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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2045
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 5 1961-1964
Copyright © 2007 by The Endocrine Society

BRIEF REPORT

In Vitro Glycoxidized Low-Density Lipoproteins and Low-Density Lipoproteins Isolated from Type 2 Diabetic Patients Activate Platelets via p38 Mitogen-Activated Protein Kinase

Catherine Calzada, Laurent Coulon, Déborah Halimi, Elodie Le Coquil, Valérie Pruneta-Deloche, Philippe Moulin, Gabriel Ponsin, Evelyne Véricel and Michel Lagarde

Institut National de la Santé et de la Recherche Médicale, U870, F-69008 Lyon, France; Institut National des Sciences Appliquées de Lyon, Régulations Métaboliques, Nutrition, et Diabètes, F-69621 Villeurbanne, France; Institut National de la Recherche Agronomique, U1235, F-69008 Lyon, France; University of Lyon 1, F-69003 Lyon, France; and Hospices Civils de Lyon, F-69003 Lyon, France

Address all correspondence and requests for reprints to: Dr. Catherine Calzada, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 870/Institut National des Sciences Appliquées de Lyon, Régulations Métaboliques, Nutrition, et Diabètes/Institut Multidisciplinaire de Biochimie des Lipides, Bât. Louis Pasteur, 20 av. Albert Einstein, 69621 Villeurbanne, France. E-mail: catherine.calzada{at}insa-lyon.fr.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Platelet hyperactivation contributes to the increased risk for atherothrombosis in type 2 diabetes and is associated with oxidative stress. Plasma low-density lipoproteins (LDLs) are exposed to both hyperglycemia and oxidative stress, and their role in platelet activation remains to be ascertained.

Objective: The aim of this study was to investigate the effects of LDLs modified by both glycation and oxidation in vitro or in vivo on platelet arachidonic acid signaling cascade. The activation of platelet p38 MAPK, the stress kinase responsible for the activation of cytosolic phospholipase A2, and the concentration of thromboxane B2, the stable catabolite of the proaggregatory arachidonic acid metabolite thromboxane A2, were assessed.

Results: First, in vitro-glycoxidized LDLs increased the phosphorylation of platelet p38 MAPK as well as the concentration of thromboxane B2. Second, LDLs isolated from plasma of poorly controlled type 2 diabetic patients stimulated both platelet p38 MAPK phosphorylation and thromboxane B2 production and possessed high levels of malondialdehyde but normal {alpha}-tocopherol concentrations. By contrast, LDLs from sex- and age-matched healthy volunteers had no activating effects on platelets.

Conclusions: Our results indicate that LDLs modified by glycoxidation may play an important contributing role in platelet hyperactivation observed in type 2 diabetes via activation of p38 MAPK.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
OXIDATIVE STRESS HAS been identified as one of the factors closely associated with platelet hyperactivation in type 2 diabetic patients (1), even in the absence of any vascular complications (2). It remains to be determined whether oxidative stress is inherent to platelets and/or is a consequence of circulating factors that could influence platelet function. Among them, low-density lipoproteins (LDLs) are submitted to both glycation and oxidation in diabetes (3). In this context, the aims of our study were: 1) to determine the effect of in vitro glycoxidized LDLs on platelets and compared it with control, glycated, or oxidized LDL; and 2) to investigate the effect of LDLs isolated from plasma of type 2 diabetic patients, compared with healthy volunteers on platelets. Anti-/prooxidant status of LDL was assessed and the activation of platelet p38 MAPK as well as the formation of thromboxane (Tx) B2 were determined.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
In vitro experiments

LDL isolation from healthy subjects. LDLs were isolated from plasma by density gradient ultracentrifugation (density, 1.019–1.063 g·ml–1) (4). The concentration of protein was estimated using the Bradford assay (5). LDLs were stored at 4 C in the dark under nitrogen and used within 2 d after preparation.

In vitro LDL modification. LDLs (3.5 mg protein per milliliter) in PBS (pH 7.2) were all incubated at 37 C in the dark under nitrogen in the presence of sodium azide (1.5 mmol/liter): 1) control LDLs prepared by incubating LDLs with EDTA (1 mmol/liter) and butylated hydroxytoluene (BHT; 5 µmol/liter) for 5 d; 2) glycated LDLs LDL incubated with 50 mmol/liter D-glucose for 5 d in the presence of EDTA (1 mmol/liter) and BHT (5 µmol/liter); 3) oxidized LDLs prepared by incubating LDLs for 5 d and then treating them with 1 µmol/liter CuSO4 for 1 h at 37 C; and 4) glycoxidized LDL consisting of LDLs incubated with 50 mmol/liter D-glucose for 5 d and treating them with 1 µmol/liter CuSO4 for 1 h at 37 C.

LDL preparations were then dialyzed against PBS before their interaction with platelets.

LDL characterization

LDL concentrations of {alpha}-tocopherol were determined by reversed-phase HPLC (6). Briefly, LDL samples, containing tocol (6-hydroxy-2-methyl-2-phytylchroman) and {delta}-tocopherol as internal standards, were extracted with 4 volumes of hexane after the addition of 1 volume of ethanol. Tocopherol isomers were separated onto a Nucleosil C18 column, 5 µm (4 x 150 mm), and detected fluorometrically (excitation 295 nm, emission 340 nm).

Overall lipid peroxidation was evaluated by quantitation of malondialdehyde (MDA) by reversed-phase HPLC (7). LDL samples were mixed with thiobarbituric acid (10 mmol/liter) and acetic acid in the presence of BHT (5 mmol/liter) and incubated at 95 C for 1 h. Thiobarbituric acid-MDA adduct was extracted with ethyl acetate, separated on a Nucleosil C18 column 5 µm (4.6 x 250 mm), and detected fluorometrically (excitation 515 nm, emission 553 nm).

The degree of glycation was determined by the trinitrobenzene sulfonic acid assay (8). LDL glycation was expressed as the percentage of relative reduction of the detected amino groups of lysine of modified LDLs, compared with control LDLs.

Diabetic and control subjects

Ten type 2 diabetic patients (five men and five women, aged 58 ± 2 yr) from the Department of Endocrinology and Metabolic Diseases were matched for sex and age to 10 healthy subjects (five men and five women, aged 54 ± 2 yr). Exclusion criteria for diabetic patients were smoking, antioxidant/vitamin supplementation, antiaggregating drugs, and insulin treatment. Six of 10 were on metformin or sulfamides, three of 10 on glitazones, and seven of 10 took lipid-lowering drugs (statins). The patients had poorly controlled diabetes (fasting glycemia 11.9 ± 2.0 mmol/liter; glycated hemoglobin A1C 8.8 ± 0.6%). They had mild dyslipidemia with mild hypertriglyceridemia (triglycerides 2.0 ± 0.2 mmol/liter), normal LDL-cholesterol (2.7 ± 0.3 mmol/liter), and normal high-density lipoprotein-cholesterol (1.3 ± 0.1 mmol/liter). Control subjects were in good health as assessed by medical history, and exclusion criteria were any pathology including diabetes and antiaggregatory drugs. Written informed consent was obtained from all participants.

Interaction between platelets and LDL

Platelet isolation and incubation with LDL. Blood was collected at the regional blood center from healthy volunteers who had not ingested any aspirin or other nonsteroidal antiinflammatory drugs in the previous 10 d. Platelets were prepared (9) and incubated in the presence or absence of LDL (500 µg/ml) for 2 h at 37 C.

Platelet p38 MAPK activation. Platelets were lysed (10), proteins (25 µg) were denatured for 10 min at 100 C, electrophoresed in 12% Tris-HCl polyacrylamide gels at 25 mA for 135 min, and transferred to nitrocellulose membranes (100 V, 30 min). The membranes were incubated with either 1:1000 anti-p38 MAPK or antiphospho-p38 MAPK polyclonal antibodies (Cell Signaling Technologies, Beverly, MA), washed, and incubated with 1:2000 goat antirabbit horseradish peroxidase conjugate for 1 h. p38 MAPK and phospho-p38 MAPK were visualized by enhanced chemiluminescence, and bands were quantified by densitometry with an ImageMaster VDS-CL camera (Amersham Biosciences, Buckinghamshire, UK).

Platelet TxB2 measurement. Platelet TxB2 was quantified by enzyme immunoassay (Amersham Biosciences). Coefficient of variation was lower than 10%.

Statistical analysis

Results are expressed as the mean ± SD. Comparisons between groups were performed using a Wilcoxon test. Statistical significance was established at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effects of in vitro modified LDLs isolated from healthy subjects on platelets

Glycoxidized LDLs were compared with LDLs incubated solely with glucose or CuSO4 to differentiate the specific effects of glycation or oxidation from combined effects. The incubation of LDLs with 50 mmol/liter glucose had no significant effects on LDL {alpha}-tocopherol (7.70 ± 1.84 nmol/mg protein in glycated LDL vs. 8.53 ± 2.27 nmol/mg protein in control LDL, n = 8) and MDA level, compared with control LDLs (0.19 ± 0.16 nmol/mg protein vs. 0.16 ± 0.17 nmol/mg protein, respectively, n = 5). As expected, the addition of CuSO4 to LDLs resulted in a decreased {alpha}-tocopherol (6.73 ± 3.27 nmol/mg protein) and an increased MDA level (0.27 ± 0.26 nmol/mg protein), compared with control LDLs. Glycoxidation of LDLs led to neither significant additional decreases of {alpha}-tocopherol (7.03 ± 2.34 nmol/mg protein) nor further increases of peroxide levels (0.27 ± 0.33 nmol/mg protein), compared with oxidized LDLs. Their percentage of glycation was 34 ± 6%, similar to that measured in glycated LDLs (35 ± 9%).

The incubation of platelets with control LDLs or glycated LDLs had no significant effect on the activation of p38 MAPK (Fig. 1AGo) and TxB2 concentration (Fig. 1BGo), compared with platelets alone. The addition of oxidized LDLs to platelets increased p38 MAPK phosphorylation by 2-fold and TxB2 concentration LDL by 69%. Finally, glycoxidized LDLs had the most pronounced effect on platelet p38 MAPK phosphorylation (3-fold increase) and TxB2 level (2.4-fold increase), compared with platelets alone.


Figure 1
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  		FIG. 1. Effect of in vitro modified LDL on platelet p38 MAPK phosphorylation (A) and TxB2 concentration (B). Platelets were incubated for 2 h at 37 C in the absence (0) or presence of 500 µg/ml control LDL, glycated LDL (LDL + 50 mmol/liter glucose), oxidized LDL (LDL + 1 µmol/liter CuSO4) or glycoxidized LDL (LDL + 50 mmol/liter glucose + 1 µmol/liter CuSO4). Data are means ± SD of five to eight experiments. a, P < 0.05 vs. platelets.

 
Effect of LDL isolated from type 2 diabetic patients on platelets

MDA levels increased by 6-fold in LDLs from type 2 diabetic patients, compared with LDLs from control subjects (1.74 ± 2.58 nmol/mg protein vs. 0.29 ± 0.29 nmol/mg protein, respectively, n = 10), whereas LDL {alpha}-tocopherol concentrations did not differ between patients and control subjects (10.25 ± 1.94 nmol/mg protein vs. 10.26 ± 3.25 nmol/mg protein in LDL, respectively, n = 10). The incubation of platelets with LDLs from diabetic patients resulted in a 2.2-fold increase of phosphorylated p38 MAPK amount (Fig. 2AGo) and a 2-fold increased basal concentration of TxB2, compared with platelets (578 ± 164 pmol/109 platelets vs. 267 ± 22 pmol/109 platelets) (Fig. 2BGo). It is worth noting that neither significant phosphorylation of p38 MAPK nor increase of TxB2 concentration was observed in platelets incubated with LDLs from healthy volunteers.


Figure 2
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  		FIG. 2. Effect of LDL from control subjects and type 2 diabetic patients on platelet p38 MAPK phosphorylation (A) and TxB2 concentration (B). Platelets were incubated for 2 h at 37 C in the absence (0) or presence of LDL (500 µg/ml). Results are means ± SD (n = 10). a, P < 0.05 vs. platelets; b, P < 0.05 vs. platelets + LDL from control subjects.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our results show that LDLs modified by glycoxidation in vitro or in vivo activate platelets, whereas control or glycated LDLs have no effect. Previous studies on the interaction between platelets and LDLs have shown that LDLs do not induce platelet activation per se (11) but may increase the sensitivity of platelets to different agonists (12) via an increased phosphorylation of p38 MAPK (13). Such a discrepancy between results could be ascribed to differences in the degree of LDL oxidation. We show that the incubation of LDLs with glucose did not change their anti-/prooxidant status, corroborating that glycation alone is insufficient to promote LDL oxidation (14). Moreover, glycated LDLs had no stimulating effects on platelets. Although one study reported that glycated LDL caused an increased platelet aggregating response to ADP, the presence of high levels of thiobarbituric acid reactive substances in those LDLs suggests that the effects were likely due to the combination of LDL glycation and oxidation (15). We also show that copper-oxidized LDLs increased p38 MAPK phosphorylation by 2-fold and TxB2 concentration by 69%, confirming that LDLs defined as minimally or mildly oxidized may activate platelets (12, 16). Finally, glycoxidized LDLs, reflecting at best the state that may occur in LDLs from diabetic patients, were the most effective triggers of platelet arachidonic acid signaling cascade. Although the presence of glucose has been shown to accelerate copper-induced LDL oxidation (17), we did not observe any significant differences between oxidized LDLs and glycoxidized LDLs in terms of vitamin E and MDA levels. This could be related to the copper to LDL ratios used, which were much lower in our study than in the above published studies, as suggested by Knott et al. (14).

Supporting our in vitro results, we present new data indicating that LDLs isolated from poorly controlled type 2 diabetic patients increase platelet p38 MAPK phosphorylation and TxB2 formation, whereas those isolated from healthy control subjects do not. It has been shown that LDLs isolated from type 1 diabetic patients increased platelet aggregation and TxB2 release (18). Concerning the agents in LDLs from diabetic patients responsible for platelet activation in our experiments, their identification seem rather difficult because LDLs represent a heterogenous mixture of particles modified to different degrees of glycation and oxidation. However, an important feature is that LDLs from selected patients possessed high concentrations of MDA, which supports data reporting increased levels of plasma lipid peroxides in type 2 diabetic patients (19). No modification of vitamin E was observed, ruling out an involvement of this antioxidant. Moreover, LDLs isolated from selected diabetic patients can be defined as glycoxidized LDLs because the percent of hemoglobin A1C (8.8%) is known to be correlated with the percent of glycated LDLs (20). In conclusion, LDLs from type 2 diabetic patients, as well as glycoxidized LDL in vitro, activate platelet arachidonic acid signaling cascade. The effect of LDL appears to be related to the combination of hyperglycemia and lipid peroxidation independently of vitamin E status. Thus, it suggests that glycoxidized LDLs may act as one of the triggers of platelet activation occurring in type 2 diabetes.


   Acknowledgments
 
We gratefully thank nurses for expert blood-drawing assistance.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale and Agence Nationale de la Recherche ANR 2005 "Cardiovasculaire, Obésité et Diabète." C.C. is supported by Centre National de la Recherche Scientifique.

Disclosure Statement: The authors have nothing to declare.

First Published Online March 6, 2007

Abbreviations: BHT, Butylated hydroxytoluene; LDL, low-density lipoprotein; MDA, malondialdehyde; Tx, thromboxane.

Received September 18, 2006.

Accepted February 27, 2007.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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  11. Meraji S, Moore CE, Skinner O, Bruckdorfer KR 1992 The importance of oxidation or glycosylation of low-density lipoproteins in relation to platelet activation. Platelets 3:155–162
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This Article
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