CETP expression reverses the reconstituted HDL-induced increase in VLDL.

Abstract

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.

Full text

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This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 52, 2011
1533
Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc.
Dyslipidemia is an important risk factor for cardiovascu-
lar disease (CVD). Current treatment mainly focuses on
lowering of LDL-cholesterol (LDL-C), e.g., by statins. LDL-
C-lowering treatment results in a signifi cant reduction in
the morbidity and mortality of CVD, but it cannot prevent
the majority of cardiovascular events ( 1, 2 ). Prospective epi-
demiological studies have demonstrated a strong inverse
correlation between HDL-cholesterol (HDL-C) and CVD
( 3 ), and recent studies revealed that high HDL-C levels are
indeed protective against plaque progression ( 4 ). Although
the exact mechanisms by which HDL protects are unclear,
HDL has been shown to have antioxidant, antithrombotic
and anti-infl ammatory properties, and to mediate reverse
cholesterol transport (RCT) via the hepatobiliary route
( 5 ). Therefore, new strategies to raise HDL-C are currently
being developed to prevent and treat CVD.
Various therapeutic strategies are currently under de-
velopment 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) ( 6 ). Although still in early
stage of development, infusion of rHDL seems to be a
promising strategy for the treatment of CVD. Recent re-
views have demonstrated that infusion of rHDL improves
atherosclerotic plaque characteristics both in animal mod-
els and humans ( 7–9 ). For example, rHDL, composed of
recombinant human apoAI Milano and phosphatidylcholine,
rapidly mobilized tissue cholesterol and reduced the lipid
and macrophage content of atherosclerotic plaques after
a single injection into apoE-defi cient mice ( 10 ). Moreover,
it prevented the progression of aortic atherosclerosis as
well as promoted the stabilization of plaques after six
Abstract Human data suggest that reconstituted HDL
(rHDL) infusion can induce atherosclerosis regression.
Studies in mice indicated that rHDL infusion adversely af-
fects 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 pro-
tein (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. Collec-
tively, we conclude that CETP protects against the rHDL-
induced increase in VLDL. We anticipate that studies
evaluating the anti-atherosclerotic effi cacy of rHDL in mice
that are naturally defi cient for CETP should be interpreted
with caution, and that treatment of atherogenic dyslipi-
demia by rHDL should not be combined with agents that
aggressively reduce CETP activity. — Wang, Y., J. F. P. Berbée,
E. S. Stroes, J. W. A. Smit, L. M. Havekes, J. A. Romijn, and
P. C. N. Rensen. CETP expression reverses the reconstituted
HDL-induced increase in VLDL. J. Lipid Res. 2011. 52:
1533–1541.
Supplementary key words cholesterol • transgenic mice • triglycer-
ides • very low density lipoprotein production • cholesteryl ester trans-
fer protein • high density lipoprotein
This work was supported by Netherlands Heart Foundation (NHS) Grant
2007B81 (P.C.N.R.). P.C.N.R. is an established investigator of the Netherlands
Heart Foundation (Grant 2009T038). This work performed within the frame-
work of the Leiden Center for Cardiovascular Research Leiden University Medi-
cal Center-Netherlands organization for Applied Scientifi c Research-Biosciences.
Manuscript received 20 April 2011 and in revised form 20 May 2011.
Published, JLR Papers in Press, May 23, 2011
DOI 10.1194/jlr.M016659
CETP expression reverses the reconstituted
HDL-induced increase in VLDL
Yanan Wang , 1, * Jimmy F. P. Berbée , * Erik S. Stroes , § Johannes W. A. Smit , *
Louis M. Havekes , * ,†, ** Johannes A. Romijn , * and Patrick C. N. Rensen *
Departments of General Internal Medicine, Endocrinology, and Metabolic Diseases,* and Cardiology, †
Leiden University Medical Center , Leiden, The Netherlands ; Department of Vascular Medicine, §
Academic Medical Center , Amsterdam, The Netherlands ; and Netherlands Organization for Applied
Scientifi c Research-Biosciences,** Gaubius Laboratory , Leiden, The Netherlands
Abbreviations: CE, cholesteryl ester; CETP, cholesteryl ester transfer
protein; CVD, cardiovascular disease; E3L, APOE*3-Leiden; HDL-C,
HDL-cholesterol; rHDL, reconstituted HDL; HL, hepatic lipase; LDL-C,
LDL-cholesterol; LXR, liver X receptor; PL, phosphatidylcholine;
RTC, reverse cholesterol transport; TC, total cholesterol; TG, triglyceride.
1 To whom correspondence should be addressed.
e-mail: Y.Wang@lumc.nl
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1534 Journal of Lipid Research Volume 52, 2011
Plasma lipid and lipoprotein analysis
Plasma was obtained via tail vein bleeding and assayed for TC,
TG, and phospholipids using the commercially available enzy-
matic kits 236691, 11488872 (Roche Molecular Biochemicals,
Indianapolis, IN) and phospholipids B (Wako Chemicals, Neuss,
Germany), respectively. The distribution of lipids over plasma li-
poproteins was determined using fast protein liquid chromatog-
raphy (FPLC). Plasma was pooled per group, and then 50 ? l 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 fl ow rate of 50 ? l/min in PBS, 1 mM
EDTA, pH 7.4. Fractions of 50 ? l were collected and assayed for
TC, TG, and phospholipid as described above.
Plasma human apoAI concentration
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 over-
night 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 Bio-
medical; 11H-G1b) was added and incubated for 2 h at 37°C.
Horseradish peroxidase was detected by incubation with tetra-
methylbenzidine (Organon Teknika, Boxtel, The Netherlands)
for 15 min at room temperature. Human apoAI (Academy Bio-
medical; 11P-101) was used as a standard.
weeks of administration ( 11 ). 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 sig-
nifi cantly improved the plaque characterization index and
coronary score on quantitative coronary angiography ( 12 ).
In addition, a single dose of rHDL led to acute changes in
plaque characteristics with a reduction in lipid content,
macrophage size, and infl ammatory mediators ( 13 ).
Although rHDL seems to benefi cially modulate athero-
sclerosis in mice and humans, differences have been ob-
served with respect to modulation of VLDL levels.
Infusion of rHDL into apoE-defi cient mice increased (V)
LDL-C in both acute and chronic studies ( 10, 11 ), whereas
rHDL did not adversely affect (V)LDL-C in clinical stud-
ies ( 12, 13 ). This discrepancy may be explained by the
fact that, in contrast to mice ( 14 ), humans express CETP
( 15 ), a crucial factor involved in the metabolism of both
(V)LDL and HDL by mediating the transfer of triglycer-
ides (TG) and cholesteryl esters (CE) between these lipo-
proteins. Therefore, the aim of this study was to elucidate
the role of CETP in the effect of rHDL on VLDL metabo-
lism. 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 fl anking regions ( 16 ),
resulting in E3L.CETP mice. This allows distinguishing
between the effect of rHDL administration on VLDL me-
tabolism in the absence and presence of CETP-mediated
lipid transfer.
MATERIALS AND METHODS
Animals
Hemizygous human CETP transgenic ( CETP ) mice, expressing
human CETP under the control of its natural fl anking regions
( 16 ), were purchased from the Jackson Laboratory (Bar Harbor,
ME) and crossbred with hemizygous E3L mice ( 17 ) at our Institu-
tional Animal Facility to obtain E3L.CETP mice ( 18 ). In this study,
female mice were used, housed under standard conditions in con-
ventional cages with free access to food and water. At the age of 12
weeks, mice were fed a semisynthetic Western-type diet, contain-
ing 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 intrave-
nous injection of rHDL (CSL-111; CSL Behring AG, Bern, Swit-
zerland) (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.
Reconstituted HDL
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 fi nal apoAI and PL concentrations were 20 and 86 mg/ml,
respectively.
Fig. 1. Effect of rHDL on plasma human apoAI and phospho-
lipids in E3L and E3L.CETP mice. E3L (A, C) and E3L.CETP (B,
D) mice were fed a Western-type diet for three weeks. Subse-
quently, they received a single intravenous injection of rHDL
(250 mg/kg in 250 µl PBS) or vehicle. Blood was drawn at the
indicated time points, and plasma was assayed for human apoAI
(A, B) and phospholipids (C, D). Values are means ± SD
(n = 8-10); * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the
control group.
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CETP reverses the rHDL-induced increase in VLDL1535
emulsions were obtained by adding 200 µCi of glycerol tri[ 3 H]
oleate and 20 µCi of [ 14 C]cholesteryl oleate to 100 mg of emul-
sion lipids before sonication (isotopes obtained from GE Health-
care, Little Chalfont, UK). Mice were fasted for 4 h, sedated with
In vivo clearance of VLDL-like emulsions
Glycerol tri[ 3 H]oleate- and [1 ? ,2 ? (n)- 14 C]cholesteryl oleate-
double labeled VLDL-like emulsion particles (80 nm) were pre-
pared as described by Rensen et al. ( 19 ). In short, radiolabeled
Fig. 2. Effect of rHDL on the lipoprotein distribu-
tion 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).
Fig. 3. Effect 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.
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1536Journal of Lipid Research Volume 52, 2011
that consists of human apoAI and PL, plasma levels of hu-
man 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 phos-
pholipid were cleared at a similar rate and disappeared from
plasma after approximately 24 h. At 1 h after injection, lipopro-
teins in plasma were separated, and the distribution of human
apoAI and phospholipid was determined ( Fig. 2 ). rHDL ap-
peared 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 represent-
ing HDL. In addition, phospholipid derived from rHDL se-
lectively 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 trans-
fer 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.
Infusion of rHDL affects plasma levels of endogenous
lipids differentially in E3L and E3L.CETP mice
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
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[ 3 H]oleate and 20 µCi of [ 14 C]cholesteryl oleate.
In vitro LPL activity assay
The effect of rHDL on LPL activity was determined essentially
as described ( 20 ). First, glycerol tri[ 3 H]oleate-labeled VLDL-like
emulsion particles (200 µg of TG, corresponding to a fi nal con-
centration 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 se-
rum (20 µl, corresponding to a fi nal concentration of 5% v/v) in
a total volume of 75 µl of phosphate-buffered saline. Subse-
quently, 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 (fi nal concentration 3.5 U/ml, Sigma) in 200 µl of
120 mg/ml free fatty acid-free BSA (Sigma), corresponding with
a fi nal concentration of 60 mg/ml, was added (37°C). At t = 15,
30, 60, 90, and 120 min, [ 3 H]oleate generated during lipolysis by
LPL was extracted. Hereto, 50 µl samples were added to 1.5 ml
extraction liquid (CH 3 OH: CHCl 3 : 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 centrifuga-
tion (10 min at 1,000 g ), 3 H radioactivity in 0.5 ml of the aqueous
phase was counted. After taking the last samples, 50 µl of the in-
cubations were also directly counted, representing the total
amount of radioactivity in the assay. Lipolysis rate (i.e., LPL activ-
ity) was calculated by linear regression between incubation time
and percentage of [ 3 H]oleate released.
Hepatic VLDL-TG and VLDL-apoB production
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 Trans 35 S 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 ( 21 ). 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 ultracentrifu-
gation. 35 S-apoB was measured in the VLDL fraction, and VLDL-
apoB production rate was shown as dpm.h ? 1 ( 22 ).
Statistical analysis
All data are presented as means ± SD. Data were analyzed us-
ing the unpaired Student’s t -test. P values less than 0.05 were con-
sidered statistically signifi cant.
RESULTS
Infusion of rHDL transiently increases plasma apoAI and
phospholipid levels in both E3L and E3L.CETP mice
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 intrave-
nous injection of rHDL. To assess the kinetics of rHDL
Fig. 4. Effect of rHDL on plasma cholesterol and triglycerides 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. Blood was drawn at the indicated time points,
and plasma was assayed for total cholesterol (A, B) and triglycer-
ides (C, D). Values are means ± SD (n = 8-10); * P < 0.05, ** P < 0.01,
*** P < 0.001 compared with the control group.
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CETP reverses the rHDL-induced increase in VLDL1537
plasma clearance of the VLDL-like emulsion particles, in-
cluding glycerol tri[ 3 H]oleate and [ 14 C]cholesteryl oleate,
in both E3L mice ( Fig. 6A, C ) and E3L.CETP mice ( Fig.
6B, D ). An in vitro LPL activity assay confi rmed 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., 3 H-activity) as well as the resulting
core remnants (i.e., 14 C-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.
At long term, rHDL raises VLDL in E3L mice and
decreases VLDL in E3L.CETP mice
To determine the mechanism underlying the divergent
long-term effects of rHDL, infusion on plasma was also ob-
tained 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 sig-
nifi cantly 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 fl ux of HDL to the liver
can increase the availability of substrate for hepatic VLDL
synthesis and subsequently VLDL-TG secretion ( 23 ), we
speculated that rHDL may have increased the VLDL
types ( Fig. 4 ). At 1 h after injection, rHDL signifi cantly
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 signifi -
cantly increased plasma cholesterol in E3L mice (+26%,
P < 0.01) ( Fig. 4A ) but actually decreased plasma choles-
terol in E3L.CETP mice ( ? 22%, P < 0.01) ( Fig. 4B ). In addi-
tion, whereas rHDL caused a signifi cant 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
signifi cantly increase plasma TG at any time point in E3L.
CETP mice ( Fig. 4D ).
At short term, rHDL raises HDL-C in E3L and E3L.CETP
mice, and increases VLDL mainly in E3L mice
To investigate the mechanism underlying the early ef-
fects of rHDL infusion on plasma lipids, plasma was ob-
tained 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 effl ux
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 be-
tween 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[ 3 H]
oleate [ 14 C]cholesteryl oleate double-labeled VLDL-like
emulsion particles ( Fig. 6 ). Indeed, rHDL decreased the
Fig. 5. Effect of rHDL on lipoprotein distribution
of cholesterol and triglycerides 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 in-
travenous injection of rHDL (250 mg/kg in 250 µl
PBS) or vehicle. 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 total
cholesterol (A, B) and triglycerides (C, D).
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1538Journal of Lipid Research Volume 52, 2011
This fi nding is in line with previous observations showing
that rHDL, composed of human apoAI Milano and PL, also
increased the VLDL-TC pool in apoE-defi cient mice at 1 h
after injection ( 10 ). These effects cannot be explained
simply by transfer of lipid compounds from rHDL to
VLDL, since rHDL does not contain cholesterol or triglyc-
erides. Rather, we speculated that infusion of a substantial
amount of rHDL may interfere with endogenous VLDL
catabolism. Indeed, rHDL decreased the plasma clearance
of VLDL-like emulsion particles, including the clearance
of both glycerol tri[ 3 H]oleate and [ 14 C]cholesteryl oleate.
Apparently, rHDL competes with endogenous VLDL for
binding to triacylglycerol hydrolases, resulting in impaired
hydrolysis of TG within VLDL. Indeed, rHDL dose-de-
pendently inhibited LPL activity in an in vitro assay. At an
rHDL concentration of 0.625 mg/ml, resulting in an
rHDL:TG ratio similar to the in vivo situation, rHDL in-
hibited LPL activity by as much as 80%. Sucrose and so-
dium cholate, both present in rHDL, did not inhibit LPL
activity in vitro and are thus unlikely to inhibit LPL in vivo.
As a consequence of LPL inhibition by rHDL in vivo, the
clearance of core remnants is attenuated, and plasma
VLDL levels are increased. Our fi nding that normalization
of elevated phospholipid levels at 8 h after injection also
normalized plasma TG levels in E3L mice is consistent with
such a mechanism.
In addition to increasing VLDL at 1 h after injection,
rHDL caused a second raise in VLDL-TC and VLDL-TG in
E3L mice at 24 h after administration. Because competi-
tion of rHDL for VLDL clearance mechanisms can be ex-
cluded at this time point because rHDL has been cleared
from the circulation, we hypothesized that rHDL may have
caused an increase in hepatic VLDL production. Indeed,
we observed that rHDL increased the production rate of
production. Therefore, the effect of rHDL on VLDL pro-
duction 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 secre-
tion 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.
DISCUSSION
In this study, we investigated the role of CETP in the ef-
fect of rHDL on VLDL metabolism by using E3L mice with
or without human CETP expression. In both E3L and E3L.
CETP mice, rHDL caused a similar transient increase in
plasma human apoAI and phospholipid levels, and it in-
duced a transient increase in the endogenous HDL-C
pool. However, rHDL caused an increase in VLDL in E3L
mice, at both 1 h and 24 h after injection, which was pre-
vented by CETP expression in E3L.CETP mice.
We observed that rHDL caused a rapid increase in VLDL-TC
and VLDL-TG in E3L mice within 1 h after administration.
Fig. 7. Effect of rHDL on in vitro LPL activity. Glycerol tri[ 3 H]
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). [ 3 H]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 regres-
sion between incubation time and percentage of [ 3 H]oleate gener-
ated. Values are means ± SD (n = 3); ** P < 0.01, *** P < 0.001
compared with control incubations containing vehicle.
Fig. 6. Effect 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[ 3 H]oleate- and [ 14 C]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 3 H and
14 C-activity was determined. Values are means ± SD (n = 8); * P <
0.05, ** P < 0.01, *** P < 0.001 compared with the control group.
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CETP reverses the rHDL-induced increase in VLDL 1539
E3L.CETP mice, similar to E3L mice. Expression of CETP
thus clearly prevents the adverse effects of rHDL on VLDL
levels. These data are in line with various human studies in
which infusion of rHDL was used as an experimental treat-
ment of CVD ( 12 ), diabetes ( 25 ), and infl ammation ( 26 ).
In these clinical studies, no specifi c adverse VLDL-increas-
ing effects were reported, which is in line with our data
that human CETP may protect against rHDL-induced ele-
vation of VLDL.
It is interesting to speculate on the mechanism(s) un-
derlying the protective effect of CETP on the rHDL-in-
duced raise in VLDL. It is well known that CETP mediates
the transfer of CE from HDL particles to LDL and VLDL
particles in exchange for TG, and that this reciprocal neu-
tral lipid transfer approaches equilibrium under physio-
logical conditions. However, under conditions of increased
VLDL levels as observed at 1 and 24 h after administration
of rHDL to E3L mice, the increase in VLDL results in ele-
vated acceptor activity for CETP, which would result in an
increased net rate of TG transfer from VLDL particles to
both HDL and LDL particles ( 27 ). Both TG-enriched HDL
and LDL particles are avidly bound to hepatic lipase (HL)
that effectively hydrolyzes TG (as well as phospholipid) to
form small, dense LDL and HDL, respectively ( 28 ), thereby
effectively eliminating TG from plasma.
On the basis of our data and literature studies, we pro-
pose the following mechanism by which CETP has a pro-
tective effect on rHDL-induced increase in VLDL. Infusion
of rHDL initially decreases VLDL clearance via blocking of
LPL-mediated lipolysis and, at a later stage, increases VLDL
VLDL-TG without affecting the production rate of VLDL-
apoB. Since each VLDL particle contains a single molecule
of apoB, this indicates that rHDL infusion increases the
lipidation of hepatic apoB rather than increasing the num-
ber of VLDL particles produced. Interestingly, it has been
observed previously that increasing the fl ux of HDL to the
liver by hepatic overexpression of scavenger receptor class
B member 1 also increases the VLDL-TG production rate,
and that HDL-derived cholesterol can be resecreted from
the liver within VLDL particles ( 23 ). Therefore, we postu-
late that, in a similar manner, infusion of rHDL causes an
increased net fl ux of lipids to the liver. We showed that
rHDL transiently enhanced HDL-C, which may at least
partly be attributed by effl ux of cholesterol from periph-
eral tissues. This increased HDL-C may subsequently be
taken up by the liver via SR-BI, which could then be rese-
creted within VLDL. However, it is even more likely that a
high fl ux of rHDL-associated phospholipid to the liver en-
hances the amount of hepatic TG available for secretion as
VLDL-TG. Indeed, it has been demonstrated that HDL-
associated phosphatidylcholine that has been taken up by
hepatocytes is converted to TG after phosphatidylcholine-
phospholipase C-mediated hydrolysis of phosphatidyl-
choline, resulting in diacylglycerol that is subsequently
converted into TG by DGAT2 ( 24 ).
Although rHDL infusion caused a clear hyperlipidemic
side effect in E3L mice, there was no increase in VLDL in
E3L.CETP mice at any time point, although rHDL induced
a signifi cant decrease of VLDL clearance at short term and
tended to increase VLDL production at long term in
Fig. 8. Effect of rHDL on lipoprotein distribution
of cholesterol and triglycerides at 24 h after injec-
tion 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. 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 total
cholesterol (A, B) and triglycerides (C, D).
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1540Journal of Lipid Research Volume 52, 2011
( 32 ). However, other CETP inhibitors, such as dalcetrapib
( 33 ) and anacetrapib ( 34 ), have now progressed into phase
III clinical trials without showing any off-target toxicity.
Additionally, niacin effectively increases HDL by 25-30%
( 35 ) and improves carotid intima-media thickness ( 36 ).
We recently showed that the HDL-raising effect of niacin
is caused by reducing the hepatic CETP expression and
plasma CETP protein ( 37 ). Since the HDL-raising effects
of CETP inhibitors and niacin both depend on reducing
CETP activity in plasma, whereas CETP activity now ap-
pears crucial to prevent the raise in VLDL as induced by
rHDL infusion, combining rHDL with these HDL-raising
agents could reveal adverse VLDL effects and, as a conse-
quence, counteract the potentially protective effect of
rHDL in CVD.
In conclusion, our results show that rHDL infusion in-
duces an increase in VLDL levels, which is prevented by
CETP expression. Therefore, studies that evaluate the
anti-atherosclerotic effi cacy of rHDL in mouse models that
are naturally defi cient for CETP should be interpreted
with caution, and treatment of atherogenic dyslipidemia
as a risk factor for CVD by rHDL should not be combined
with agents that aggressively reduce CETP activity.

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