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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 9 4238-4242
Copyright © 2004 by The Endocrine Society

Special Feature

Ala12Ala Genotype of the Peroxisome Proliferator-Activated Receptor {gamma}2 Protects against Atherosclerosis

Theodora Temelkova-Kurktschiev, Markolf Hanefeld, Giulia Chinetti, Christophe Zawadzki, Stephan Haulon, Agata Kubaszek, Carsta Koehler, Wolfgang Leonhardt, Bart Staels and Markku Laakso

Center for Clinical Studies, Technical University Dresden (T.T.-K., M.H., C.K., W.L.), Dresden, Germany; UR 545, Institut National de la Santé et de la Recherche Médicale, Institut Pasteur de Lille and Université de Lille II (G.C., B.S.), Lille, France; Equipe d’Accueil 2693, Université de Lille II (C.Z.), Lille, France; Chirurgie Vasculaire (S.H.), Lille, France; and Department of Medicine, University of Kuopio (A.K., M.L.), Kuopio, Finland

Address all correspondence and requests for reprints to: Dr. Theodora Temelkova-Kurktschiev, Center for Clinical Studies, Technical University Dresden, Fiedlerstrasse 34, 01307 Dresden, Germany. E-mail: theodora{at}gwtonline-zks.de.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
A mutation in the peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2) gene with a cytosine to guanine substitution results in an exchange of proline (Pro) with alanine (Ala) in exon B (codon 12) of this gene. This polymorphism has been associated with high insulin sensitivity and low body weight, but no data have been published to date about its effect on early atherosclerosis. We investigated the relationship of the Pro12Ala polymorphism to early atherosclerosis, measured by the intima-media thickness (IMT). A total of 622 subjects were included, aged 40–70 yr, who were participants of the RIAD (Risk factors in Impaired glucose tolerance for Atherosclerosis and Diabetes) study and were at risk of developing type 2 diabetes. Altogether, 449 of the subjects had the common genotype (Pro12Pro), 162 had the Pro12Ala genotype, and 11 the Ala12Ala genotype. IMT was significantly decreased in subjects with the Ala12Ala genotype compared with subjects with the other two genotypes. Body mass index, free fatty acid levels, and leukocyte count were lower in subjects with the Ala12Ala genotype compared with subjects with the Pro12Pro or Pro12Ala genotypes. In multivariate analysis, the Ala12Ala genotype was a significant independent determinant of IMT. Furthermore, we demonstrated specific expression of the PPAR{gamma}2 gene in human atherosclerotic lesions as well as in cultured primary macrophages and foam cells. In conclusion, our data suggest that the Ala12Ala genotype of the PPAR{gamma}2 gene may protect from early atherosclerosis in subjects at risk for diabetes.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE PEROXISOME PROLIFERATOR-activated receptor {gamma} (PPAR{gamma}) is a member of the nuclear receptor superfamily of transcription factors that regulate adipocyte differentiation and glucose metabolism (1, 2). PPAR{gamma} is highly expressed in adipose tissue, adrenal gland, spleen, and large colon (3, 4, 5, 6). Recent studies indicate that PPAR{gamma} is expressed in cells of the monocyte/macrophage lineage and suggest that PPAR{gamma} could have an effect on atherogenesis (7, 8, 9, 10, 11, 12, 13). PPAR{gamma} is expressed in macrophage foam cells of human atherosclerotic lesions, and its expression is highly correlated with oxidation-specific epitopes (10). PPAR{gamma} activation may have antiatherogenic effects (7, 8, 11), and it may exert antiinflammatory effects by negatively regulating the expression of proinflammatory genes (7, 8). Furthermore, PPAR{gamma} activation has been found to inhibit gene expression and migration in human vascular smooth muscle cells (11). These studies along with the observation of decreased carotid intima-media thickness (IMT) in diabetic patients treated with the PPAR{gamma} agonist troglitazone (14) suggest that PPAR{gamma} activation might prevent atherogenesis.

A cytosine to guanine substitution in the PPAR{gamma}2 gene results in an exchange of proline (Pro) to alanine (Ala) in exon B (codon 12) of this gene (15). Because this mutation is very close to the N-terminal end of the protein, in the ligand-independent activation domain, it may cause conformational changes and consequently affect its function. There are data on the impact of the polymorphism on insulin sensitivity, development of type 2 diabetes, body weight, and lipoprotein metabolism (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27), but no epidemiological data have been published on its effect on atherosclerosis.

The measurement of IMT of the common carotid artery (CCA) is a generally accepted method to monitor the early stages of atherosclerosis (28, 29, 30, 31). Carotid IMT has been shown to be related to cardiovascular risk factors and to be a strong independent predictor of future myocardial infarction and stroke (30, 31).

The aim of this study was to examine the association of the Pro12Ala polymorphism of the PPAR{gamma}2 gene with carotid IMT in a population at high risk for diabetes and to determine whether PPAR{gamma}2 is expressed in human atherosclerotic lesions and macrophages.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
A total of 622 subjects (284 men and 338 women), who were participants of the RIAD Study, a prospective survey on the Risk factors in Impaired glucose tolerance for Atherosclerosis and Diabetes (32), and in whom the measurement of IMT was available, were included in this study. In brief, subjects from 40–70 yr of age were examined who had risk factors for the development of type 2 diabetes, such as a family history of type 2 diabetes, obesity, and/or hyper/dyslipoproteinemia. Known diabetes, medication affecting glucose tolerance, liver and kidney diseases, thyroid gland functional disorders, and acute infections were exclusion criteria. Written consent was obtained from all participants. The basic characteristics of the study subjects are shown in Table 1Go.


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  		TABLE 1. Basic characteristics (mean and SEM)

 
Ultrasound measurement

B-Mode ultrasound of the CCA was performed with Acuson 128XP computed sonography system using a 10-MHz linear array transducer, as previously described (32). The patients were examined in the supine position. In brief, the thickness of the intima-media complex was assessed as described by Pignoli et al. (28). To avoid variability during the cardiac cycle, the images were frozen in the end-diastolic phase. IMT was determined only from the far wall of the artery, because it is known to have a higher precision than the near arterial wall (33, 34). Measurements were conducted in plaque-free portions of the 10-mm linear segment proximal to the carotid bulb. For each patient two measurements were performed bilaterally, and the values were averaged, which presented the mean of IMT of the CCA.

Laboratory examination

Venous blood was collected after an overnight fast of at least 10 h in EDTA monovettes, and plasma was immediately separated by centrifugation (4000 rpm for 8 min at 4 C). Genomic DNA was isolated from leukocytes by salt extraction. Exon B of the PPAR{gamma}2 gene was amplified by PCR with published primers, and the Pro12Ala polymorphism was screened by the single strand conformation polymorphism as previously described in detail (17). A standard oral glucose tolerance test was performed with 75 g glucose (Glucodex, Rougier, Inc., Chambly, Canada). Plasma glucose was measured by the hexokinase method. Insulin was measured by RIA (Pharmacia Biotech, Uppsala, Sweden). Total cholesterol and triglycerides were measured enzymatically on a Ciba Corning Express Plus analyzer (Boehringer, Mannheim, Germany). High density lipoprotein cholesterol was determined after precipitation with dextran sulfate on a Ciba Corning Express Plus analyzer (Boehringer). Free fatty acids (FFAs) were analyzed by enzyme colorimetric assay with a test kit (Boehringer). Complete blood cell counts were performed with standard techniques. Urine was collected as fresh morning urine samples. Albuminuria was measured by nephelometry (Nephelometer BNII, Behring, Marburg, Germany).

Tissue and cell culture

Human atherosclerotic plaques removed during carotid endarterectomy were collected into RNAlater (Ambion, Inc., Austin, TX) until RNA extraction. Mononuclear cells isolated from blood of healthy normolipidemic donors by Ficoll gradient centrifugation were suspended in RPMI 1640 medium containing gentamicin (40 mg/ml), glutamine (0.05%), and 10% pooled human serum. Mature monocyte-derived macrophages were used for experiments after 10 d of culture. Cholesterol-loaded macrophages were obtained by 24-h incubation with acetylated low density lipoprotein (AcLDL; 50 µg protein/ml) (13).

RNA extraction and analysis

RNA from human macrophages and atherosclerotic plaques was extracted using TRIzol (Invitrogen Life Technologies, Cergy Pontoise, France). Total RNA was reverse transcribed using random hexameric primers and Superscript reverse transcriptase (Invitrogen Life Technologies). PCR was performed using PPAR{gamma}2-specific primers (5'-CCC AGA AAG CGA TTC CTT CAC-3' and 5'-AGC TGA TCC CAA AGT TGG TGG-3') and cyclophilin-specific primers (5'-GCA TAC GGG TCC TGG CAT CTT GTC C-3' and 5'-ATG GTG ATC TTC TTG CTG GTC TTG C-3'). The resulting products were separated on a 1% agarose gel and stained with ethidium bromide.

Statistics

Data analyses were conducted using the SPSS/PC+ programs. Data are presented as the mean ± SEM. The distribution of values was assessed by the Kolmogorov-Smirnov test for homogeneity of variances. Variables that were not normally distributed were logarithmically transformed. Carotid IMT and cardiovascular risk factors in subjects with the Pro12Pro, Pro12Ala, and Ala12Ala genotypes were analyzed by ANOVA and covariance. Because systolic and diastolic blood pressures were not normally distributed after logarithmical transformation, the groups were compared by the Kruskal-Wallis test. P < 0.05 was considered statistically significant. Multivariate analysis was conducted by multiple linear regression.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Altogether, 449 (72.2%) of the subjects had the wild-type allele (Pro12Pro), 162 (26.0%) were heterozygous for the Ala allele (Pro12Ala), and 11 (1.8%) were homozygous for the Ala allele. The genotype distribution was in Hardy-Weinberg equilibrium.

The subjects of the three genotype groups, Pro12Pro, Pro12Ala, and Ala12Ala, did not differ with respect to age or gender (Table 2Go). Subjects with the Pro12Pro or Pro12Ala genotype had higher body mass index (BMI) and 2-h insulin level than subjects with the Ala12Ala genotype. No differences among the three genotype groups were observed in waist/hip ratio, systolic and diastolic blood pressures, fasting and 2-h glucose levels, fasting insulin levels, homeostasis model assessment insulin resistance, glucose tolerance (impaired glucose tolerance/diabetes), lipids and lipoproteins, or albuminuria. Significantly lower FFAs and leukocyte count were found in subjects with the Ala12Ala genotype compared with other genotypes even after adjustment for age and gender.


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  		TABLE 2. Cardiovascular risk factors according to the genotypes of the PPAR{gamma}2 gene

 
As shown in Fig. 1Go, subjects with the Ala12Ala genotype exhibited a significantly lower mean IMT of the common carotid artery than subjects in the other two genotype groups. This finding remained statistically significant even after adjustment for age, gender, and the use of lipid-lowering and antihypertensive drugs. No difference was found between the Pro12Pro genotype and the Pro12Ala genotype in mean IMT. In multivariate linear regression analysis age, total cholesterol, diabetes status (0 = no; 1 = yes), high density lipoprotein cholesterol (negatively), male gender, leukocyte count, and the Ala12Ala genotype (negatively) were independent determinants of mean carotid IMT (Table 3Go).



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  		FIG. 1. IMT (millimeters) of the CCA in subjects with the Pro12Pro, Pro12Ala, and Ala12Ala genotypes. *, P < 0.05 compared with the Ala12Ala genotype after adjustment for age, gender, and use of lipid-lowering and antihypertensive medications.

 

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  		TABLE 3. Association of cardiovascular risk factors with average IMT of the CCA (multivariate linear regression analysis)

 
To determine the presence of PPAR{gamma}2 in human atherosclerotic lesions, RNA expression analysis was performed on lipid-rich atherosclerotic plaques obtained after carotid endarterectomy. PPAR{gamma}2 gene expression was detected in atheroclerotic lesions obtained from four patients (Fig. 2AGo). To determine whether PPAR{gamma}2 is expressed in human macrophages and foam cells, which are abundant in atherosclerotic lesions, RT-PCR analysis was performed on RNA of macrophages from different healthy donors. Expression of PPAR{gamma}2 was observed in all differentiated macrophages as well as in macrophage-derived foam cells, suggesting that in atherosclerotic lesions, PPAR{gamma}2 may be expressed in the lipid-rich macrophages (Fig. 2BGo).



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  		FIG. 2. Expression of PPAR{gamma}2 in human atherosclerotic lesions and in primary macrophages. A, RT-PCR analysis of PPAR{gamma}2 from lipid-rich atherosclerotic lesions obtained by endarterectomy from four patients (no. 1–4). B, RT-PCR analysis of PPAR{gamma}2 from macrophages isolated from four healthy donors (A–D) in the absence (cont) or presence of AcLDL to induce foam cell formation (AcLDL). Quantification by optical densitometry of PPAR{gamma}2 mRNA was performed, and results were normalized to cyclophilin as an internal control.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The novel finding in our study was that IMT of the common carotid artery, a marker of early atherosclerosis, was significantly decreased in subjects with the Ala12Ala genotype of the PPAR{gamma}2 gene. We also demonstrated specific expression of the PPAR{gamma}2 gene in human atheroscleroticlesions as well as in cultured primary macrophages and foam cells. Thus, our data, reported in Fig. 2Go, indicate that PPAR{gamma}2 is present in human differentiated macrophages in vitro as well as in AcLDL-loaded macrophages, an experimental model to mimic foam cell formation. These results suggest that PPAR{gamma}2 could be an important player in the pathology of atherosclerosis. Furthermore, our data indicate a direct protective effect of the Ala12Ala genotype on IMT, because this genotype was found to be a significant determinant of IMT in multivariate regression analysis independently of several other cardiovascular risk factors, including diabetes, insulin resistance, free fatty acids, dyslipidemia, and inflammatory markers.

Subjects with the Pro12Ala genotype had similar mean carotid IMT compared with subjects with the Pro12Pro genotype. This may indicate that the Pro12Ala polymorphism of the PPAR{gamma}2 gene has an effect on atherosclerosis only in its homozygous form. This conclusion is in agreement with previous studies of the effects of the Pro12Ala and Ala12Ala genotypes on the risk of obesity, type 2 diabetes, or quantitative traits related to the metabolic syndrome. In only two of the 40 previous studies published to date (MEDLINE) have the effects of the Ala12Ala genotype been separately reported, probably due to a low frequency of this genotype (<2%). However, in both of these studies, the Ala12Ala genotype differed from the Pro12Pro and Pro12Ala genotypes with respect to its effect on BMI (35) or on blood pressure or triglycerides (36). Carriers of the Pro12Pro and the Pro12Ala genotypes had similar clinical characteristics, and carriers of the Ala12Ala genotype clearly had a different phenotype. Therefore, it is possible that the effect of the Pro12Ala polymorphism on the levels of cardiovascular risk factors or on early atherosclerosis does not depend linearly on the number of Ala alleles. Because the number of carriers with the Ala12Ala genotype was quite low in our study, additional studies in other populations are needed to confirm our findings.

Our study supports previous studies that have reported lower BMI, lower total triglycerides, 2-h insulin level, and homeostasis model assessment of insulin resistance index in subjects with the Ala12Ala genotype, although these findings were not always statistically significant in the present study (16, 17, 18, 19, 20, 21, 22, 23, 24, 25). Similarly to a previous study (17), the levels of FFAs were lower in subjects with the Ala12Ala genotype.

Interestingly, we found a significantly lower leukocyte count in subjects with the Ala12Ala genotype than in subjects with the Pro12Ala genotype or subjects with the Pro12Pro genotype. Thus, the Ala12Ala genotype of the PPAR{gamma}2 gene may also have an antiinflammatory effect, although we cannot exclude the possibility that this variant also has an effect on bone marrow. In vitro studies have shown that PPAR{gamma} activation inhibits the expression of genes that become up-regulated during macrophage differentiation and activation (7). In addition, PPAR{gamma} agonists have been shown to inhibit the production of monocyte inflammatory cytokines (8). Because inflammation is known to be an independent risk factor for atherosclerosis (37, 38), it could be at least in part responsible for the vasoprotective effect of the Ala12Ala genotype. Additional studies are needed to confirm our findings by using additional inflammatory parameters, such as highly sensitive C-reactive protein, cytokine levels, etc. Furthermore, PPAR{gamma} exerts antiatherogenic effect by facilitating the removal of cholesterol from macrophages via cholesterol transport proteins such as ATP binding cassette transporter A1 (13, 14).

In conclusion, we demonstrated that PPAR{gamma}2 is expressed in human atherosclerotic lesions. We also showed that subjects with the Ala12Ala genotype of the PPAR{gamma}2 gene have a significantly lower carotid intima-media thickness, which could be due to a direct antiatherogenic effect of this polymorphism as well as to an indirect effect through its association with a lower level of inflammatory parameters and insulin resistance.


   Footnotes
 
This work was supported by a grant from the European Community (QLG1-CT-1999-00674; to M.L.) and by FEDER-Conseil Régional Nord Pas-de-Calais (Génopole 01360124) and the European Community (QLRT-1999-01007; to B.S. and G.C.).

Abbreviations: AcLDL, Acetylated low-density lipoprotein; Ala, alanine; BMI, body mass index; CCA, common carotid artery; FFA, free fatty acid; IMT, intima-media thickness; PPAR, peroxisome proliferator-activated receptor; Pro, proline.

Received December 10, 2003.

Accepted June 11, 2004.


   References
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 Introduction
 Subjects and Methods
 Results
 Discussion
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Peroxisome Proliferator-Activated Receptor-{gamma}2 P12A Polymorphism and Risk of Coronary Heart Disease in US Men and Women
Arterioscler. Thromb. Vasc. Biol., August 1, 2005; 25(8): 1654 - 1658.
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J. C. Florez
Phenotypic Consequences of the Peroxisome Proliferator-Activated Receptor-{gamma} Pro12Ala Polymorphism: The Weight of the Evidence in Genetic Association Studies
J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4234 - 4237.
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