Glycated Phosphatidylethanolamine Promotes Macrophage Uptake
of Low Density Lipoprotein and Accumulation of Cholesteryl Esters and
Triacylglycerols*
Amir
Ravandi
§,
Arnis
Kuksis§¶, and
Nisar A.
Shaikh
From the
Department of Laboratory Medicine and
Pathobiology, § Banting and Best Department of Medical
Research, University of Toronto and
Spectral Diagnostics Inc.,
Toronto, Ontario M5G 1L6, Canada
 |
ABSTRACT |
Non-enzymatic glycation of low
density lipoprotein (LDL) has been suggested to be responsible for the
increase in susceptibility to atherogenesis of diabetic individuals.
Although the association of lipid glycation with this process has been
investigated, the effect of specific lipid glycation products on LDL
metabolism has not been addressed. This study reports that
glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL
lipid glycation product, promotes LDL uptake and cholesteryl ester (CE)
and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation
of THP-1 macrophages at a concentration of 100 µg/ml protein LDL
specifically enriched (10 nmol/mg LDL protein) with synthetically
prepared Glc-PtdEtn resulted in a significant increase in CE and TG
accumulation when compared with LDL enriched in non-glucosylated
PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the
macrophages contained 2-fold higher CE (10.11 ± 1.54 µg/mg cell
protein) and TG (285.32 ± 4.38 µg/mg cell protein) compared
with LDL specifically enriched in non-glucosylated PtdEtn (CE,
3.97 ± 0.95, p < 0.01 and TG, 185.57 ± 3.58 µg/mg cell protein, p < 0.01). The
corresponding values obtained with LDL containing glycated protein and
lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 ± 1.35 and TG, 280.78 ± 3.98 µg/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe,
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when
compared with control LDL. Competition studies revealed that acetylated
LDL is not a good competitor for DiI Glc-PtdEtn LDL (5-6%
inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results
indicate that glucosylation of PtdEtn in LDL accounts for the entire
effect of LDL glycation on macrophage uptake and CE and TG accumulation
and, therefore, the increased atherogenic potential of LDL in hyperglycemia.
 |
INTRODUCTION |
LDL1 glycation has been
proposed to play central role in the atherosclerosis of diabetic
hyperglycemia (1). The effect of protein glycation in LDL modification
and its oxidation has been extensively investigated. Several groups of
investigators have shown that glycated LDL is capable of inducing foam
cell formation in a variety of cell culture systems (2-5).
Furthermore, LDL from diabetic patients has been shown to increase
accumulation of CE in macrophages, and the extent of accumulation has
been correlated with the extent of LDL glycation (6, 7). In all these
studies it was presumed that the glycated apoB is responsible for the
entire altered activity.
By using antibodies to advanced glycation end products (AGE), Bucala
et al. (8) have, however, shown that the lipid component of
glycated LDL contains most of the AGE present and that the relative
amount of this AGE antigen was proportional to the susceptibility of
LDL to peroxidation. Since lipid glycation products were not isolated
or identified, the significance of lipid glycation in LDL uptake by
macrophages could not be investigated or a differentiation attempted
between lipid and protein glycation in this process.
We have previously demonstrated that the amino phospholipids of plasma
and red blood cells (RBC) from diabetic subjects show a 10-fold
increase in glycated PtdEtn over control subjects (9, 10) and that
incorporation of Glc-PtdEtn into LDL (11) facilitates peroxidation of
LDL lipids. The present study demonstrates that the presence of
Glc-PtdEtn in LDL results in an increased uptake of LDL and a dramatic
increase in neutral lipid accumulation in THP-1 macrophages.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
THP-1 cells were obtained from the American
Type Tissue Culture Collection (TIB 202) and were propagated in RPMI
1640, 10% fetal calf serum/penicillin (100 units/ml)/streptomycin (100 µg/ml) at 37 °C, 5% CO2. Cells were plated at a
density of 1 × 106 cells/ml in 10% fetal calf serum
medium containing 10
7 M phorbol myristate
acetate (PMA) for 72 h. The cells were then washed extensively
with serum-free RPMI medium and incubated with or without lipoproteins
as indicated for each experiment. In all experiments, cell viability
exceeded 90% as determined by trypan blue exclusion.
Synthesis and Isolation of Glc-PtdEtn--
Glc-PtdEtn was
prepared and purified as described previously (9). Briefly, PtdEtn (2 mg) dissolved in 1 ml of methanol was transferred to a 15-ml test tube,
and the solvent evaporated under nitrogen. Four ml of 0.1 M
phosphate buffer containing 0-400 mM glucose and 0.1 mM EDTA were added and sonicated at low power for 5 min at
room temperature, and the mixture was incubated under nitrogen at
37 °C for various periods. Lipids were extracted into chloroform/methanol (2:1, v/v) as described by Folch et al.
(12), and the solvents evaporated under nitrogen. Samples were
redissolved in chloroform/methanol (2:1, v/v) and kept at
20 °C
until analysis. Glc-PtdEtn (2 mg) was purified by preparative TLC
(20 × 20-cm glass plates) coated with Silica Gel H (250-µm
thick layer). The chromatoplates were developed using
chloroform/methanol, 30% ammonia (65:35:7, by volume) as described
(9). Phospholipids were identified by co-chromatography with
appropriate standards, visualizing any lipid bands under ultraviolet
light after spraying the plate with 0.05% 2,7-dichlorofluorescein in
methanol. Both glucosylated and non-glucosylated lipids were
recovered by scraping the gel from appropriate areas of the plate and
extracting it twice with the developing solvent.
Lipoprotein Isolation and Oxidation--
LDL (1.019-1.069 g/ml)
was obtained by density gradient ultracentrifugation (13) from plasma
of fasted normolipidemic individuals. LDL (2 mg of protein/ml) was
subsequently dialyzed against 0.1 M phosphate buffer (pH
7.4) containing 0.1 mM EDTA for 24 h (three buffer
changes). LDL samples were sterilized by passing through a 0.22-micron
filter (Millipore, Milford, MA), kept at 4 °C, and used within 1 week. Lipoprotein concentration was determined by the method of Lowry
et al. (14) and expressed as mg/ml. Oxidation of LDL (5 mg
of protein/5 ml) was performed by dialysis against 5 µM
CuSO4·5H2O in 0.1 M phosphate
buffer (pH 7.4), for 12 h at 37 °C in the dark. LDL (2 mg of
protein/ml) in 1 mM EDTA, containing 0.1 mg/ml
chloramphenicol and 3 mM NaN3, was incubated
with 50 mM glucose at 37 °C for 1 week under nitrogen to
obtain glycated LDL. Acetylated LDL (AcLDL) was prepared by the method
of Basu et al. (15).
Enrichment of LDL with PtdEtn--
Glucosylated and
non-glucosylated PtdEtn was incorporated into LDL essentially as
described by Engelmann et al. (16) for enriching human
plasma lipoproteins with phospholipids. Glc-PtdEtn (1 mg) in
chloroform/methanol (2:1, v/v) was transferred to a 15-ml test tube,
the solvent evaporated under nitrogen, and the lipids were dispersed by
vortexing in 1.5 ml of buffer containing 50 mM Tris/HCl, 1 mM dithiothreitol, and 0.03 mM EDTA (pH 7.4). The solutions were sonicated in a bath sonicator for 5 min at 1-min
intervals while being kept on ice under a stream of nitrogen. The
liposome mixture was centrifuged at 3,500 × g and the
supernatant collected and passed through a 0.45-µm filter. The
liposomal mixture (1 ml) was added to fresh plasma (4 ml) containing 3 mM NaN3 under gentle mixing. The mixture was
incubated under nitrogen at 37 °C for 24 h. Lipoproteins were
isolated as described above.
LC/ES/MS of Lipoprotein Phospholipids--
Normal phase high
performance liquid chromatography (HPLC) of phospholipids was performed
on a 5-µm Spherisorb column (250 × 4.6 mm inner diameter,
Alltech Associates, Deerfield, IL). The column was installed into a
Hewlett-Packard model 1090 liquid chromatograph and eluted with a
linear gradient of 100% solvent A (chloroform/methanol/30% ammonium
hydroxide, 80:19.5:0.5, by volume) to 100% solvent B
(chloroform/methanol/water/30% ammonium hydroxide, 60:34:5.5:0.5, by
volume) in 14 min and then at 100% solvent B for 10 min (17). The flow
was set at 1 ml/min. The peaks were monitored by on-line ES/MS. Normal
phase HPLC with on-line electrospray mass spectrometry (LC/ES/MS) was
performed by splitting the HPLC flow by 1/50 resulting in 20 µl/ml
being admitted to a Hewlett-Packard model 5988B quadrupole mass
spectrometer equipped with a nebulizer-assisted electrospray interface
(HP 59987A) (18). Tuning and calibration of the system was achieved in
the mass range of 400-1500 by using the standard phospholipid mix
dissolved in the HPLC solvent A and flow-injected at 50 µl/min into
the mass spectrometer. Capillary voltage was set at 4 kV, the end plate
voltage at 3.5 kV, and the cylinder voltage at 5 kV in the positive
mode of ionization. In the negative mode, the voltages were 3.5, 3, and
3.5 kV, respectively. Both positive and negative ion spectra were taken
in the mass range 400-1100 atomic mass units. The capillary exit
voltage was set at 120 V in the positive and 160 V in the negative ion
mode. Nitrogen gas was used as both nebulizing gas (40 pounds/square
inch) and drying gas (60 pounds/square inch, 270 °C). Phospholipids
were quantified on basis of standard curves established for each
phospholipid class. The equimolar ion intensities of different species
of each phospholipid class varied by less than 5% in each of the ion
modes (18). The LC/ES/MS response to different phospholipid classes varied greatly and required the regular use of standards.
Labeling of Lipoproteins and Liposomes--
LDL was labeled with
the fluorescent probe
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI) according to the method of Stephan and Yurachek (19). Briefly, to
10 ml of LDL solution (1 mg/ml protein) containing 1 mM
EDTA in 0.1 M PBS, 300 µl of DiI solution in
Me2SO (30 mg/ml) was slowly added under gentle agitation.
The solution was incubated for 8 h at 37 °C under nitrogen and
in the dark. The LDL was reisolated by ultracentrifugation. Under these
conditions the average labeling efficiency was 20-25 ng of DiI/µg of
LDL protein.
Quantitative Spectrofluorometry of DiI LDL--
To study the
uptake of DiI-labeled LDL, cells seeded in 24-well culture dishes were
incubated for 4 h at 37 °C with increasing concentrations of
the lipoprotein (10-200 µg/ml) (20). Specific uptake of DiI LDL in
all preparations was determined at 10 µg of lipoprotein/ml with
50-fold excess of unlabeled lipoprotein. Afterward, the cells were
washed twice with PBS containing 0.4% bovine serum albumin and twice
with PBS alone. To each well 1 ml of lysis buffer was added (1 g/liter
SDS, 0.1 M NaOH). Cells were incubated at room temperature
under gentle shaking for 1 h. This allowed both direct
fluorescence and protein measurement. The fluorescence of each well was
measured in duplicate by a Shimadzu spectrofluorometer (RF5000U). The
excitation and emission wavelengths were set at 520 and 575 nm,
respectively. The detection range for the fluorescence was linear from
0.05 to 20 µg/ml LDL protein. Protein determinations were made in
duplicate using the method of Lowry et al. (14) with bovine
serum albumin dissolved in lysis buffer as standard. Fluorescence
microscopy was performed as described previously (21).
Determination of Cellular CE and TG Accumulation--
The THP-1
cells were exposed for 24 h to control and modified LDL
preparations. After incubation the cells were washed once with ice-cold
PBS containing 0.4% bovine serum albumin and twice with PBS alone.
Cells were scraped from the culture flask into PBS and sonicated. The
cellular lipids were extracted with chloroform/methanol (2:1 v/v) and
analyzed by gas-liquid chromatography (22). For this purpose the lipid
extract was digested with phospholipase C (Clostridium
welchii), and the digestion mixture was extracted with
chloroform/methanol (2:1 v/v) containing 100 µg of
tridecanoylglycerol as internal standard. The lipid extracts were then
reacted for 30 min at 20 °C with SYLON BFT plus 1 part dry pyridine.
This procedure converts the free fatty acids into trimethyl silyl
esters and the free sterols, diacylglycerols, and ceramides into
trimethyl silyl ethers, leaving the cholesteryl esters and
triacylglycerols unmodified. The free cholesterol, cholesterol esters,
and triacylglycerols were quantitated using a non-polar capillary
column as described previously (23).
Statistical Analysis--
Cellular uptake and neutral lipid
accumulation assays were done in triplicate, and statistical
significance was performed with analysis of variance.
 |
RESULTS |
LDL Phospholipid Analysis--
Fig.
1 shows the results of the LC/ES/MS
analysis of phospholipids from control LDL (Fig. 1A) and
from LDL preparations containing Glc-PtdEtn (Fig. 1B). The
Glc-PtdEtn is well resolved from PtdEtn and from other phospholipids
seen in the negative ion profile, where the choline phospholipids yield
minimal response. The added glucosylated 16:0-18:2 Gro-PEtn
(m/z 876) [M
1]
was easily detected
by single ion mass chromatogram (Fig. 1C). By using
appropriate correction factors, it was estimated that the total PtdEtn
content of LDL had been increased from 3 to 6% of total phospholipid
(20-50% of total PtdEtn) as a result of the enrichment.

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Fig. 1.
LC/ES/MS analysis of LDL phospholipids.
A, control LDL; B, LDL enriched with 16:0-18:2
Glc Gro-PEtn; C, single ion plot of ion 876 representing the
Glc-PtdEtn present in LDL enriched with the glycated phospholipid. LDL
total lipid extract was dissolved in chloroform/methanol (2:1 v/v), and
20 µl of the sample containing 10 µg of lipid was analyzed.
LC/ES/MS conditions. Normal phase 5-µm Spherisorb column (250 × 4.6 mm inner diameter) eluted with a linear gradient of 100% solvent A
(chloroform/methanol/30% ammonium hydroxide, 80:19.5:0.5, by volume)
to 100% solvent B (chloroform/methanol/water/30% ammonium hydroxide,
60:34:5.5:0.5, by volume) in 14 min, then at 100% B for 10 min. at 1 ml/min. The effluent was split 1:50 resulting in 20 µl/ml being
admitted into the mass spectrometer and scanning in the negative ion
mode from 400 to 1100 atomic mass units. Lyso PtdCho,
lyso-phosphatidylcholine; SM, sphingomyelin.
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Stimulation of CE and TG Accumulation by Glc-PtdEtn LDL--
Fig.
2 shows that incubation of Glc-PtdEtn LDL
(10-30 nmol of Glc-PtdEtn/mg of LDL protein) with THP-1 cells leads to
a specific increase in CE and TC accumulation when compared with LDL
containing non-glycated PtdEtn (10.11 ± 1.54 versus
3.97 ± 0.35 µg/mg cell protein, p < 0.01). The
supplementation of LDL with non-glucosylated PtdEtn did not have any
significant effect on CE accumulation. In order to differentiate
between the contributions of LDL lipid and protein glycation on CE
accumulation, these results were compared with those obtained with LDL
that had been glycated in both protein and lipid moieties by incubation
with glucose (50 mM, 7 days, 37 °C). The glycated LDL
preparation caused a CE accumulation (11.95 ± 1.38 µg/mg cell
protein) that was comparable to that obtained when macrophages were
incubated with Glc-PtdEtn LDL (10.11±1.54 µg/mg cell protein).

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Fig. 2.
Cholesteryl ester deposition in THP-1
macrophages due to Glc-PtdEtn. Cellular accumulation of free
(FC), esterified (CE), and total (TC)
cholesterol in human THP-1 macrophages incubated with 100 µg/ml
control LDL ( ), PtdEtn LDL ( ), Glc-PtdEtn + LDL ( ), and
glycated LDL ( ). Significance of comparing to control
*p < 0.01 (n = 3). THP-1 cells (1 × 106/ml) were cultured in the presence of PMA for 72 h prior to incubation with the specific lipoproteins. The cells were
incubated with the lipoproteins for 24 h. Cells were scraped from
the culture plates and the lipids extracted with the
chloroform/methanol (2:1 v/v). Subsequent to phospholipase C digestion
the lipid extract was analyzed by gas chromatography as described under
"Experimental Procedures."
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Fig. 3 shows that incubation of THP-1
cells with Glc-PtdEtn LDL also causes a significant accumulation of TG
(285.32 ± 4.38 µg/mg cell protein) which again was
indistinguishable from the accumulation obtained by incubation with LDL
glycated in lipid and protein (280.78 ± 3.98 µg/mg cell
protein). LDL supplemented with non-glucosylated PtdEtn did not
stimulate TG accumulation (185.57 ± 3.42 µg/mg cell protein).
Furthermore, the increase in TG content of the macrophages seen at 50 µg/ml LDL was only slightly lower than that seen at 100 µg/ml LDL
indicating a leveling off in the response. Interestingly, the TG levels
in THP-1 cells represented the major neutral lipid pool contributing up
to 85% of the cell neutral lipid content.

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Fig. 3.
Triacylglycerol accumulation in THP-1
macrophages due to Glc-PtdEtn. THP-1 cells activated with PMA (72 h) were incubated with 50 and 100 µg/ml of different forms of LDL for
24 h. The cells were scraped from the plates after extensive
washing. TG levels were measured as described in legend for Fig. 2.
Tridecanoylglycerol (25 µg/ml) was used as an internal standard for
the gas chromatographic analysis. Significance of comparing to control
*p < 0.01 (n = 3). , LDL + PtdEtn;
, glycated LDL; , LDL + Glc PtdEtn.
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Stimulation of CE and TG Accumulation by OxLDL
Glc-PtdEtn--
Previous work done in our laboratory had shown that
Glc-PtdEtn is more susceptible to oxidation and also facilitates the
oxidation of other LDL phospholipids as well as the CE present in the
lipid core of the molecule (11). In the present study we investigated the effects of Glc-PtdEtn on LDL oxidation and on foam cell formation. We oxidized Glc-PtdEtn-enriched LDL by copper and measured the CE and
TG deposition in THP-1 cells. Fig.
4A shows the effect of
incubation of oxidized PtdEtn LDL and oxidized Glc-PtdEtn-enriched LDL
upon the accumulation of free cholesterol, CE, and TC by THP-1 cells in
comparison to LDL. The greatest increase is seen in the CE. The
oxidized PtdEtn LDL caused a 4-fold increase in the CE content of the
cells (18.36 ± 1.95 µg/mg cell protein) compared with control
LDL (3.97 ± 1.95 µg/mg cell protein). There was a significant
further increase after incubation with oxidized Glc-PtdEtn LDL
(28.36 ± 3.25 µg/mg cell protein). Fig. 4B shows the
effect of incubation of oxidized PtdEtn-enriched LDL and oxidized
Glc-PtdEtn-enriched LDL upon the accumulation of TG by THP-1 cells in
comparison to LDL enriched with PtdEtn. The incubation with LDL
oxidized after enrichment with Glc-PtdEtn (350.91 ± 26.32 µg/mg
cell protein) gave significantly greater accumulation than the
incubation with oxidized LDL enriched with PtdEtn (310 ± 21.84 µg/mg cell protein), both values being significantly greater than
those obtained for non-oxidized LDL or LDL enriched in PtdEtn. This
increase in neutral lipid deposition correlated with the increase in
PtdCho oxidation products due to the presence of Glc-PtdEtn in LDL
during oxidation. The phospholipids from oxidized Glc-PtdEtn LDL were
analyzed by LC/ES/MS. The total positive ion current (Fig.
5A) shows the separation of
Glc-PtdEtn LDL phospholipids after oxidation with copper ions. The
major phospholipid oxidation products were identified as the PtdCho
hydroperoxides (PtdCho OOH), PtdCho core aldehydes (PtdCho Ald), and
PtdCho isoprostanes (PtdCho IsoP) in the positive mode of ionization.
Fig. 5B shows representative mass chromatograms. In the
absence of reference standards the structures proposed for the
isoprostanes and core aldehydes containing hydroxyl groups must remain
tentative. The pro-oxidative effect of Glc-PtdEtn resulted in a 4-fold
increase in the production of total hydroperoxides, core aldehydes, and
isoprostanes, when compared with LDL enriched in PtdEtn (Fig.
6).

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Fig. 4.
Effect of oxidation of Glc-PtdEtn LDL on
accumulation of neutral lipids in THP-1 cells. CE (A)
and TG (B) in THP-1 macrophages were measured subsequent to
oxidation of Glc-PtdEtn LDL. LDL ( ), oxidized PtdEtn LDL ( ), and
Glc-PtdEtn LDL ( ). Compared with oxidized PtdEtn LDL
*p < 0.01. LDL was initially enriched in PtdEtn or
Glc-PtdEtn and subsequently oxidized by dialysis against 5 µM CuSO4·5H2O in 0.1 M phosphate buffer (pH 7.4) for 12 h at 37 °C in
the dark.
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Fig. 5.
LC/ES/MS analysis of oxidized PtdCho in
oxidized Glc-PtdEtn LDL. Positive total ion current profile of
oxidized Glc-PtdEtn LDL (A). Single ion plots of
representative PtdCho oxidation products (B). Glc-PtdEtn LDL
was oxidized by incubation with 5 µM CuSO4 in
0.1 M PBS for 12 h at 37 °C. The total lipid
extract of the oxidized LDL was dissolved in chloroform/methanol 2:1,
and 20 µl of the sample containing 10 µg lipid was analyzed.
Structural assignments for aldehydes and hydroperoxides are according
to reference standards. Ions 832 and 830 were identified on the basis
of retention time and molecular weight.
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Fig. 6.
Increased oxidative susceptibility of LDL due
to Glc-PtdEtn. Levels of PtdCho hydroperoxides (PtdCho
OOH), PtdCho aldehydes, and PtdCho isoprostanes in oxidized LDL
enriched with PtdEtn ( ) or Glc-PtdEtn ( ). Levels represent sum
of four of the most abundant ions for each class of oxidized
PtdCho.
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Cellular Uptake of DiI Labeled Glc-PtdEtn LDL--
In order to
quantitate the rate of uptake of LDL, we utilized DiI-labeled
lipoproteins. After a 4-h incubation with cells, the fluorescence
intensity was measured as an indicator of total cell-associated
lipoprotein. Cells incubated with DiI LDL enriched with Glc-PtdEtn
(10-200 µg/ml) showed a much more rapid increase in cell-associated
lipoprotein when compared with control LDL enriched with PtdEtn (Fig.
7). The uptake of DiI LDL enriched in
Glc-PtdEtn occurred at the same rate as that for glycated LDL showing
that the presence of Glc-PtdEtn in LDL can mimic the properties of
glycated LDL. The uptake of all LDL preparations tended to level off in
the range of 100-200 µg/ml LDL.

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Fig. 7.
Concentration-dependent uptake of
DiI-labeled GlcPtdEtn LDL by THP-1 macrophages. Cells (1 × 106/ml) were incubated with increasing concentrations of
labeled PtdEtn LDL ( ), Glc-PtdEtn LDL ( ), and glycated LDL ( )
for 4 h. Cells were washed with PBS, and the fluorescence was
measured subsequent to cell lysis by SDS solution (1 g/liter SDS, 0.1 M NaOH). Cell protein was also measured from the same
solution.
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Fig. 8 shows the time course (4, 6, 10, and 20 h) of macrophage accumulation of fluorescently labeled
lipoprotein. At all time points the average accumulation was
consistently higher for the LDL preparation containing Glc-PtdEtn when
compared with control LDL. The accumulation of Glc-PtdEtn LDL was
parallel to that of glycated LDL at all time points. To explore the
specificity Glc-PtdEtn LDL interaction with macrophages, we
investigated the ability of AcLDL, PtdEtn LDL, glycated LDL, and LDL + Glc-PtdEtn at increasing concentrations to compete with DiI-labeled
Glc-PtdEtn (50 µg/ml) for uptake by macrophages (Fig.
9). After the 4-h incubation the AcLDL
and control LDL were only able to inhibit 5-6% of the cell association. Glycated LDL led to 80% inhibition of Glc-PtdEtn LDL
uptake, whereas the unlabeled Glc-PtdEtn resulted in 93% inhibition of
uptake after correction for nonspecific association. Fluorescence microscopy analysis of LDL uptake by THP-1 cells (Fig.
10) showed increased fluorescence in
cells incubated with DiI Glc-PtdEtn LDL compared with cells incubated
with the DiI PtdEtn LDL. The distribution of fluorescence in cells
incubated with DiI-labeled Glc-PtdEtn LDL (Fig. 10C) shows a
punctated pattern believed to be indicative of receptor-mediated
endocytosis from coated pits and vesicles to endosomes and lysosomes
(24). This pattern was also observed for glycated LDL and for AcLDL as
already reported (24). Cells incubated with DiI PtdEtn LDL showed a
diffuse pattern of fluorescence with less intensity, indicative of lack
of uptake.

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Fig. 8.
Time course of uptake of Glc-PtdEtn LDL by
THP-1 macrophages. 50 µg/ml DiI-labeled Glc-PtdEtn LDL ( ) and
PtdEtn LDL ( ), compared with that of glycated LDL ( ). Subsequent
to PMA activation for 72 h THP-1 cells cultured in 24-well plates
were incubated with DiI-labeled lipoproteins. At each time point cells
were lysed, and fluorescence was measured.
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Fig. 9.
Competitive inhibition of Glc-PtdEtn LDL
uptake by THP-1 macrophages. Macrophages were incubated 4 h
with 50 µg/ml DiI-labeled Glc-PtdEtn LDL together with varying
concentrations of unlabeled AcLDL ( ), glycated LDL ( , LDL
incubated with 50 mM glucose, 1 week), and unlabeled LDL
enriched with Glc-PtdEtn ( ). Values shown are means of triplicate
incubations that varied by less than 10%.
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Fig. 10.
Uptake of Glc-PtdEtn LDL by THP-1
macrophages. THP-1 cells (1 × 106 cells/ml)
activated with PMA were incubated with 50 µg/ml, DiI-labeled PtdEtn
LDL (A), glycated LDL (B), Glc-PtdEtn LDL
(C), and AcLDL (D). After the incubation for
3 h at 37 °C cells were washed with cold PBS, fixed with
formaldehyde in PBS, and then analyzed by fluorescence
microscopy.
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Increase in Negative Charge of PtdEtn Due to Glycation--
Fig.
11 shows that the glycation of PtdEtn
increases the negativity of the molecule. Injection of equimolar
amounts of PtdEtn and Glc-PtdEtn, chromatographed on normal phase
silica column, resulted in a 1.5-fold higher response for Glc-PtdEtn
when compared with PtdEtn. The calculated molar response of Glc-PtdEtn
was comparable to that of other anionic phospholipids, such as PtdIns
and PtdSer (Table I).

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Fig. 11.
Increase in negative charge of PtdEtn after
glycation. LC/ES/MS analysis of synthetic PtdEtn and Glc-PtdEtn
total negative ion profile for equimolar amounts of glucosylated and
non-glucosylated PtdEtn (A), single ion plots for 16:0-18:2
Gro-PEtn (m/z 714) and 16:0-18:2 Gro Glc PEtn
(m/z 876) (B), and ion spectra averaged over both
glucosylated and non-glucosylated PtdEtn (C).
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Table I
LC/ES/MS relative ionization intensities in negative ionization mode
for anionic phospholipids as compared with PtdEtn
Equimolar amounts of each phospholipid was injected in the LC/ES/MS
system. The ratios were calculated by measuring the peak area for each
phospholipid compared with that of PtdEtn. PtdEtn,
phosphatidylethanolamine; Glc-PtdEtn, glucosylated
phosphatidylethanolamine; PtdSer, phosphatidylserine; PtdIns,
phosphatidylinositol; CL, cardiolipin.
|
|
 |
DISCUSSION |
This study demonstrates that incorporation of Glc-PtdEtn into LDL
at a level commonly achieved in hyperglycemia can fully account for the
increased uptake of glycated LDL by macrophages. Furthermore, the
resulting increased accumulation of CE and TG by macrophages exceeds
the amounts of neutral lipids anticipated to be transferred to the
cells on the basis of uptake of the lipoprotein. This increase was
mainly due to accumulation of TG which contributed over 85% of the
cell total neutral lipid (TC + TG). The glycated subfraction of LDL,
which is elevated in diabetic subjects, has been reported to promote CE
deposition and to increase the rate of CE synthesis in macrophages
(25), but TG levels had not been measured. Although there was a
significant increase in CE deposition in our THP-1 cells, the major
neutral lipid accumulated was TG. In this connection it may be noted
that recent studies have shown that THP-1 cells contain a large and
metabolically active TG pool but a relatively inactive pool of CE (26).
A large and active TG pool has also been seen in other human macrophage
cultures, but the regulation of TG metabolism in these cells is not known.
The general effects of glycation on LDL interaction with macrophages
have been extensively investigated (4, 5). Non-enzymatic glycation of
LDL has been shown to increase the fluidity of the phospholipid
monolayer, to result in an altered lipid composition (27), and,
possibly, in an increased susceptibility to oxidation. These
alterations in biological and physicochemical properties have been
attributed solely to ApoB glycation without consideration of LDL
phospholipid glycation.
The inability of AcLDL to compete with Glc-PtdEtn LDL suggests the
participation of receptors other than the LDL or scavenger receptor.
Due to the increased negative charge of Glc-PtdEtn compared with PtdEtn
and structural similarities to acidic glycerophospholipids, it is
possible that receptors for Glc-PtdEtn uptake could be found in the
family of receptors that identify anionic phospholipids such as CD 36 (28). It has been shown that CD 36 present in photoreceptor outer
segment cells binds PtdSer- and PtdIns-rich liposomes resulting in
their uptake (29). LC/ES/MS shows that glucosylation of PtdEtn
increases the negative charge of the amino phospholipid, and with
structural and physicochemical similarities to acidic
glycerophospholipids, Glc-PtdEtn could increase uptake of LDL by CD 36.
In fact, macrophages have already been shown to specifically
phagocytose oxidized RBC in which the outer leaflet of the phospholipid bilayer exposes anionic phospholipids, specifically PtdSer. Sambrano et al. (30) have demonstrated that peroxidation of RBC
results in disruption of the asymmetry of the plasma membrane
phospholipid bilayer resulting in PtdSer exposure and recognition of
oxidized RBC by macrophages and their subsequent phagocytosis.
Inhibition of uptake of oxidized RBC by OxLDL showed that the scavenger
receptors on macrophages were responsible for a major part of the
oxidized RBC recognition (31).
Recently another receptor, SRB1, has been shown to have the capability
of binding anionic phospholipids. The inhibition of the selective
uptake of high density lipoprotein CE in liver parenchymal cells by
modified LDL, in particular OxLDL and ionic phospholipids, has
suggested that in liver the SRB1 is responsible for the efficient uptake of high density lipoprotein CE (32). The concept that negatively
charged molecules can form complexes with LDL is not restricted to
negatively charged phospholipids. Basu et al. (15) has
demonstrated that complexes containing LDL and large molecular weight
dextran sulfates are avidly metabolized by macrophages via a receptor,
which appears to be distinct from the AcLDL receptor. Besides the
effects of PtdSer on macrophage uptake, association of other negatively
charged phospholipids, like cardiolipin, with LDL increases its uptake
and the deposition of cholesteryl esters by macrophages (33). It has
also been claimed that an alteration in the composition of LDL
phospholipids caused by phospholipase D (34) and phospholipase
A2 (35) can influence the metabolism of LDL by macrophages,
but it is not clear whether the effect is related to a relative
increase in acidic phospholipids or lysophospholipids, which would be
anticipated to result from the action of these enzymes.
Another possible receptor for Glc-PtdEtn is the newly characterized AGE
receptor (36). This receptor has been demonstrated to have specificity
toward protein glycation products (37). Many of the binding assays
performed with this receptor have only focused on the protein glycation
products and the interaction of Glc-PtdEtn with AGE receptor-advanced
glycation end products has not been investigated.
The present study demonstrates that peroxidation increases the neutral
lipid deposition due to LDL beyond the extent achieved by glucosylation
of PtdEtn alone. We have previously shown that glycation of LDL PtdEtn
promotes the oxidation of both surface and core components of LDL (11).
The increase in susceptibility of LDL in the presence of Glc-PtdEtn to
oxidation was characterized by an increase in products of phospholipid
oxidation such as PtdCho core aldehydes, which have been specifically
shown to induce increased monocyte-endothelial interactions
in vitro (38).
This is in agreement with the observation that both apolipoprotein and
lipid in glycated LDL are more susceptible to oxidation in the presence
of Fe3+ than non-glycated LDL (39). It was suggested that
Fe3+ could be coordinated with the endiol group in Amadori
compounds and could be converted to ferryl iron with a high redox
potential. It was postulated, on the basis of the known chemistry of
Schiff base and Amadori products, that during the nucleophilic addition of glucose to protein amino groups, as in the early glycation, the
glycation products of proteins deposited in the arterial wall could
themselves generate free radicals capable of oxidizing lipids (40).
This possibility is supported by the demonstration that both Schiff
base and Amadori glycation products generate free radicals in a ratio
of 1:1.5 and that these radicals cause increased peroxidation of
membrane lipids (40). In similar studies Kobayashi et al.
(41) demonstrated that oxidation of glycated LDL results in increased
binding and degradation by cultured bovine aortic endothelial cells
compared with normal or oxidized LDL.
The present study demonstrates that glucosylation of the PtdEtn
component of LDL exerts a dramatic effect on LDL metabolism which
mimics that of total LDL glycation. This is the first direct evidence
that glycation of LDL lipids results in increased LDL uptake and in CE
and TG accumulation in macrophages. In conclusion, this study suggests
that the presence of Glc-PtdEtn in LDL plays an important role in the
alteration of the biological activity of LDL and reconciles diverse
observations regarding the binding of glycated and peroxidized LDL to
macrophages and other cells and may account for the increased
atherogenesis in diabetes.
 |
FOOTNOTES |
*
This work was supported by the Heart and Stroke Foundation
of Ontario, Toronto, Canada (to A. K.), and Spectral Diagnostics Inc.,
Toronto, Canada (to N. A. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Banting and Best
Dept. of Medical Research, University of Toronto, 112 College St.,
Toronto, Ontario M5G 1L6, Canada. Tel.: 416-978-2590; Fax: 416-978-8528; E-mail: arnis.kuksis{at}utoronto.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
LDL, low
density lipoprotein;
AGE, advanced glycation end products;
AcLDL, acetylated LDL;
OxLDL, oxidized LDL;
LC/ES/MS, liquid chromatography
with on-line electrospray mass spectrometry;
Glc-PtdEtn, glucosylated
phosphatidylethanolamine;
Gro-PEtn, glycerophosphoethanolamine;
PtdCho, phosphatidylcholine;
PtdEtn, phosphatidylethanolamine;
PtdIns, phosphatidylinositol;
PtdSer, phosphatidylserine;
RBC, red blood cells;
HPLC, high performance liquid chromatography;
DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate;
PBS, phosphate-buffered saline;
PMA, phorbol myristate acetate;
CE, cholesteryl ester;
TG, triacylglycerol;
TC, total cholesterol.
 |
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