© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Short-Term Feeding of Atherogenic Diet to Mice Results in Reduction of HDL and Paraoxonase That May Be Mediated by an Immune Mechanism
From the Division of Cardiology and UCLA Undergraduate Student Research Program, University of California, Los Angeles, and the Department of Medicine (J.L.W.), University of California, San Diego.
Correspondence to Alan M. Fogelman, MD, 10833 Le Conte Ave, Box 951736, Los Angeles, CA 90095-1736. E-mail afogelman{at}mednet.ucla.edu
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Abstract |
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Abstract—Short-term feeding (up to 7 days) of an atherogenic diet to C57BL/6 low density lipoprotein receptor–deficient mice did not result in decreased hepatic paraoxonase (PON) mRNA but caused a dramatic decrease in plasma PON activity and mass. The decreased activity and mass were temporally related to an increase in plasma and high density lipoprotein (HDL) lipid hydroperoxides and to a decrease in HDL cholesterol and native apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II). As the native apoA-I protein disappeared from the circulation, higher molecular weight forms of apoA-I appeared, some of which contained epitopes recognized by an antibody (EO6) that recognizes oxidized phospholipids. After mice consumed an atherogenic diet for 1 or 3 days, switching the mice to a low fat chow diet for 3 days resulted in a return to baseline levels of lipid hydroperoxides but only a small return toward baseline for HDL cholesterol, with no significant increase in apoA-I mass or PON activity and mass. After mice consumed an atherogenic diet for 3 days, switching to the chow diet for 3 days did not significantly alter the high molecular weight forms of apoA-I or the signal generated by EO6. In marked contrast, after mice consumed an atherogenic diet for 7 days, switching to the chow diet for 3 days resulted in a dramatic increase in native apoA-I to baseline levels, with virtual disappearance of the high molecular weight forms of apoA-I, including the form recognized by EO6. After mice consumed an atherogenic diet for 7 days, switching to the chow diet for 3 days also resulted in significant increases in HDL cholesterol and PON mass and activity, although baseline levels were not reached. IgG and IgM antibodies were found to be associated with apoA-I in control animals, were minimally decreased after the 3-day atherogenic diet, were dramatically decreased after the 7-day atherogenic diet, and returned to near or above baseline levels after a return to the chow diet for 3 days. We conclude that the atherogenic diet rapidly induces lipid hydroperoxide formation and apoA-I oxidation with the formation of high molecular weight forms of apoA-I. Concomitant with these changes in apoA-I levels, HDL cholesterol and PON activity and mass declined without changes in mRNA levels for apoA-I or PON, suggesting increased clearance of these altered HDL particles. We further conclude that between the third and seventh day of the atherogenic diet, an as-yet-unidentified mechanism for clearing the high molecular weight forms of apoA-I is induced and that this mechanism may be related to the clearance of immune complexes.
Key Words: diet • paraoxonase • HDL • apolipoproteins
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Introduction |
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We previously reported that feeding an atherogenic diet to mice (C57BL/6J) that were genetically susceptible to atherosclerosis resulted in a decrease in hepatic paraoxonase (PON) mRNA levels after 5 weeks on the diet.1 Mice that were genetically resistant to atherosclerosis (C3H/HeJ) did not show a decrease in hepatic PON mRNA levels.1 Recombinant inbred strains of mice that were created by crossbreeding the C57BL/6J and C3H/HeJ parental strains for generations showed a heterogeneous response in hepatic PON mRNA levels on being fed the atherogenic diet.1 On analysis, there was a significant inverse correlation between the level of hepatic PON mRNA in these new recombinant inbred strains placed on an atherogenic diet and in the development of aortic atherosclerotic lesions.1 Thus, these data demonstrate a genetically determined inverse relationship between the dietary responsiveness of hepatic PON mRNA and atherosclerosis.
In another study,2 we demonstrated that the injection of
mildly oxidized LDL or oxidized
L-
-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine
(Ox-PAPC) into the atherosclerosis-sensitive C57BL/6J
mice significantly reduced PON activity within 24 hours. In contrast,
injection of mildly oxidized LDL or Ox-PAPC into the
atherosclerosis-resistant C3H/HeJ mice did not
cause a decrease in PON activity under these
conditions.2
Previously, we also demonstrated that in rabbits and humans PON activity changed dramatically over the course of a few days in response to the induction of an acute phase reaction.3 Because studies in mice1 4 and humans5 6 7 8 have suggested that PON may be important in determining susceptibility to atherosclerosis, we performed studies to determine whether short-term dietary interventions could alter PON activity. To accentuate the effects of the diet and to determine the mechanisms involved, we chose to feed an atherogenic diet to LDL receptor–deficient mice with an atherosclerosis-susceptible C57BL/6J genetic background. As reported in the present study, periods of feeding the atherogenic diet for as little as 3 days resulted in profound changes in plasma lipid hydroperoxides, HDL cholesterol, apoA-I, apoA-II, and PON levels.
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Methods |
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Materials
The atherogenic diet (TD 90221) and the rodent chow containing 0.5% sodium cholate (TD 99057) were purchased from Teklad, and the rodent chow diet (Purina Rodent Chow) was from Ralston-Purina Co. Trizol reagent was from BRL Life Technologies, Inc. Taqman reverse transcription (RT)–polymerase chain reaction (PCR) kits and rodent GAPDH primers and probe were from Perkin-Elmer, Inc. All other Taqman primers and probes were from IDT Technologies. NuPage polyacrylamide gels were purchased from Novex, Inc. Cholesterol-20 and a cholesterol calibrator were from Sigma Chemical Co. Enhanced chemiluminescence reagents were from Amersham Pharmacia, Inc. Monospecific polyclonal antibodies to mouse apoA-I, mouse apoA-II, and mouse PON were a kind gift from Dr Aldons J. Lusis (University of California, Los Angeles).
Mice
LDL receptor knockout mice on a C57BL/6 background were
purchased from Jackson Laboratories (Bar Harbor, Me). All animals were
female and aged 4 to 6 months at the time of the experiments. The
atherogenic diet contained 15.75% fat, 1.25% cholesterol,
and 0.5% sodium cholate. The rodent chow diets contained 4% fat in
the absence or presence of 0.5% sodium cholate. The protocol used to
test the diets entailed feeding the mice the atherogenic diet for 1, 3,
or 7 days, followed by the chow diet for 3 days. Mice fed the
atherogenic diet for 1, 3, or 7 days were identified as groups 1.0,
3.0, and 7.0, respectively. Mice fed the atherogenic diet for 1, 3, or
7 days, followed in each case by 3 days on the rodent chow diet, were
identified as groups 1.3, 3.3, and 7.3, respectively. Groups of mice
were also maintained for 7 days on either the rodent chow diet or the
rodent chow diet containing 0.5% sodium cholate to serve as
controls.
Lipoproteins
With the mice under mild isoflurane anesthesia,
blood was obtained from the retro-orbital sinus, with heparin used as
an anticoagulant (2.5 U/mL blood); the regulations set forth by the
University of California Animal Research Committee were followed. LDL
and HDL were isolated by fast performance liquid
chromatography (FPLC) with the use of methods
previously described.9 HDL cholesterol levels
in the plasma of mice were determined after heparin and manganese
chloride precipitation of VLDL and LDL. The concentration of
lipoproteins reported in the present study is based on
cholesterol content, unless otherwise specified.
Determination of mRNA Levels
Total cellular RNA was isolated from tissue with the use of
Trizol reagent according to the manufacturer’s protocol. Levels of
mRNA abundance were determined by Taqman real-time RT-PCR as described
previously.10 11 12 The RT-PCR reaction was as follows:
50°C for 2 minutes and 60°C for 30 minutes, followed by 40 cycles
of 95°C for 5 minutes, 94°C for 20 seconds, and 62°C for 1
minute. The mRNAs of interest were normalized to GAPDH mRNA with the
use of rodent GAPDH Taqman primers and probe (Perkin Elmer, Inc). The
sequences used for detection of murine apoA-I mRNA were as follows:
sense 5'-ttcacgggctttcctaccag-3', antisense 5'-gccagatgaatcccatccct-3',
and the Taqman hydrolysis probe
5'-6FAM-ctggaacctgccatggtgccca-TAMRA-3'. The sequences used for
detection of murine PON mRNA were as follows: sense
5'-tctgggtgggatgccatc-3', antisense 5'-tctgagccgggaggattct-3', and the
Taqman hydrolysis probe
5'-6FAM-caacggaatgaggatctttttctatga-cgca-TAMRA-3'. The sequences
used for detection of murine SR-B1 mRNA were as follows: sense
5'-gggacttccgggcagatg-3', antisense 5'-cctccgggctgaagaattc-3', and the
Taqman hydrolysis probe 5'-6FAM-acccttcatgacacccgaatcctcg-TAMRA-3'. The
sequences used for detection of murine cubilin mRNA were as follows:
sense 5'-tgctgtcttccaatctgaggag-3', antisense 5'-gtccgcaccggctaatga-3',
and the Taqman hydrolysis probe
5'-6FAM-cagcccagggcttctctg-catcc-TAMRA-3'.
SDS-PAGE and Western Blotting
Polyacrylamide gel electrophoresis in SDS was performed
as previously described.9 FPLC-isolated HDLs (25 µg of
total cholesterol) from each group of mice were run on 2%
to 16% NuPage gels (Novex, Inc) with the use of MOPS buffer at 125 V
for 45 minutes according to the manufacturer’s protocol. The proteins
were then transferred to nitrocellulose at 200 V for 2 hours with the
use of a Novex transfer apparatus. Protein bands were
visualized by a chemiluminescence procedure and quantified by
densitometry (ZeroD Scan, Stratagene). The resulting signals were
shown to be in the linear portion of the response curve with the use of
apolipoprotein standards run in parallel at various
dilutions.9
Antibodies
Monospecific polyclonal antisera to mouse apoA-I, mouse apoA-II,
and mouse PON were used.4 9 13 14 Quantitative estimates
of the levels of the apolipoproteins were performed as previously
described by using apolipoproteins isolated from mouse
lipoproteins.13 14
Immunoassays
Autoantibody titers against malondialdehyde-modified LDL, an
important epitope of oxidized LDL, were determined by ELISA as
previously described,15 except that a highly sensitive
chemiluminescent detection system was used.16 In brief,
malondialdehyde-modified LDL in 50 mmol/L Tris-buffered saline
(TBS) at 5 µg/mL was plated in flat-bottomed high-binding Microfluor
microtiter plates (Dynex Technologies, Inc) and incubated overnight at
4°C. The plates were then washed with PBS containing 0.27 mmol/L
EDTA, 0.02% NaN3, 0.05% Tween 20, and 0.001%
aprotinin, with use of a microtiter plate washer. Fifty microliters of
a 1:400 dilution of plasma in TBS/3% BSA was incubated with each well
for 1 hour at room temperature. After 3 additional washes, the amount
of bound immunoglobulin was detected by the addition of alkaline
phosphatase–linked secondary antibodies against murine IgG or IgM,
which were then detected by chemiluminescence
techniques.16 To measure the amount of plasma
immunoglobulin specifically associated with HDL (ie, apoA-I), 5 µg/mL
of an affinity-purified goat anti-mouse IgG (or anti-mouse IgM) was
used to coat the bottom of microtiter wells. Varying dilutions of
murine plasma in 1% BSA/TBS were then incubated in the wells for 90
minutes at room temperature. After 3 independent wash steps with an
automated plate washer, a 1:10 000 dilution of a rabbit anti-mouse A-I
antibody was added for a 1-hour incubation at room temperature. After 4
additional washes with TBS, the amount of rabbit IgG (or IgM) bound was
detected by use of the alkaline phosphatase–linked secondary antibody
as described above. In similar assays, we sought to detect the amount
of apoA-I found on particles containing EO6 epitopes. Microtiter
wells were coated with 5 µg/mL of purified EO6 and incubated with
varying dilutions of plasma, and then the amount of apoA-I bound was
detected by use of the rabbit anti-mouse A-I antibody as described
above. For each of these assays, the data are expressed as relative
light units measured over 100 ms, and each data point is the average of
triplicate determinations.
Other Methods
The protein content of lipoproteins was determined by a
modification17 of the Lowry assay.18 Lipid
hydroperoxide levels were measured by the Auerbach
assay.19 PON activity was measured as previously described
with paraoxon used as the substrate.20
Cholesterol concentrations of FPLC-isolated LDL and HDL
used for electrophoresis and for Auerbach and PON assays were
determined with a cholesterol-20 kit from Sigma with the
use of 20 µL of LDL or HDL. Statistical significance was determined
by ANOVA and the Statview 6.0 statistical program. Significance is
defined as P<0.05.
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Results |
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Plasma Lipid Hydroperoxide Levels in Mice on the Atherogenic Diet
As shown in Figure 1
, plasma lipid
hydroperoxide levels increased after 1 day on the atherogenic diet and
continued to increase to almost 10-fold above baseline by 7 days on the
diet. In each instance (ie, after 1, 3, or 7 days), switching the
atherogenic diet to a chow diet for 3 days resulted in a return to or
below baseline in plasma lipid hydroperoxide levels. Adding 0.5%
cholate to the rodent chow diet for 7 days did not significantly change
plasma lipid hydroperoxide levels. The maximum levels of lipid
hydroperoxides in LDL differed in that they were seen after day 3 of
the atherogenic diet and actually declined by day 7 (data not shown).
The lipid hydroperoxide levels in HDL paralleled the data in Figure
1 for plasma, with dramatic elevations in lipid hydroperoxide
levels in HDL after the 7-day atherogenic diet (data not shown). The
majority of plasma lipid hydroperoxides were associated with HDL in all
instances (data not shown).
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PON Activity in Mice on the Atherogenic Diet
PON activity in serum declined slightly (P<0.01) when
0.5% cholate was added to the rodent chow diet for 7 days (Figure 2). However, there was a progressive and
dramatic decline in PON activity after days 1, 3, and 7 of the
atherogenic diet. At 7 days, PON activity was only 
25% of control
values (Figure 2). Changing the atherogenic diet to chow for 3
days did not change PON activity if the atherogenic diet had been fed
for only 1 or 3 days (Figure 2). However, after 7 days of the
atherogenic diet, switching to the chow diet for 3 days resulted in a
significant increase in PON activity (Figure 2). The majority of
PON activity in plasma resides in HDL,21 and similar
results were seen in HDL PON activity (data not shown).
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HDL Cholesterol Levels in Mice Fed the Atherogenic
Diet
Plasma HDL cholesterol levels fell slightly
(P<0.01) when the mice were fed rodent chow plus 0.5%
cholate for 7 days, and these levels were similar to the levels seen
after 1 day of the atherogenic diet (Figure 3). There was a progressive decline in
plasma HDL cholesterol after 3 and 7 days of the
atherogenic diet (Figure 3). After 7 days of the atherogenic
diet, HDL cholesterol levels were only 15% of control
values (Figure 3). Changing the atherogenic diet to a chow diet
for 3 days did not significantly increase HDL cholesterol
levels if the atherogenic diet had been fed for 1 day. However, after
the mice were fed the atherogenic diet for 3 or 7 days and then
switched to the chow diet for 3 days, there was a significant increase
in HDL cholesterol levels, although the levels of HDL
cholesterol remained substantially below the levels on the
chow or chow plus cholate diets (Figure 3).
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FPLC profiles of lipoprotein cholesterol confirmed the data
in Figure 3
and further demonstrated the increase in VLDL and
LDL-like particles induced by the atherogenic diet compared with chow
or chow plus cholate diets and the corresponding decrease in HDL (data
not shown).
PON Protein and Apolipoprotein Levels and the Atherogenic
Diet
As shown in Figure 4A, PON protein
did not decline until the atherogenic diet was fed for 3 days but
continued to decline afterward, such that PON protein was only 15%
of baseline after 7 days of the atherogenic diet, a value similar to
that seen for HDL cholesterol (Figure 3). Changing
to a chow diet for 3 days after 3 days of the atherogenic diet did not
produce an increase in PON protein (Figure 4A). However, if the
diet was switched to chow for 3 days after the 7-day atherogenic diet,
there was a dramatic increase in PON protein to approximately two
thirds of the baseline value (Figure 4A).
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PON is known to be tightly bound to apoA-I.22 23 As shown
in Figure 4B, the changes in apoA-I protein paralleled
closely the changes seen in PON protein. Switching to the chow diet for
3 days after the 7-day atherogenic diet resulted in a dramatic return
of apoA-I levels to baseline (Figure 4B).
ApoA-II levels paralleled the changes seen in apoA-I levels, except
that after changing to the chow diet for 3 days after the 7-day
atherogenic diet, there was only a small increase in apoA-II levels
(data not shown) compared with the dramatic increase in apoA-I levels
(Figure 4B).
Levels of ApoA-I and PON mRNA Abundance in Liver Are Not Affected
by the Atherogenic Diet
Despite the dramatic changes in plasma PON activity (Figure
2) and PON protein (Figure 4A), there were no changes in
hepatic PON mRNA levels (data not shown). Similarly, despite the
dramatic changes in apoA-I protein levels (Figure 4B), there
were no significant changes in apoA-I mRNA levels (data not shown).
Native ApoA-I Disappears With an Atherogenic Diet
As shown in Figure 5, the 3-day
atherogenic diet resulted in a dramatic decrease in native apoA-I in
HDL and a dramatic increase in high molecular weight proteins in HDL
that were recognized by a mouse apoA-I antibody in a Western blot.
Changing to a chow diet for 3 days after the 3-day atherogenic diet did
not result in a significant change in these high molecular weight
proteins in HDL that were recognized by antibody to apoA-I (Figure
5). However, after the 7-day atherogenic diet, there was a
dramatic decrease in these high molecular weight proteins in HDL that
were recognized by an antibody to apoA-I (Figure 5). Despite the
decrease in these high molecular weight proteins in HDL after the 7-day
atherogenic diet, there was no return of native apoA-I (Figure
5). However, changing to a chow diet for 3 days after the 7-day
atherogenic diet resulted in a dramatic disappearance of the high
molecular weight proteins and reappearance of native apoA-I in HDL
(Figure 5).
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Oxidized Phospholipid Epitopes Appear in HDL After 3-Day
Atherogenic Diet
To look for the presence of oxidized phospholipids in HDL, we used
the monoclonal antibody E06. This antibody has been well characterized
and recognizes oxidized phospholipids that have been shown to play a
role in atherosclerosis15 16 24 The EO6
antibody recognized an epitope in HDL that mildly increased after the
3-day atherogenic diet (data not shown). Interestingly, after switching
to the chow diet for 3 days after the 3-day atherogenic diet, the
signal for this epitope showed little change (data not shown). After
the 7-day atherogenic diet, the signal for the EO6 epitope in HDL was
maximal (data not shown). On changing to the chow diet for 3 days after
the 7-day atherogenic diet, the signal for the EO6 epitope dramatically
declined (data not shown).
Search for Mechanisms for Clearance of HDL
Because scavenger receptor B1 (SR-B1) has been implicated in the
removal of cholesteryl esters from HDL,25 26 27 we
determined hepatic SR-B1 protein levels. After the 7-day atherogenic
diet with or without a further 3 days of chow diet, there was no
significant change in SR-B1 protein levels (data not shown).
Recent publications28 29 have reported that disruption of the gene for the ATP-binding cassette transporter-1 (abc-1) reproduced the manifestations of Tangier disease. Alterations of this gene in patients with Tangier disease were also seen.30 31 32 It has been concluded that abc-1 is critical for the export of cholesterol from cells to nascent HDL and that its absence results in low levels of HDL cholesterol. The low levels of HDL cholesterol are presumed to occur because of increased clearance of the nascent HDL compared with mature HDL.30 31 32 A chow diet supplemented with 0.5% cholate caused a small but significant increase in hepatic mRNA levels for abc-1 (data not shown). This increase was comparable to the small increase seen after a 1-day atherogenic diet (data not shown). The 3- and 7-day atherogenic diets progressively increased hepatic mRNA levels for abc-1, and changing to the chow diet for 3 days after the 3- or 7-day atherogenic diet resulted in a significant decline in abc-1 hepatic mRNA levels but not to baseline levels (data not shown).
Cubilin, the intrinsic factor–vitamin B12 receptor, is expressed in the terminal ileum of the intestine and in the kidney. Cubilin has been shown to be a high-affinity apoA-I and HDL receptor, which facilitates the endocytosis of HDL.33 34 We examined whether the expression of cubilin in the kidney was affected by the atherogenic diet. Expression of mRNA for cubilin in the kidney was not changed after the 7-day atherogenic diet (data not shown).
Autoantibodies to oxidized lipids have been found in a variety of
species.15 16 Because dramatic changes in the levels of
apoA-I and the apoA-I–associated enzyme PON were detected between days
3 and 7 of the atherogenic diet, we reasoned that an immune complex
mechanism might account for some of the observed changes. First, we
measured the titer of autoantibodies to a general epitope of oxidized
LDL, malondialdehyde-modified LDL, in the plasma of control mice, mice
fed the atherogenic diet for 3 or 7 days, and mice fed the atherogenic
diet for 3 or 7 days and then switched to the chow diet for 3 days. The
titers of the autoantibodies were similar in all groups (data not
shown). Next, we determined the presence of specific apoA-I–IgG and
apoA-I–IgM complexes at the various time points. As shown in Figure 6, these complexes were found in the
control animals on a chow diet. After the 3-day atherogenic diet, there
was a small decrease in the level of these complexes, which returned to
above baseline on switching to the chow diet for 3 days. After the
7-day atherogenic diet, there was a dramatic decrease in the level of
these complexes, which returned toward baseline after switching to the
chow diet for 3 days. Similar results were seen with apoA-I–EO6
complexes. Please note that in the experiments shown in Figure
6, the relative amount of apoA-I associated with a fixed amount
of EO6 at the different time points was measured. As noted previously,
when the amount of EO6 staining protein found in HDL was measured at
the various time points, the signal was maximal after the 7-day
atherogenic diet and virtually disappeared on changing to the chow diet
for 3 days (data not shown).
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Discussion |
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The data in the present study indicate that short-term feeding of an atherogenic diet to LDL-receptor–deficient mice with an atherosclerosis-susceptible genetic background (C57BL/6J) results in dramatic changes in lipid hydroperoxides, HDL cholesterol, apoA-I, apoA-II, PON protein, and PON activity. Dueland et al35 have reported that cholate is required for the decline in HDL cholesterol levels that have been seen in susceptible mice fed the atherogenic diet used in the present study. The studies of Dueland et al, like those previously reported by this laboratory,1 36 were carried out for months. Our data demonstrate that the cholate component of the atherogenic diet had only minimal effects on HDL cholesterol levels in the short-term feeding studies that we performed and, thus, could not account for the dramatic changes in HDL in these studies.
These acute changes induced by the atherogenic diet include a virtual
disappearance of HDL cholesterol (Figure 3), HDL
apoA-I (Figure 4B), HDL apoA-II (data not shown), HDL PON
activity (Figure 2), and PON protein (Figure 4) after the
7-day atherogenic diet. The return of these parameters
after switching to a chow diet for 3 days was minimal after the 3-day
atherogenic diet. In contrast, there was a much more robust return
toward normal after changing to the chow diet for 3 days after the
7-day atherogenic diet.
After the 3-day chow diet that followed the 3-day atherogenic diet,
there was virtually no decline in the high molecular weight proteins in
HDL that were recognized by antibody to apoA-I (Figure 5). In
contrast, lipid hydroperoxide levels, which progressively increased
with the atherogenic diet from day 1 to day 7, returned to baseline
levels after switching to the chow diet for 3 days at each time point
studied (Figure 1). By day 7 of the atherogenic diet, there was
a dramatic decrease in the high molecular weight proteins in HDL
recognized by the antibody to apoA-I (Figure 5), and there was
an increase in the EO6 signal (data not shown) with no return of native
apoA-I (Figure 5). On changing to the chow diet for 3 days after
the 7-day atherogenic diet, there was a dramatic decrease in the high
molecular weight proteins in HDL recognized by the antibody to apoA-I
(Figure 5) and in the signal for EO6 (data not shown) and a
dramatic return of native apoA-I (Figure 5).
Despite the dramatic changes in apoA-I protein (Figures 4 and
5), PON activity (Figure 2), and protein (Figure
4), there was no change in hepatic mRNA for apoA-I or PON (data
not shown).
These data suggest that between days 3 and 7 of the atherogenic diet, a mechanism was induced for clearing the abnormal high molecular weight proteins in HDL recognized by the antibody to apoA-I, including the proteins containing the oxidized phospholipid epitope recognized by the EO6 antibody. The abc-1 transporter has been demonstrated to be induced with cholesterol loading and reduced with cholesterol removal by apolipoproteins.30 31 32 37 38 Because hepatic mRNA levels for the abc-1 transporter were progressively induced from day 1 to 7 of the atherogenic diet and because these mRNA levels fell only modestly on switching to the chow diet, we suspect that the abc-1 protein does not account for the observed changes. We suspect that abc-1 mRNA levels were induced to compensate for the increased cellular cholesterol that must have resulted from the atherogenic diet.
The failure of SR-B1 protein levels to change suggests that this pathway is not the mechanism for the clearance of the abnormal HDL proteins. SR-B1 has been shown to remove cholesteryl esters, but not apoA-I, from plasma.25 26 27 We also examined the levels of mRNA for cubilin in the kidney because cubilin has been implicated in the regulation of apoA-I levels.33 34 However, we found no significant change in the level of mRNA for cubilin in mice fed the atherogenic diet for 7 days.
The changes in the levels of apoA-I immune complex detected after the
atherogenic diet and the switch to the chow diet (Figure 6) are
consistent with the presence of immune complexes at baseline,
followed by enhanced clearance between days 3 and 7. The return to
baseline or above 3 days after switching from the atherogenic to the
chow diet may imply the involvement of the same mechanism that mediated
the clearance of lipid hydroperoxides in plasma, LDL, and HDL, because
these returned to baseline after switching to the chow diet at each
time point studied (Figure 1).
These data suggest that in the LDL receptor–negative mice, there are
always detectable levels of oxidized lipids in HDL and that these are
associated with autoantibodies complexed to the modified HDL. In
response to the atherogenic diet, there is an increase in oxidized
lipids per HDL particle, which, in turn, may lead to an increased
number of antibodies per particle. When some threshold is exceeded,
this could lead to enhanced plasma clearance of the immune complexes.
In turn, this could cause decreased absolute plasma levels of HDL
cholesterol, apoA-I, apoA-II, and the associated oxidized
lipids detected by EO6 as well as decreased levels of
apoA-I–associated IgG, IgM, and EO6 epitopes (Figure 6). This
scenario is very similar to that previously demonstrated by studying
the turnover and tissue sites of degradation of nonenzymatically
glycated LDL in normal and immunized rabbits.39 In rabbits
fed normal chow, the plasma clearance of glycated LDL was retarded in
proportion to the extent of lysine derivatization.39 In
contrast, in animals immunized with the glycated LDL, the clearance of
the glycated LDL was greatly accelerated.39 For example,
when only 5% of the lysine residues were derivatized, glycated LDL
clearance was retarded by 20% when injected into a control
nonimmunized rabbit, but when the same glycated LDL was injected into
the immunized animals, there was an acceleration of 2- to 3-fold in its
clearance.39 However, when 10% of the lysine residues
were derivatized, the injected glycated LDL was cleared from the plasma
of the immunized animals 50- to 100-fold faster than
control.39 In the present study, switching back to the
chow diet led to a rapid decrease in the content of lipid
hydroperoxides per HDL particle and, presumably, a decrease in the
number of antibodies bound per particle, with a net result that the
rapid immune-mediated clearance was decreased with a consequent net
increase in the apoA-I–IgG and apoA-I–IgM complexes found in the
plasma (Figure 6). Further elucidation of the details of the
mechanism of clearance of HDL and its associated proteins after a
short-term atherogenic diet will likely yield new insights into
lipoprotein metabolism.
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Acknowledgments |
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This work was supported by US Public Health Service grants HL-30568 and HL-56989 (J.L.W.), a Tobacco-Related Diseases Research Program (TRDRP) grant from the State of California, and the Laubisch, Castera, and M.K. Grey funds at UCLA. We thank Dr Lawrence W. Castellani and Sarada Charugundla for assistance with the HDL cholesterol determinations and Drs Erik Avaniss-Aghajani, Jinliang Li, and Irwin Kurland for advice and assistance with Taqman RT-PCR. We also thank Dr Aldons J. Lusis for providing the mouse apolipoprotein and PON antibodies.
Received February 11, 2000; accepted April 28, 2000.
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References |
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