© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Decreased Protection by HDL From Poorly Controlled Type 2 Diabetic Subjects Against LDL Oxidation May Be Due to the Abnormal Composition of HDL
From the Metabolic Research Group, Veterans Affairs Medical Center and Department of Internal Medicine (M.S.G., J.W.A., S.R.B.), and the Department of Internal Medicine (D.R.V.d.W.), College of Medicine, University of Kentucky, Lexington.
Correspondence to James W. Anderson, MD, Endocrine Section, Room 402B, VAMC, Cooper Drive Division, 2250 Leestown Rd 111C, Lexington, KY 40511. E-mail jwandersmd{at}aol.com
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Abstract |
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Abstract—High plasma triglyceride concentrations in diabetic subjects increase their risk for developing coronary heart disease. Numerous studies have shown that the high density lipoprotein (HDL) composition is abnormal in type 2 diabetic subjects. One study has shown that HDL (lipoprotein A-I) isolated from subjects with non–insulin-dependent diabetes mellitus exhibits a decreased capacity to induce cholesterol efflux. The current study examined the effect of HDL2 and HDL3 subfractions from poorly controlled type 2 diabetic and control subjects on THP-1 macrophage–mediated low density lipoprotein (LDL) oxidation. The composition and protective effects of HDL2, but not of HDL3, differed significantly between control and diabetic subjects. HDL2 from diabetics were triglyceride enriched and cholesterol depleted compared with those from controls. Control HDL2 inhibited LDL oxidation, as assessed by lipid peroxides and electrophoretic mobility, significantly (P<0.05) more than did diabetic HDL2 in both the fasting and postprandial state. In addition, HDL2 from diabetics did not protect against apolipoprotein B-100 fragmentation in LDL. Cross-linking in apolipoprotein A-I, oxidized in the presence of LDL, was extensive in HDL2 from diabetics compared with that from controls. Serum triglyceride concentrations were negatively correlated with protection by HDL2 (r=-0.673, P<0.05) in diabetic but not in control subjects. HDL2-associated platelet-activating factor acetylhydrolase activity was positively correlated with protection by HDL2 in control (r=0.872, P<0.002) but not in diabetic subjects. In conclusion, compositional alterations in HDL2 from poorly controlled type 2 diabetic subjects may reduce its antiatherogenic properties.
Key Words: diabetes • hypertriglyceridemia • atherosclerosis • oxidized LDL • HDL
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Introduction |
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Diabetic individuals have a significantly higher risk for atherosclerosis than do nondiabetic individuals.1 Persons with type 2 diabetes have significantly increased serum triglyceride (TG) and decreased HDL cholesterol concentrations. High plasma TG concentrations among diabetic subjects appear to be an independent predictive factor of mortality from coronary artery disease (CAD).2 Over the last decade, the abnormal HDL composition in type 2 diabetes has been well studied and is characterized by TG enrichment, cholesterol depletion, and decreased apo A-I concentrations.3 4 5 6 In subjects with type 2 diabetes, total serum TG concentrations were found to be inversely correlated with HDL cholesterol and positively correlated with HDL-TG concentrations.6 Such abnormalities in HDL might contribute to the well-established high risk for atherosclerosis in diabetes.
Type 2 diabetic subjects also have a decreased lipoprotein lipase activity7 that might cause decreased clearance of TG-rich lipoproteins, leading to accumulation of these lipoproteins in the plasma. Elevated levels of TG-rich lipoproteins may increase the cholesteryl ester transfer protein (CETP) activity8 that may in turn cause TG enrichment of HDL at the expense of core cholesteryl esters.9 10 Increased CETP activity has been reported in type 2 diabetes.10 11
In type 2 diabetic subjects, enhanced LDL oxidation occurs in vivo, since high titers of autoantibodies to oxidatively modified LDL are present in the plasma.12 Oxidative modification of LDL appears central to foam cell formation, the earliest lesion of atherosclerosis.13 HDL may protect from cardiovascular disease by a number of mechanisms, 1 of which is by inhibiting LDL oxidation in the subendothelial space.14 15 16 HDL prevents the formation of minimally modified LDL in cocultures of artery wall cells by facilitating hydrolysis of active, oxidized phospholipids to lysophospholipids, thereby destroying the biologically active lipids in minimally modified LDL.17 Platelet-activating factor acetylhydrolase (PAF-AH) and paraoxonase (PON) are 2 enzymes associated with HDL that are known to protect LDL from oxidation.15 16 18
Cavallero and colleagues19 showed that LpA-I (HDL containing only apo A-I) isolated from subjects with non–insulin-dependent diabetes mellitus (NIDDM) exhibited a decreased capacity to induce cholesterol efflux in both the fasting and postprandial state and hence, had a decreased ability to accomplish their antiatherogenic role. The ability of compositionally abnormal HDL particles from type 2 diabetic subjects to perform their antiatherogenic function of protecting LDL against oxidation has not been critically examined. In our study, we examined the protection exhibited by HDL2 and HDL3 from subjects with poorly controlled type 2 diabetes against macrophage-mediated oxidation of LDL compared with nondiabetic healthy controls. We found that the HDL2 subfraction from diabetic subjects was abnormal in composition, as previously reported,3 4 5 6 and also exhibited decreased protection against LDL oxidation when compared with controls.
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Methods |
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Materials
Total cholesterol and free cholesterol kits (COD-PAP) and the total phospholipid kit (phospholipids B) were purchased from Wako Chemicals. 1-Palmitoyl-2-[(7-nitrobenzoxadiazoyl)amino]caproyl phosphatidylcholine (C6NBD PC) was purchased from Avanti Polar Lipids. Rabbit anti-human apo A-I and goat anti-human apo A-II were purchased from Calbiochem. Electrophoresis reagents were purchased from Bio-Rad. FCS, phorbol 12-myristate 13-acetate, peroxidase-conjugated anti-rabbit IgG and anti-goat IgG, paraoxon, glucose enzymatic kit (glucose oxidase), and the TG enzymatic kit (INT reagent) were purchased from Sigma Chemical Co.
Study Protocol
Subjects
Ten poorly controlled type 2 diabetic men who had serum
cholesterol concentrations 
240 mg/dL and serum TG
concentrations 300 mg/dL were recruited from the Veterans
Administration outpatient clinic, and 10 matched male control subjects
were also recruited from the Veterans Administration Medical Center and
University of Kentucky Medical Center staff. Nine subjects were white
and 1 was African-American in each group. Medications taken by the
diabetic subjects at the time of the study were as follows:
sulfonylurea (all 10 subjects), biguanide (3 subjects), nitrates (4
subjects), loop diuretics (3 subjects), ß-blockers (2
subjects), 
-blockers (1 subject), and
angiotensin-converting enzyme inhibitors (5
subjects). Two of the diabetic subjects had no diabetic complications,
1 subject had cerebral artery occlusion, 4 subjects had CAD, and 3
subjects had hypertension. Except for 1 diabetic subject who smoked
<1/2 pack of cigarettes per day, all other subjects were
nonsmokers. None of the subjects took any antioxidant supplements
or probucol.
Test Meal
We gave our subjects a fat load to see whether there was any
difference between fasting and postprandial HDLs in their
antiatherogenic role of protecting LDL against oxidation. Each
individual arrived at 8 AM after an overnight fast. Fifty
milliliters of blood was collected for lipid analysis and
lipoprotein separation from serum. Immediately after the blood was
drawn, the test meal was ingested within 10 minutes. The test meal
consisted of 2 bowls of cornflakes, a half pint of whole milk, a half
pint of whipping cream, and 2 teaspoons of granulated sugar. This meal
provided 
80 g of fat and 900 calories. During the next 6 hours, the
patients were told not to consume any food or drink other than water or
unsweetened black coffee or tea. Fifty milliliters of blood was
collected 6 hours after ingestion of the test meal for lipoprotein
isolation and lipid analysis. The test meal was well tolerated
by all of the subjects except 1 of the diabetic subjects, who
experienced nausea and vomiting.
Serum Measurements
Serum glucose concentrations were determined by using an
enzymatic kit (glucose oxidase). Glycosylated hemoglobin
(HbA1c) values were determined with an
ion-exchange mini-column chromatographic procedure (Bio-Rad
Laboratories); normal values are 4.2% to 6.4%. Total
cholesterol was measured with an enzymatic kit from Wako
Chemicals (COD-PAP) and total TGs by an enzymatic kit from Sigma (INT
reagent). HDL cholesterol was quantitated after
precipitation of apo B–containing lipoproteins with
magnesium/phosphotungstic acid. HDL3
cholesterol was quantitated after a dual-precipitation
method with combinations of magnesium/phosphotungstic acid and dextran
sulfate. HDL2 cholesterol was
calculated as the difference between total HDL cholesterol
and HDL3 cholesterol.20
LDL cholesterol was calculated by the Friedewald
formula.21
LDL and HDL Subfraction Preparation
HDLs from the study subjects and LDL from a healthy donor were
isolated by sequential
ultracentrifugation.22 LDL was isolated
from the same healthy donor for every oxidation experiment. In brief,
the density of serum was adjusted to 1.09 g/mL and centrifuged
for 11 hours, 15 minutes at 50 000 rpm in a VTi50 rotor at 4°C. To
isolate LDL, the density of the VLDL-LDL fraction was adjusted to 1.3
g/mL, and gradient ultracentrifugation was carried out
at 50 000 rpm for 2.5 hours at 4°C. To isolate HDL, the density of
the bottom fraction was adjusted to 1.25 g/mL with solid KBr and
centrifuged in a VTi50 rotor at 50 000 rpm for 20 hours at
4°C. Gradient ultracentrifugation was carried out at
4°C for 15 hours at 50 000 rpm for fractionation of
HDL.23 LDL, HDL2, and
HDL3 were dialyzed extensively overnight against
3 changes of PBS (pH 7.4), sterilized through a 0.45-µm-filter unit
(Millipore), and stored at 4°C under N2.
Aliquots of HDL subfractions were frozen at -70°C for
analysis of HDL phospholipid, cholesteryl ester, and free
cholesterol concentrations.
HDL Subfraction Composition
Total cholesterol and free cholesterol
concentrations were measured by enzymatic methods with the use of
commercially available kits (COD-PAP), and cholesteryl esters were
calculated as the difference between total and free
cholesterol. TG concentrations were determined by using an
enzymatic kit (INT reagent). Total phospholipids were measured by an
enzymatic method that uses phospholipase D–choline oxidase and
peroxidase (phospholipids B).
Total protein contents of the HDL subfractions were measured by the Lowry method.24 Apo A-I and A-II in HDL subfractions were quantitated by SDS–polyacrylamide gradient gel electrophoresis (PAGE) followed by Western blotting.23 In brief, electrophoresis was carried out on a 5% to 20% acrylamide/SDS gel and a 25 mmol/L Tris, 192 mmol/L glycine electrophoresis buffer (pH 8.4) containing 0.1% SDS.23 The proteins were transferred onto a 0.2-µm nitrocellulose membrane by using a 25 mmol/L Tris, 192 mmol/L glycine transfer buffer (pH 8.4) containing 15% (vol/vol) methanol.23 The blots were blocked for 1 hour with 5% (wt/vol) nonfat dry milk in PBS (pH 7.4). The blots were then exposed to the primary and secondary antibodies for 1 hour each. The primary antibody for apo A-I was rabbit anti-human apo A-I, and for apo A-II, goat anti-human apo A-II. The secondary antibody was a peroxidase-conjugated anti-rabbit IgG and anti-goat IgG, respectively. The antibodies were then detected with the enhanced chemiluminescence (ECL) detection reagents (Amersham), and the blots were exposed to x-ray film. The bands were scanned with a densitometer.
HDL Subfraction Enzyme Activities
PON and PAF-AH activities were measured in fresh HDL samples
within 2 days of isolation. HDL PON activity was measured with a
1.0 mmol/L paraoxon substrate in 50 mmol/L Tris-HCl buffer
(pH 7.4) containing 1.0 mmol/L CaCl2 in a
total volume of 800 µL.25 One hundred micrograms of
HDL2 or HDL3 was added to
start the reaction, and the increase in absorbance at 412 nm was
recorded continuously for 10 minutes. The amount of 4-nitrophenol
formed was calculated from the molar extinction coefficient of 12 800
M-1 cm-1. The blank
contained substrate without HDL. One unit of PON activity is defined as
1 nmol of 4-nitrophenol formed per minute under the above assay
conditions.
PAF-AH activity was measured by using a fluorescent substrate,
C6NBD PC, by a modification of the method of
Steinbrecher and Pritchard.26 Four micromoles of the
substrate C6NBD PC was incubated with 10 µg of
HDL2 or HDL3 protein in 1
mL of PBS (pH 7.4) at 37°C for 90 minutes. The reaction was
terminated by vortexing with 1 mL of methanol and 1 mL of chloroform
for 1 minute, and the mixture was then centrifuged at
2500g for 10 minutes. The fluorescence of the
aqueous phase was measured at 470 nm excitation and 533 nm emission in
a fluorospectrometer. The mass of fluorescent substrate
hydrolyzed was calculated by using a standard curve generated by using
C6NBD fatty acid diluted in methanol.
Cell Culture
Dr Mark Kindy (Department of Biochemistry) provided the THP-1
monocytic cells. THP-1 cells were grown in suspension in RPMI-1640
medium containing 10% FCS, 100 U/mL streptomycin, and 100 U/mL
penicillin. Cells were maintained at a density of
106 mL-1 by pelleting the
cells twice weekly. Forty-eight hours before cell-mediated oxidation
studies, 2x106 cells were seeded per well in a
12-well tissue-culture plate and induced to differentiate into
macrophages by the addition of 10-7
mol/L phorbol 12-myristate 13-acetate.27
Macrophage-Mediated Oxidation of Lipoproteins
The differentiated macrophages were washed 3 times with
PBS before incubation with lipoproteins. Macrophage cells were
incubated with 100 µg of LDL protein, with or without 100 µg of
HDL2 or HDL3 protein, in 1
mL of serum-free Ham's F-10 medium containing gentamicin (50 µg/mL)
at 37°C in a 5% CO2 incubator for 24
hours.28 Additionally, 100 µg of
HDL2 or HDL3 protein was
incubated separately with the cells. Oxidation was arrested after 24
hours by the addition of 200 µmol/L EDTA and 20 µmol/L
BHT, and the medium was spun at 1200 rpm for 5 minutes at 4°C.
Aliquots of the medium were used for determining lipid peroxides,
electrophoretic mobility, apo B-100 fragmentation in LDL, and apo A-I
cross-linking in HDL subfractions.
Assessment of Oxidative Damage
Lipid peroxides in the oxidized lipoproteins were determined by
mixing 0.1 mL of the medium containing the modified lipoproteins with
1.0 mL of iodine-color reagent, incubating the mixture for 30 minutes
at room temperature in the dark, and reading the absorbance at 365
nm.29 The concentration of lipid peroxides was calculated
using a molar absorption coefficient of 2.46x104
M-1 cm-1. Lipid peroxides
present in HDL samples oxidized separately were used as a
correction factor for the lipid peroxides formed in the LDL samples
containing HDL during oxidation.
Electrophoretic mobility was determined using 1.0% agarose gels. Oxidized LDL was subjected to electrophoresis in 0.05 mol/L barbital buffer (pH 8.6) at 90 V for 45 minutes.30 The gel was dried and stained in Sudan black B. Electrophoretic mobility was measured as the distance from the origin to the median point of the lipoprotein peak distribution. The increase in the electrophoretic mobility of each oxidized LDL sample was calculated as a relative ratio to that of native LDL and expressed as relative electrophoretic mobility.
Apo B-100 Fragmentation
Fragmentation and aggregation of apo B-100 in the medium
containing 1 µg of LDL, oxidized in the absence or presence of
HDL2 or HDL3, was
determined using 5% to 20% SDS-polyacrylamide gradient gels
under reducing conditions as detailed above. The gels were fixed for 1
hour and stained with AgNO3 (Bio-Rad).
Apo A-I Cross-Linking
Apolipoprotein A-I cross-linking in macrophage-modified
HDL2 or HDL3 was determined
by subjecting the medium containing 1 µg of HDL protein to SDS-PAGE
under reducing conditions,23 followed by Western blotting
with an antibody against apo A-I.
Data Analysis
Statistical analysis for comparison between groups was
performed using a 2-way ANOVA followed by post hoc testing with
Tukey's multiple comparisons test by using SYSTAT
statistics software.31 Results are expressed as mean±SEM.
Correlation coefficients were determined by simple linear regression
analysis. Values of P<0.05 were considered to be
statistically significant.
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Results |
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Subject Characteristics
Table 1
provides the clinical
characteristics and also the serum lipid profiles of the diabetic and
control subjects in both the fasting and postprandial states. The
diabetic subjects were poorly controlled and had high
HbA1c and fasting serum glucose concentrations.
Although body mass index did not differ between the diabetic and
control subjects, the diabetic subjects were significantly older
(P<0.002). Diabetic and control subjects had similar serum
total and LDL cholesterol concentrations. Diabetic subjects
had slightly but not significantly higher serum TG concentrations,
compared with controls, in both the fasting and postprandial states.
However, HDL cholesterol and HDL2
cholesterol concentrations were significantly decreased in
the diabetics in both the fasting (P<0.01) and postprandial
(P<0.005) states. Serum TG concentrations were
significantly increased postprandially after a fat load in both the
control (P=0.014) and diabetic (P=0.001)
subjects.
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Composition of HDL2 and HDL3
Table 2 illustrates composition of
HDL2 and HDL3 from control
and diabetic subjects. Two-way ANOVA indicated that diabetic subjects
exhibited significant compositional abnormalities in their
HDL2 fraction in both fasting and postprandial
states compared with controls. Diabetics had (1) a decrease in
HDL2 free cholesterol concentrations
and (2) an increase in HDL2-TG
concentrations in both fasting (P<0.05) and postprandial
(P<0.05) states compared with controls. Apo A-I
concentrations were significantly decreased in
HDL2 from diabetic subjects compared with
controls in both fasting (P<0.05) and postprandial
(P<0.05) states. There was a slight but nonsignificant
increase in the HDL2 apo A-II concentrations in
diabetics compared with control subjects. PAF-AH and PON activities in
HDL2 were not significantly different between
control and diabetic subjects.
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Two-way ANOVA indicated that the only significant differences in HDL3 composition were associated with the high-fat meal and not due to diabetes. We found no difference in the HDL3 composition between the control and diabetic subjects. In both control and diabetic subjects, HDL3 had a significant increase in proteins (P<0.01) and phospholipids (P<0.05) but a significant decrease in cholesteryl esters (P<0.01) postprandially compared with the fasting state. Free cholesterol concentrations in HDL3 were significantly increased in the postprandial state (P<0.05) in control but not in diabetic subjects. There was no significant difference in either apo A-I and A-II concentrations or PON and PAF-AH activities in HDL3 from control and diabetic subjects.
Protection Against Macrophage-Mediated LDL
Oxidation
Figure 1 illustrates lipid peroxide
accumulation in LDL oxidized in the absence and presence of
HDL2 or HDL3 from control
and diabetic subjects in both fasting and postprandial states. Lipid
peroxides in LDL oxidized alone with the cells was taken as 100%.
Lipid peroxides in LDL oxidized in the presence of
HDL2 from diabetic subjects were significantly
higher, both in the fasting (53.3±8.3% LDL response versus
29.2±8.1%; P=0.049) and postprandial (63.3±8.8% versus
35.3±7.7%; P=0.01) states compared with controls. Lipid
peroxide accumulation in LDL oxidized in the presence of
HDL3 was not significantly different between
control and diabetic subjects. HDL2 was
significantly more protective than HDL3 both in
fasting (29.2±8.1% versus 63.0±8.4%; P=0.002) and in
postprandial (35.3±7.7% versus 57.9±7.7%; P<0.02)
states in controls only but not in diabetic subjects. We found a
significant increase in the electrophoretic mobility of LDL oxidized in
the presence of HDL2 from diabetic compared with
control subjects, in both fasting (1.39±0.05 versus 1.22±0.06;
P<0.05) and postprandial (1.52±0.06 versus 1.32±0.07;
P<0.05) states (Figure 2).
Again, HDL2 was more efficient in reducing the
electrophoretic mobility of LDL compared with
HDL3, both in fasting (1.22±0.06 versus
1.49±0.07; P=0.001) and postprandial (1.32±0.07 versus
1.53±0.07; P=0.05) states in controls only but not in the
diabetic subjects. During oxidation, there was no difference in either
the lipid peroxides or the electrophoretic mobility of
HDL2 or HDL3 from control
and diabetic subjects, in both the fasting and postprandial state (data
not shown).
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Figure 3 illustrates the fragmentation of
apo B-100 in LDL when oxidized in the absence or presence of
HDL2 from control and diabetic subjects.
HDL2 from control subjects, in both fasting and
postprandial states, protected the apo B-100 in LDL from fragmentation
during oxidation to a significant extent. However, the apo B-100 band
in LDL oxidized in the presence of HDL2 from
diabetic subjects was almost completely fragmented.
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Figure 4 illustrates that
HDL2 from controls, when oxidized in the presence
of LDL, had only minimal apo A-I cross-linking in the fasting state,
which was amplified in the postprandial state. However, both fasting
and postprandial HDL2 from diabetic subjects,
oxidized in the presence of LDL, showed extensive apo A-I cross-linking
when compared with control HDL2.
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Linear Regression Analysis
Simple linear regression analysis was performed to assess
which components in the control and diabetic HDL2
might be associated with their protective effects against LDL
oxidation. Protection by control HDL2 against
lipid peroxide accumulation in LDL was strongly and positively
correlated with its associated PAF-AH activity both in the fasting
(r=0.872, P=0.002) (Figure 5a) and postprandial (r=0.818,
P=0.007) state (data not shown), whereas diabetic
HDL2-associated PAF-AH activity was not
correlated with its protection in either the fasting (Figure 5b)
or postprandial state (data not shown). We found that protection by
control HDL2 against lipid peroxide accumulation
in LDL was positively correlated with PAF-AH activity when expressed
either per milligram of apo A-I or per milligram of HDL protein. No
such correlation existed in the diabetic subjects, even when the PAF-AH
activity was expressed either per milligram of apo A-I or per milligram
of HDL protein. We did not see any correlation between
HDL2-associated PON activity and protection by
HDL2 from control or diabetic subjects against
lipid peroxide accumulation in LDL. In controls, PAF-AH activity was
significantly higher in HDL2 than in
HDL3 (437 versus 247 nmol ·
min-1 · mg-1 apo
A-I, P<0.05), whereas PON activity was significantly higher
in HDL3 than in HDL2 (7.66
versus 1.35 nmol · min-1 ·
mg-1 apo A-I, P<0.05).
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Correlation analysis indicated that protection by
HDL2 from control subjects against lipid peroxide
accumulation in LDL was not correlated with serum TG,
HDL2-TG, cholesterol, phospholipid,
or apoprotein concentrations. In contrast, protection by
HDL2 from diabetic subjects was inversely
correlated with serum TG concentrations in both the fasting
(r=-0.673, P<0.05) and postprandial
(r=-0.798, P<0.02) states (Figure 6a and 6b). Similarly, protection by
HDL2 from lipid peroxide accumulation in the
diabetic subjects in the fasting state was inversely correlated with
HDL2-TG concentration (r=-0.636,
P<0.05) (Figure 6c) and positively correlated with
HDL2 free cholesterol concentration
(r=0.820, P<0.005) (Figure 6d). In the
diabetic subjects, we did not see any correlation between the level of
glycemic control and HDL2 and
HDL3 composition, PON and PAF-AH activities, or
protection against LDL oxidation (data not shown).
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Discussion |
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Type 2 diabetic subjects present an increased risk for CAD that is related to their degree of hypertriglyceridemia.3 Compositional abnormalities in HDL are well characterized in NIDDM.3 4 5 6 To determine whether abnormal HDL particles in NIDDM are able to maintain antiatherogenic functions, we examined the protective effects of HDL2 and HDL3 from poorly controlled type 2 diabetic and nondiabetic control subjects in both fasting and postprandial states against macrophage-mediated LDL oxidation.
Control subjects had a slight increase in phospholipid, free cholesterol, and protein concentrations in both HDL2 and HDL3 postprandially compared with the fasting state. Alimentary lipemia resulted in an increase in concentrations of phospholipids and proteins in HDL, particularly in the HDL2 subfraction.32 The magnitude of postprandial lipemia determines the proportion of TGs in postprandial HDL2.33 In our study, HDL2 from diabetic subjects were enriched in TGs and depleted of free cholesterol and apo A-I compared with that in controls. Other studies34 35 reported similar compositional abnormalities in type 2 diabetes. Similar to our results, Cavallero et al3 reported a significant postprandial decrease in free cholesterol and cholesteryl ester concentrations and an increase in TG concentrations in HDL2 from type 2 diabetics. Increased cholesteryl ester transfer rates mediated by CETP in type 2 diabetes10 11 36 appear important for the TG enrichment and cholesterol depletion seen in HDL2. In the diabetic subjects, we found a positive correlation between serum TG concentrations and HDL2-TG concentrations (data not shown), as previously reported by Biesbroeck and colleagues.6 Postprandially, we found a significant TG enrichment of HDL2 in diabetic subjects only, although both control and diabetic subjects had significant increases in their serum TG concentrations postprandially compared with the fasting state. Abbott et al37 have shown that serum PON activity is lower in type 2 diabetic subjects than in controls. However, we did not see a significant difference in HDL PON or PAF-AH activity between diabetic and control subjects.
Effects of the individual HDL2 and HDL3 subfractions against LDL oxidation in relation to their PAF-AH and PON activities are poorly understood. When we examined the protective effects of HDL subfractions against LDL oxidation from control subjects, HDL2 was significantly more efficient than HDL3 in inhibiting LDL oxidation in all parameters assayed. Navab and colleagues38 have shown that virtually all HDL-mediated inhibition of monocyte transmigration induced by minimally modified LDL can be accounted for by HDL2, whereas HDL3 had no effect. The greater protective effect of HDL2 seen in our study may be due to a higher PAF-AH activity associated with HDL2 compared with HDL3. In our study, HDL2 and HDL3 were not good substrates for macrophage oxidation compared with LDL. This may be due to a decreased lipid content in HDL. Other studies have also shown that unlike LDL, HDL is not susceptible to modification by endothelial cells14 or in cocultures of artery wall cells.38
In comparing the protective effects of HDL from control and diabetic subjects against LDL oxidation, no differences between HDL3 from control and diabetic subjects were observed. On the other hand, HDL2 fractions from diabetic subjects exhibited decreased protection against LDL oxidation compared with controls in both fasting and postprandial states in most of the parameters assayed. There was increased lipid peroxide accumulation and electrophoretic mobility; also, their apo B-100 was almost completely fragmented when LDL was oxidized in the presence of HDL2 from diabetic compared with control subjects. In controls, in both fasting and postprandial states, HDL2 exhibited significantly greater protection compared with HDL3, but this difference between HDL2 and HDL3 was not seen in diabetic subjects. This suggests that HDL2 from diabetic subjects may have lost its efficiency to protect LDL from oxidation. Correlation analysis indicated that the increase in serum TG concentrations might mediate these effects by altering the composition of HDL2. Decreased free cholesterol and increased TG concentrations in HDL2 from diabetic subjects may contribute importantly to the decreased protection exhibited by diabetic HDL2.
Of importance, TG-enriched HDL2 from diabetic subjects, though not susceptible to oxidation, exhibited decreased protection against LDL oxidation compared with HDL2 from controls. To determine whether diabetic HDL2 was being more structurally altered in the presence of LDL compared with control HDL2, we studied the apo A-I cross-linking in HDL2. When HDL2 was oxidized in the presence of LDL, there was more extensive apo A-I cross-linking in HDL2 from diabetic subjects compared with controls. The greater apo A-I cross-linking seen in HDL2 from diabetic subjects compared with controls, when oxidized along with LDL, suggests that compositionally altered diabetic HDL2 may not be as efficient as control HDL2 in inhibiting lipid peroxide generation in LDL. Therefore, the large amounts of aldehydes formed during LDL oxidation may have modified the apo A-I in diabetic HDL2. Aldehydes such as 4-hydroxynonenal and malondialdehyde generated during LDL oxidation can cause apo A-I cross-linking in HDL.39
Two possible mechanisms for the decreased protection exhibited by HDL2 from diabetic subjects include the following: (1) The decreased free cholesterol concentration seen in diabetic HDL2 may alter its surface fluidity, perhaps thereby facilitating the lipid peroxides generated during LDL oxidation to "seed" the HDL2. Once seeded with lipid peroxides, the TG-enriched HDL2 from diabetic subjects may be more susceptible to oxidation and become less protective. (2) The lipid peroxidation products transferred from oxidized LDL may directly inactivate the PAF-AH activity in HDL2. Dentan and colleagues40 have demonstrated that 4-hydroxynonenal generated during LDL oxidation may inhibit PAF-AH activity, either by a direct modification of amino acid side chains in PAF-AH or by a modification of the phospholipid environment of the enzyme at the surface of the lipoprotein particle. In conclusion, our study demonstrates that HDL2 from subjects with poorly controlled NIDDM, in both fasting and postprandial states, exhibits decreased protection against macrophage-mediated LDL oxidation, and this may contribute to accelerated atherosclerosis in type 2 diabetes.
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Acknowledgments |
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This research was supported in part by the High Carbohydrate Fiber Nutrition Foundation (to J.W.A.) and by National Institutes of Health (Bethesda, Md) grant RO1HL59376 (to D.R.V.d.W.). We gratefully acknowledge Dr Frederick DeBeer for his support. We thank Dr Marcielle DeBeer and Darin Cecil for their advice and technical assistance.
Received April 9, 1998; accepted January 19, 1999.
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