corp. BSA (fraction V), sodium oleate ({approx}99% pure), sodium palmitate ({approx}99% pure), and all reagents for salt and buffer solutions were obtained from Sigma Chemical Co. Stock solutions of the fatty acid salts were prepared in deionized water.

Isolation of CETP and LTIP
Partially purified LTIP and CETP were isolated from lipoprotein-deficient human plasma by hydrophobic and ion exchange chromatography as previously described.9 24 During purification, CETP 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 1.0% BSA in a total volume of 0.7 mL.25 26 LTIP activity was determined by the capacity of samples to suppress a standard quantity of CETP under these conditions.

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,27 extensively dialyzed against 0.9% NaCl, 0.02% EDTA, pH 7.4, and stored at 4°C. Lipoproteins were quantitated on the basis of their total cholesterol content. In selected experiments, total lipoproteins were isolated from plasma by ultracentrifugation after adjusting the solvent density to 1.21 g/mL with solid NaBr. In some instances, before isolation from plasma, lipoproteins were radiolabeled with [3H]CE or with [3H]TG and [14C]CE by a lipid dispersion technique.28 Under these labeling conditions, lipoproteins typically contained >=1.6x103 dpm [3H] and >=5x102 dpm [14C] per microgram cholesterol. LDL and HDL were bound to Sepharose as previously described.10

CETP and LTIP Activity Assays
Lipid transfer assays between [3H]CE-VLDL, [3H]CE-LDL, or [3H]TG-LDL (donor) and HDL (acceptor) were carried out as previously described.25 26 Radiolabeled VLDL or LDL and unlabeled HDL (10 µg cholesterol of each unless indicated otherwise) were incubated with or without CETP at 37°C for 1.5 hours in 1.5 mL microfuge tubes (Sarstedt Inc). Assays were concluded by selectively precipitating the donor lipoprotein26 and counting the radioactivity in the supernatant (acceptor). Assays in the reverse direction, ie, from [3H]CE-HDL to LDL or VLDL, were terminated in the same manner, but the pellet containing the acceptor was washed once with "equilibrated top phase" (the supernatant derived from precipitated assay blanks not containing labeled lipoprotein) and its radiolabel content determined. CE transfer assays between [3H]CE-VLDL and unlabeled LDL, and the reverse from labeled LDL to VLDL, were as described above, except the donor and acceptor lipoproteins were separated by ultracentrifugation at d=1.019 g/mL. Transfer assays with the d<1.21 g/mL lipoprotein fraction were performed by the addition of tracer amounts of radiolabeled lipoproteins to the total lipoprotein fraction. Transfer was assessed by label transfer into lipoprotein fractions isolated by ultracentrifugation.

All assays were carried out in a shaking water bath (Bellco Glass Inc) at 37°C. In experiments assessing the effect of oleate on CE transfer, the 1% BSA normally present in the transfer assay25 26 was omitted but added after the incubation time to aid in the reproducibility of the precipitation procedures used to separate donor and acceptor lipoproteins. In all assays, the radiolabel content of the acceptor fraction was determined after separation of the donor and acceptor as referenced above. The fraction of radiolabeled, donor lipid that was transferred (kt) to the acceptor particle was calculated as described before,25 and reported as percent lipid transfer (%kt). Assay blanks without CETP were carried out at each oleate concentration and subtracted to determine the facilitated transfer. LTIP activity was determined from the decrease in CETP activity induced by LTIP and is reported either as percent inhibition (percent decrease in CETP activity due to LTIP) or as the absolute decrease in %kt units caused by LTIP.

Analytical Procedures
Protein was quantitated by the method of Lowry et al29 as modified by Peterson,30 with BSA as standard. Total cholesterol of lipoproteins was assayed by a colorimetric, enzymatic method using a kit from Sigma Chemical Co. Lipid phosphorus was determined by the method of Bartlett31 ; PL mass was calculated assuming an average molecular weight of 800. FFA concentrations of plasma and isolated lipoproteins were determined by a NEFA kit from Wako Diagnostics. Agarose gel electrophoresis was performed as previously described, and lipoprotein proteins and lipids were visualized by Coomassie blue or fat red B staining, respectively.32


*    Results
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 *Results
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Oleate Modification of CETP and LTIP Activities
In our initial studies, we have confirmed that oleate stimulates the transfer of CE from HDL to LDL (Fig 1ADown). However, we also observed that increased oleate stimulates CE transfer in the reverse direction, ie, from LDL to HDL (Fig 1BDown). In marked contrast to the effects on CETP activity, oleate decreased the capacity of LTIP to inhibit CETP activity (Fig 1Down). This suppression of LTIP activity was dependent on the direction of lipid transfer. Oleate (15 µmol/L) almost completely prevented the suppression of CETP activity by LTIP in LDL-to-HDL transfers, whereas this effect on LTIP activity was only partial in HDL-to-LDL transfers.



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  		Figure 1. Effect of low and high oleate concentration on CETP and LTIP activities in CE transfers between LDL and HDL. Radiolabeled LDL or HDL was incubated with unlabeled HDL or LDL, respectively, in the presence or absence of 15 µmol/L sodium oleate. A, CE transfer from HDL to LDL facilitated by CETP (18 µg protein) in the absence (solid bars) or presence (hatched bars) of LTIP (20 µg protein). B, Same as A, except data are for CE transfers from LDL to HDL. Assays were performed as described in "Methods." Values are mean±SE. Results are representative of three similar experiments.

The above results suggest that CETP and LTIP are differentially regulated by FFA levels and that the effect of FFAs on LTIP activity may be dependent on the nature of the lipoprotein participants in the transfer process. To characterize the relationships between CETP and LTIP activities and lipoprotein FFA levels more fully, oleate titration studies were performed in assays in which a single donor and acceptor lipoprotein pair was present. In this way, the six possible donor-acceptor pairs among VLDL, LDL, and HDL were examined. Assay concentrations of 0-30 µmol/L were studied. The amount of oleate incorporated into the lipoproteins was determined in separate, identical studies by assessing the partition of 3H-oleate into the lipoprotein fraction after ultracentrifugal separation. The incorporation of oleate was determined at each fatty acid concentration; on average 58.7±2.2%, 72.8±2.9%, and 74.0±1.4% of the oleate was incorporated into lipoproteins in assays containing VLDL-LDL, VLDL-HDL, and LDL-HDL, respectively. Essentially the same incorporation of oleate into VLDL-LDL was observed when the lipoproteins were separated by manganese precipitation26 instead of ultracentrifugation. Oleate in solution was 1 to 2 µmol/L over the 0 to 30 µmol/L oleate concentration range studied. The remaining, non–lipoprotein-associated oleate was tightly bound to the plastic assay tubes.

The effect of oleate on CETP activity was varied and highly dependent on the lipoproteins involved in the CE transfer reaction. The results could be grouped into three types of responses. CETP-mediated CE transfers from VLDL to LDL and from LDL to VLDL (Fig 2ADown) were stimulated 50% to 80% above control by low levels (5 to 10 µmol/L) of oleate but were progressively suppressed by lipoprotein FFA concentrations achieved by the addition of >=20 µmol/L oleate. In distinction, CE transfers from VLDL to HDL (Fig 2CDown) and from LDL to HDL (Fig 2EDown) were stimulated 40% to 60% by low (10 µmol/L) oleate with little additional effect noted up to 30 µmol/L FFA. Oleate elicited a concentration-dependent increase in CE transfer from HDL to either VLDL (Fig 2CDown) or LDL (Fig 2EDown) with 30 µmol/L oleate, causing a twofold to threefold increase in transfer activity.



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  		Figure 2. Response of CETP and LTIP activities to increasing oleate concentration. [3H]CE transfer from the indicated donor lipoprotein to the designated acceptor lipoprotein (10 µg cholesterol each) was determined as described in "Methods." CE transfer due to CETP (18 µg protein) is shown in A, C, and E. In the absence of added oleate, CETP activity was 7.98 and 7.47 %kt for transfers from LDL to VLDL and from VLDL to LDL, respectively (A), 14.33 and 6.72 %kt for transfers from HDL to VLDL and from VLDL to HDL, respectively (C), and 5.11 and 15.24 %kt for transfers from HDL to LDL and LDL to HDL, respectively (E). The inhibition of CE transfer caused by LTIP (20 µg protein) is shown in B, D, and F. Without added oleate, LTIP caused 63.8% inhibition of CETP activity from LDL to VLDL and 79.5% inhibition for transfers from VLDL to LDL (B), 34.7% inhibition of CETP activity from HDL to VLDL and 22.0% inhibition for transfers from VLDL to HDL (D), and 54.1% inhibition of CETP activity for transfers from HDL to LDL and 42.8% inhibition for transfers from LDL to HDL (F). Values are mean±SE.When not visible, error bars are contained within the symbol. Results are representative of at least three similar experiments for each donor-acceptor pair, except for LDL to VLDL assays, which were performed twice.

LTIP activity changes due to increasing oleate were distinct from those observed above for CETP. The response of LTIP to oleate enrichment was characterized by two different responses, one in which LTIP activity was completely blocked by oleate and another in which oleate only partially suppressed LTIP activity. LTIP activity in CE transfer assays from VLDL to LDL, LDL to VLDL (Fig 2BUp), VLDL to HDL (Fig 2DUp), and LDL to HDL (Fig 2FUp) was abruptly and almost completely suppressed by oleate concentrations exceeding 4 wt% ({approx}10 to 15 µmol/L oleate). In all situations, LTIP activity was completely suppressed at oleate concentrations lower than those necessary to impair CETP activity. The inactivation of CETP or LTIP activity by oleate (Fig 2AUp and 2BUp) was not prevented by the addition of 10 mmol/L EDTA to the assay, suggesting that the previously reported oxidative inactivation of CETP by oleate33 was not the mechanism of inactivation in this instance.

In contrast to the effects of oleate on LTIP activities when VLDL or LDL was the donor, when HDL was the donor of CE to LDL or VLDL (Fig 2DUp and 2FUp), oleate was less effective in suppressing LTIP, achieving {approx}50% inactivation at the highest oleate concentration. This suppression was progressive over the initial portion of the oleate concentration curve but tended to plateau at higher FFA concentrations.

Barter34 suggested that oleate may uncouple the heteroexchange of CE and TG, perhaps by altering the mechanism by which CETP functions. This, however, does not seem to occur at the oleate concentrations tested herein. Oleate (30 µmol/L) stimulated both TG and CE transfer from HDL to LDL to a similar extent, resulting in only a small change in the ratio of CE to TG transferred, from 0.95 without oleate to 1.12 with 30 µmol/L oleate present. The ability of LTIP to suppress TG and CE transfers was decreased by oleate, but the extent of this suppression was the same for both lipids (53.6% and 55.7% inhibition without oleate versus 33.0% and 31.1% in the presence of 30 µmol/L oleate for TG and CE transfers, respectively). Similar results to these were also observed for TG transfer and LTIP inhibition in transfer assays from HDL to VLDL and from LDL to HDL (data not shown). No evidence for significant uncoupling of TG and CE transfer by oleate up to 30 µmol/L was observed.

Modulation of LTIP Activity by Palmitate
The suppression of LTIP activity by oleate was not unique to this FFA. Compared with oleate, palmitate was more effective in blocking LTIP activity in LDL-to-HDL transfer assays (Fig 3Down). This greater effectiveness for palmitate compared with oleate was also observed for HDL-to-LDL and VLDL-to-HDL transfers (data not shown). In general, approximately half as much palmitate was required to elicit the LTIP activity suppression caused by a given oleate level. Furthermore, in contrast to its effects on LTIP activity, palmitate elicited smaller changes in CETP activity than that caused by equivalent oleate (data not shown). For VLDL-to-HDL and LDL-to-HDL transfer assays, complete LTIP activity suppression occurred at palmitate concentrations that did not stimulate CETP activity.



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  		Figure 3. Comparison of the effects of oleate and palmitate on LTIP activity. The capacity of oleate or palmitate to diminish the ability of LTIP to inhibit CETP-mediated CE transfer from LDL to HDL was measured as described in "Methods." See legend to Fig 2Up for experimental details. Values are mean±SD.

Oleate Modification of CETP and LTIP Activities in a Physiological Mixture of Lipoproteins
To assess the effect of oleate on CETP and LTIP activities in a more physiological setting, plasma was supplemented with oleate, and then the lipoprotein fraction isolated by ultracentrifugation. Tracer amounts of [3H]CE-LDL or -HDL were added to the isolated lipoprotein fraction to measure the transfer of CE in the presence of CETP and LTIP. As shown in Fig 4ADown and 4BDown, the responses of CETP and LTIP activities to oleate were similar to those described in Fig 2Up in many respects. However, in general, CETP activities were stimulated to a lesser degree, and only transfers from HDL to LDL were markedly stimulated by oleate addition. LTIP activities, as observed before, were progressively suppressed by oleate; nearly complete inhibition of all LTIP activities occurred at oleate concentrations similar to those noted above (Fig 2Up). In contrast to that observed in Fig 2DUp and 2FUp, LTIP activity in transfers from HDL to VLDL or LDL was as sensitive to oleate addition as it was in reactions not involving HDL as the lipid donor. Overall, these data show a more uniform sensitivity of LTIP activity to oleate among the different CE transfers measured than that observed above in assays with low concentrations of isolated lipoproteins. Nonetheless, these data support the observation that LTIP activity is largely suppressed by oleate concentrations that are neutral or stimulatory to CETP.



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  		Figure 4. Effect of plasma supplementation with oleate on CETP and LTIP activities with isolated lipoproteins. Fresh plasma was supplemented with sodium oleate and the mixture incubated for 1 hour at 37°C. The lipoprotein fraction (d<1.21 g/mL) was isolated by ultracentrifugation and dialyzed against 0.9% NaCl, 0.02% EDTA, pH 7.4, and complete lipid composition was determined. Lipoproteins (100 µg cholesterol) were incubated with [3H]LDL or [3H]HDL (5 µg cholesterol) and CETP alone (28 µg protein, A) or with LTIP (164 µg protein, B). After incubation (1.5 hours for [3H]HDL or 2 hours for [3H]LDL), 0.5 mL of plasma was added, the lipoprotein fractions were isolated by sequential ultracentrifugation, and the extent of radiolabeled CE transfer was determined. The FFA content of lipoproteins was determined by the extent of [3H]oleate incorporation under conditions identical to those in the transfer assays.

Oleate Effect on LTIP-Lipoprotein Interactions
FFAs are known to increase the binding of CETP to all classes of lipoproteins.14 15 To better characterize the mechanism by which oleate suppresses LTIP activity, the effect of oleate on the binding of LTIP to LDL- and HDL-Sepharose was measured. Oleate only modestly decreased the binding of LTIP to isolated LDL or HDL at FFA concentrations at which LTIP activity is completely inhibited (TableDown). Oleate concentrations higher that those studied above (Figs 1 through 3UpUpUp) were also ineffective in disrupting the binding of LTIP to LDL or HDL. These results suggest that oleate does not suppress LTIP activity by disrupting its physical interaction with substrate lipoproteins.


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  		Table 1. Effect of Oleate on LTIP Binding to LDL- and HDL-Sepharose

FFA Distribution in Native and FFA-Enriched Plasma
The above studies suggest that lipoprotein FFA concentrations3 4 wt% profoundly suppress LTIP activity. To assess the conditions under which this level of FFA enrichment would occur in lipoproteins, sodium oleate was added to plasma to achieve final concentrations up to 2 mmol/L. This physiological range of FFAs led to a progressive increase in the lipoprotein FFA level (Fig 5Down). Lipoprotein (d<1.21-g/mL fraction) FFA levels remained low until the fatty acid:albumin mole ratio exceeded 1 (0.63 mmol/L), and then increased nonlinearly. Albumin contamination in the d<1.21-g/mL fraction was less that 0.65% of its plasma concentration, as assessed by protein staining of agarose electrophoresis gels, and could account for less than 6% of the oleate recovered in the lipoprotein fraction. The addition of an equimolar mixture of sodium palmitate and sodium oleate, which more closely approximates the FFA composition of plasma,35 resulted in even greater enrichment of lipoproteins with FFAs than caused by oleate alone, with up to 18% of the added FFA incorporated into the lipoprotein fraction (Fig 5Down, inset). The lipoprotein FFA levels necessary to suppress LTIP activity by 50% to 100% (based on Figs 2Up and 4Up) are shown by the striped zone in Fig 5Down.



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  		Figure 5. Incorporation of FFAs into plasma lipoproteins. Fresh plasma was supplemented with sodium oleate or an equimolar mixture of sodium oleate and sodium palmitate at the indicated final concentrations and incubated for 1 hour at 37°C. The d<1.21-g/mL lipoprotein fraction was isolated by ultracentrifugation and extensively dialyzed against 0.9% NaCl, 0.02% EDTA, pH 7.4. The FFA and lipid phosphorus content were determined as described in "Methods." Native plasma (nonfasted) contained 0.20 µg FFA per 100 µg phospholipid. The hatched area illustrates the lipoprotein FFA concentrations at which LTIP activity was suppressed by 50% on the basis of data shown in Figs 2Up and 4Up. Inset: the percent of plasma FFA recovered in the d<1.21-g/mL fraction. Data are representative of three similar experiments.

The effects of fatty acid enrichment on VLDL, LDL, and HDL were readily observed by changes in their REM (Fig 6Down). The relative mobility of each lipoprotein was linearly related to the total FFA content of the d<1.21-g/mL fraction of plasma regardless of whether fatty acid enrichment was mediated by oleate or oleate+palmitate. The linear response of each lipoprotein class indicates that FFAs in the d<1.21-g/mL fraction partition among VLDL, LDL, and HDL in a constant ratio over the FFA levels studied. The changes in lipoprotein REM, and their linear correlation with the FFA content of the d<1.21-g/mL fraction, provide a means of determining whether the lipoprotein fractions isolated by ultracentrifugation contain the same FFA levels as lipoproteins in unfractionated plasma. As seen in the inset to Fig 6Down, the REM of LDL in the d<1.21-g/mL fraction was linearly related to the REM of LDL in unfractionated plasma. Similar results were obtained with VLDL and HDL (not shown). This indicates that the FFA content of lipoproteins in the d<1.21 g/mL fraction is very similar to than in native plasma.



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  		Figure 6. Alteration of lipoprotein electrophoretic mobilities by FFAs. The REMs of LDL (squares), VLDL (circles), and HDL (triangles) in the d<1.21-g/mL fraction from plasma supplemented with oleate (solid symbols) or equimolar oleate+palmitate (open symbols) were determined by agarose gel electrophoresis. FFA was added up to 2 mmol/L final concentration. LDL, VLDL, and HDL mobilities in the absence of added oleate were 0.83, 1.23, and 2.66 relative to a transferrin standard, respectively. The results are representative of three similar experiments. Inset: a plot of the REM of LDL in unfractionated plasma compared with the REM of LDL isolated by ultracentrifugation from the same plasma (slope=1.11, y intercept=-0.13, r=.996). Results are the mean of triplicate determinations. Isolated LDL was dialyzed versus 0.9% NaCl, 0.02% EDTA before analysis. Similar results were obtained for VLDL and HDL (not shown).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study we report that LTIP activity is suppressed by elevated lipoprotein FFA levels. This effect on LTIP differed markedly from the effects that FFA had on CETP activity. At the 4 wt% FFA level at which the majority of LTIP activity was blocked, CE transfer mediated by CETP was uniformly increased by 40 to 50% in all transfer pathways. The loss of LTIP activity was not due simply to the increased CETP activity, since essentially the same conclusions are reached when LTIP activity is expressed as the absolute amount of transfer inhibited instead of the percent of lipid transfer inhibited. This result is consistent with the observation that LTIP activity, expressed as percent inhibition, is unaffected by threefold to fourfold changes in CETP concentration.28 36 37 Overall, these results demonstrate that CETP and LTIP activities are differentially affected by lipoprotein FFA levels; LTIP activity is the more sensitive barometer of lipoprotein FFA levels. Since LTIP plays an important role in modifying CETP activity,9 37 these results suggest that elevated FFAs may alter the overall flux of neutral lipids between lipoproteins by inactivating LTIP.

The effect of FFAs on CETP activity has been previously studied.34 In the present studies, the FFA-stimulated transfer of CE from HDL to VLDL and LDL has been confirmed. In addition, we have observed that limited FFA levels stimulate all transfer reactions among the three major lipoprotein classes. Both CE and TG flux were stimulated by oleate to similar degrees, indicating that no uncoupling of TG-CE transfer was observed at these FFA levels. As FFAs increased, the transfer of CE from HDL to other lipoproteins was stimulated to a greater degree than for the reverse pathways (to HDL). This is similar to the studies of Barter and colleagues19 20 in which FFA concentrations of at least 8 to 9 wt% FFA (relative to PL) stimulated the unidirectional flux of CE. These investigators suggested that this reflects a switch in CETP transfer mechanisms.

Although a full understanding of the mechanism by which FFAs suppress LTIP activity remains to be determined, binding studies of LTIP to LDL and HDL demonstrated that the association of LTIP with these lipoproteins is not diminished by increased oleate. Since the FFA-mediated increase in CETP binding to lipoproteins is well established,13 14 15 these results suggest that the suppression of LTIP activity is not due to a disruption in lipoprotein-LTIP binding but to an increased binding affinity of lipoproteins for CETP due to the increased negative surface change that results from FFA enrichment. Presumably LTIP is unable to disrupt these tighter interactions. Alternatively, FFAs may create new CETP "binding sites" that are not an interaction site for LTIP. Either mechanism is consistent with the dose-dependent suppression of LTIP activity and increased CETP activity seen when VLDL and LDL are the lipid donors and when HDL is the donor but the oleate-induced increase in CETP activity is low. The partial suppression of LTIP activity by oleate when HDL is the donor suggests that LTIP activity is relatively insensitive to oleate when the transfer mechanism switches from an exchange (shuttle) to a unidirectional (ternary complex) mechanism at higher FFA concentrations.19 20 34 Since LTIP functions by disrupting the binding of CETP to the lipoprotein surface,10 this explanation would require that the binding of CETP to lipoproteins is qualitatively different when CETP promotes shuttle transfer versus ternary complex transport.

The portion of plasma FFAs that is associated with lipoproteins has been debated.35 We have demonstrated a linear relationship between the FFA content of the total lipoprotein fraction and the increased electrophoretic mobility of lipoproteins in this fraction. Additionally, the FFA-induced shift in REM of isolated lipoproteins was very similar to the mobilities of lipoproteins in native plasma. We conclude that the FFA content of isolated lipoproteins (d<1.21-g/mL fraction) closely reflects the distribution of these compounds in plasma. This is consistent with the findings of Shafrir,38 who demonstrated that the high salt used to isolate lipoproteins by ultracentrifugation minimally affects the distribution of FFAs. Based on the basal valance values for plasma lipoproteins,39 the change in electrophoretic mobilities caused by FFAs (Fig 6Up), and typical plasma concentrations of lipoproteins,40 41 42 it is estimated that 7%, 48%, and 45% of the FFAs in the lipoprotein fraction are associated with VLDL, LDL, and HDL, respectively. These values closely approximate the distribution of phospholipid among plasma lipoproteins.10 40

Based on our studies with oleate, we estimate that plasma FFA concentrations of 0.8 to 1.0 mmol/L result in lipoprotein FFA levels that are sufficient to block the majority of LTIP activity. Notably, since palmitate partitions into lipoproteins more readily than oleate, slightly lower FFA levels may be sufficient to suppress LTIP activity in vivo. Therefore, under basal conditions (0.3 to 0.5 mmol/L FFA35 ), LTIP is maximally active and remains active during periods of carbohydrate consumption, which lower plasma FFA levels.43 However, after fat ingestion, overnight fasting, or extended exercise,43 44 the elevated FFAs that result from the increased lipolysis would suppress LTIP activity.

Elevated FFAs are common in a number of diseases, such as in diabetes, obesity, and nephrotic syndrome,35 and in normal aging and coronary heart disease.45 Under these circumstances, FFA-induced suppression of LTIP activity may exist under basal conditions, suggesting that these subjects may be functionally LTIP deficient. FFA levels are lowered by insulin.35 Therefore, the improvement in lipid transfer profiles toward normal noted in diabetics after insulin therapy may reflect, in part, alterations in LTIP activity.46

In normal plasma, VLDL TG is the rate-limiting step in the mass transfer of CE from LDL and HDL to VLDL.4 LTIP preferentially suppresses lipid transfer to and from LDL, thus allowing greater mass transfer along the VLDL-HDL pathway.9 37 When plasma FFA levels are elevated, the resulting suppression of LTIP would permit LDL to participate in mass transfer reactions to a greater extent. We suggest that in the presence of lipolytic activity, the enhanced lipid transfer between VLDL and LDL that results from LTIP suppression may facilitate the maturation of VLDL remnants to LDL-sized particles.1 Additionally, the reduced flux of CE from HDL would impair its ability to support lecithin:cholesterol acyltransferase activity and to mediate free cholesterol efflux from cells.9 47 During periods of energy demand, which typify conditions in which plasma FFAs are increased, such alterations may be beneficial.

In conclusion, we report that LTIP activity is remarkably sensitive to the FFA content of lipoproteins. The increased FFA content of plasma, which is typical during postprandial lipemia, fasting, and exercise, is sufficient to increase lipoprotein FFA levels to the point where LTIP activity is suppressed. These results suggest that changes in plasma lipid transfer activity that occur under these conditions15 16 are complex, reflecting both a direct stimulation of CETP activity and an inactivation of LTIP activity. The suppression of LTIP activity, while contributing to the rise in total CETP activity, also alters the relative participation of LDL and HDL in the CETP-mediated remodeling of lipoproteins. These studies illustrate another mechanism by which CETP activity may be controlled by lipid metabolism and metabolic status.


*    Selected Abbreviations and Acronyms
 
CE = cholesteryl ester
CETP = CE transfer protein
FFA = free fatty acid
LTIP = lipid transfer inhibitor protein
PL = phospholipid
REM = relative electrophoretic ability
TG = triglyceride


*    Acknowledgments
 
This research was supported by grant HL29582 from the National Heart, Lung, and Blood Institute, National Institutes of Health. This work was also supported by an Established Investigatorship (to R.E. Morton) from the American Heart Association and funds contributed in part by the Iva D. Savage Award of the AHA Maryland Affiliate, Inc.

Received January 31, 1997; accepted March 18, 1997.


*    References
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