Content uploaded by Christian Dina
Author content
All content in this area was uploaded by Christian Dina on Aug 22, 2014
Content may be subject to copyright.
Abstract
Aim/hypothesis. Plasminogen activator inhibitor-1
(PAI-1) is a main regulator of the endogenous fibrino-
lytic system and modulates the thrombosis progression.
We analyzed genetic contributions of PAI-1 mutations
to the metabolic syndrome and to its complications.
Methods. PAI-1 promoter and coding sequences were
screened for mutations. Genotypes were determined
for 1067 unrelated individuals of a French Caucasian
cohort, selected for diabetes and obesity. Association
between PAI-1 polymorphisms and phenotypes related
to metabolic syndrome were statistically studied.
Results. There were five variants identified: two com-
mon polymorphisms, −765 4G/5G and −844A>G, in
the promoter, and three new non-synonymous SNPs,
Ala15Thr, Val17Ile and Asn195Ile. In obese non-dia-
betic subjects, the two promoter polymorphisms were
associated with higher fasting glucose concentrations
(p=0.006 andp=0.0004, for −765 4G/5G and −844
A>G, respectively) and insulin (p=0.05 and p=0.008,
for −765 4G/5G and −844 A>G, respectively). More-
over, the −844 A>G SNP was associated with lower
triglyceride (p=0.002) and higher HDL cholesterol
concentrations (p=0.02) in lean subjects. In addition,
the two promoter and Ala15Thr polymorphisms
showed a trend towards association with CHD in dia-
betic subjects (–765 4G/5G: 0.56/0.51, p=0.05;
−844A>G: 0.63/0.57, p=0.02; Ala15Thr: 0.91/0.88,
p=0.04). The SNPs Ala15Thr, located in the PAI-1
signal peptide, and rare the Asn195Ile, located in a β-
sheet structure, could influence conformation of these
two structures.
Conclusions/interpretation. Our results support the
hypothesis that PAI-1 polymorphisms probably inter-
act with known environmental risk factors (chronic
hyperglycaemia, obesity, etc.) to induce a more severe
insulin-resistant metabolic profile in overweight sub-
jects, and to further increase risk for CHD in diabetic
subjects. [Diabetologia (2003) 46:1284–1290]
Keywords PAI-1, obesity, metabolic syndrome, CHD,
fasting glucose, insulin, triglycerides, HDL, Type 2
diabetes mellitus.
Received: 3 March 2003 / Revised: 7 April 2003
Published online: 11 July 2003
© Springer-Verlag 2003
Corresponding author: Dr. P. Froguel, CNRS 8090-Institute of
Biology, Institut Pasteur de Lille, 1 Rue du Professeur Cal-
mette, B.P. 447, 59021 Lille, France
E-mail: froguel@mail-good.pasteur-lille.fr
Abbreviations: PAI-1, Plasminogen activator-inhibitor 1; SNP,
single nucleotide polymorphism; T2D, Type 2 diabetes.
Diabetologia (2003) 46:1284–1290
DOI 10.1007/s00125-003-1170-0
PAI-1 polymorphisms modulate phenotypes associated with the
metabolic syndrome in obese and diabetic Caucasian population
C. Lopes2, C. Dina2, E. Durand1, P. Froguel1, 2
1 CNRS Institute of Biology, Institut Pasteur de Lille, Lille, France
2 Hammersmith Genome Centre, Imperial College, London, UK
cose Tolerance (IGT) or chronic hyperglycaemia
(Type 2 diabetes, T2D), central obesity, and complex
dyslipidaemia with hypertriglyceridaemia and hypo-
HDL-cholesterolaemia [1, 2]. In addition, a decreased
fibrinolytic capacity has been shown to contribute to
the high prevalence of Coronary Heart Disease (CHD)
in individuals and cohorts with metabolic syndrome
[3, 4], for which CHD has been generally attributed to
increased type 1 plasminogen activator inhibitor (PAI-
1) activity.
PAI-1, a member of the serine protease inhibitor
(SERPIN) family, is a main regulator of the endogenous
fibrinolytic system. It inhibits fibrinolysis activity of the
The metabolic insulin resistance syndrome associates
a cluster of risk factors for Coronary Arterial Disease
(CAD), including hyperinsulinaemia, Impaired Glu-
C. Lopes et al.: PAI-1 polymorphisms modulate phenotypes associated with the metabolic syndrome 1285
tissue-type plasminogen activator, tPA, which produces
active plasmin from plasminogen, that then cleaves fi-
brin [5]. Thus, PAI-1 determines in part fibrinolysis ac-
tivity and modulates the progression of thrombosis [6,
7]. PAI-1 is expressed and secreted in a variety of tis-
sues, including liver, spleen [8] and adipocytes [9]. PAI-
1 synthesis is regulated by various agents, including in-
sulin [10], very-low-density lipoprotein, VLDL [11],
low-density lipoprotein and glucose [12].
The human PAI-1 gene is located at chromosome
7q22 [13] in a region that shows a linkage with lipid
concentrations in Mexican Americans [14, 15]. More-
over, it has been shown that PAI-1 activity correlates
with features of the insulin resistance syndrome [6], in
particular, plasma insulin and triglyceride concentra-
tions in subjects with CHD [16], T2D [17, 18] and
obesity [19, 20]. Interestingly, obese and diabetic
ob/ob mice deprived of the PAI-1 gene showed re-
duced adiposity [21].
In light of these data, PAI-1 is a good candidate
gene and that might contribute to the pathological fea-
tures associated to the metabolic syndrome. To verify
this role of PAI-1, we have screened the promoter and
coding sequence of PAI-1 gene and identified five po-
tentially functional polymorphisms: two known pro-
moter polymorphisms, the −765 4G/5G and the −844
A>G, and three previously unknown non-synonymous
single nucleotide polymorphisms (SNPs). We geno-
typed these five polymorphisms in a case-control co-
hort to determine if an association is present between
PAI-1 genotypes and metabolic parameters linked to
diabetes and obesity. Possible functional effects of
these variants were also analyzed.
Methods
Subjects. A case-control cohort of 1067 unrelated French Cau-
casians was stratified for diabetic status, fulfilling the 1999
WHO criteria for diabetes mellitus (Diabetic/normoglycaemic
subjects 675/391; mean age 63.6±11.6/62.6±10.3 years; mean
BMI 28.0±5.8/32.9±11.0 kg/m2; sex ratio Male to Female
378–297/132–259) and for obesity, defined as BMI greater
than or equal to 30 (Obese/non obese subjects 343/690; mean
age 62.6±9.5/63.7±11.8 years; mean BMI 39.9±6.1/
24.6±2.9 kg/m2; sex ration Male to Female 110–233/381–308).
In this cohort, biochemical variables were measured in the
fasting state. Glycaemia (mmol/l), triglyceridaemia, total- and
HDL-cholesterolaemia (mmol/l) were measured enzymatically
(Roche, Boehringer, Meylan, France) and insulinaemia (mU/l)
was measured immunologically (IRMA, Immunotech, BioRad-
Pasteur IRMA, France). Coronary Heart Disease (CHD), diag-
nosed by physicians, was defined as presence of myocardial
infarction, angina pectoris, bypass or the presence of a patho-
logical Q wave on a current electrocardiogram. Informed con-
sent was given by each participant. The protocol has been ap-
proved by the Hotel Dieu ethical Committee.
Screening and genotyping PAI-1 polymorphisms. DNA was ex-
tracted from EDTA whole-blood samples using the Puregene
kit (Gentre, Minneapolis, Minn., USA).
We scanned 1 kb of the PAI-1 promoter and the entire cod-
ing sequence (1.2 Kb) for SNPs by direct sequencing in 48
randomly selected unrelated Type 2 diabetic French Caucasian
subjects. The protocol was carried out using a 3700 DNA se-
quencer (Applied Biosystems, Foster City, Calif., USA) as pre-
viously described [22].
Identified polymorphisms of the PAI-1 gene were geno-
typed using LightCycler technology (Roche, Manheim,
Germany) based on hybridization probes labelled with fluores-
cent dyes. Polymorphisms were identified by differences of
fluorescence resonance energy transfer (FRET) during melting
curve analysis [23].
Statistical analysis. In order to use parametric methods, skew-
ness of quantitative trait distributions were normalized by loge-
transformation. Chi-square tests were used to analyze devia-
tion of the genotype frequency from the distribution that would
be expected if alleles were in Hardy-Weinberg equilibrium and
to test differences in genotype frequencies between groups.
Log-normalized quantitative variables were compared using
the ANOVA tests and, when positive, with the non parametric
rank, Wilcoxon and Kruskal-Wallis tests. Calculations were
carried out using SSPS 10.0 program (SSPS, Chicago, Ill.,
USA). In each test, a pvalue of less than 0.05 was considered
statistically significant. Haplotype frequencies and standard-
ized linkage disequilibrium (D’) were determined with the
PM+EH+ software.
Results
Genetic screening of the promoter and coding se-
quences of PAI-1 gene in a French Caucasian cohort
resulted in the identification of five variants. Of them,
Ala15Thr, Val17Ile and Asn195Ile, were previously
uncharacterized and are non synonymous polymor-
phisms. The Ala15Thr and Val17Ile are both deter-
mined by substitution of a guanine to adenine in exon
2, and lead to a change of alanine to threonine at posi-
tion 15, and of valine to isoleucine at position 17, re-
spectively. The Asn195Ile, an asparagine to isoleucine
change at position 195, is determined by an adenine to
thymine substitution in exon 4 (Table 1).
The two other variants, both in the promoter, were
the common single-base-pair guanine deletion/inser-
tion, 4G/5G, at −675 [24, 25] and the SNP −844 A>G
[26]. The two known promoter polymorphisms
showed similar frequencies as previously reported in
other Caucasian populations [24, 26].
All the polymorphisms were in Hardy-Weinberg
equilibrium (Table 1), except the −844 A>G variant
which differed significantly (χ2=5.49, p=0.02), as was
previously described [26].
The Val17Ile and Asn195Ile variants (Table 1)
were rare (14 heterozygous subjects for Val17Ile and 1
heterozygous individual for Asn195Ile, among 532
genotyped subjects). Due to their very low frequen-
cies, genetic statistical analyses for these SNPs were
not relevant. The other three, −675 4G/5G, −844 A>G
and Ala15Thr, showed a very high linkage disequilib-
rium,with a standardized linkage disequilibrium (D′)
for each polymorphism pair: −675 4G/5G/−844A>G,
1286 C. Lopes et al.: PAI-1 polymorphisms modulate phenotypes associated with the metabolic syndrome
D′=0.9772; −675 4G/5G/Ala15Thr, D′=0.9997; −844
A>G/Ala15Thr, D′=0.9207.
Genotypic and allelic frequencies of the three vari-
ants did not significantly differ between T2D and non-
diabetic individuals (data not shown).
Chi-square tests with obesity and analysis of vari-
ance of BMI with regard to the −844A>G polymor-
phism showed an increased obesity of non-diabetic ho-
mozygous AA subjects (Obese/Lean AA: 0.36/0.26,
p=0.04; BMI: 34.7±11.4/31.8±10.9, p=0.02). A similar,
although not significant trend was observed for −675
4G/5G polymorphism in non-diabetic subjects homozy-
gous for the 4G allele (4G4G) (Obese/Lean 4G4G:
0.29/0.21, p=0.06; BMI: 34.5±11.0/32.2±11.0, p=0.08).
Given the possible association between PAI-1 and
BMI analysis of variance of glucose and lipid metabo-
lism associated traits were carried out in obese and
lean individuals without therapy (non-diabetic or non-
hyperlipidaemic subjects) (Table 2; Table 3). Differ-
ences were observed in fasting glycaemia and insu-
linaemia in non-diabetic obese individuals for the two
promoter variants (Table 2). Homozygotes for the
most frequent alleles (4G andAalleles for 4G/5G and
−844A>G polymorphisms, respectively) showed high-
ly increased levels of fasting glucose and insulin
(glycaemia: p=0.006 and p=0.0004, for 4G/5G and
−844 A>G polymorphisms, respectively; insulinae-
mia:p=0.05 and p=0.008, for 4G/5G and −844A>G
polymorphisms, respectively), which might suggest an
increased insulin-resistance in these carriers. In con-
trast, the Ala15Thr variant did not show association
with these parameters (Table 2).
To analyze lipid profiles, we selected individuals
without lipid-lowering therapy. A decreased level of
triglycerides (p=0.09, p=0.0027 and p=0.09, for
4G/5G, −844A>G and Ala15Thr polymorphisms, re-
spectively) and an increased level of HDL (p=0.27,
p=0.027 and p=0.01, for 4G/5G, −844A>G and
Ala15Thr polymorphisms, respectively) were ob-
served in lean individuals homozygous for the most
frequent alleles (Table 3), while no significant associ-
ations were observed in obese individuals.
Table 1. Genotypic and allelic frequencies for the five variants
of PAI-1 gene. n=1067 for 4G/5G, −844 A>G and Ala15Thr
variants; n=532 for Val17Ile and Asn195Ile variants. For each
variant, the most frequent allele is indicated first. For the three
non-synonymous polymorphisms, the corresponding codon se-
quences were indicated in brackets. Significance of χ2to test
discordance from the Hardy-Weinberg equilibrium was indi-
cated (HWE)
Polymorphisms Genotypes Alleles HWE
4G5G 4G4G 4G5G 5G5G 4G 5G
0.27 0.52 0.21 0.53 0.47 ns
−844 A>G AA AG GG A G
0.33 0.52 0.15 0.59 0.41 0.02
Ala15Thr (GCC>ACC) GG GA AA G A
0.80 0.19 0.01 0.90 0.10 ns
Val17Ile (GTC>ATC) GG GA AA G A
0.97 0.03 0.00 0.99 0.01 ns
Asn195Ile (AAC>ATC) AA AT TT A T
0.998 0.002 0.00 0.999 0.001 ns
Table 2. Association analysis for glucose metabolism parame-
ters in non-diabetic subjects. Means ± standard deviation of
glycaemia and insulin in non-diabetic obese (Fat, n=173) and
non-obese (Lean, n=141) individuals. ANOVA tests were used
with loge-normalized values. Significant differences were ob-
served between homozygous individuals for more frequent al-
leles of the two promoter variants and individuals carrying the
less frequent variants, following a dominant genetic model
SNP Population Genotype 11 12+22 pvalue
Glycaemia (mmol/l) 4G5G Lean 5.1±0.5 5.12±0.4 0.9
Fat 5.5±0.4 5.3±0.4 0.006
−844 A>G Lean 5.1±0.5 5.12±0.4 0.9
Fat 5.5±0.4 5.2±0.4 0.0004
Ala15Thr Lean 5.1±0.4 5.1±0.5 0.6
Fat 5.3±0.4 5.3±0.5 0.9
Insulin (mU/l) 4G5G Lean 6.7±3.4 8.1±5.4 0.8
Fat 13.5±9 10.8±5.7 0.05
−844 A>G Lean 6.4±3.8 8.2±5.4 0.2
Fat 13.6±8.3 10.5±5.7 0.008
Ala15Thr Lean 7.8±5.1 7.9±5.6 0.8
Fat 11.9±7.2 10.±5.3 0.3
C. Lopes et al.: PAI-1 polymorphisms modulate phenotypes associated with the metabolic syndrome 1287
Of the 675 T2D subjects studied, 229 had a clinical
history of coronary heart disease (CHD). The allelic
frequency for the three polymorphisms differed be-
tween the two groups (Table 4), with and without
CHD: the diabetic group with CHD had a higher fre-
quency of carrier of the most frequent alleles [4G/5G:
p=0.05; -844A>G: OR=1.31 (1.04–1.65), p=0.02;
Ala15Thr: OR=1.48 (1.02–2.16), p=0.04]. Attribut-
able risk for CHD due to the presence of the risk al-
leles for −844A>G and Ala15Thr polymorphisms was
estimated at 15% and 30%, respectively.
Haplotype analyses were carried out for the three
common polymorphisms in regard to diabetic status,
obesity and coronary complications. Differences be-
tween haplotype frequencies in T2D individuals with
or without coronary complications were observed for
−844 A>G and the Ala15Thr polymorphisms (A-Ala:
0.625/0.563; A-Thr: 0.002/0.007; G-Ala: 0.291/0.318;
G-Thr: 0.082/0.112; p=0.029, for CHD/non CHD dia-
betic patients respectively). The CHD at risk haplo-
type was determined by the two most frequent alleles
(haplotype A-Ala).
Phenotypes of the 14 subjects carrying the rare
Val17Ile variant were analyzed and compared to the
remaining population, but no differences were ob-
served (data not shown).
The Asn195Ile variant was identified in only one
diabetic individual. The genotypes of other members
of this family were determined and analyzed for co-
segregation with diabetes and quantitative traits
(Fig. 1). Carriers of the rare allele showed higher con-
centrations of triglycerides compared to the other
members. When expressed in standard deviation
Table 3. Association analysis for lipid metabolism parameters.
Means ± standard deviation of triglyceride and HDL cholester-
ol in non-obese individuals (n=485; 355 diabetic and 130 non-
diabetic subjects). Significant or indicative differences were
observed for triglyceride and HDL cholesterol, between homo-
zygous individuals for more frequent alleles of the variants and
individuals carrying the less frequent variants, following a
dominant genetic model
Trait SNP Genotype 11 12+22 pvalue
Triglyceride (mmol/l) 4G5G 1.29±0.9 1.39±0.9 0.09
−844 A>G 1.24±0.8 1.43±0.9 0.0027
Ala15Thr 1.33±0.8 1.55±1.1 0.09
HDL (mmol/l) 4G5G 1.49±0.4 1.45±0.4 0.27
−844 A>G 1.53±0.5 1.43±0.4 0.027
Ala15Thr 1.48±0.4 1.37±0.4 0.01
Table 4. Association analysis for coronary complication in dia-
betes. Genotype and allelic frequencies in diabetic individuals
with (CHD; n=229) and without (NoCHD; n=406) coronary
complications. Significant or indicative differences were
shown for the three variants
Polymorphisms Genotypes pvalue Alleles pvalue
4G5G 4G4G 4G5G+5G5G 4G 5G
CHD 0.31 0.69 0.56 0.44
NoCHD 0.26 0.75 0.1 0.51 0.49 0.05
−844 A>G AA AG+GG A G OR=1.31 [1.04–1.65]
CHD 0.38 0.62 0.63 0.37
NoCHD 0.32 0.68 0.13 0.57 0.43 0.02
Ala15Thr GG GA+AA G A OR=1.48 [1.02–2.16]
CHD 0.83 0.17 0.91 0.09
NoCHD 0.76 0.24 0.02 0.88 0.12 0.04
Fig. 1. Co-segregation analysis for the N195I polymorphism of
PAI-1. In genealogical tree, diabetic subjects are indicated in
black. Individual 2, pointed by arrow, was included in the dia-
betic cohort. The father was not analyzed, and his phenotypes
are unknown. For each individual (1–6), genotype was indicat-
ed (N or I amino acid), diabetic status (T2D), BMI, fasting glu-
cose (Gly) and insulin (Ins), triglyceride (Tri) and correspond-
ing standardized value, Z score (Ztri) and HDL cholesterols
unites (Z-score), we observed a difference of 0.8 stan-
dard deviation units.
The potential effects of these new non-synonymous
SNPs on PAI-1 protein conformation were predicted
using algorithms available at ProtScale (http://www.
expasy.org/cgi-bin/protscale.pl) (data not shown).
The Ala15Thr and the Val17Ile are located in the
signal peptide consisting of the first 23 amino acids of
PAI-1 [27]. The alanine to threonine change in posi-
tion 15 could result in a lower propensity to form
α-helix [28] and in decreased hydrophobicity [29]. In
contrast, no effects of the Val 17 to Ile 17 change were
observed on these two parameters. The Asn195Ile
SNP is part of a β-sheet structure (184–195 AA) and
the rare allele Ile 195 might result in a higher β-sheet
propensity [28] and in increased hydrophobicity [29].
Discussion
In addition to the two already reported polymorphisms
in the promoter sequence of PAI-1, we have identified
three missense mutations in the coding region. Only
the Ala15Thr missense mutation, located in the signal
peptide, was prevalent enough (10%) to allow associa-
tion studies in our population. This variant and the
two promoter mutations are in high but not in full LD
with each other, they were not associated with T2D.
However, our data indicated that the two promoter
polymorphisms were associated with obesity and
could modulate BMI in non-diabetic subjects. More-
over, presence of the most frequent alleles of these
polymorphisms is associated with a more severe insu-
lin-resistant profile in obese subjects, who have higher
fasting glucose, insulin and triglyceride, and lower
HDL cholesterol than non-carrier obese subjects.
Moreover, the most frequent alleles for PAI-1 poly-
morphisms also conferred a higher risk for CHD in di-
abetic individuals, a population known to be already
at risk for premature atherosclerosis.
Unexpectedly, in healthy non-obese middle-aged
subjects, the PAI-1 most frequent alleles seemed to be
associated with lower triglyceride and higher HDL
cholesterol concentrations, suggesting that these al-
leles might be “protective” for metabolic cardiovascu-
lar risk in the absence of fat excess. A similar dual ef-
fect was already reported for the angiotensin convert-
ing enzyme gene (ACE), for which the deletion (D)
allele was more frequently found in subjects with
myocardial infarction [30, 31], but was also more
prevalent in centenarians [32] and in elite athletes in
power-oriented performances [33, 34]. Given the role
of PAI-1 in the control of fibrinolysis, it could be an
advantage to have relatively increased PAI-1 levels at
a certain age, if not associated with potent metabolic
disturbances.
We have studied three polymorphisms for associa-
tion with three-disease status (T2D, Obesity and
CHD) and five quantitative traits (BMI, fasting glu-
cose, insulin, triglycerides and HDL cholesterol). Us-
ing Bonferroni correction, a global pvalue of 0.05,
will be represented by a pvalue of at least 0.0021 in
one single test. Under this condition, only the associa-
tion between the −844 A>G polymorphism and the
fasting glycaemia in obese non-diabetic subjects was
significant. However, there was a strong LD between
the 3 PAI-1 polymorphisms and a strong correlation
between fasting glucose and diabetes (RSpearman=
−0.74, p<0.0001) and between BMI and obesity
(RSpearman=−0.82, p<0.0001), and between triglyce-
rides and HDL cholesterol (RPearson=−0.39, p<0.0001).
Therefore, a less conservative correction could be ap-
plied (one variant block and five phenotypes, uncor-
rected p=0.01). Accordingly, we have associations be-
tween the two promoter variants and higher levels of
fasting glucose and between the −844 A>G SNP and
higher levels of insulin, in obese non-diabetic sub-
jects, and between this SNP and lower levels of tri-
glycerides in lean untreated subjects. Nevertheless, fu-
ture studies are needed to replicate our results in other
populations.
Previous studies suggested an association between
PAI-1 −844 A>G and the 4G/5G polymorphisms and
vascular complications in non-insulin-dependent dia-
betic individuals [35, 36]. Moreover, fasting insulin
levels, triglycerides and BMI have also been associat-
ed with the 4G/5G in subjects with hyperlipidaemia
and premature coronary disease or non-insulin-depen-
dent diabetes in Caucasians [24, 37, 38], but not in all
tested populations [39, 40]. Recently, an association
was found for the 4G/5G variant with obesity in a
Scandinavian population [41], but these results were
not further confirmed [42, 43]. These findings togeth-
er with our data illustrate the difficulty to analyze gen-
otype-phenotype correlation in complex traits where-
by environmental factors strongly modulate the possi-
ble effects of gene polymorphisms on human diseases.
Importantly, the −675 4G/5G has been found to
correlate with higher plasma PAI-1 activity in patients
with myocardial infarction [24, 25] and in subjects
with deep vein thrombosis [26]. In contrast, another
study failed to show any effect of the −844A>G SNP
on plasma PAI-1 level [26]. Expression studies
showed that the −675 4G/5G polymorphism affects
the binding of nuclear proteins regulating PAI-1 tran-
scription [24, 25]. Although both alleles bind a tran-
scriptional activator, the 5G allele also binds a repres-
sor protein to an overlapping binding site, increasing
the basal level of PAI-1 gene transcription [24, 25].
Furthermore, the −844 SNP that is part of an Ets nu-
clear protein consensus sequence binding site could
also be implicated in the regulation of the PAI-1 gene
[26, 42].
The other three identified SNPs are responsible for
amino acid changes and could interfere with PAI-1 ac-
tivity. In particular, the Ala15Thr SNP, which is asso-
1288 C. Lopes et al.: PAI-1 polymorphisms modulate phenotypes associated with the metabolic syndrome
5. Kruithof EK, Tran-Thang C, Ransijn A, Bachmann F
(1984) Demonstration of a fast-acting inhibitor of plasmi-
nogen activators in human plasma. Blood 64:907–913
6. Juhan-Vague I, Alessi MC (1997) PAI-1, obesity, insulin
resistance and risk of cardiovascular events. Thromb
Haemost 78:656–660
7. Alessi MC, Peiretti F, Morange P, Henry M, Nalbone G,
Juhan-Vague I (1997) Production of plasminogen activator
inhibitor 1 by human adipose tissue: Possible link between
visceral fat accumulation and vascular disease. Diabetes
46:860–867
8. Loskutoff DJ, Samad F (1998) The adipocyte and hemo-
static balance in obesity: Studies of PAI-1. Arterioscler
Thromb Vasc Biol 18:1–6
9. Shimomura I, Funahashi T, Takahashi M et al. (1996) En-
hanced expression of PAI-1 in visceral fat: Possible con-
tributor to vascular disease in obesity. Nat Med 2:800–803
10. Alessi MC, Juhan-Vague I, Kooistra T, Declerck PJ, Collen
D (1988) Insulin stimulates the synthesis of plasminogen
activator inhibitor 1 by the human hepatocellular cell line
Hep G2. Thromb Haemost 60:491–494
11. Stiko-Rahm A, Wiman B, Hamsten A, Nilsson J (1990) Se-
cretion of plasminogen activator inhibitor-1 from cultured
human umbilical vein endothelial cells is induced by very
low density lipoprotein. Arteriosclerosis 10:1067–1073
12. Nordt TK, Klassen KJ, Schneider DJ, Sobel BE (1993)
Augmentation of synthesis of plasminogen activator inhibi-
tor type-1 in arterial endothelial cells by glucose and its
implications for local fibrinolysis. Arterioscler Thromb
13:1822–1828
13. Klinger KW, Winqvist R, Riccio A et al. (1987) Plasmino-
gen activator inhibitor type 1 gene is located at region
q21.3-q22 of chromosome 7 and genetically linked with
cystic fibrosis. Proc Natl Acad Sci USA 84:8548–8552
14. Arya R, Blangero J, Williams K et al. (2002) Factors of in-
sulin resistance syndrome-related phenotypes are linked to
genetic locations on chromosomes 6 and 7 in nondiabetic
mexican-americans. Diabetes 51:841–847
15. Duggirala R, Blangero J, Almasy L et al. (2000) A major
susceptibility locus influencing plasma triglyceride concen-
trations is located on chromosome 15q in mexican ameri-
cans. Am J Hum Genet 66:1237–1245
16. Landin K, Tengborn L, Smith U (1990) Elevated fibrinogen
and plasminogen activator inhibitor (PAI-1) in hypertension
are related to metabolic risk factors for cardiovascular dis-
ease. J Intern Med 227:273–278
17. Juhan-Vague I, Roul C, Alessi MC, Ardissone JP, Heim M,
Vague P (1989) Increased plasminogen activator inhibitor
activity in non insulin dependent diabetic patients-relation-
ship with plasma insulin. Thromb Haemost 61:370–373
18. Potter van Loon BJ, Kluft C, Radder JK, Blankenstein MA,
Meinders AE (1993) The cardiovascular risk factor plasmi-
nogen activator inhibitor type 1 is related to insulin resis-
tance. Metabolism 42:945–949
19. Vague P, Juhan-Vague I, Aillaud MF et al. (1986) Correlation
between blood fibrinolytic activity, plasminogen activator in-
hibitor level, plasma insulin level, and relative body weight in
normal and obese subjects. Metabolism 35:250–253
20. Landin K, Stigendal L, Eriksson E et al. (1990) Abdominal
obesity is associated with an impaired fibrinolytic activity
and elevated plasminogen activator inhibitor-1. Metabo-
lism 39:1044–1048
21. Schafer K, Fujisawa K, Konstantinides S, Loskutoff DJ
(2001) Disruption of the plasminogen activator inhibitor 1
gene reduces the adiposity and improves the metabolic pro-
file of genetically obese and diabetic ob/ob mice. FASEB J
15:1840–1842
C. Lopes et al.: PAI-1 polymorphisms modulate phenotypes associated with the metabolic syndrome 1289
ciated with CHD in diabetic individuals, is located in
the central hydrophobic core of the PAI-1 signal pep-
tide. The most frequent risk allele of this SNP increas-
es hydrophobicity and α-helix propensity, indicating
that it could stabilize the α-helix conformation of the
signal peptide. These two properties are known to be
important in signal peptide function and therefore the
mutations might modulate the secreted PAI-1 level. In
this regard, a similar amino acid change has been
shown to be a functional mutation, interfering with the
translocation to the membrane of the thyrotropin re-
ceptor [44].
Interestingly, we also identified a PAI-1 non-synon-
ymous SNP, Asn195Ile that seemed to co-segregate
with a higher level of triglyceride in a single pedigree.
This SNP, included in a β-sheet structure, caused al-
teration of hydrophobicity and β-sheet propensity,
where the rare allele could promote a more stable con-
formation.
Our results support the hypothesis that PAI-1 poly-
morphisms contribute to increased body fat in non-
diabetic subjects. PAI-1 SNPs probably interact with
known environmental risk factors (chronic hyperglyca-
emia, obesity, etc.) to induce a more severe insulin-re-
sistant metabolic profile in overweight subjects, and to
further increase the risk for CHD in diabetic subjects.
It is not yet clear whether the association between
PAI-1 activity and overweight- and obesity-associated
metabolic traits is directly due to PAI-1 action or it is
only a consequence of the increased body fat that se-
cretes excess PAI-1. A reduced adiposity has been ob-
served in obese and diabetic ob/ob mice that are also
deprived of the PAI-1 gene [21], suggesting a primi-
tive effect of PAI-1 on adiposity. Our results are in
agreement with this hypothesis, as potential functional
PAI-1 SNPs modulate obesity-associated phenotypes
in human.
Acknowledgements. This work was supported by a grant from
E. Lilly though Lilly Consortium for Diabetes and Obesity (M.
McCarty, P. Froguel, R. Leibel, M. Lathrop, J. Caro and E.
Ravussin). We thank B. Neve and M. Rachidi for critical read-
ing of the manuscript.
References
1. Reaven GM (1988) Role of insulin resistance in human
disease. Diabetes 37:1595–1607
2. DeFronzo RA, Ferrannini E (1991) Insulin resistance. A
multifaceted syndrome responsible for niddm, obesity, hy-
pertension, dyslipidemia, and atherosclerotic cardiovascu-
lar disease. Diabetes Care 14:173–194
3. Juhan-Vague I, Thompson SG, Jespersen J (1993) Involve-
ment of the hemostatic system in the insulin resistance syn-
drome. A study of 1500 patients with angina pectoris. The
ECAT angina pectoris study group. Arterioscler Thromb
13:1865–1873
4. Hamsten A, Faire U de, Walldius G et al. (1987) Plasmino-
gen activator inhibitor in plasma: Risk factor for recurrent
myocardial infarction. Lancet 2:3–9
35. Mansfield MW, Stickland MH, Grant PJ (1995) Plasmino-
gen activator inhibitor-1 (PAI-1) promoter polymorphism
and coronary artery disease in non-insulin-dependent
diabetes. Thromb Haemost 74:1032–1034
36. Nagi DK, McCormack LJ, Mohamed-Ali V, Yudkin JS,
Knowler WC, Grant PJ (1997) Diabetic retinopathy, pro-
moter (4 g/5 g) polymorphism of PAI-1 gene, and PAI-1
activity in Pima indians with type 2 diabetes. Diabetes
Care 20:1304–1309
37. Panahloo A, Mohamed-Ali V, Lane A, Green F, Humphries
SE, Yudkin JS (1995) Determinants of plasminogen activa-
tor inhibitor 1 activity in treated NIDDM and its relation to
a polymorphism in the plasminogen activator inhibitor 1
gene. Diabetes 44:37–42
38. Mansfield MW, Stickland MH, Grant PJ (1995) Environ-
mental and genetic factors in relation to elevated circulat-
ing levels of plasminogen activator inhibitor-1 in caucasian
patients with non-insulin-dependent diabetes mellitus.
Thromb Haemost 74:842–847
39. McCormack LJ, Nagi DK, Stickland MH et al. (1996) Pro-
moter (4 g/5 g) plasminogen activator inhibitor-1 genotype
in Pima indians: Relationship to plasminogen activator in-
hibitor-1 levels and features of the insulin resistance syn-
drome. Diabetologia 39:1512–1518
40. Viitanen L, Pihlajamaki J, Halonen P et al. (2001) Associa-
tion of angiotensin converting enzyme and plasminogen ac-
tivator inhibitor-1 promoter gene polymorphisms with fea-
tures of the insulin resistance syndrome in patients with pre-
mature coronary heart disease. Atherosclerosis 157:57–
64
41. Hoffstedt J, Andersson IL, Persson L, Isaksson B, Arner P
(2002) The common −675 4 g/5 g polymorphism in the
plasminogen activator inhibitor −1 gene is strongly associ-
ated with obesity. Diabetologia 45:584–587
42. Henry M, Chomiki N, Scarabin PY et al. (1997) Five
frequent polymorphisms of the PAI-1 gene: Lack of associ-
ation between genotypes, PAI activity, and triglyceride lev-
els in a healthy population. Arterioscler Thromb Vasc Biol
17:851–858
43. Freeman MS, Mansfield MW (2002) Comment to: J. Hoff-
stedt et al. (2002) the common −675 4 g/5 g polymorphism
in the plasminogen activator inhbitor-1 gene is strongly as-
sociated with obesity. Diabetologia 45:1602–1603
44. Abramowicz MJ, Duprez L, Parma J, Vassart G, Heinrichs C
(1997) Familial congenital hypothyroidism due to inactivat-
ing mutation of the thyrotropin receptor causing profound
hypoplasia of the thyroid gland. J Clin Invest 99:3018–
3024
1290 C. Lopes et al.: PAI-1 polymorphisms modulate phenotypes associated with the metabolic syndrome
22. Boutin P, Wahl C, Samson C, Vasseur F, Laget F, Froguel P
(2000) Big dye terminator cycle sequencing chemistry: accu-
racy of the dilution process and application for screening mu-
tations in the TCF1 and GCK genes. Hum Mutat 15:201–203
23. Blomeke B, Sieben S, Spotter D, Landt O, Merk HF (1999)
Identification of N-acetyltransferase 2 genotypes by con-
tinuous monitoring of fluorogenic hybridization probes.
Anal Biochem 275:93–97
24. Dawson SJ, Wiman B, Hamsten A, Green F, Humphries S,
Henney AM (1993) The two allele sequences of a common
polymorphism in the promoter of the plasminogen activa-
tor inhibitor-1 (PAI-1) gene respond differently to interleu-
kin-1 in HepG2 cells. J Biol Chem 268:10739–10745
25. Eriksson P, Kallin B, van ‘t Hooft FM, Bavenholm P,
Hamsten A (1995) Allele-specific increase in basal tran-
scription of the plasminogen- activator inhibitor 1 gene is
associated with myocardial infarction. Proc Natl Acad Sci
USA 92:1851–1855
26. Grubic N, Stegnar M, Peternel P, Kaider A, Binder BR
(1996) A novel g/a and the 4 g/5 g polymorphism within the
promoter of the plasminogen activator inhibitor-1 gene in
patients with deep vein thrombosis. Thromb Res 84:431–443
27. Pannekoek H, Veerman H, Lambers Het al. (1986) Endo-
thelial plasminogen activator inhibitor (PAI): a new mem-
ber of the serpin gene family. EMBO J 5:2539–2544
28. Deleage G, Roux B (1987) An algorithm for protein
secondary structure prediction based on class prediction.
Protein Eng 1:289–294
29. Kyte J, Doolittle RF (1982) A simple method for display-
ing the hydropathic character of a protein. J Mol Biol
157:105–132
30. Cambien F, Poirier O, Lecerf L et al. (1992) Deletion poly-
morphism in the gene for angiotensin-converting enzyme is
a potent risk factor for myocardial infarction. Nature
359:641–644
31. Tiret L, Kee F, Poirier O et al. (1993) Deletion polymor-
phism in angiotensin-converting enzyme gene associated
with parental history of myocardial infarction. Lancet
341:991–992
32. Schachter F, Faure-Delanef L, Guenot F et al. (1994) Ge-
netic associations with human longevity at the APOE and
ACE loci. Nat Genet 6:29–32
33. Woods D, Hickman M, Jamshidi Y et al. (2001) Elite
swimmers and the D allele of the ACE I/D polymorphism.
Hum Genet 108:230–232
34. Nazarov IB, Woods DR, Montgomery HE et al. (2001) The
angiotensin converting enzyme I/D polymorphism in rus-
sian athletes. Eur J Hum Genet 9:797–801
