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Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1642-1649
doi: 10.1161/hq1001.096722
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1642.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Markedly Elevated Lipid Transfer Inhibitor Protein in Hypercholesterolemic Subjects Is Mitigated by Plasma Triglyceride Levels

Richard E. Morton; Valéria Nunes; Lahoucine Izem; Eder Quintão

From the Department of Cell Biology (R.E.M., L.I.), Lerner Institute, Cleveland Clinic Foundation, Cleveland, Ohio, and the Lipids Laboratory (V.N., E.Q.), University of São Paulo Medical School, São Paulo, Brazil.

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


*    Abstract
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 *Abstract
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Abstract—— Lipid transfer inhibitor protein (LTIP, apolipoprotein F) regulates the interaction of cholesteryl ester transfer protein (CETP) with lipoproteins and is postulated to enhance the ability of CETP to stimulate reverse cholesterol transport. The factors that regulate LTIP levels and control its biosynthesis are unknown. Here, we demonstrate that plasma LTIP is dramatically increased (3-fold) in hypercholesterolemic subjects with normal to mildly elevated plasma triglyceride (TG) levels compared with control subjects. LTIP in these subjects is not correlated with the extent of hypercholesterolemia or with low density lipoprotein (LDL), high density lipoprotein, or CETP levels. However, unlike CETP, LTIP levels correlate negatively with plasma TG levels. This association does not appear to reflect decreased LTIP synthesis, inasmuch as conditions that stimulate TG synthesis and secretion (200 µmol/L oleate) do not reduce LTIP secretion by SW872 or Caco-2 cells. In contrast, native or acetyl LDL stimulates LTIP secretion 2-fold. Importantly, although plasma LTIP typically resides on LDL, up to 25% of LTIP is bound to very low density lipoprotein when this lipoprotein is enriched in cholesteryl esters, as occurs in hypercholesterolemia. In summary, LTIP levels are markedly elevated by hypercholesterolemia; however, plasma TG levels attenuate this response. We hypothesize that this arises from an increased association of LTIP with very low density lipoprotein, leading to a more rapid clearance of the inhibitor from circulation.


Key Words: hypercholesterolemia • cholesteryl ester transfer protein • lipid transfer inhibitor protein • triglycerides • lipoprotein metabolism


*    Introduction
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 up arrowAbstract
 *Introduction
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Cholesteryl ester transfer protein (CETP) mediates the flux of cholesteryl esters (CEs) and triglycerides (TGs) between lipoproteins.1,2 CETP facilitates a heteroexchange reaction, resulting in the net transfer of CEs toward CE-poor particles (VLDLs) with the reciprocal movement of TGs to CE-rich particles (LDLs or HDLs).3,4 Strong in vitro evidence demonstrates an important role for CETP in facilitating VLDL catabolism to LDLs and in promoting HDL metabolism and reverse cholesterol transport.3–5 However, the overall impact of CETP activity on atherogenesis has remained controversial, inasmuch as CETP can potentially facilitate processes that would appear to be proatherogenic and antiatherogenic.

CETP activity is regulated by another plasma component, lipid transfer inhibitor protein (LTIP). We recently purified and cloned LTIP and demonstrated its identity with apoF.6 Although LTIP was first identified simply by its capacity to suppress CETP activity in binary lipid transfer assays,7 it now appears that LTIP plays a more complex role in regulating CETP. CETP has little preference for interacting with different lipoprotein classes under steady-state conditions,8 and within a mixture of lipoproteins, CETP mediates transfer events between lipoprotein classes at rates that are largely determined by their relative concentrations.9 This finding contrasts with that seen in plasma, in which HDL appears to be a preferred CETP substrate.10–12 We have recently demonstrated that LTIP activity accounts for this discrepancy.9 This is hypothesized to occur because LTIP preferentially suppresses the interaction of CETP with LDL.13 Because VLDL concentrations are rate limiting in normal plasma to the CE-TG exchange process,14 the suppression of transfers with LDL results in a stimulation of lipid exchange between VLDL and HDL.9,15 Therefore, LTIP is a regulator of CETP function in that it controls the rate of individual lipid transfer reactions. We have proposed that LTIP augments the antiatherogenic capacities of CETP by stimulating reverse cholesterol transport.9,15

CETP synthesis is strongly upregulated by cholesterol,16,17 and elevated CETP levels are commonly observed in hypercholesterolemic subjects.18,19 This appears to be an adaptive response to enhance mechanisms responsible for sterol homeostasis. Because the beneficial actions of CETP are likely to be enhanced by LTIP, it seems reasonable that LTIP levels may be increased by similar stimuli. At present, nothing is known about the factors that influence LTIP biosynthesis. The purpose of the present study is to examine the response of LTIP to hyperlipidemia. The data show that LTIP levels are markedly increased in hypercholesterolemic subjects and also demonstrate an intriguing relationship between LTIP and plasma TG levels.


*    Methods
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 *Methods
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Lipoprotein Isolation and Derivation
Lipoproteins were isolated from fresh human plasma by sequential ultracentrifugation.20 In some instances, lipoproteins were labeled with cholesteryl[1{alpha},2{alpha}(n)-3H]oleate (50 Ci/mmol, Amersham Corp) before isolation.7 Labeled and unlabeled lipoproteins were extensively dialyzed against 0.9% NaCl, 0.01% EDTA, and 0.02% NaN3, pH 7.4, and stored at 4°C. LDL was biotinylated with NHS-biotin, as previously described,21 or acetylated by incubation with acetic anhydride.22

Human Subjects
Brazilian Subjects
Twenty-three patients with primary hypercholesterolemia were studied. None of the patients had diabetes mellitus or renal, hepatic, or other secondary causes of hyperlipidemia. Some (14 of 23) of these subjects are a subset of a previously reported group.23 Additionally, 46 normolipidemic control subjects in good clinical health and without any known cardiovascular risk factors were studied. For reasons explained below, these controls are reported as 2 separate groups (control 1 and control 2, Table 1). None of these subjects had taken drugs known to interfere with lipid metabolism for at least 2 months. Venous blood samples were drawn after an overnight fast, and plasma was obtained by low-speed centrifugation. Antibiotics and protease inhibitors were added.23 Plasma cholesterol and TG levels were measured with commercially available kits, and LDL cholesterol, apoB, and apoA-I levels were assayed as previously described.23 For most subjects, plasma was stored at -70°C until further analysis. Lipoproteins were isolated from a subset of fresh plasma samples as described above.


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  		Table 1. Characteristics of Study Groups


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  		Table 1A. (continued)

Subjects From the United States
Blood was collected from 7 healthy normolipidemic subjects. After centrifugation to isolate plasma, LDL and HDL cholesterol levels were measured by the LDL-Direct method (Isolabs, Inc). A pool of plasma was made by combining equal volumes of each donor plasma. After the addition of phenylmethylsulfonyl fluoride and paraoxon (1 mmol/L final for each), aliquots were frozen at -80°C.

All subjects signed a formal written consent approved by the ethics committees from the respective institutions. See Table 1 for characterization of these subject groups.

Preparation of Lipoprotein-Deficient Plasma
Lipoprotein-deficient plasma fractions were prepared by a modified divalent cation precipitation method.24 Briefly, 100 µL of freshly prepared 6.5% dextran sulfate (Mr {approx}5x105) was added to 1 mL plasma, mixed briefly, and incubated for 30 minutes on ice. Subsequently, 61 µL of 2 mol/L MnCl2 was added, mixed briefly, and incubated for 60 minutes on ice. After centrifugation (51 500g) for 25 minutes at 4°C, the supernatant was transferred to a second tube, and 122 µL of 15% BaCl2 was added, mixed briefly, and incubated on ice for 30 minutes. After centrifugation as described above, the supernatant was dialyzed against freshly prepared 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.02% EDTA, and 0.02% azide, pH 7.4 buffer (Tris/saline buffer). The dialysis consisted of two 2-hour steps versus 1 L of buffer each and then finally (overnight) versus 2 L of buffer. During each step, performed at 4°C, 1 g Chelex-100 (Bio-Rad Laboratories) was added to the dialysis buffer. The following day, dialysates were filtered through 0.45-µm syringe-type filters (Whatman Inc) to remove particulate material. Samples were stored on ice and assayed the same day for LTIP activity. This method quantitatively removed lipoproteins from normal and hyperlipidemic samples, as determined by lipid assay.

Assay of LTIP Activity in Lipoprotein-Deficient Plasma
LTIP activity in lipoprotein-deficient plasma was determined by measuring its capacity to suppress exogenous CETP.10,25 This assay was modified slightly as follows. Samples (30 to 40 µL) were combined with 500 µL of "assay mix," without or with partially purified CETP25 (sufficient to facilitate {approx}20% CE transfer) and Tris/saline buffer (pH 7.4 at 37°C) to yield a final assay volume of 700 µL. The assay mix consisted of the following: 10 µg [3H]CE LDL, 30 µg unlabeled LDL, 40 µg HDL, 14 µL of 100 mmol/L EDTA (pH 7.4), 200 µL of 3.5% BSA, 100 µL H2O, and sufficient Tris/saline buffer to make 500 µL. All components were kept on ice until use. After gentle mixing, samples were incubated at 37°C for 2 hours. Subsequently, tubes were placed in ice water for 15 minutes, and then the donor and acceptor lipoproteins were separated by selective precipitation.26 LTIP assays were performed over a 3-week period and used the same assay reagents. All samples were incubated in quadruplicate; duplicate samples received exogenous CETP, whereas the second duplicate was incubated without the addition of CETP.

LTIP activity was calculated by comparing the activity of exogenously added CETP in tubes containing lipoprotein-deficient plasma (after subtracting the CETP activity measured in samples without added CETP) to tubes containing exogenous CETP alone. To facilitate the comparison of values between assays and to correct for the nonlinear response of LTIP in this assay,7,10,21 these percent inhibition values were then read against a dose-response curve generated in each assay by using lipoprotein-deficient plasma prepared from pooled normolipidemic plasma (distinct from the plasma pool described in Table 1). An aliquot of this "standard" plasma was processed alongside each group of plasma samples. Because LTIP activity declines on long-term storage at -70°C, LTIP activities are reported as normalized values (ie, relative to the mean value of a control group of the same storage age) to facilitate comparison of samples stored for different times. The hypercholesterolemic samples are storage-age–matched to those in control 1 (21 subjects); hypercholesterolemic LTIP values are compared with only this control group to simplify data analysis.

Electrophoresis and Immunoblot Analyses
Samples were separated on 7.5% SDS-PAGE gels, electrotransferred, and reacted with a 1:500 dilution of anti-LTIP antibody 2 antisera as previously described.6 To measure LTIP on intact lipoproteins, 2 methods were used. Lipoproteins were resolved by agarose gel electrophoresis, transferred to nitrocellulose, and reacted with an equal volume mix of anti-LTIP antibody 1 and antibody 2 IgG.6 Alternatively, aliquots (5 µL) of isolated lipoproteins containing <=1.6 µg protein were applied to nitrocellulose with a slot-blot apparatus. After 30 minutes, wells were washed with 50 µL PBS, and then the membrane was blocked and reacted with anti-LTIP antisera as described before.6 Reaction products were visualized with enhanced chemiluminescent reagent (NEN Life Science Products, Inc). X-ray film images were captured by a ScanMaker III (Microtek Laboratory Inc) scanner and quantified with NIH Image software.

Cell Culture
SW872 adipocytes (American Type Culture Collection No. HTB 92) were grown in 100-mm dishes containing DMEM/F-12 medium with 5% calf serum and antibiotics. At confluence, cells were washed twice with serum-free medium and then incubated with the indicated agent in OptiMEM (GIBCO-BRL). After 24 hours, cells were washed twice in medium and then incubated in serum-free medium for 48 hours. This conditioned medium was centrifuged to remove cell debris and then assayed for LTIP activity and mass. LTIP activity was determined in a transfer assay containing [3H]LDL as donor and biotinylated LDL (10 µg cholesterol each) as acceptor.21 LTIP protein in conditioned medium (concentrated 10 times) was detected by SDS-PAGE/Western blot as described above, except that blots were reacted with 1:700 dilutions of anti-LTIP antibody 1 and antibody 2 antisera. CETP activity in conditioned medium was measured by its capacity to promote CE transfer from LDL to HDL1 during long-term incubation (18 hours). CE transfer activity was linear with the dose of medium and due fully to CETP, as assessed by monoclonal antibody against the transfer protein. The percentage of CE transfer was calculated as previously described.1

Cholesterol synthesis was measured by determining the extent of [14C]acetate incorporation into cholesterol. Cells, preincubated for 24 hours in medium containing 5% lipoprotein-deficient serum, received 0.5 µCi [14C]acetate (NEN Life Science Products, Inc) with or without 100 µg LDL in DMEM/F-12 medium for 24 hours. Afterward, cells were washed, and lipids were extracted27 and fractionated by thin-layer chromatography.3 Measurements of TG synthesis followed the same approach but monitored the incorporation of [9,10-3H(n)]oleic acid (NEN).

Other Methods
Protein, total and free cholesterol, CE, and TGs were quantified by published methods or with commercial kits, as previously described.9 CETP mass was determined by radioimmunoassay28 in the laboratory of Dr Ruth McPherson (University of Ottawa Heart Institute, Ottawa, Canada).

A nonparametric Mann-Whitney unpaired test was used to evaluate the difference between groups, unless indicated otherwise. Reported probability values are based on 2-tailed calculations. Results were considered significant at P<0.05. Linear correlation coefficients were determined by the Pearson (parametric) method.


*    Results
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 *Results
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LTIP Activity in Hypercholesterolemic Subjects
Increased CETP mass and activity in response to hypercholesterolemia are well documented.4,19 We have proposed that LTIP modulates the activity and function of CETP such that the pathways associated with reverse cholesterol transport are enhanced.9,15 This suggests that LTIP levels may be increased under conditions of cholesterol excess, such as hypercholesterolemia. To investigate this possibility, LTIP activity was measured in lipoprotein-deficient plasma fractions from control and hyperlipidemic subjects. Plasma cholesterol and TG levels in hypercholesterolemic subjects average 160% and 192% of control values, respectively, although the mean TG level of this group was within accepted normal ranges (Table 1). LTIP levels in control subjects recruited in Brazil varied nearly 4-fold (Figure 1), similar to that previously reported for normolipidemic subjects.25 A pool of plasma from 7 normolipidemic individuals recruited at the Cleveland Clinic had very similar LTIP levels (Figure 1). The 5 data points presented for this plasma pool were determined on separately prepared lipoprotein-deficient plasma fractions. Variability in these assay values (118.5±15.2% [mean±SD], n=5) reflects the combined reproducibility of the lipoprotein-deficient plasma preparation and the LTIP assay. Notably, compared with LTIP activity in control subjects, LTIP activity in hypercholesterolemic subjects was increased 3-fold (300.9±90.2% [mean±SD], P<0.0001; n=14; Figure 1). LTIP activity did not correlate with subject age or weight (body mass index) in either the hypercholesterolemic or control group. Furthermore, LTIP activity in male and female control subjects was similar (100±12.8% [n=7] versus 91.1±8.7% [n=4], respectively). Thus, it is not apparent, within the limitations of sample size, that the differences in age, body mass index, and sex between these 2 study groups contribute significantly to the large differences in LTIP activity observed.



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  		Figure 1. LTIP activity in control and hypercholesterolemic subjects. LTIP activity was measured in lipoprotein-deficient plasma fractions prepared from control group 1 (n=13) and hypercholesterolemic (n=14) subjects as described in Methods. A pool of control subjects (control pool, see Table 1) was prepared from 7 normolipidemic subjects. LTIP activity values are presented relative to the mean value of the control samples (percentage of control). Control lipoprotein-deficient plasma (30 µL) suppressed exogenous CETP by 22.9%. Each data point is the mean of duplicate determinations. Bars reflect the mean value for each group. LTIP activity in hypercholesterolemic subjects was 3-fold higher than that in control subjects (P<0.0001).

Although not suitable for immunoassays, the anti-LTIP antibodies available6 do permit an estimation of LTIP mass by Western blotting. Among 9 individuals, selected from various ongoing studies to represent a wide range in LTIP activities, immunodetectable LTIP protein in lipoprotein-deficient plasma increased nearly linearly with LTIP activity (Figure 2A). Among 4 control and 4 hypercholesterolemic subjects that expressed LTIP activities similar to the means of their respective groups, LTIP mass was significantly increased in hypercholesterolemia (Figure 2B). According to the data in Figure 2A, the increase in LTIP immunoreactivity was consistent with the fold increase in LTIP activity noted in this hyperlipidemic group. This higher level of LTIP protein, measured in lipoprotein-deficient plasma, was also readily observed in whole plasma (Figure 2C). These data show that LTIP protein recovery in lipoprotein-deficient plasma is nearly quantitative.



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  		Figure 2. Immunoblot of LTIP. Lipoprotein-deficient plasma samples (0.5 µL equivalent) were reduced, denatured, and fractionated on SDS-PAGE as described in Methods. Proteins were transferred to polyvinylidine difluoride and reacted with anti-LTIP antisera. A, LTIP immunoreactivity, determined by image analysis, vs LTIP activity. Nine samples containing widely varying levels of LTIP activity were evaluated for relative LTIP protein content by Western blot. B, LTIP immunoreactivity in control and hyperlipidemic lipoprotein-deficient plasmas. Representative control and hypercholesterolemic subject samples were analyzed for LTIP protein content. Bars are mean±SD (n=4); mean values were significantly different (P<0.0001). Inset shows the LTIP immunoblot for each subject. C, LTIP immunoreactivity in lipoprotein-deficient plasma vs plasma. Ten microliters of plasma (diluted 1:40) and lipoprotein-deficient plasma are compared on an equal protein basis. Hyperchol. indicates hypercholesterolemic; LPDP, lipoprotein-deficient plasma.

Relationship Between LTIP Activity and Other Plasma Parameters
Although hypercholesterolemic subjects as a group displayed a 3-fold increase in LTIP activity, there was considerable heterogeneity within this group. Linear regression analysis demonstrated that whereas LTIP activity was increased in hypercholesterolemia, there was no apparent correlation between LTIP activity and plasma cholesterol levels (Table 2). However, LTIP was negatively correlated with the extent of hyperlipidemia (plasma cholesterol+TGs). This association was largely due to plasma TGs, inasmuch as LTIP levels were strongly and inversely correlated with this lipid (Table 2). No correlation between LTIP activity and HDL or LDL levels or apoA-I, apoB, or CETP concentration was apparent with this sample size.


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  		Table 2. Pearson Correlation Coefficients for LTIP vs Lipoprotein and Lipid Variables

The relationship between plasma LTIP activity and TG levels is shown graphically in Figure 3A. LTIP activity decreased with a slope of -1.32, resulting in a 40% reduction in LTIP as plasma TG levels doubled. This negative relationship, based on LTIP activity, was also evident at the protein level in lipoprotein-deficient plasma and whole plasma (Figure 3B). These data further demonstrate that LTIP recovery in lipoprotein-deficient plasma is unaffected by hyperlipidemia. In contrast to that seen for LTIP, plasma CETP mass increased by >50% over the same TG range. Although this correlation did not reach statistical significance for this limited sample population (r=0.495, P=0.072; Figure 3C), these data clearly demonstrate that the relationship of CETP and LTIP with TG levels in these subjects is distinct and opposite. The negative correlation between LTIP activity and TG levels, noted in hypercholesterolemic subjects, was also observed in control subjects (Figure 3D) but fell just short of reaching statistical significance (r=-0.315, P=0.061).



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  		Figure 3. Relation of LTIP and CETP to plasma TG levels. A, Plasma LTIP activities in hypercholesterolemic subjects are plotted vs plasma TG levels. B, LTIP immunoblots of LPDP and plasma from hypercholesterolemic subjects with low or high TG levels are shown. C, Plasma CETP mass in the same hypercholesterolemic subjects shown in panel A are plotted vs plasma TG levels. The CETP mass values are a subset of a previously reported group.23 The linear correlations shown have coefficient (r) and probability (P) values of -0.645 and 0.013 (A), respectively, and 0.495 and 0.072 (C), respectively. D, LTIP activities in normolipidemic subjects (control groups 1 and 2, n=36) are plotted against plasma TG concentrations (r=-0.315, P=0.061). Mean CETP inhibition induced by these lipoprotein-deficient plasma fractions (30 µL) was 26.3%.

The strong negative correlation between LTIP activity levels and plasma TG levels in hypercholesterolemic subjects may result from at least 2 possible mechanisms. LTIP synthesis may be suppressed under conditions of increased TG-rich lipoprotein biosynthesis. Alternatively, the plasma lifetime of LTIP may be shortened because of the increased clearance of the protein from circulation. The possible involvement of these 2 mechanisms in the LTIP-TG relationship is examined below.

Regulation of LTIP Biosynthesis in Cultured Cells
Several cell types involved in lipoprotein/lipid synthesis, which are known to secrete CETP, were tested for LTIP secretion. Although HepG2 cells have been reported to secrete CETP inhibitor activity,29 we have been unable to detect LTIP protein synthesis by these cells. In contrast, LTIP activity and protein were readily measurable in the conditioned medium from SW872 adipocytes and Caco-2 intestinal epithelial cells. LTIP activity accumulated in the medium in a near-linear fashion (<=72 hours) for both cell types (data not shown). Compared with serum-starved control cells, SW872 cells incubated with LDL-containing medium increased the secretion of LTIP activity 2-fold (Figure 4A). LTIP mass in conditioned medium was increased to a similar extent (Figure 4A, inset). The addition of LDL suppressed cholesterol biosynthesis 3-fold (293±21 versus 114±15 cpm/µg cell protein). Similarly, the addition of acetylated LDL (100 µg) to SW872 cells increased LTIP secretion (181±51% of control). CETP secretion was stimulated by LDL (Figure 4B) and acetyl LDL.30 Limited studies with Caco-2 cells showed similar changes in LTIP secretion after LDL or acetyl LDL supplementation (not shown). Thus, independent of the receptor-mediating lipoprotein uptake, LTIP secretion is stimulated by cholesterol delivery to cells. Like CETP, LTIP appears to be positively regulated by cholesterol.



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  		Figure 4. Secretion of LTIP and CETP by cultured SW872 adipocytes. A, Conditioned medium (48 hours) was collected from adipocytes after treatment of cells for 24 hours with the agent indicated. Bars are the mean±SE of the LTIP activities measured in the medium from separate experiments: control (none), n=5; LDL (100 µg/mL), n=5; and oleate (200 µmol/L), n=3. Probability values for treatment groups vs the no-addition control group were calculated by the paired Student t test. NS indicates not significant. Inset shows Western blot of LTIP protein contained in conditioned medium (concentrated 10 times). Compared with LTIP isolated from plasma (leftmost lane), LTIP in conditioned medium has a slightly higher apparent molecular mass, suggesting differences in glycosylation. B, Conditioned medium (200 µL) from cells incubated as described in panel A was assayed for CETP content by determining lipid transfer activity. The percentage of CE transfer was calculated as previously described and is expressed as %kt. See Methods for details. Results are the mean±SD of 2 or 3 determinations and are representative of 3 separate experiments. Probability values for treatment groups vs the no-addition control group were calculated by the Student t test.

To evaluate the potential connection between LTIP secretion and TG biosynthesis, cells were incubated with oleate (200 µmol/L) before the conditioned medium was collected for LTIP activity measurements. This level of oleate maximally stimulates TG synthesis and TG-rich lipoprotein secretion in cultured cells.31,32 On the basis of acetate incorporation studies, 200 µmol/L oleate induced a >10-fold increase in TG synthesis (165±8 versus 2259±420 cpm/µg cell protein). However, there was no significant change in the amount of LTIP activity or protein secreted by oleate-treated cells (Figure 4A). CETP secretion, on the other hand, was increased 2-fold under these conditions (Figure 4B). In Caco-2 cells, in which fatty acids stimulate lipoprotein biosynthesis,33 oleate also failed to significantly alter LTIP secretion (not shown). Thus, there appears to be no clear connection between LTIP synthesis and TG biosynthesis. Overall, these data do not account for the negative relationship between LTIP and TG levels in hypercholesterolemic subjects but do suggest a causal link between sterol metabolism and the high LTIP activity in these subjects.

Effect of Plasma TG Levels on LTIP-Lipoprotein Association
LTIP is primarily associated with LDL in plasma.6,13 However, small variable amounts of LTIP are detected on VLDL in normolipidemic individuals,6,13 suggesting that the distribution of LTIP among plasma lipoproteins may be more heterogeneous than previously appreciated, especially in dyslipidemic subjects. Redistribution of LTIP from LDL to other apoB-containing particles could shorten its plasma residence time because of the more rapid clearance of these particles. To assess how variations in plasma VLDL levels affect this distribution, lipoproteins were isolated from various donors by ultracentrifugation, and LTIP was quantified by immunoblot. Representative data are shown in Figure 5A. Among 14 normolipidemic control subjects, there was no apparent relationship between the amount of LTIP recovered in the VLDL fraction and VLDL concentration expressed as TGs (Figure 5B). For hypercholesterolemic subjects, the fraction of LTIP on VLDL increased with plasma VLDL TG levels, but this correlation failed to reach statistical significance (r=0.620, P=0.075; Figure 5B). This finding is consistent with our observation that supplementation of plasma with excess exogenous VLDL does not induce LTIP redistribution (data not shown). However, there was a significant correlation between LTIP associated with VLDL and its chemical composition. In hypercholesterolemic subjects (r=0.874, P=0.0021; Figure 5C) and normolipidemic control subjects (r=0.666, P=0.0093; Figure 5D), there is a significant positive correlation between the percentage of LTIP on VLDL and the CE content (CE/protein) of VLDL. On average, the CE/protein ratio of hypercholesterolemic VLDL was significantly higher than that in control VLDL (1.68±0.42 versus 1.23±0.35, respectively; P=0.014). The LTIP content of hypercholesterolemic VLDL, but not control VLDL, also correlated with VLDL CE/TG (r=0.696, P=0.037). Variations in LDL properties (LDL TG/protein or LDL size, as determined by chromatography) did not correlate with the LTIP content of either normal or hypercholesterolemic VLDL. This suggests that changes in VLDL, not LDL, composition lead to LTIP redistribution.



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  		Figure 5. LTIP association with VLDL. A, Representative immuno–slot blot of LTIP in VLDL and LDL isolated from hypercholesterolemic (n=4) and control (n=3) subjects. Results from control subjects are shown in lanes 3, 5, and 6 (anti-LTIP blot, left to right) and lanes 2 and 4 (nonimmune blot). For detailed studies, LTIP levels in VLDL and LDL were quantified by image analysis of duplicate blots for 9 hypercholesterolemic (closed symbols) and 14 control (open symbols) samples. B, Percentage of LTIP on VLDL vs VLDL TG concentration is shown. The solid line shows the regression for hypercholesterolemic subjects (r=0.620, P=0.075). There was no apparent correlation for control subjects. C and D, For the same subjects, the percentage of LTIP on VLDL vs VLDL CE/protein content is shown in panel C (hypercholesterolemic subjects; r=0.874, P=0.0021) and panel D (control subjects; r=0.666, P=0.0093). E, Plasma aliquots were incubated for 0 to 24 hours at 37°C in the presence of 1 mmol/L paraoxon. After incubation, lipoproteins were isolated, and LTIP was quantified (triplicate readings) after separation by agarose gel electrophoresis (see Methods). Inset, Immunoreactive LTIP recovered in the VLDL fraction (4 µL plasma equivalent) at each time point is shown.

The foregoing data suggest that VLDL composition, not VLDL mass per se, determines the redistribution of LTIP. To further investigate this connection, plasma was incubated in vitro to permit CETP to mediate the enrichment of VLDL with CE.3,4 Analysis of the VLDL isolated from plasma incubated for varying times shows that its LTIP content increases severalfold over time and that this is well correlated with changes in VLDL CE (Figure 5E). However, it is important to note that the magnitude of this in vitro–induced redistribution of LTIP was highly dependent on the donor plasma studied. For some plasma samples, although the enrichment of VLDL with CE occurred as expected, the redistribution of LTIP was near detection limits. This suggests that the CE/protein ratio of VLDL from native plasma may be a marker for another feature of VLDL (such as particle size) that increases its affinity for LTIP. The ability of in vitro remodeling reactions to replicate this feature may be highly dependent on the properties of the VLDL fraction initially present. Nonetheless, these studies strongly suggest that a significant portion of plasma LTIP can be recovered in the VLDL fraction and that the properties of VLDL determine this association.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
CETP has similar affinities for VLDL, LDL, and HDL8 and promotes lipid transfer among different lipoprotein classes with little preference.9 However, LTIP, presumably because of its primary association with LDL,13 retards lipid transfer events involving LDL,13 and the addition of LTIP to physiological mixtures of lipoproteins restores the apparent preference of CETP for HDL that is seen in human plasma.9 Thus, LTIP provides directionality to CETP activity. Because LTIP reduces the heteroexchange of CEs and TGs between LDL and VLDL, the CETP-mediated net CE flux from HDL to VLDL is stimulated.9 On the basis of this, we have hypothesized that LTIP promotes reverse cholesterol transport by facilitating the transfer of HDL-derived CEs to VLDL, which is then rapidly cleared from circulation by the liver after conversion to remnants.15

Very little is known about factors regulating the levels of LTIP. Plasma LTIP activity is markedly decreased in uremic patients on continuous peritoneal dialysis10 and may be decreased or increased in uremic patients on hemodialysis.10,34 However, the mechanisms underlying this altered LTIP activity are not known. Furthermore, the response of LTIP to altered lipid status is not known. Given its hypothesized role in augmenting reverse cholesterol transport, it seems likely that LTIP levels may be increased in conditions in which the need to eliminate cholesterol is enhanced. As a first step in understanding the factors that regulate LTIP, we have measured LTIP levels in hypercholesterolemic subjects. In the present study, we report for the first time that plasma LTIP (activity and mass) is increased 3-fold above control levels in individuals with elevated cholesterol. However, the elevated LTIP levels do not correlate with plasma cholesterol levels in these subjects and are unrelated to LDL, HDL, or CETP concentrations. This contrasts with the strong negative correlation between LTIP levels and plasma TG concentration. These data demonstrate that LTIP levels are strongly influenced by plasma TG concentrations within the normal and mildly elevated range.

Two possible mechanisms underlying the negative association of LTIP and TG were investigated. We report that cells of adipose and intestinal origin synthesize LTIP. LTIP biosynthesis and secretion is upregulated by cholesterol delivery to cells. This is similar to that observed for CETP30,35 (and also measured in the present study), suggesting that LTIP has a positive sterol response element within its promoter. This novel finding provides a possible mechanism whereby LTIP is increased in hypercholesterolemia. However, LTIP synthesis was not significantly altered by conditions that stimulate triglyceride synthesis. This contrasts with CETP secretion, which is increased in this situation (Izem and Morton30, Faust and Albers,36 and present study). These results show that LTIP biosynthesis is not decreased under conditions in which TG-rich lipoprotein secretion is enhanced31,33 and does not support this process as a plausible mechanism for the negative association of plasma LTIP and TG levels.

An alternative mechanism for the negative correlation of LTIP and plasma TG levels is that a portion of LTIP redistributes to VLDL as its concentration increases. Increased association with VLDL could decrease LTIP levels, because this lipoprotein is more rapidly cleared from circulation. We observed that LTIP is variably associated with the VLDL fraction when evaluated in multiple individuals but that this is poorly correlated with the concentration of VLDL (expressed as TGs) in plasma. However, we report the interesting finding that LTIP association with VLDL is well correlated with the CE content of the VLDL fraction. This correlation was highly significant for hypercholesterolemic and control subjects. This suggests that LTIP association with VLDL is determined by specific properties of VLDL. Elevated CE content of VLDL can result either from an increase in CE within the nascent VLDL particle or from elevated remodeling by CETP-mediated lipid transfer processes. Both of these processes are likely in hypercholesterolemia.14,37 However, the success of in vitro attempts to induce the redistribution of LTIP from LDL to VLDL by enriching VLDL in CE have not been consistently successful and are highly dependent on the properties of the starting plasma. This strongly suggests that the CE content of plasma VLDL may generally reflect other properties of VLDL (such as particle size) that are not easily manipulated in vitro with transfer reactions. Nonetheless, these studies clearly show that the distribution of LTIP among VLDL and LDL is variable and dependent on the physicochemical properties of VLDL.

We suggest that the redistribution of LTIP to VLDL particles may explain the negative correlation of LTIP and plasma TG levels in hypercholesterolemic subjects. We demonstrate that LTIP progressively associates with VLDL as it becomes enriched in CE. Although a similar percentage of LTIP is associated with VLDL in hypercholesterolemic and control subjects for a given VLDL CE content (Figure 5C and 5D), the 3-fold higher LTIP levels in hypercholesterolemic subjects results in a significant increase in total LTIP associated with VLDL. Additionally, the CE content is significantly higher in hypercholesterolemic VLDL than in control VLDL, as has been reported in other hyperlipidemic situations.38,39 This may occur for 2 reasons: First, plasma CETP concentrations are increased.19,23 Second, because the rates of net lipid transfer in plasma are strongly influenced by the concentration of VLDL TG,14 CETP-mediated remodeling of VLDL composition is likely to be increased in these subjects and progressively enhanced as TG concentrations increase. Because VLDL and its remnants are more rapidly cleared from circulation than LDL, we hypothesize that the negative correlation of LTIP with plasma TG levels can be explained by the increased turnover of LTIP from the plasma compartment that is due to the redistribution of LTIP to particles with shorter plasma lifetimes. If such a mechanism is verified, this may provide an explanation for the profoundly decreased plasma LTIP levels in patients undergoing continuous ambulatory peritoneal dialysis. These patients, because of the infusion of glucose, have significantly increased VLDL synthesis and flux40 as well as VLDL levels that are elevated in their CE content.10

In summary, we report the novel finding that plasma LTIP levels are markedly increased in hypercholesterolemic subjects and that the extent of this increase is partially mitigated by plasma elevated TG concentrations. In cultured cells, LTIP synthesis and secretion are increased by cholesterol delivery, suggesting that the high levels of LTIP in hypercholesterolemia reflect the presence of a positive sterol response element in the LTIP gene. The negative correlation of LTIP levels with plasma TG levels may be related to a partial redistribution of LTIP from its normal localization on LDL to VLDL, resulting in a more rapid turnover of LTIP from the plasma compartment. This plausible testable hypothesis is under investigation. Increased turnover of LTIP could compromise the antiatherogenic potential of this protein,15 providing another link between elevated plasma TG levels and coronary heart disease.


*    Acknowledgments
 
This research was supported by grant HL-60934 from the National Heart, Lung, and Blood Institute, National Institutes of Health, and a grant from S. Paulo State Research Foundation (FAPESP, No. 99/10735-9). The authors are grateful to Drs Alexandre Carrilho, Eliana C. de Faria, and Águeda Zaratin for the selection and care of the patients.

Received May 14, 2001; accepted July 16, 2001.


*    References
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 *References
 

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