Postprandial apoE Isoform and Conformational Changes
Associated with VLDL Lipolysis Products Modulate
Laura J. den Hartigh1*, Robin Altman1,2, Romobia Hutchinson1, Jitka Petrlova2,3,
Madhu S. Budamagunta2, Sarada D. Tetali4, Jens O. Lagerstedt3, John C. Voss2, John C. Rutledge1
1Division of Cardiology, Department of Internal Medicine, University of California Davis, Davis, California, United States of America, 2Department of Biochemistry and
Molecular Medicine, University of California Davis, Davis, California, United States of America, 3Department of Experimental Medicine, Lund University, Lund, Sweden,
4Department of Plant Sciences, University of Hyderabad, Hyderabad, India
Objective: Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and
circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the
interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation.
Methods and Results: We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte
cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than
apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of
apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically,
compared to apoE4, apoE bearing the E3-like R112RSer mutation displays increased self association when exposed to LPS,
consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors
attenuated LPS-induced TNFa secretion from monocytes to a greater extent than postprandial VLDL, an effect partially
reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not
from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4.
Conclusion: Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular
Citation: den Hartigh LJ, Altman R, Hutchinson R, Petrlova J, Budamagunta MS, et al. (2012) Postprandial apoE Isoform and Conformational Changes Associated
with VLDL Lipolysis Products Modulate Monocyte Inflammation. PLoS ONE 7(11): e50513. doi:10.1371/journal.pone.0050513
Editor: Andrea Cignarella, University of Padova, Italy
Received July 11, 2012; Accepted October 22, 2012; Published November 28, 2012
Copyright: ? 2012 den Hartigh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors would like to acknowledge the Richard A. and Nora Eccles Harrison Endowed Chair in Diabetes Research, NHLBI HL055667, and the
Training Program in Biomolecular Technology (T32- GM08799) at the University of California, Davis for their financial support. This publication was also made
possible by the University of California, Davis Alzheimer’s Disease Center (grant AG010129) and grant AG029246 from the National Institute on Aging (NIA),
a component of the NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Postprandial activation of monocytes has been implicated as
a pro-inflammatory and pro-atherogenic condition [1,2], and the
repetitive injury of vascular cells during postprandial states could
be an important mechanism in the development of atherosclerotic
cardiovascular disease. Lipopolysaccharide (LPS), a form of
endotoxin, is a principal component of gram-negative bacterial
cell walls and induces a potent host inflammatory response. Recent
studies have shown that consumption of a meal high in fat
increases circulating endotoxin levels , presumably from gut
flora absorbed with dietary lipids [4,5]. Furthermore, plasma
lipoproteins provide an important defense mechanism against
LPS-induced inflammation by neutralizing and clearing LPS-
associated lipoproteins by the liver .
Consumption of a meal high in saturated fat results in a transient
rise in circulating triglycerides contained in chylomicrons (exog-
enous pathway) and very low-density lipoproteins (endogenous
pathway). Circulating postprandial triglyceride peaks about 3–4
hours after the meal and usually returns to fasting levels by 6 hours
post-consumption . Postprandial VLDL have been implicated
as atherogenic lipoproteins [8–10], and the effects of circulating
VLDL lipolysis on vascular function have recently been under
intense investigation [11–13]. Lipolysis of VLDL by lipoprotein
lipase results in the release of smaller remnant particles and free
fatty acids, monoglycerides, diglycerides, and phospholipids (de-
fined herein as lipolysis products) in close proximity to endothelial
cells and circulating monocytes . While it is known that
lipoproteins such as VLDL can neutralize LPS, the mechanism by
which they do so remains unclear. Further, the ability of
lipoproteins to neutralize LPS has not been compared between
the fasted and fed states, and it is not known if lipoprotein lipolysis
products influence binding and neutralization of LPS to prevent
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ApoE exists as three major isoforms: apoE2, apoE3, and apoE4.
All are exchangeable between circulating lipoproteins, most
commonly associated with chylomicrons, VLDL, and high-density
lipoprotein (HDL). ApoE3 is the most prevalent isoform in the
general population, and differs from apoE4 by a single amino acid
substitution at position 112, cysteine (apoE3) to arginine (apoE4).
Approximately 15% of the population carries at least one apoE4
allele, which has been correlated with increased risk of athero-
sclerosis and Alzheimer’s disease [15,16]. In addition, mechanisms
by which apoE4 and apoE3 promote pro- and anti-inflammatory
conditions, respectively, remain unclear . ApoE4 associates
primarily with VLDL in the postprandial state, and lipolysis of
VLDL induces a distinct conformational change in the apoE4
protein . Although it is thought that apolipoproteins such as
apoE can bind to LPS , isoform-specific variability has not
The purpose of this study was to determine if apoE isoforms
differentially protect monocytes from LPS-induced activation, and
how fasting and postprandial VLDL lipolysis products containing
apoE3 or apoE4 influence this process. Furthermore, we in-
vestigated the hypothesis that apoE3 attenuates LPS-induced
cellular activation by binding directly to LPS with a higher affinity
than apoE4. We have determined that apoE3 protects THP-1
monocyte-like cells from LPS-induced inflammatory cytokine
secretion, while apoE4 exacerbates monocyte activation. In
addition, apoE from fasting VLDL lipolysis products is more
protective against LPS-induced monocyte activation than post-
prandial VLDL lipolysis products. Finally, we show that the C-
terminal domain of apoE3 interacts with LPS, providing
a mechanism for its sequestration from monocytes.
Two cohorts of human subjects were recruited for blood
donation. The first consisted of healthy volunteers, 18–55 years of
age and representative of both genders, recruited from the
University of California, Davis campus. The second included
subjects of either apoE3/E3 (n=3) or apoE4/E4 (n=3) genotype
recruited from the greater Sacramento, California area by the
Alzheimer’s Disease Center of the University of California, Davis.
Both studies were approved by the Human Subjects Research
Committee of the University of California, Davis. The study aims
and protocol were explained to each participant, and informed
written consent was obtained.
THP-1 human monocytes were purchased from American
Type Culture Collection and maintained in suspension between
56104and 86105cells/ml in RPMI 1640 medium, and used in
experiments at a concentration of 16106cells/mL. All monocyte
experiments included treatment with 0.5 mg/mL LPS derived
from Escherichia coli (Sigma-Aldrich, L2654). Monocyte viability
was monitored after all treatments using trypan blue exclusion and
a Live/Dead Viability/Cytotoxicity kit from Molecular Probes.
ApoE3- and ApoE4-DMPC Preparations
Small unilamellar dimyristoyl phosphatidylcholine (DMPC,
10 mg/mL) vesicles were made by sonication in a water bath
and extruded using a mini-extruder (Avanti Polar Lipids,
Alabaster, AL), as described previously . Recombinant apoE3
and apoE4 (purchased from EMD Biosciences) were added to
DMPC vesicles at a lipid:protein ratio of 4:1 (w/w), vortexed
briefly for 10 seconds, and incubated for 1 hour at 25uC to allow
protein lipidation, as described previously .
Quantitative Real-Time PCR
THP-1 monocytes (16106cells per mL) were treated with the
indicated combinations of DMPC vesicles (200 mg/mL), apoE3
(50 mg/mL), apoE4 (50 mg/mL), and LPS (0.5 mg/mL) for 3
hours at 37uC. Cells were pelleted and RNA isolated using TRIzol
reagent as described previously . Total RNA was quantified
using a Nanodrop-1000 system. Total RNA (2 mg) was used to
make cDNA with a Superscript II RNase H-reverse transcriptase
kit according to the manufacturer’s guidelines (Invitrogen).
Quantification of mRNA from gene transcripts of tumor necrosis
factor-a (TNFa), interleukin 1-b (IL-1b), and beta-actin was
performed using the GeneAmp 7900 HT sequence detection
system (Applied Biosciences), as described previously . Primers
for the genes of interest were designed using Primer Express
(Applied Biosystems) and synthesized by Integrated DNA Tech-
nologies. The primer sequences were as follows: TNFa (sense, 59-
To determine if apoE3 and apoE4 bind to LPS, an ELISA
protocol was developed. After determining the optimal LPS
coating concentration, 96-well plates were coated with LPS at
5 mg/ml and incubated at 4uC overnight for apoE capture.
Unbound LPS was removed by gentle washing with 1X PBS
+0.05% Tween-20. Plates were blocked for 1 hour at room
temperature with 1 X PBS +10% fetal bovine serum (FBS), and
recombinant apoE isoforms, with or without prior complexing to
DMPC, were added at the indicated final concentrations for 2
hours at room temperature to allow binding to coated LPS. After
washing away unbound apoE3 and apoE4, a monoclonal apoE
antibody (EMD Millipore) was added for 1 hour at room
temperature followed by secondary antibody (anti-mouse IgG-
HRP, 1:3000) for one hour. TMB substrate solution (BD
Biosciences) was prepared by mixing equal volumes of reagent A
and reagent B and added to each well for 30 minutes to detect the
peroxide-labeled secondary antibody. The colorimetric reaction
was stopped by addition of 1 M phosphoric acid, and the
absorbance was read within 10 minutes. ApoE3 and apoE4
adhesion to LPS was represented as a dose-dependent increase in
absorbance values at 450 nm. Control wells, with and without
LPS coating, containing either apoE alone, apoE+DMPC, apoE
vehicle, apoE antibody, anti-mouse antibody, or substrate solution
contained no measureable absorbance (data not shown), indicating
specific LPS-apoE interactions were measured. DMPC alone
background controls did not exceed absorbance levels above the
lowest apoE concentrations used.
ApoE Mutant Cloning and Purification
The gene encoding human apoE4 protein containing the
W264RCys (apoE4-W264C) substitution was cloned into the
commercial vector pET151/D-Topo (Invitrogen) according to the
manufacturer’s instructions. The N-terminal fusion tag was
removed by introducing flanking BamHI sites by PCR mutagen-
esis, followed by BamHI digestion and then ligation, so that the
first residue of apoE (Lys) in the expressed protein is preceded by
the amino acid sequence M-G-S. To generate the apo E3-like
Effects of ApoE Isoforms on Monocyte Inflammation
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protein containing the same Cys substitution at position 264, the
R112RS mutation in the apoE4-W264C template was introduced
by PCR mutagenesis. Fidelity of cloning and mutations was
confirmed by DNA sequencing.
For protein expression, apoE4-W264C and apoE3-like-W264C
were transformed into BL21(DE3)AI Escherichia coli cells (Invitro-
gen). Cells were grown in LB broth (1 L) to mid-log phase at 37u,
then induced with 0.2% arabinose followed by incubation for 3
hours at 37u. Cells were harvested by centrifugation, and inclusion
bodies isolated and washed as described previously . To purify
the protein, washed inclusion bodies were dissolved in 8 M urea,
and the sample filtered through a 0.2 micron filter and then
separated on a SuperDex size exclusion column containing
100 mM tris-(2-carboxyethyl)phosphine (TCEP) to maintain re-
duced disulfides. The fractions compiling the predominant protein
peak were pooled, and the protein spin labeled with 0.4 mM
MTS-SL (Toronto Research Chemicals) for 30 minutes. Dena-
tured samples were scanned and double-integrated to determine
labeling efficiency, which measures at least 95% at position 264
under the TCEP labeling conditions. The protein was re-folded
and separated from free spin- label using an ion exchange column
(GE Health Sciences) connected to a Pharmacia FPLC chroma-
tography system. The pool of labeled apoE protein was loaded
onto the column, washed, and then eluted using a NaCl gradient.
The major eluted peaks were pooled, concentrated using spin
concentrators (Millipore), and the protein concentration was
determined using the Pierce BCA kit (Thermo Scientific).
Electron Paramagnetic Resonance (EPR) Spectroscopy
EPR measurements were carried out in a JEOL TE-100 X-
band spectrometer fitted with a loop-gap resonator as described
previously . LPS was added to the nitroxy spin-labeled protein
(4 mg/mL) at a final concentration of 30 mg/mL for 2 hours prior
to EPR measurements. Appropriate vehicle controls were used for
all samples. Approximately 5 mL of the protein was loaded into
a sealed quartz capillary tube. The spectra were obtained by
averaging two 2-minute scans with a sweep width of 100 G at
a microwave power of 4 mW and modulation amplitude
optimized to the natural line width of the attached spin probe.
All the spectra were recorded at room temperature.
Isolation of Very Low-Density Lipoproteins (VLDL) and
All studies were performed at the same time of day to eliminate
any diurnal variables. Following a 12-hour fast, subjects were fed
a moderately high fat (40% calories from fat) meal as described
previously . Blood was drawn by venipuncture from subjects
pre- and post-prandially (3.5 hours following meal consumption)
into K2-EDTA vacutainer tubes and centrifuged at 1,200 g for 10
minutes to obtain cell-free plasma. Plasma was treated with 0.01%
sodium azide as a preservative and subjected to lipoprotein
isolation as described previously , with minor modifications.
Chylomicrons were removed from postprandial plasma by
centrifuging for 30 minutes at 63,000 g prior to VLDL isolation.
Lipid samples were dialyzed overnight at 4uC in 0.9% NaCl and
0.01% EDTA and quantified using an autoanalyzer for total
triglyceride and apoE content. Purity of lipid fractions was
visualized by TITAN lipoprotein gel electrophoresis (gels obtained
from Helena Laboratories). Lipolysis of VLDL was allowed by
addition of lipoprotein lipase (LpL, 2 U/mL, obtained from
Sigma-Aldrich, St. Louis, MO) for 30 minutes at 37uC prior to
treatment of monocytes. Remnants were further isolated by
density gradient ultracentrifugation of hydrolyzed VLDL, and
characterized for total triglyceride, apoE, and apoB content using
a PolyChem Analyzer using manufacturer’s reagents (MedTest
DX, Cortlandt Manor, NY).
Tumor Necrosis Factor-a ELISA
THP-1 monocytes were treated with VLDL, hydrolyzed VLDL,
or VLDL remnants for 3 hours. All treatments included LPS at
0.5 mg/mL. A monoclonal apoE antibody (EMD Biosciences) was
used for blocking experiments by incubating it with hydrolyzed
VLDL at 4uC for 15 minutes at a final dilution of 1:1000. Total
secreted TNFa protein was quantified using an ELISA kit from
BD Biosciences according to the manufacturer’s instructions, and
normalized to the LPS control(s).
All statistical analyses were performed using SigmaStat software.
All results are reported as mean 6 SEM, as indicated. A student t-
test was used to elucidate significant differences between apoE3
and apoE4 binding affinity to LPS, with group comparisons
determined significant for P,0.01, as indicated. One-way
ANOVA was used to determine significance for all other
experiments, with pairwise comparisons performed using the
Holm-Sidak method. Statistical significance was reported for
P,0.05 or P,0.01, as indicated.
ApoE3 and apoE4 Differentially Alter Monocyte Gene
Expression in Response to LPS
To determine if apoE3 and apoE4 influence LPS-induced
cytokine expression, THP-1 cells were treated with recombinant
apoE3 and apoE4 complexed with DMPC. As shown in Figure 1A,
ApoE3-DMPC attenuated LPS-induced TNFa and IL-1b gene
expression to 58% and 40% of the LPS+DMPC control,
respectively. Conversely, apoE4-DMPC increased TNFa and IL-
1b gene expression to 340% and 240%, respectively. There was no
significant effect on expression of either gene upon treatment with
DMPC alone, apoE vehicle, DMPC+apoE vehicle, or either apoE
isoform without LPS (not shown). Secreted TNFa showed a similar
trend towards an attenuated response with ApoE3-DMPC and an
enhanced response with ApoE4-DMPC (Figure 1B). These results
suggest that apoE3 is protective against LPS-induced cellular
injury, while apoE4 potentiates it, which is consistent with our
previous studies in human endothelial cells .
ApoE3 Binds to LPS with Greater Affinity than apoE4
To determine if apoE3 and/or apoE4 directly bind to LPS, an
apoE ELISA was developed. Human recombinant apoE3 and
apoE4 were titrated in equal concentrations between 0 and
1000 ng/mL in separate wells of an LPS-coated ELISA plate.
Binding is represented as the absorbance output values at 450 nm.
ApoE3 bound LPS more avidly than apoE4 in the linear range of
the titration, between 0 and 200 ng/mL (Figure 2A). ApoE3
complexed to DMPC resulted in similar levels of LPS binding
(Figure 2B), suggesting that LPS also interacts with lipid-bound
apoE3. Appropriate ELISA controls, such as no LPS, resulted in
no measureable absorbance detected. These data indicate that
both lipid-free and lipid-bound apoE3 has higher affinity for LPS
ApoE3 Exhibits Greater C-terminal Domain Interactions
than apoE4 when Associated with LPS
The C-terminal region of apoE has been identified as the
protein’s principal lipid binding domain , with this distinction
Effects of ApoE Isoforms on Monocyte Inflammation
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attributed to a stronger interaction between amino acids in the N-
and C-terminal domain of apoE4 . As shown by Tetali et al.
, a decrease in the self-association of lipid-free apoE4 protein is
observed via the decreased broadening of the EPR spectrum
resulting from a diminished magnetic coupling between spin labels
attached to position Trp264 in the C-terminal domain of the
protein . Upon lipid binding, the self-association of the C-
terminal domain is disrupted, resulting in larger EPR amplitude
due to the decreased dipolar broadening. We therefore in-
vestigated the effect of LPS on the lipid-induced structural
conversion of apoE4. Since apoE3 contains a Cys at position
112, specific spin labeling at position 264 is not possible. However,
previous work has shown that the ArgRSer substitution at position
112 imparts E3-like behavior to apoE4 . We constructed the
mutant apoE3-like W264C+Arg112RSer to evaluate whether the
LPS binding differences observed between the isoforms by ELISA
are reflected by a differential conformation of the apoE C-terminal
As shown in Figure 3, spin labeled apoE4-264C and the apoE3-
like W264+Arg112RSer proteins were examined by EPR
spectroscopy in the absence and presence of 30 mg/mL LPS.
Figure 3A shows the spectra of each protein (normalized to the
same number of spins) in the absence of LPS. Consistent with
greater domain interaction in the E4 isoform, the spectrum of the
spin-label attached to apoE4-264C is slightly broader compared to
its attachment at the same location in the E3-like protein . The
spectra are nearly identical for each apoE mutant protein.
Figure 3B compares the same protein samples after treatment
with LPS. Remarkably, in contrast to lipids in the form of VLDL
or synthetic emulsions , both proteins display increased C-
terminal interaction in the presence of LPS (apparent by the
increased spectral broadening, see inset). Consistent with the
higher affinity of apoE3 for LPS, the spectral effect with the E3-
like protein is substantially larger. This indicates that the spectral
difference observed with LPS treatment is significantly higher for
the E3-like protein than it is for apoE4. When spin labels were
placed on sites in the N-terminal apoE4 domain (position 57, 76,
or 77), no LPS-dependent changes are observed (data not shown).
Figure 1. Differential effects of apoE3 and apoE4 on LPS-induced cytokine expression and secretion. ApoE3 or apoE4 were complexed
with extruded DMPC vesicles, then LPS was added for treatment of THP-1 cells. (A) TNFa and IL-1b gene expression and (B) TNFa protein secretion
were quantified and normalized to the LPS+DMPC control for each treatment, (n=5). *P,0.05.
Figure 2. ApoE3 binds LPS with greater affinity than apoE4.
LPS-coated wells were incubated with increasing concentrations of
apoE3 or apoE4 alone (A) or complexed to DMPC (B). LPS-bound apoE
was detected using an apoE antibody with secondary HRP, with
adhesion directly proportional to absorbance (n=6). Dashed line, DMPC
Effects of ApoE Isoforms on Monocyte Inflammation
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Fasting VLDL Lipolysis Products Attenuate LPS-induced
Monocyte TNFa Secretion in a Dose-dependent Manner
We have previously shown that the postprandial state induces
structural changes in apoE isoforms, with a minor role attributed
to triglyceride-rich lipoprotein lipolysis products . In the
circulation, VLDL particles become hydrolyzed by lipoprotein
lipase (LpL) into smaller remnant particles and fatty acids,
monoglycerides, diglycerides, and phospholipids. Additionally,
lipoproteins such as VLDL have been shown to interact with
circulating LPS by an unknown mechanism [19,30]. To determine
the effects of these lipolysis products on monocyte activation by
LPS, lipolysis of fasting and postprandial VLDL from normal
donors over a range of triglyceride (TG) concentrations was
performed by addition of LpL (2 U/mL) for 30 minutes at 37uC.
LPS (0.5 mg/mL) was added to each VLDL or VLDL+LpL
sample, and was immediately incubated with THP-1 monocytes
for 3 hours. A dose-dependent decrease in TNFa secretion was
observed from monocytes treated with fasting VLDL+LPS
(Figure 4A, black bars), and also from monocytes treated with
postprandial VLDL+LPS (Figure 4B, black bars). However,
lipolysis of fasting VLDL significantly enhanced the attenuation
from LPS stimulation beyond that for fasting VLDL (Figure 4A,
grey bars), while lipolysis of postprandial VLDL showed no
statistically significant difference from postprandial VLDL atten-
uation (Figure 4B, grey bars). A VLDL dose of 200 mg TG/dL
was chosen for subsequent experiments based on the differences in
attenuation between fasting and postprandial VLDL lipolysis
products at this dose.
VLDL lipolysis products attenuated the LPS-induced inflam-
matory response more than VLDL, despite the abundance of fatty
acids released upon lipolysis, some of which may be pro-
inflammatory. To determine if the remnant lipoproteins were
capable of attenuating LPS-induced TNFa secretion more than
VLDL or VLDL lipolysis products, remnants were isolated after
the lipolysis reaction and used for monocyte treatment. Figure 4C
shows that remnants from both fasting and postprandial VLDL
were no more protective than VLDL, and less protective than all
lipolysis products, suggesting that lipolysis of fasting VLDL renders
apoE more capable of sequestering LPS. In addition, postprandial
VLDL remnants were more protective than fasting VLDL
To ensure that the different effects of VLDL lipolysis products
from the fasting and postprandial state were not due to variations
in apolipoprotein or LPS content, total apoE protein, apoB
protein, and endotoxin were measured and normalized to
triglyceride content (Table 1). Using an autoanalyzer from
Polymedco, we found that the fasting and postprandial VLDL
lipolysis samples had an average total apoE content of 3.460.5
and 3.560.6 mg apoE/g TG, respectively. After isolating the
VLDL remnants, apoE content dropped to 0.5360.23 and
0.8460.32 mg apoE/g TG for fasting and postprandial samples,
respectively. The endotoxin levels in all VLDL samples averaged
0.2560.2 endotoxin units per mL, which is regarded as below the
acceptable endotoxin levels for cell cultures, using a Limulus
Amebocyte Lysate (LAL) kit from Cambrex.
Blocking apoE Associated with Fasting VLDL Lipolysis
Products Partially Reversed the Attenuation of LPS-
induced TNFa Release from Monocytes
To determine if apoE present on hydrolyzed fasting or
postprandial VLDL is involved in the attenuation of LPS-induced
monocyte activation, apoE was blocked prior to addition of LPS
using a monoclonal apoE antibody. Blocking apoE on fasting
VLDL lipolysis products pooled from normal donors reversed the
attenuation of the LPS-induced TNFa secretion from monocytes
from 17% to 53% of the LPS control (Figure 5A). Blocking apoE
associated with lipolysis of postprandial VLDL did not significantly
change the level of TNFa secreted (44% to 49%), implying that
apoE associated with fasting VLDL lipolysis products has a greater
potential to reduce LPS-induced inflammation than apoE on
Figure 3. Interaction of apoE C-terminal domain is increased by LPS. EPR spectra of spin-labeled apoE4-264C and E3-like apoE-
W264C+Arg112RSer in the absence (A) and presence (B) of LPS. LPS induces spectral broadening of both apoE4 and the E3-like proteins (inset), but
the magnitude of the effect is greater with E3-like protein (B).
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We further evaluated the protective effects of native apoE3 and
apoE4 on VLDL obtained from healthy donors with apoE3/E3
and apoE4/E4 genotypes. As shown in Figure 5B, the monocyte
responses to fasting and postprandial VLDL lipolysis products
containing apoE3 were similar to those from normal pooled
subjects containing unknown (but presumably apoE3) apoE
isoforms. Blocking apoE on apoE3-containing VLDL partially
reversed the attenuation of TNFa secretion. However, the reduced
inflammatory response to fasting and postprandial apoE4-contain-
ing VLDL lipolysis products was less striking (Figure 5C).
Furthermore, blocking apoE on apoE4-VLDL lipolysis products
had no effect on TNFa secretion levels, suggesting that apoE4 is
not involved in the protective effect of apoE4-containing VLDL
Since blocking apoE only partially reversed the attenuated
TNFa secretion, the contributions of other apolipoproteins present
on VLDL were examined. Using monoclonal antibodies, we
blocked apoB-100, apoCII, and apoCIII from pooled VLDL
samples (Figure 6). Blocking all of these apolipoproteins resulted in
a reversal of the attenuation of the LPS inflammatory response,
similar to blocking apoE. However, the effect was the same when
blocking the apolipoproteins on fasting VLDL lipolysis products as
on postprandial VLDL lipolysis products. Therefore, no differen-
tial effect was seen between fasting and postprandial samples after
blocking apoB-100, apoCII, or apoCIII. This implies that apoE,
but not the other apolipoproteins, undergoes a functional change
in the postprandial period.
Triglyceride-rich lipoproteins are known to bind and neutralize
LPS, thereby preventing sepsis-induced host death [31,32]. It has
been suggested that apolipoproteins, and in particular apoE, play
a role in reducing LPS-induced lethality . The distribution and
structural conformation of apoE on lipoproteins undergo dramatic
changes during the postprandial period, which may alter its ability
to interact with LPS. The objective of this study was to compare
the effects of apoE3 and apoE4 on LPS-induced activation of
A differential effect of apoE isoforms on endotoxemic in-
flammation has been suggested by previous studies. Microglial
cells from mice homozygous for the apoE4 allele display pro-
inflammatory characteristics including increased cytokine expres-
sion and altered cell morphology , while transgenic mice
expressing apoE4 have an increased NFkB-dependent pro-
inflammatory expression profile [34,35]. Conversely, macrophages
expressing apoE3 are less responsive to LPS than those expressing
apoE2 or apoE4 , presumably by reducing TLR4-mediated
signaling through JNK . Zhu et al. have suggested that apoE3
attenuates LPS-induced cytokine expression from these macro-
phages by enhancing cholesterol clearance, thus rearranging
Figure 4. Fasting VLDL lipolysis products attenuate LPS-
induced TNFa release. (A-B) Monocytes treated with fasting or
postprandial VLDL lipolysis products and LPS exhibited dose-de-
pendent attenuation of TNFa secretion (n=6). A: fasting VLDL; B:
postprandial VLDL. *P,0.01 from control, #P,0.05 from the same
concentration dose without LpL. (C) Postprandial remnants (VLDL rem)
attenuate LPS-induced TNFa secretion more than fasting remnants.
*P,0.05 from control, #P,0.05 from fasting.
Table 1. ApoE and apoB content of VLDL lipolysis products and remnants (n=3).
Fasting samples Postprandial samples
ApoE content mg/g TG
ApoB content mg/g TG
ApoE content mg/g TG
ApoB content mg/g TG
VLDL remnants 0.5360.23
Effects of ApoE Isoforms on Monocyte Inflammation
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plasma membrane cholesterol domains necessary for adequate
TLR4 signaling . Using a synthetic phospholipid emulsion, we
have shown that apoE3 reduces LPS-induced monocyte activa-
tion, while apoE4 potentiates it. A similar observation has been
made previously in human aortic endothelial cells, in which the
inflammatory response to TNFa was blunted by apoE3 and
augmented by apoE4 . It is apparent that there are several
potential mechanisms involving both direct and indirect LPS
action by which apoE3 and apoE4 exert opposing effects on the
inflammatory response, presumably by differentially regulating
NFkB-mediated cell signaling, and which warrant further studies.
Figure 5. Blocking apoE from fasting VLDL lipolysis products
reverses the attenuation of LPS-induced TNFa release. Mono-
cytes were treated with apoE-blocked VLDL lipolysis products from (A)
pooled normal (n=6), (B) apoE3/E3 subjects (n=3), and (C) apoE4/E4
subjects (n=3). Blocking fasting samples reversed the attenuated TNFa
release, except for apoE4/E4. Different letters represent significant
differences between groups; P,0.01 for a vs. b and a vs. c; P,0.05 for
b vs. c.
Figure 6. Blocking apoB-100, apoCII, and apoCIII on VLDL
lipolysis products reverses the attenuation of LPS-induced
TNFa secretion. Monocytes were treated with (A) apoB-100, (B)
apoCII, or (C) apoCIII-blocked fasting and postprandial VLDL lipolysis
products from pooled normal subjects (n=3). Blocking each apolipo-
protein from both fasting and postprandial samples reversed the
attenuated TNFa release. Different letters represent significant differ-
ences between groups; P,0.01 for a vs. b and c; P,0.05 for a vs. d.
Effects of ApoE Isoforms on Monocyte Inflammation
PLOS ONE | www.plosone.org7 November 2012 | Volume 7 | Issue 11 | e50513
In contrast to apoE3, apoE4 induced an inflammatory response
from monocytes when treated in combination with LPS. We
investigated whether the binding kinetics between apoE isoforms
and LPS were different. Using an ELISA that we developed, there
was significantly more direct binding of LPS to apoE3 than to
apoE4 within the linear range of the dose-response (between 0 and
200 ng/mL). This suggests that LPS directly interacts with apoE3
preferentially over apoE4, which could explain the protection from
LPS-induced inflammation associated with apoE3. However, the
lower binding capacity of apoE4 to LPS does not explain the
augmented inflammatory response above that for the LPS+DMPC
control, which may be due to additional interactions between
apoE4 and specific cellular signaling pathways.
Previous EPR studies have demonstrated that apoE self-
associates via its C-terminal domain . LPS induces substantial
broadening of the EPR spectrum from labels attached at the C-
terminal domain of the apoE3-like protein. A similar, but smaller,
spectral change of apoE4 occurs when incubated with LPS. This
suggests that LPS stabilizes the interaction between C-termini, to
a greater extent in the apoE3-like mutant than apoE4. Examina-
tion of three different sites in the N-terminal domain did not reflect
any conformational changes, indicating the C-terminus primarily
binds to LPS. Thus position 264 serves as an especially useful
marker for studying the influences of LPS on apoE. Given that the
C-terminal domain represents the region of the protein with the
highest lipid-binding affinity, factors that stabilize protein-protein
interactions may alter the distribution of apoE on lipoprotein
particles, and as such have profound effects on lipid metabolism or
downstream processes linked to inflammation .
After consumption of a high fat meal, VLDL released from the
liver undergoes lipolysis in close proximity to the endothelial
surface of the vascular wall, exposing passing monocytes to high
concentrations of lipolysis products. Here we report a protective
effect of hydrolyzed VLDL on subsequent monocyte activation by
LPS, with protection being greater when the lipolysis substrate is
fasting VLDL rather than postprandial VLDL. There was no
significant difference in secreted TNFa attenuation between
fasting and postprandial VLDL treatment without lipolysis. Our
observation that LPS interacts with the C-terminal lipid binding
domain of apoE, combined with the enhanced protection provided
by hydrolyzed apoE-containing VLDL against LPS, suggests that
lipolysis of VLDL exposes the C-terminal domain of apoE, which
could explain the added protection provided by VLDL lipolysis
products compared with whole VLDL. Furthermore, apoE3 was
found to be involved in this protective effect associated with fasting
VLDL lipolysis products, but not involved in protecting monocytes
from LPS when associated with postprandial VLDL lipolysis
products. The total apoE content of both fasting and postprandial
VLDL used in each experiment was similar, and therefore any
variations in apoE-LPS interactions could be attributed to changes
in isoform or conformation. Our data suggest that apoE becomes
more available for LPS neutralization when VLDL is hydrolyzed,
particularly from fasting VLDL. In contrast, the remnant particles
isolated following hydrolysis of postprandial VLDL were more
protective against LPS-induced inflammation than fasting rem-
nants, supported by higher apoE content. Our previous studies
also have shown that apoE4 preferentially associates with VLDL
while apoE3 associates with HDL in postprandial plasma .
This change in apoE distribution could explain the reduced
capacity for postprandial VLDL lipolysis products to attenuate
LPS-induced inflammation [18,40]. The conformation of apoE,
and therefore its affinity for LPS, could become altered
postprandially, which could account for the reduction in post-
prandial protection from LPS-induced injury.
Several explanations could account for the difference between
fasting and postprandial VLDL and protection from LPS-induced
monocyte inflammation. First, our previous study showed that
lipoprotein lipase itself could exert pro- or anti-inflammatory
effects on endothelial cells depending on the specific stimulating
agent . However, this seems unlikely in this case given that
LpL alone did not attenuate LPS-induced inflammation. Second,
lipolysis products from VLDL, such as free fatty acids, could
counteract the actions of LPS. We have recently shown that
lipolysis of postprandial VLDL generates approximately two-fold
more non-esterified fatty acids than lipolysis of fasting VLDL ,
and that these fatty acids alone induce pro-inflammatory gene
expression from THP-1 monocytes . Third, VLDL lipolysis
products may differ in their composition when released from
fasting or postprandial VLDL, yielding a net greater anti-
inflammatory effect when monocytes are treated with fasting
VLDL lipolysis products and LPS. Using laser trapping Raman
spectroscopy, we previously showed that individual fasting VLDL
are rich in unsaturated lipid modes, while postprandial VLDL
contain highly ordered saturated modes . This compositional
difference between fasting and postprandial VLDL suggests that
lipolysis of fasting VLDL would release more unsaturated fatty
acids than postprandial VLDL. Other studies have shown that
treatment of monocytes with polyunsaturated fatty acids (PUFA)
decreases cellular activation with LPS co-stimulation , while
saturated fatty acids have been implicated as pro-inflammatory
, suggesting that the composition of the VLDL lipolysis
products could contribute to an overall pro-inflammatory or anti-
inflammatory response. We have recently characterized post-
prandial VLDL lipolysis products from VLDL samples pooled
from normal human subjects . As expected, these contain high
levels of saturated (348.48 nmol/mg TG), monounsaturated
(227.55 nmol/mg TG), and polyunsaturated (204.8 nmol/mg
TG) non-esterified fatty acids, of which C16:0, C18:1n9/1n7t,
and C18:2n6 were the most abundant. With more unsaturated
fatty acids released per mg triglyceride, it seems plausible that
these could contribute to inflammatory protection by activating
anti-inflammatory and LPS-counteracting signaling pathways such
as those controlled by PPARc.
In conclusion, apoE3 prevents LPS-induced inflammatory
responses from monocytes, while apoE4 exaggerates them. We
have shown for the first time that LPS binds directly to apoE3,
which could explain some of the anti-inflammatory properties
associated with apoE3. Further, apoE3 plays a role in blunting the
inflammatory response to LPS activation, especially when
associated with hydrolyzed fasting VLDL. Our findings provide
insights into complex postprandial lipoprotein metabolism expe-
rienced multiple times per day in persons consuming the typical
Western diet. This knowledge of the pro- and anti-inflammatory
actions of apoE and VLDL and their lipolysis products will
facilitate the development of therapeutic strategies to prevent and
attenuate atherosclerotic cardiovascular disease.
The authors would like to acknowledge Jodi Ensunsa and Roberta Holt of
the Department of Nutrition at the University of California, Davis, for their
assistance in meal preparation and phlebotomy, and Theresa Tonjes of the
Clinical Nutrition Research Unit at the University of California, Davis for
her assistance in the quantification of lipoproteins and lipolysis products.
Conceived and designed the experiments: LJD SDT JOL JCV JCR.
Performed the experiments: LJD RA RH JP MSB. Analyzed the data: LJD
Effects of ApoE Isoforms on Monocyte Inflammation
PLOS ONE | www.plosone.org8 November 2012 | Volume 7 | Issue 11 | e50513
RA JP MSB JCV. Contributed reagents/materials/analysis tools: LJD Download full-text
JCV. Wrote the paper: LJD.
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Effects of ApoE Isoforms on Monocyte Inflammation
PLOS ONE | www.plosone.org9 November 2012 | Volume 7 | Issue 11 | e50513