Plasma lipases and lipid transfer proteins increase phospholipid but not free cholesterol transfer from lipid emulsion to high density lipoproteins.
© Nunes et al; licensee BioMed Central Ltd. 2001
Received: 29 November 2000
Accepted: 20 February 2001
Published: 20 February 2001
Plasma lipases and lipid transfer proteins are involved in the generation and speciation of high density lipoproteins. In this study we have examined the influence of plasma lipases and lipid transfer protein activities on the transfer of free cholesterol (FC) and phospholipids (PL) from lipid emulsion to human, rat and mouse lipoproteins. The effect of the lipases was verified by incubation of labeled (3H-FC,14C-PL) triglyceride rich emulsion with human plasma (control, post-heparin and post-heparin plus lipase inhibitor), rat plasma (control and post-heparin) and by the injection of the labeled lipid emulsion into control and heparinized functionally hepatectomized rats.
In vitro, the lipase enriched plasma stimulated significantly the transfer of 14C-PL from emulsion to high density lipoprotein (p<0.001) but did not modify the transfer of 3H-FC. In hepatectomized rats, heparin stimulation of intravascular lipolysis increased the plasma removal of 14C-PL and the amount of 14C-PL found in the low density lipoprotein density fraction but not in the high density lipoprotein density fraction. The in vitro and in vivo experiments showed that free cholesterol and phospholipids were transferred from lipid emulsion to plasma lipoproteins independently from each other. The incubation of human plasma, control and control plus monoclonal antibody anti-cholesteryl ester transfer protein (CETP), with 14C-PL emulsion showed that CETP increases 14C-PL transfer to human HDL, since its partial inhibition by the anti-CETP antibody reduced significantly the 14C-PL transfer (p<0.05). However, comparing the nontransgenic (no CETP activity) with the CETP transgenic mouse plasma, no effect of CETP on the 14C-PL distribution in mice lipoproteins was observed.
It is concluded that: 1-intravascular lipases stimulate phospholipid transfer protein mediated phospholipid transfer, but not free cholesterol, from triglyceride rich particles to human high density lipoproteins and rat low density lipoproteins and high density lipoproteins; 2-free cholesterol and phospholipids are transferred from triglyceride rich particles to plasma lipoproteins by distinct mechanisms, and 3 - CETP also contributes to phospholipid transfer activity in human plasma but not in transgenic mice plasma, a species which has high levels of the specific phospholipid transfer protein activity.
There have been plenty of epidemiological, clinical and experimental evidence that plasma high density lipoproteins levels are inversely correlated with the risk of atherosclerosis [1,2]. The contribution of enzymes and proteins associated with HDL to its process of generation and maturation have been extensively studied, both in vitro and in vivo.
The plasma cholesteryl ester transfer protein (CETP) modulates HDL levels and composition. It mediates the transfer of cholesteryl ester (CE) from HDL to triglyceride (TG) rich lipoproteins (LP), while TG is transferred in the opposite direction, to HDL. One way CE transfer from HDL to LDL may also occur . CETP also promotes phospholipid (PL) transfer to human HDL [4,5,6]. CETP activity has been directly correlated with LDL cholesterol levels and inversely correlated with HDL cholesterol levels in human plasma [3,7].
PLTP, a specific phospholipid transfer protein, has been identified in human plasma [8,9] and in plasma of other vertebrate species . It promotes the PL transfer from VLDL to HDL . In addition to PL, PLTP transfers free cholesterol (FC) from PL/FC vesicles to HDL, although with a low efficiency . Both, CETP and PLTP, can promote HDL remodelling. While CETP, together with hepatic lipoprotein lipase, stimulates the generation of small alpha-HDL, PLTP favours the emergence of large alpha-HDL particles . Significantly higher levels of HDL-cholesterol were observed in human PLTP transgenic mice . Furthermore, overexpression of human PLTP produced by recombinant adenovirus injection into mice, resulted in increased levels of prebeta-HDL, increased fractional catabolic rate and liver uptake of CE and PL from HDL .
After intravascular hydrolysis of TG rich LP by lipoprotein lipase (LPL), surface remnant components such as FC, PL and apoproteins may provide substrates for generation or modification of plasma HDL. Net transfer of PL and FC from chylomicrons and VLDL to HDL has previously been demonstrated in rats [16,17] and in human plasma after a fat meal [18,19] or during lipolysis . The contribution of the lipolysed LP components to HDL formation has been reinforced by several studies where the activity of the enzyme lipoprotein lipase LPL was shown to correlate with HDL cholesterol levels in human plasma [20,21,22]. However, changes in the HDL-cholesterol concentration have not been observed in mice overexpressing LPL  or in LPL heterozygous knockout mice .
The metabolism of HDL in rats and mice differs significantly from that in humans. Part of the species differences observed in mice and in rats may result from their high levels of circulating lipases [25,26], lack of CETP  and high levels of PLTP . Clee et al.  have shown that, in double transgenic mice overexpressing LPL and CETP, HDL cholesterol levels were significantly influenced by the LPL activity while no such correlation was observed in the absence of CETP expression.
In this study we have further evaluated the influence of plasma lipases and CETP on the free cholesterol and phospholipid transfer from triglyceride rich lipid emulsion similar to chylomicrons [29,30] to human, rat and mouse lipoproteins, in vitro and in vivo.
Influence of plasma lipases on the 3H-free cholesterol (FC) and 14C-phospholipid (PL) transfer from lipid emulsions to the human plasma lipoproteins.
Influence of plasma lipases on the distribution of 14C-free cholesterol (FC) and 3H-phospholipid (PL) from lipid emulsions to plasma lipoproteins of hepatectomized rats in vivo.
(53 - 74)
(27 - 61)
(43 - 55)
(14 - 22)
LDL + remnants
(11 - 24)
(10 - 26)
(29 - 38)
(33 - 42)
(15 - 23)
(29 - 47)
(14 - 22)
(40 - 51)
Influence of CETP on the 14C-phospholipid (PL) transfer from lipid emulsions to the human plasma lipoproteins.
4 (0 - 6)
0 (-5 - 1)
30a (26 - 38)
18a,b (16 - 27)
Several studies, using different experimental approaches, have shown that the transfer of surface components of TG-rich lipoproteins during their intravascular metabolism is important to determine both level and chemical composition of the HDL subfractions. The present work has confirmed and extended previous observations showing that human and rat plasma lipoprotein lipases stimulate PLTP mediated PL transfer from TG-rich particles to HDL but do not influence the FC transfer process. Some of the previous studies [11,35,36,37,38] that used purified exogenous lipases in in vitro incubations with isolated LP displayed a potent stimulation of the PL transfer to HDL. In order to prepare a model that would mirror a physiological system more closely, we have used whole plasma and maximal endogenous lipases activity through heparin administration in vivo as well as in vitro. It is possible that other proteins released by heparin in the vascular bed may have played a role in stimulating PL transfer. The role of circulating lipases was confirmed through the use of a lipase inhibitor (THL), which abolished the stimulating effect of lipase-enriched plasma on the PL transfer to HDL (Tab. 1). According to these experiments it is likely that during fasting state, where no circulating lipases are detectable, all PL transfer results from the action of the lipid transfer proteins, PLTP and CETP, while in a post-prandial state, when lipases expectedly are more active, the PL transfer to HDL could be raised by 60% or more due to a greater substrate supply .
Noteworthy the in vitro PL transfer to HDL in the basal plasmas of rat (fig. 1, 82%) and mouse (fig. 2, 57%) was higher than in the human's (Tab. 1, 31%) and could be ascribed to the high levels of circulating lipases [26,39,40] and PLTP  found in those species. This could also explain the positive correlation between the lipoprotein lipase activity and HDL concentration in human plasma [20,21,22] but not in mice with genetically modified expression of LPL [23,24,28].
The in vivo studies showed that the EM PL transfer to HDL did not differ in control and in LPL stimulated (heparin treated) hepatectomized rats. Instead, the LDL density fraction was PL enriched in the heparinized animals. This could be explained by several and not exclusive possibilities. First, the lipolysis stimulation by heparin generates more remnants of the EM that would float in the same density range as LDL (1.006 - 1.063). Second, the rat plasma fraction smaller than HDL that appeared in the in vitro incubations with post-heparin plasma (Fig. 1) could also occur in vivo and float in the same density range as LDL (1.006 -1.063). In this regard, O'Meara et. al.  had shown that small HDL particles from heparinized hypertriglyceridemic subjects, identified in non-denaturing gel electrophoresis and by electron microscopy, floated after ultracentrifugation in a less dense range. Those authors had considered it an aberrant result of the ultracentrifugation technique. Third, the diminished availability of PL donor particles in the plasma of the heparinized rats (yield of 50%) as compared to control rats (yield of 90%) would be responsible for the apparent lack of stimulation of the PL transfer to the HDL fraction. Finally, other in vivo metabolic fates of PL would compete for the transfer process to plasma HDL particles in LPL stimulated animals.
The emulsion FC transfer to HDL was about 40% that of PL in both, in vitro (Tab. 1) and in vivo (Tab. 2) studies and it was not influenced by increased rate of intravascular lipolysis. These results suggest that the FC transfer is a slower, probably passive, process distinct from the facilitated PL transfer mechanism [8,9]. Also, these results challenge the possibility that new HDL particles are made from the EM surface peeling off during lipolysis because the relative PL/FC ratio was higher in HDL than in the CM+VLDL and emulsion fraction. Others also have shown that FC transfer to HDL is a slow process: FC increases in the HDL fraction only 5 to 8 h after a fat meal  or only after 2 h incubation of HDL with VLDL and purified rat heart LPL .
We have also confirmed previous studies  claiming that PL transfer from TG-rich particles to human HDL is facilitated by CETP, since its partial inhibition significantly reduced the PL transfer (Table 3). However, human CETP expressed in the transgenic mice plasma had no effect whatsoever on the PL transfer to mouse plasma lipoproteins or to human HDL. This lead us to admit that the mouse plasma PL transfer activity is so potent that some additional protein (CETP) with PL transfer activity would be irrelevant in an already saturated in vitro system.
In summary, the present findings indicate unequivocally the importance of the intravascular lipolytic mechanisms for the PLTP and CETP facilitated PL, but not FC, transfer process from TG-rich particles to HDL. PL enriched HDL would be more efficient in promoting FC efflux from cell membranes, hence accelerating the reverse cholesterol transport. These may provide the basis for the mechanism that accounts for the inverse correlation between HDL and TG plasma levels found in epidemiological studies in human populations as well as in several circumstances where plasma lipid levels are modified by pharmacological and dietary means.
Materials and Methods
Cholesterol (FC), cholesteryl oleate (CO) and triolein (TO) were obtained from NuCheck Prep (Elysian, MN, USA) and lecithin (PL) from Lipid Products (Surrey,UK). They were more than 99% pure as tested by thin layer chromatography. Lipid mixtures (2% FC, 6% CO, 23%PL and 69%TO by weight) together with 130 μCi of L-α-dioleoyl [1-14C]-phosphatydylcholine and 25 μCi of [1,2-3H(N)]-cholesterol (New England Nuclear, Boston, MA, USA) were sonicated in 2.785 M NaCl solution (d=1.101 g/ml) utilizing a Branson Cell Disruptor (Branson Ultrasonics Corp., Danbury, CT, USA), model B30, 1 cm probe, with continuous output of 70-80 W, at aproximately 55°C, for 30 minutes, under N2 flow. Triglyceride-rich particles were purified after discontinuous gradient ultracentrifugation of NaCl solutions with densities 1.065, 1.020 and 1.006 g/ml. A first step of 12000 rpm for 15 min in a SW41 Beckman rotor at 22°C was performed to discard the floating coarse lipid. After replacing the 1.006 solution, the gradient was again centrifuged at 36000 rpm, for 30 min at 22°C and the emulsion particles were recovered from the top layer. The lipid emulsion composition achieved was: 1% FC, 4% CO, 14% PL and 81% TO. These particles resemble native chylomicrons . By gel filtration (FPLC), 3H-FC, 14C-PL and triglyceride co-eluted as only one peak corresponding to the plasma VLDL size fraction (fractions # 13 to 17) on a HR 10/30 superose 6 column (Pharmacia Biotech, Uppsala, Sweden).
Sources of plasma
Human blood samples from 12 fasted healthy volunteers (5 men and 7 women, total cholesterol and triglycerides < 200 mg/dl), were drawn on EDTA, pre (basal) and 10 min after an I.V. bolus injection of heparin (100 U/kg BW). Male Wistar rats, approximately 300 g, had their carotid arteries cannulated under pentobarbital anesthesia. After recovery, they received saline (control) or heparin (LPL stimulated) and after 10 min they were exsanguinated on EDTA. Adult male C57Bl6 and human CETP transgenic mice (line 5203), derived from the colony of Dr AR Tall's Laboratory (Columbia University, NY, USA) were bled with heparinized hematocrit capillary tubes in the retro-orbital plexus under ketamine anesthesia (Vetarnacol, Konig, SP, Brazil). All plasmas were obtained by centrifugation at 2000 rpm in a Sorval RT6000B refrigerated centrifuge and freshly used.
Free cholesterol (FC) and phospholipid (PL) transfer assay
Control and treated plasmas (post-heparin, post-heparin + lipases inhibitor and control + CETP monoclonal antibody, TP2, provided by Dr. AR Tall) were incubated with lipid emulsion (∼ 700 μg of triolein/ml) labeled with 3H-FC (106 dpm/ml) and 14C-PL (2 × 105 dpm/ml) at zero (on ice, "time zero") and for 30 minutes at 37°C. Plasma lipoproteins were next separated by fast protein liquid chromatography (FPLC) as described by Jiao et al. . Briefly, plasma samples (200 μl) were fractionated on a HR 10/30 Superose 6 column (Pharmacia Biotech, Uppsala, Sweden) using a constant flow of 0.5 ml/min of tris-buffered saline, pH 7.2. Sixty fractions of 0.5 ml were automatically collected. 3H- and 14C- dpm of each FPLC fraction was determined by liquid scintilation in a beta counter Beckman LS6000TA. As we have measured only radioactive FC and PL, the term "transfer" is used to describe either net transfer or exchange process among LP. Total cholesterol was also determined in the fractions #10 to 40 by an enzymatic assay in an automatic analyzer Cobas (F. Hoffman-La Roche, Basileia, Switzerland) using Boehringer Mannheim reagents (Mannheim, Germany).
In Vivo studies
Male Wistar rats, weighing ∼ 300 g were anesthetised with pentobarbital ip (50 mg/Kg BW). The right carotid artery was cannulated with a PE 50 siliconized catheter and after laparostomy, the liver hilum was ligated. Physiologic solution (control) or heparin (250 U/Kg BW) in a final volume of 0.25 ml was injected through the carotid catheter. After 10 minutes, labeled lipid emulsion (4 × 105 dpm of 3H-PL and 7 × 105 dpm of 14C-FC) was injected intra-arterially. After 30 minutes, the animals were exsanguinated by the carotid catheter and plasma lipoproteins were immediately separated by ultracentrifugation in a discontinuous gradient. Plasmas were adjusted to density (d) 1.21 g/ml with solid KBr and overlayed with solutions of d=1.063 and 1.006 g/ml and centrifuged for 24 h in a SW 41 rotor, at 4°C, 100000 × g, in a L8 Beckman ultracentrifuge. Lipoproteins fractions were collected from the top to bottom by vacuum as follow: 1.5 ml VLDL (d<1.006), 2.5 ml LDL (d = 1.006 - 1.063) and 7.5 ml HDL (d>1.063). Radioactivity was determined in aliquots from each lipoprotein fraction.
Intravascular Lipases Activity
Total lipase activity was determined according to Ehnholm & Kuusi . Briefly, overnight fasted human plasmas, collected pre (basal) and 10 minutes after heparin I.V. injection (100 U/Kg body weight), were incubated with a 3H-triolein/arabic gum substrate ([9,10 3H (N)]-triolein, New England Nuclear, Boston, MA) in 0.2 M Tris-HCl buffer, pH 8.5, 37ºC, during 1 hour. Hepatic lipase (HL) activity was determined in tubes where the lipoprotein lipase (LPL) was inhibited by 2 M NaCl. The hydrolyzed labeled free fatty acids were extracted with methanol / chloroform / heptane (1.4 : 1.25 : 1), 0.14 M K2CO3 / H3BO3, pH 10.5, dried under N2, and their radioactivity was determined in a liquid scintillation solution in a LS6000 Beckman Beta Counter. The LPL activity was calculated as the difference between the total lipase and the hepatic lipase activities.
Cholesteryl ester transfer protein activity assay
A mixture of human VLDL and LDL protein (100 μg) were incubated with 10000 dpm of human HDL3 labeled with [14C]-cholesteryl ester (CE)  and 5 μl of diluted CETP transgenic mice plasma or undiluted human plasma as the source of CETP in a final volume of 100 μl. Blanks were prepared with tris/saline/EDTA buffer (10 mM/140 mM/1 mM), pH 7.4, and negative controls with non-transgenic mice plasma. The incubations were carried out at 37°C for 2 or 4 hours. After these periods, the apo B containing lipoproteins were precipitated using a mixture of 1.6% dextran sulfate / 1 M MgCl2 solution (1:1) and the radioactivity was measured in the remaining supernatant in scintillation solution Ultima Gold (Eastman Kodak Co., NY) in a LS6000 Beckman Beta Counter. The % CE transferred from [14C]-CE-HDL to VLDL+LDL was calculated as: (dpm in the blank tube - dpm in the plasma sample / dpm in the blank tube) × 100.
All comparisons were analysed by the non-parametric Mann-Whitney test using the GraphPad Prism, version 2.01 (1996) program. Differences were considered significant when p<0.05.
We are grateful to Dr. Alan R. Tall for kindly providing some human CETP transgenic mice and the CETP monoclonal antibody, TP2. This study was supported by Brazilian grants from FAPESP, CNPq and Pronex/FINEP. V.S.N was a MSc Student in the program of the Dept of Biochemistry, Escola Paulista de Medicina da Universidade Federal de São Paulo, SP, Brasil.
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