CETP expression reverses the reconstituted HDL-induced increase in VLDL
2011; Elsevier BV; Volume: 52; Issue: 8 Linguagem: Inglês
10.1194/jlr.m016659
ISSN1539-7262
AutoresYanan Wang, Jimmy F.P. Berbée, Erik S.G. Stroes, Johannes W. A. Smit, Louis M. Havekes, Johannes A. Romijn, Patrick C.N. Rensen,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoHuman data suggest that reconstituted HDL (rHDL) infusion can induce atherosclerosis regression. Studies in mice indicated that rHDL infusion adversely affects VLDL levels, but this effect is less apparent in humans. This discrepancy may be explained by the fact that humans, in contrast to mice, express cholesteryl ester transfer protein (CETP). The aim of this study was to investigate the role of CETP in the effects of rHDL on VLDL metabolism by using APOE*3-Leiden (E3L) mice, a well-established model for human-like lipoprotein metabolism. At 1 h after injection, rHDL increased plasma VLDL-C and TG in E3L mice, but not in E3L mice cross-bred onto a human CETP background (E3L.CETP mice). This initial raise in VLDL, caused by competition between rHDL and VLDL for LPL-mediated TG hydrolysis, was thus prevented by CETP. At 24 h after injection, rHDL caused a second increase in VLDL-C and TG in E3L mice, whereas rHDL had even decreased VLDL in E3L.CETP mice. This secondary raise in VLDL was due to increased hepatic VLDL-TG production. Collectively, we conclude that CETP protects against the rHDL-induced increase in VLDL. We anticipate that studies evaluating the anti-atherosclerotic efficacy of rHDL in mice that are naturally deficient for CETP should be interpreted with caution, and that treatment of atherogenic dyslipidemia by rHDL should not be combined with agents that aggressively reduce CETP activity. Human data suggest that reconstituted HDL (rHDL) infusion can induce atherosclerosis regression. Studies in mice indicated that rHDL infusion adversely affects VLDL levels, but this effect is less apparent in humans. This discrepancy may be explained by the fact that humans, in contrast to mice, express cholesteryl ester transfer protein (CETP). The aim of this study was to investigate the role of CETP in the effects of rHDL on VLDL metabolism by using APOE*3-Leiden (E3L) mice, a well-established model for human-like lipoprotein metabolism. At 1 h after injection, rHDL increased plasma VLDL-C and TG in E3L mice, but not in E3L mice cross-bred onto a human CETP background (E3L.CETP mice). This initial raise in VLDL, caused by competition between rHDL and VLDL for LPL-mediated TG hydrolysis, was thus prevented by CETP. At 24 h after injection, rHDL caused a second increase in VLDL-C and TG in E3L mice, whereas rHDL had even decreased VLDL in E3L.CETP mice. This secondary raise in VLDL was due to increased hepatic VLDL-TG production. Collectively, we conclude that CETP protects against the rHDL-induced increase in VLDL. We anticipate that studies evaluating the anti-atherosclerotic efficacy of rHDL in mice that are naturally deficient for CETP should be interpreted with caution, and that treatment of atherogenic dyslipidemia by rHDL should not be combined with agents that aggressively reduce CETP activity. Dyslipidemia is an important risk factor for cardiovascular disease (CVD). Current treatment mainly focuses on lowering of LDL-cholesterol (LDL-C), e.g., by statins. LDL-C-lowering treatment results in a significant reduction in the morbidity and mortality of CVD, but it cannot prevent the majority of cardiovascular events (1Baigent C. Keech A. Kearney P.M. Blackwell L. Buck G. Pollicino C. Kirby A. Sourjina T. Peto R. Collins R. et al.Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins.Lancet. 2005; 366: 1267-1278Abstract Full Text Full Text PDF PubMed Scopus (5768) Google Scholar, 2LaRosa J.C. Deedwania P.C. Shepherd J. Wenger N.K. Greten H. DeMicco D.A. Breazna A. Comparison of 80 versus 10 mg of atorvastatin on occurrence of cardiovascular events after the first event (from the Treating to New Targets [TNT] trial).Am. J. Cardiol. 2010; 105: 283-287Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Prospective epidemiological studies have demonstrated a strong inverse correlation between HDL-cholesterol (HDL-C) and CVD (3Gordon D.J. Probstfield J.L. Garrison R.J. Neaton J.D. Castelli W.P. Knoke J.D. Jacobs Jr, D.R. Bangdiwala S. Tyroler H.A. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies.Circulation. 1989; 79: 8-15Crossref PubMed Scopus (2664) Google Scholar), and recent studies revealed that high HDL-C levels are indeed protective against plaque progression (4Johnsen S.H. Mathiesen E.B. Fosse E. Joakimsen O. Stensland-Bugge E. Njolstad I. Arnesen E. Elevated high-density lipoprotein cholesterol levels are protective against plaque progression: a follow-up study of 1952 persons with carotid atherosclerosis the Tromso study.Circulation. 2005; 112: 498-504Crossref PubMed Scopus (88) Google Scholar). Although the exact mechanisms by which HDL protects are unclear, HDL has been shown to have antioxidant, antithrombotic and anti-inflammatory properties, and to mediate reverse cholesterol transport (RCT) via the hepatobiliary route (5Rader D.J. Alexander E.T. Weibel G.L. Billheimer J. Rothblat G.H. The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis.J. Lipid Res. 2009; 50: S189-S194Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). Therefore, new strategies to raise HDL-C are currently being developed to prevent and treat CVD. Various therapeutic strategies are currently under development to raise HDL levels, including cholesteryl ester transfer protein (CETP) inhibition, niacin, upregulation of apoAI expression, and infusion of apoAI mimetics or reconstituted HDL (rHDL) (6Duffy D. Rader D.J. Update on strategies to increase HDL quantity and function.Nat. Rev. Cardiol. 2009; 6: 455-463Crossref PubMed Scopus (159) Google Scholar). Although still in early stage of development, infusion of rHDL seems to be a promising strategy for the treatment of CVD. Recent reviews have demonstrated that infusion of rHDL improves atherosclerotic plaque characteristics both in animal models and humans (7Tardif J.C. Heinonen T. Noble S. High-density lipoprotein/apolipoprotein A-I infusion therapy.Curr. Atheroscler. Rep. 2009; 11: 58-63Crossref PubMed Scopus (32) Google Scholar–9Nissen S.E. Tsunoda T. Tuzcu E.M. Schoenhagen P. Cooper C.J. Yasin M. Eaton G.M. Lauer M.A. Sheldon W.S. Grines C.L. et al.Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes: a randomized controlled trial.JAMA. 2003; 290: 2292-2300Crossref PubMed Scopus (1566) Google Scholar). For example, rHDL, composed of recombinant human apoAIMilano and phosphatidylcholine, rapidly mobilized tissue cholesterol and reduced the lipid and macrophage content of atherosclerotic plaques after a single injection into apoE-deficient mice (10Shah P.K. Yano J. Reyes O. Chyu K.Y. Kaul S. Bisgaier C.L. Drake S. Cercek B. High-dose recombinant apolipoprotein A-I(milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential implications for acute plaque stabilization.Circulation. 2001; 103: 3047-3050Crossref PubMed Scopus (345) Google Scholar). Moreover, it prevented the progression of aortic atherosclerosis as well as promoted the stabilization of plaques after six weeks of administration (11Shah P.K. Nilsson J. Kaul S. Fishbein M.C. Ageland H. Hamsten A. Johansson J. Karpe F. Cercek B. Effects of recombinant apolipoprotein A-I(Milano) on aortic atherosclerosis in apolipoprotein E-deficient mice.Circulation. 1998; 97: 780-785Crossref PubMed Scopus (226) Google Scholar). Recent clinical trials assessed the effect of rHDL that consisted of human apoAI and phosphatidylcholine (CSL-111) as a potential HDL-raising therapeutic strategy. Short-term infusion of CSL-111 significantly improved the plaque characterization index and coronary score on quantitative coronary angiography (12Tardif J.C. Gregoire J. L'Allier P.L. Ibrahim R. Lesperance J. Heinonen T.M. Kouz S. Berry C. Basser R. Lavoie M.A. et al.Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial.JAMA. 2007; 297: 1675-1682Crossref PubMed Scopus (623) Google Scholar). In addition, a single dose of rHDL led to acute changes in plaque characteristics with a reduction in lipid content, macrophage size, and inflammatory mediators (13Shaw J.A. Bobik A. Murphy A. Kanellakis P. Blombery P. Mukhamedova N. Woollard K. Lyon S. Sviridov D. Dart A.M. Infusion of reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic plaque.Circ. Res. 2008; 103: 1084-1091Crossref PubMed Scopus (239) Google Scholar). Although rHDL seems to beneficially modulate atherosclerosis in mice and humans, differences have been observed with respect to modulation of VLDL levels. Infusion of rHDL into apoE-deficient mice increased (V)LDL-C in both acute and chronic studies (10Shah P.K. Yano J. Reyes O. Chyu K.Y. Kaul S. Bisgaier C.L. Drake S. Cercek B. High-dose recombinant apolipoprotein A-I(milano) mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein e-deficient mice. Potential implications for acute plaque stabilization.Circulation. 2001; 103: 3047-3050Crossref PubMed Scopus (345) Google Scholar, 11Shah P.K. Nilsson J. Kaul S. Fishbein M.C. Ageland H. Hamsten A. Johansson J. Karpe F. Cercek B. Effects of recombinant apolipoprotein A-I(Milano) on aortic atherosclerosis in apolipoprotein E-deficient mice.Circulation. 1998; 97: 780-785Crossref PubMed Scopus (226) Google Scholar), whereas rHDL did not adversely affect (V)LDL-C in clinical studies (12Tardif J.C. Gregoire J. L'Allier P.L. Ibrahim R. Lesperance J. Heinonen T.M. Kouz S. Berry C. Basser R. Lavoie M.A. et al.Effects of reconstituted high-density lipoprotein infusions on coronary atherosclerosis: a randomized controlled trial.JAMA. 2007; 297: 1675-1682Crossref PubMed Scopus (623) Google Scholar, 13Shaw J.A. Bobik A. Murphy A. Kanellakis P. Blombery P. Mukhamedova N. Woollard K. Lyon S. Sviridov D. Dart A.M. Infusion of reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic plaque.Circ. Res. 2008; 103: 1084-1091Crossref PubMed Scopus (239) Google Scholar). This discrepancy may be explained by the fact that, in contrast to mice (14Jiao S. Cole T.G. Kitchens R.T. Pfleger B. Schonfeld G. Genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel-permeation chromatography.Metabolism. 1990; 39: 155-160Abstract Full Text PDF PubMed Scopus (99) Google Scholar), humans express CETP (15Ha Y.C. Barter P.J. Differences in plasma cholesteryl ester transfer activity in sixteen vertebrate species.Comp. Biochem. Physiol. B. 1982; 71: 265-269Crossref PubMed Scopus (46) Google Scholar), a crucial factor involved in the metabolism of both (V)LDL and HDL by mediating the transfer of triglycerides (TG) and cholesteryl esters (CE) between these lipoproteins. Therefore, the aim of this study was to elucidate the role of CETP in the effect of rHDL on VLDL metabolism. We used APOE*3-Leiden (E3L) transgenic mice, a unique model for human-like lipoprotein metabolism, which had been crossbred with mice expressing human CETP under control of its natural flanking regions (16Jiang X.C. Agellon L.B. Walsh A. Breslow J.L. Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.J. Clin. Invest. 1992; 90: 1290-1295Crossref PubMed Google Scholar), resulting in E3L.CETP mice. This allows distinguishing between the effect of rHDL administration on VLDL metabolism in the absence and presence of CETP-mediated lipid transfer. Hemizygous human CETP transgenic (CETP) mice, expressing human CETP under the control of its natural flanking regions (16Jiang X.C. Agellon L.B. Walsh A. Breslow J.L. Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.J. Clin. Invest. 1992; 90: 1290-1295Crossref PubMed Google Scholar), were purchased from the Jackson Laboratory (Bar Harbor, ME) and crossbred with hemizygous E3L mice (17van den Maagdenberg A.M. Hofker M.H. Krimpenfort P.J. de Bruijn I. van Vlijmen B. van der Boom H. Havekes L.M. Frants R.R. Transgenic mice carrying the apolipoprotein E3-Leiden gene exhibit hyperlipoproteinemia.J. Biol. Chem. 1993; 268: 10540-10545Abstract Full Text PDF PubMed Google Scholar) at our Institutional Animal Facility to obtain E3L.CETP mice (18Westerterp M. van der Hoogt C.C. de Haan W. Offerman E.H. Dallinga-Thie G.M. Jukema J.W. Havekes L.M. Rensen P.C. Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2552-2559Crossref PubMed Scopus (180) Google Scholar). In this study, female mice were used, housed under standard conditions in conventional cages with free access to food and water. At the age of 12 weeks, mice were fed a semisynthetic Western-type diet, containing 1% (w/w) corn oil and 15% (w/w) cacao butter (Hope Farms, Woerden, The Netherlands) with 0.25% (w/w) cholesterol (E3L mice) or 0.1% (w/w) cholesterol (E3L.CETP mice) for three weeks, aimed at yielding comparable VLDL levels between both mouse genotypes. Upon randomization according to total plasma cholesterol (TC) and TG levels, mice received a single intravenous injection of rHDL (CSL-111; CSL Behring AG, Bern, Switzerland) (250 mg/kg in 250 µl PBS) or vehicle. Experiments were performed after 4 h of fasting at 12:00 PM with food withdrawn at 8:00 AM. The institutional Ethical Committee on Animal Care and Experimentation approved all experiments. rHDL (CSL-111) consists of apoAI isolated from human plasma and phosphatidylcholine from soybean with a molar ratio of 1:150. Before infusion, rHDL was reconstituted with 50 ml of sterile water, yielding 62.5 ml of clear, pale-yellow solution, pH 7.5, and 10% (w/v) sucrose as a stabilizing agent. The final apoAI and PL concentrations were 20 and 86 mg/ml, respectively. Plasma was obtained via tail vein bleeding and assayed for TC, TG, and phospholipids using the commercially available enzymatic kits 236691, 11488872 (Roche Molecular Biochemicals, Indianapolis, IN) and phospholipids B (Wako Chemicals, Neuss, Germany), respectively. The distribution of lipids over plasma lipoproteins was determined using fast protein liquid chromatography (FPLC). Plasma was pooled per group, and then 50 μl of each pool was injected onto a Superose 6 PC 3.2/30 column (Äkta System, Amersham Pharmacia Biotech, Piscataway, NJ) and eluted at a constant flow rate of 50 μl/min in PBS, 1 mM EDTA, pH 7.4. Fractions of 50 μl were collected and assayed for TC, TG, and phospholipid as described above. Plasma human apoAI concentrations were determined using a sandwich ELISA. Goat anti-human apoAI antibody (Academy Biomedical Co., Inc., Houston, TX; 11A-G2b) was coated overnight onto Costar medium binding plate (Costar, Inc., New York, NY) (3 µg/ml) at 4°C and incubated with diluted mouse plasma (dilution, 1:100,000) for 2 h at 37°C. Subsequently, horseradish peroxidase-conjugated goat antihuman apoAI (Academy Biomedical; 11H-G1b) was added and incubated for 2 h at 37°C. Horseradish peroxidase was detected by incubation with tetramethylbenzidine (Organon Teknika, Boxtel, The Netherlands) for 15 min at room temperature. Human apoAI (Academy Biomedical; 11P-101) was used as a standard. Glycerol tri[3H]oleate- and [1α,2α(n)-14C]cholesteryl oleate-double labeled VLDL-like emulsion particles (80 nm) were prepared as described by Rensen et al. (19Rensen P.C. Herijgers N. Netscher M.H. Meskers S.C. van Eck M. van Berkel T.J. Particle size determines the specificity of apolipoprotein E-containing triglyceride-rich emulsions for the LDL receptor versus hepatic remnant receptor in vivo.J. Lipid Res. 1997; 38: 1070-1084Abstract Full Text PDF PubMed Google Scholar). In short, radiolabeled emulsions were obtained by adding 200 µCi of glycerol tri[3H]oleate and 20 µCi of [14C]cholesteryl oleate to 100 mg of emulsion lipids before sonication (isotopes obtained from GE Healthcare, Little Chalfont, UK). Mice were fasted for 4 h, sedated with 6.25 mg/kg acepromazine (Alfasan), 6.25 mg/kg midazolam (Roche), and 0.3125 mg/kg fentanyl (Janssen-Cilag), and injected with the radiolabeled emulsion particles (0.15 mg TG in 200 µl PBS) via the tail vein. At indicated time points after injection, blood was taken from the tail vein to determine the serum decay of glycerol tri[3H]oleate and 20 µCi of [14C]cholesteryl oleate. The effect of rHDL on LPL activity was determined essentially as described (20Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis.J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). First, glycerol tri[3H]oleate-labeled VLDL-like emulsion particles (200 µg of TG, corresponding to a final concentration of 0.5 mg/ml), prepared as described above, were added to the indicated amounts of rHDL (or vehicle containing sucrose or sodium cholate only) and heat-inactivated human serum (20 µl, corresponding to a final concentration of 5% v/v) in a total volume of 75 µl of phosphate-buffered saline. Subsequently, 0.1 M Tris HCl (pH 8.5) was added to a total volume of 200 µl, and incubation mixtures were equilibrated at 37°C. At t = 0, bovine LPL (final concentration 3.5 U/ml, Sigma) in 200 µl of 120 mg/ml free fatty acid-free BSA (Sigma), corresponding with a final concentration of 60 mg/ml, was added (37°C). At t = 15, 30, 60, 90, and 120 min, [3H]oleate generated during lipolysis by LPL was extracted. Hereto, 50 µl samples were added to 1.5 ml extraction liquid (CH3OH: CHCl3: heptane: oleic acid (1,410: 1,250: 1,000: 1, v/v/v/v). Samples were mixed, and 0.5 ml of 0.2 M NaOH was added. Following vigorous mixing and centrifugation (10 min at 1,000 g), 3H radioactivity in 0.5 ml of the aqueous phase was counted. After taking the last samples, 50 µl of the incubations were also directly counted, representing the total amount of radioactivity in the assay. Lipolysis rate (i.e., LPL activity) was calculated by linear regression between incubation time and percentage of [3H]oleate released. Mice were fasted for 4 h, with food withdrawn at 8:00 AM, prior to the start of the experiment. During the experiment, mice were sedated as described above. At t = 0 min, blood was taken via tail bleeding and mice were intravenously injected with 100 µl PBS containing 100 µCi Trans35S label to measure de novo total apoB synthesis. After 30 min, the animals received 500 mg of tyloxapol (Triton WR-1339, Sigma-Aldrich) per kg body weight as a 10% (w/w) solution in sterile saline to prevent systemic lipolysis of newly secreted hepatic VLDL-TG (21Aalto-Setala K. Fisher E.A. Chen X. Chajek-Shaul T. Hayek T. Zechner R. Walsh A. Ramakrishnan R. Ginsberg H.N. Breslow J.L. Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice. Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles.J. Clin. Invest. 1992; 90: 1889-1900Crossref PubMed Scopus (400) Google Scholar). Additional blood samples were taken at 15, 30, 60, and 90 min after tyloxapol injection and used for determination of plasma TG concentration. At 120 min, the animals were euthanized, and blood was collected by orbital puncture for isolation of VLDL by density gradient ultracentrifugation. 35S-apoB was measured in the VLDL fraction, and VLDL-apoB production rate was shown as dpm.h−1 (22Li X. Catalina F. Grundy S.M. Patel S. Method to measure apolipoprotein B-48 and B-100 secretion rates in an individual mouse: evidence for a very rapid turnover of VLDL and preferential removal of B-48- relative to B-100-containing lipoproteins.J. Lipid Res. 1996; 37: 210-220Abstract Full Text PDF PubMed Google Scholar). All data are presented as means ± SD. Data were analyzed using the unpaired Student's t-test. P values less than 0.05 were considered statistically significant. To investigate the role of CETP in the effect of rHDL infusion on VLDL metabolism, female E3L mice with or without human CETP expression received a single intravenous injection of rHDL. To assess the kinetics of rHDL that consists of human apoAI and PL, plasma levels of human apoAI and phospholipid was determined over time (Fig. 1) rHDL caused a transient increase in plasma human apoAI and phospholipid levels in both E3L mice (Fig. 1A, C) and E3L.CETP mice (Fig. 1B, D). Human apoAI and phospholipid were cleared at a similar rate and disappeared from plasma after approximately 24 h. At 1 h after injection, lipoproteins in plasma were separated, and the distribution of human apoAI and phospholipid was determined (Fig. 2). rHDL appeared to integrate into the endogenous HDL pool in both E3L and E3L.CETP mice, as both human apoAI (Fig. 2A, B) and phospholipid (Fig. 2C, D) eluted in fractions representing HDL. In addition, phospholipid derived from rHDL selectively integrated into (V)LDL fractions (Fig. 2C, D). The presence of rHDL-phospholipid in (V)LDL is not due to the presence of large rHDL aggregates that would elute in the void volume, since apoAI is not detected in the void volume (Fig. 3A); rather, it is explained by a time-dependent transfer of phospholipid to endogenous VLDL as evident from incubation of rHDL with plasma from E3L mice (Fig. 3B) and E3L.CETP mice (Fig. 3C) in vitro.Fig. 2Effect of rHDL on the lipoprotein distribution of human apoAI and phospholipids at 1 h after injection in E3L and E3L.CETP mice. E3L (A, C) and E3L.CETP (B, D) mice were fed a Western-type diet for three weeks. Subsequently, they received a single intravenous injection of rHDL (250 mg/kg in 250 µl PBS) or vehicle. After 1 h, blood was drawn, and plasma was pooled per group (n = 8-10). Pooled plasma was fractionated using FPLC on a Superose 6 column, and the individual fractions were assayed for human apoAI (A, B) and phospholipids (C, D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Effect of in vitro incubation of rHDL with E3L and E3L.CETP mouse plasma on phospholipid distribution. E3L and E3L.CETP mice were fed a Western-type diet for three weeks, and fresh plasma was collected. rHDL was incubated (1 h at 37°C) without mouse plasma (A) or with plasma of E3L mice (B) or E3L.CETP mice (C). Samples were pooled per group (n = 8-10) and fractionated using FPLC on a Superose 6 column, and the individual fractions were assayed for phospholipids.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although rHDL was cleared at a similar rate in E3L and E3L.CETP mice, its effects on endogenous plasma levels of cholesterol and TG were clearly different in both mouse types (Fig. 4). At 1 h after injection, rHDL significantly increased plasma cholesterol in both E3L mice (+63%; P < 0.001) (Fig. 4A) and E3L.CETP mice (+28%; P < 0.01) (Fig. 4B). However, at 24 h after injection, rHDL still significantly increased plasma cholesterol in E3L mice (+26%, P < 0.01) (Fig. 4A) but actually decreased plasma cholesterol in E3L.CETP mice (−22%, P < 0.01) (Fig. 4B). In addition, whereas rHDL caused a significant increase in plasma TG levels in E3L mice at both 1 h (+89%; P < 0.01) and 24 h after injection (+67%; P < 0.01) (Fig. 4C), rHDL did not significantly increase plasma TG at any time point in E3L.CETP mice (Fig. 4D). To investigate the mechanism underlying the early effects of rHDL infusion on plasma lipids, plasma was obtained at 1 h after injection, and lipoproteins were fractionated by FPLC (Fig. 5). rHDL increased HDL-C in both E3L mice (Fig. 5A) and E3L.CETP mice (Fig. 5B), indicating that rHDL induces a rapid cholesterol efflux from peripheral tissues into plasma. In addition, rHDL markedly increased VLDL-C (Fig. 5A) and VLDL-TG (Fig. 5C) in E3L mice, while its VLDL-increasing effect was only modest in E3L.CETP mice (Fig. 5B, D). To investigate whether the raise in VLDL was due to competition between rHDL and VLDL for binding and subsequent TG hydrolysis by LPL, we assessed the effect of rHDL on the plasma kinetics of intravenously injected glycerol tri[3H]oleate [14C]cholesteryl oleate double-labeled VLDL-like emulsion particles (Fig. 6). Indeed, rHDL decreased the plasma clearance of the VLDL-like emulsion particles, including glycerol tri[3H]oleate and [14C]cholesteryl oleate, in both E3L mice (Fig. 6A, C) and E3L.CETP mice (Fig. 6B, D). An in vitro LPL activity assay confirmed that rHDL dose-dependently decreased LPL-mediated lipolysis of VLDL-like emulsion particles (Fig. 7), whereas sucrose and sodium cholate at amounts present at the various rHDL concentrations did not (data not shown). These data indicate that rHDL competes for the binding of VLDL-like emulsion particles with LPL in both E3L and E3L.CETP mice, resulting in delayed clearance of TG-derived fatty acids (i.e., 3H-activity) as well as the resulting core remnants (i.e., 14C-activity). The fact that rHDL does not substantially raise VLDL levels in E3L.CETP mice is probably related to rapid remodeling of VLDL by CETP.Fig. 6Effect of rHDL on the plasma clearance of VLDL-like emulsion particles in E3L and E3L.CETP mice. E3L (A, C) and E3L.CETP (B, D) mice were fed a Western-type diet for three weeks, and they received a single intravenous injection of rHDL (250 mg/kg in 200 µl PBS) or vehicle. After 1 min, mice were intravenously injected with glycerol tri[3H]oleate- and [14C]cholesteryl oleate-double labeled VLDL-like emulsion particles (0.15 mg TG in 200 µl PBS). Blood was drawn at the indicated time points, and 3H and 14C-activity was determined. Values are means ± SD (n = 8); *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Effect of rHDL on in vitro LPL activity. Glycerol tri[3H]oleate-labeled VLDL-like emulsion particles were incubated at 37°C with bovine LPL (3.5 U/ml) in 0.1 M Tris HCl (pH 8.5) in the presence of heat-inactivated human serum (5%, v/v) and free fatty acid-free BSA (60 mg/ml). [3H]oleate generated during lipolysis was extracted after 15, 30, 60, 90, and 120 min of incubation. The lipolysis rate (i.e., LPL activity) was calculated by the linear regression between incubation time and percentage of [3H]oleate generated. Values are means ± SD (n = 3); **P < 0.01, ***P < 0.001 compared with control incubations containing vehicle.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the mechanism underlying the divergent long-term effects of rHDL, infusion on plasma was also obtained 24 h after administration, and lipoproteins were fractionated by FPLC (Fig. 8). In both E3L and E3L.CETP mice, the effect of rHDL on increasing HDL-C levels had disappeared (Fig. 8A, B). However, whereas rHDL still significantly raised VLDL-C (+60%) (Fig. 8A) and VLDL-TG (+86%) (Fig. 8C) in E3L mice, rHDL actually decreased VLDL-C (−25%) in E3L.CETP mice (Fig. 8B). Since it has been shown that increasing the flux of HDL to the liver can increase the availability of substrate for hepatic VLDL synthesis and subsequently VLDL-TG secretion (23Wiersma H. Nijstad N. Gautier T. Iqbal J. Kuipers F. Hussain M.M. Tietge U.J. Scavenger receptor BI facilitates hepatic very low density lipoprotein production in mice.J. Lipid Res. 2010; 51: 544-553Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), we speculated that rHDL may have increased the VLDL production. Therefore, the effect of rHDL on VLDL production was evaluated after injection of Triton WR1339 (tyloxapol) to block LPL-mediated lipolysis (Fig. 9). Indeed, at 24 h after administration of rHDL, the VLDL-TG production rate was increased in E3L mice (+36%; P < 0.01) (Fig. 9A). ApoB production was not affected (Fig. 9C), indicating that rHDL increases lipidation of VLDL particles rather than increasing the VLDL particle secretion rate. Likewise, rHDL tended to increase the VLDL-TG production rate (Fig. 9B) without affecting the apoB production rate (Fig. 9D) in E3L.CETP mice.Fig. 9Effect of rHDL on the hepatic VLDL-TG production at 24 h after injection in E3L and E3L.CETP mice. E3L (A, C) and E3L.CETP (B, D) mice were fed a Western-type diet for three weeks, and they received a single intravenous injection of rHDL (250 mg/kg in 250 µl PBS) or vehicle. At 24 h after rHDL or vehicle injection, mice were injected with Trans35S label and tyloxapol to block VLDL-TG clearance. Blood was drawn at the indicated time points, and plasma TG concentrations were determined. VLDL-TG production rate was calculated from the slopes of the TG-time curves from the individual mice (A, B). At 120 after tyloxapol injection, mice were exsanguinated, and VLDL was isolated by ultracentrifugation. 35S-activity was determined, and V
Referência(s)