Vol.3, No.4, 214-220 (2013) Journal of Diabetes Mellitus
http://dx.doi.org/10.4236/jdm.2013.34033
Insulin receptor substrate gene polymorphisms are
associated with metabolic syndrome but not with its
components
Fulden Sarac1*, Afig Berdeli2, Sefa Sarac3, Sumru Savas1, Merve Atan2, Fehmi Akcicek1
1Department of Geriatrics Medicine, Medical Faculty, Ege University, Izmir, Turkey;
*Corresponding Author:
2Department of Moleculer Medicine Laboratory, Medical Faculty, Ege University, Izmir, Turkey
3Department of Cardiology, Atatürk Training and Research Hospital, Izmir, Turkey
Received 9 October 2013; revised 3 November 2013; accepted 10 November 2013
Copyright © 2013 Fulden Sarac et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Aim: Metabolic syndrome (MetS) is a major risk
factor for both diabetes mellitus and cardio-
vascular disease (CVD). The aims of the study
were 1) to investigate the insulin receptor sub-
strate-1 (IRS-1) and insulin receptor substrate-2
(IRS-2) gene polymorphisms in patients with
MetS and 2) to examine the relationships be-
tween gene polymorphisms and components of
MetS. Patients & Methods: The study popula-
tion included 100 patients with MetS and 30 pa-
tients without MetS as control group. Metabolic
syndrome (MS) was defined as in ATP III. Entire
coding exons of IRS-1 and IRS-2 genes were
amplified by polymerase chain reaction (PCR).
Insulin resistance (IR) was estimated using the
homeostasis model assessment (HOMA). Re-
sult s: In p atients with MetS, 34 (34%), had G972R
(rs1801278) gene polymorphism and 66 (66%)
had no nucleotide substitutions at the IRS-1
gene (p < 0.0001). As for the IRS-2 gene, 18.0%
of the patients were heterozygous and 11.0%
were homozygous for the G1057D mutation,
2.0% were heterozygous for the P1031P and
P1033PG1057 mutations, 17.0% were heterozy-
gous for P1033P, 3.0% were homozygous for
P1033P and 5% were heterozygous for the G
1067D and P1033P mutations in patients with
MetS (p = 0.0001). However, none of the control
subject s had nucleotide substitution s in the IRS-
1 and IRS-2 genes. There were no correlations
between IRS-1/IRS-2 gene polymorphisms and
metabolic syndrome components such as waist
circum ference, blood pressure, triglyceride, HDL-
Cholesterol, LDL-Cholesterol and HOMA-IR lev-
els. Conclusion: Insu lin receptor substrate-1 and
2 gene polymorphisms were associated with
met abolic syndrome but not it s components.
Keyw ords: Metabolic Syndrome; Insulin Receptor
Substrat-1 Gene; Insulin Receptor Subs trat-2 Gene
1. INTRODUCTION
Metabolic syndrome (MetS) is one of the fastest
growing health problems worldwide. It is a major risk
factor for both diabetes mellitus [1] and cardiovascular
disease (CVD) [2]. The etiology is complex, determined
by the interplay of both genetic and environmental fac-
tors [3]. It is characterized by the clustering of multiple
metabolic abnormalities, including abdominal obesity,
hypertension, dyslipidemia, insulin resistance, and im-
paired glucose tolerance. Candidate gene studies have
identified linkage between MetS and a number of genes,
such as PPARgamma, adiponectin, CD36, and beta ad-
renergic receptors [2]. Recently, De la Cruz-Mosso et al.
[4] suggested that the -844 G/A PAI-1 polymorphism
was related with the risk of developing metabolic syn-
drome, obesity and atherogenic dyslipidemia, and the
HindIII C/G PAI-1 polymorphism was associated with
the increase of total cholesterol levels in Mexican chil-
dren. In addition to these genes, in a previous IRS pro-
teins [5-8] have been identified, but only IRS-1 and
IRS-2 are thought to participate in the regulation of glu-
cose homeostasis [9-11]. In a previous study, Baroni et al.
[12] demonstrated the association of the G972R variant
of the IRS-1 gene with reduced insulin sensitivity in
obese subjects, and indicate a possible interaction be-
tween the IRS-1 variant and obesity in worsening of in-

F. Sarac et al. / Journal of Diabetes Mellitus 3 (2013) 214-220 215
sulin sensitivity. Likewise, Arg972 polymorphism in
IRS-1 was observed in 15.8% of the type 2 diabetic pa-
tients and 12.9% of controls in a Turkish population [13].
In addition to the IRS-1 gene, a number of polymer-
phisms have been identified in the IRS-2 gene, the most
common of which is represented by Gly1057Asp sub-
stitution [14]. And also, the G972R polymorphism of the
IRS-1 gene was associated with insulin resistance, salt
sensitivity and non-dipper hypertension [15]. However,
not many studies exist concerned with the relationships
between IRS-1 or IRS-2 gene polymorphisms and com-
ponents of MetS. Therefore, the aims of the study were
1) to investigate the insulin receptor substrate-1 (IRS-1)
and insulin receptor substrate-2 (IRS-2) gene polymor-
phisms in patients with MetS and 2) to examine the
relationships between gene polymorphisms and com-
ponents of MetS.
2. METHODS
2.1. Study Population
The study population included 100 patients with
metabolic syndrome (MetS) (71 females, 29 males, mean
age 50.1 ± 8.8 yrs, BMI 34.4 ± 5.8 kg/m2). Thirty one
subjects without MetS (5 males, 26 females, mean age
49.4 ± 6.4 yrs, BMI 33.5 ± 4.8 kg/m2) were enrolled in
the study as the control group. The local ethics commit-
tee approved the study and all subjects gave informed
consent. Metabolic syndrome (MS) was defined as in
ATP III [16].
Anthropometric measurements were made in all sub-
jects. A two point bioelectrical impedance apparatus
calibrated for adults (Tanita TBF 300, TANITA Corp.)
was used to measure the percentage body fat (%BF) and
fat mass.
Habitual alcohol consumption and smoking were in-
quired with the following two questions: “Do you drink
alcohol at least once a month? Yes/No”.
“Do you smoke? Yes/No.” If Yes, smokers were classi-
fied into two categories as ex-smokers and non-smokers
[17].
2.2. Molecular Analysis
2.2.1. IRS1 Gene Polymorphism Genotyping
Two mililiter whole blood samples were collected into
EDTA-anticoagulated tubes by the standard vein punc-
ture method. Genomic DNA was extracted from EDTA-
anticoagulated whole blood samples employing the
QIAmp Blood DNA mini-kit (Qiagen, Hilden, Germany)
following manufacturer’s instructions. DNA concentra-
tion was determined by the Nano Drop digital spectros-
copy according to the manufacturer’s instructions and
diluted to 100 ng/µl.
2.2.2. Polymerase C hain Reaction (PCR) and
Enzyme Digest
IRS1 gene polymorphism was genotyped be the
method of Baroni et al. [18] who had designed primers
spanning a region of 198 bp using the following primers:
forward 5’-GCTTTCCACAGCTCACCTTC-3’ and re-
verse 5’-GGTAGGCCTGCAAATGCTA-3’.
PCR conditions: Amplification was carried out on a
GeneAmp PCR System 9700 ( PE Applied Biosystems,
Foster City, CA, USA) in a 25 µl reaction mixture in 0.2
ml thin-wall PCR strip tubes (Axygen Scientific, Inc.,
CA, USA) containing 1 µl genomic DNA solution,
Platinium Enhancer Buffer, 2.0 mmol MgCl2, 50 µmol/l
each of the dGTP, dATp, dTTP and dCTP (Promega, Ma-
dison, WI, USA), 5 pmol each forward and reverse
primers and 1.0 U Platinium Taq polymerase (Invitrogen,
Carlsbad, UK). The cycling conditions comprised a hot
start at 95˚C for 10 min, followed by 35 amplification
cycles at 95˚C for 30 s, 55˚C for 30 s, and 72˚C for 25 s,
followed by one elongation step at 72˚C for 5 min.
Digestion conditions: The amplified IRS-1 products
were digested by SmaI (5.0 units) in a total volume of 20
µl containing NEBuffer 4 (New England Biolabs, Bev-
erly, Ma, USA) for at least 2 h at 25˚C. The IRS-1 frag-
ments (wild-type GG-171 and 27 bp; heterozygous GA-
198, 171 and 27 bp and homozygous AA-198 bp) were
run on a 2% agarose gel containing Etidium Bromide and
visualized under ultraviolet illumination.
2.2.3. IRS2 Gene Polymorphism Genotyping
Two mililiter whole blood samples were collected into
EDTA-anticoagulated tubes by the standard vein punc-
ture method. Genomic DNA was extracted from EDTA-
anticoagulated whole blood samples employing the
QIAmp Blood DNA mini-kit (Qiagen, Hilden, Germany)
following manufacturer’s instructions. DNA concentra-
tion was determined by the NanoDrop digital spectros-
copy according to the manufacturer’s instructions and
diluted to100 ng/µl.
2.2.4. Polymerase Chain Reaction and Enzyme
Digest
IRS2 gene polymorphism was genotyped by the me-
thod of Lautier et al. Who had designed primers span-
ning a region of 198 bp using the following primers:
forward 5’-TCCTTGGACGGCCTCCTGT-3’ and 5’-AA
GGCCTCGACTCCCGACA-3’ [18].
2.2.5. Reverse Primers
PCR conditions: Amplification was carried out on a
GeneAmp PCR System 9700 (PE Applied Biosystems,
Foster City, Ca, USA) in a 25 µl reaction mixture in 0.2
ml thin-wall PCR strip tubes (Axygen Scientific Inc., CA,

F. Sarac et al. / Journal of Diabetes Mellitus 3 (2013) 214-220
216
2.3. Biochemical Analysis
USA) containing 1 µl genomic DNA solution, Platinium
Enhancer Buffer, 2.5 mmol MgCl2, 50 µmol/l each of the
dGTP, dATp, dTTP and dCTP (Promega, Madison,WI,
USA), 5 pmol each forward and reverse primers and 1.0
U Platinium Taq polymerase (Invitrogen, Carlsbad, UK).
The cycling conditions comprised a hot start at 95˚C for
10 min, followed by 35 amplification cycles at 95˚C for
30 s, 58˚C for 30 s, and 72˚C for 25 s, followed by one
elongation step at 72˚C for 5 min.
Serum concentrations of glucose, triglyceride, total
and HDL-Cholesterol were determined by enzymatic
procedures. Serum insulin was measured by chemilum-
minance.
Insulin resistance (IR) was estimated using the homeo-
stasis model assessment (HOMA) from fasting glucose
and insulin concentrations using the following formula
[19].








fasting plasma insulinμUmlfasting plasmaglucosemmoll
22.5
HOMA IR 
2.4. Statistical Analysis of patients without MetS (p = 0.001, p = 0.01, p = 0.001,
p = 0.002, respectively).
Statistical analysis was performed using the SPSS for
Windows (13.0) software package. Numerical variables
were expressed as mean ± standard deviation. The
groups were compared using the Mann Whitney test. The
relationships between metabolic syndrome risk and gene
polymorphisms were evaluated using chi-square or
Fisher’s Exact test. Also, numerical demographic and
biochemical parameters were evaluated using Mann
Whitney test according to the gene polymorphisms in
groups.
Among patients with MetS, 45.0% were smokers and
22.0% informed alcohol consumption. However, 9.0% of
control subjects were smokers and only 16.1% of them
consumed alcohol (p = 0.03, p = 0.02).
Of 100 patients with MetS, 15 (15%) had a history of
cardiovascular disease, 49 (49%) had dyslipidemia, 40
(40%) had hypertension and 15 (15%) had type 2 diabe-
tes mellitus. In the control group, 2 (6.4%) had a family
history of cardiovascular disease, 7 (22.5%) had dyslipi-
demia, 10 (32.2%) had hypertension and 1 (3.2%) had
type 2 diabetes mellitus (Table 2).
3. RESULTS In patients with MetS, 34 (34%) patients had G972R
(rs1801278) gene polymorphism and 66 (66%) had no
nucleotide substitution in the IRS-1 gene (p < 0.0001).
As to the IRS-2 gene, 44 (44%) had no nucletide substi-
tution, 18 (18%) had G1057D (rs1805097) heterozygous,
11 (11.0%) had G1057D homozygous, 2 (2%) had
P1031P heterozygous/P1033PG1057 heterozygous, 17
(17.0%) had P1033P heterozygous, 3 (3.0%) had P1033P
homozygous and 5 (5%) had P1033P heterozygous/
G1067D heterzoygous polymorphisms in MetS (p =
0.0001). However, none of control subjects had nucleo-
tide substitutions in the IRS-1 and IRS-2 genes.
The characteristics and clinical findings of the study
and control groups are shown in Table 1. Patients with
MetS had higher mean values of waist and hip circum-
ference, systolic and diastolic blood pressures than those
Table 1. Demographic characteristics of patients with meta-
bolic syndrome and control were shown.
Parameters Metabolic syndrome
(n = 100)
Control
(n = 31) p
Age (years) 50.1 ± 8.8 49.4 ± 6.40.63
Smokers, no (%) 45.0% 9.0% 0.03*
Smoking pocket/year 20.1 ± 7.5 10.1 ± 2.80.01*
Alcohol consumption, no (%) 22.0% 16.1% 0.02*
Body weight (kg) 92.0 ± 17.6 91.0 ± 8.50.26
Body Mass Index (kg/m2) 34.4 ± 5.8 33.5 ± 4.80.83
Fat mass (kg) 40.0 ± 7.6 39.5 ± 5.10.70
% Fat 42.6 ± 11.1 40.7 ± 8.80.95
Waist circumference (cm) 105.6 ± 18.2 95.2 ± 15.50.001*
Hip circumference (cm) 112.0 ± 9.8 101.1 ± 9.00.01*
Systolic Blood Pressure (mmHg) 120.0 ± 14.0 110.0 ± 17.10.001*
Diastolic Blood Pressure (mmHg) 69.9 ± 15.3 62.1 ± 14.80.002*
There were statistically significantly differences for
plasma levels of fasting insulin (p = 0.001), triglyceride
(p = 0.001), LDL-Cholesterol (p = 0.001), and HOMA-
IR (p = 0.01), between the study and control groups
(Table 3).
Demographic and biochemical parameters according
to gene polymorphisms were shown in patients with
MetS (Table 4). There were no associations between
IRS-1 and IRS-2 gene polymorphisms and blood pres-
sures, waist circumferences, fasting glucose, triglyceride
and HDL-Cholesterol levels in patients with MetS (p >
0.05).
4. DISCUSSION
*Data are expressed as mean ± SD. Metabolic syndrome (MetS) is a consequence of mul-

F. Sarac et al. / Journal of Diabetes Mellitus 3 (2013) 214-220 217
Table 2. Genetic variants of IRS-1 and IRS-2 genes were shown in women with and without metabolic syndrome.
Metabolic syndrome (n = 100) Control (n = 31) p values
No nucleotide substitution 66 (66%) 31 (100%)
IRS-1
G972R 34 (34%) 0 (0%)
p < 0.0001
No nucleotide substitution 44 (44%) 31 (100%)
G1057D heterozygous 18 (18%) 0 (0%)
G1057D homozyous 11 (11%) 0 (0%)
P1031P heterozygous/P1033PG1057 heterozygous2 (2%) 0 (0%)
P 1033 P heterozygous 17 (17%) 0 (0%)
P 1033 P homozygous 3 (3%) 0 (0%)
IRS-2
P 1033P heterozygous/G 1057D heterozygous 5 (5%) 0 (0%)
p < 0.0001
Table 3. Blood profiles of patients with metabolic syndrome
and control were shown.
Metabolic
syndrome
(n = 100)
Control
(n = 31) p
Triglyceride (mg/dl) 177.6 ± 10.8 133.6 ± 27.30.001*
Total-Cholesterol (mg/dl) 209.5 ± 45.0 199.0 ± 29.00.85
LDL-Cholesterol (mg/dl) 148.1 ± 35.6 101.3 ± 122.30.001*
HDL-Cholesterol (mg/dl) 45.3 ± 7.2 45.8 ± 5.3 0.37
Fasting glucose (mg/dl) 90.6 ± 11.0 89.5 ± 8.4 0.17
Fasting insulin (μU/ml) 9.9 ± 3.7 7.1 ± 0.7 0.001*
HOMA-IR 2.2 ± 0.89 1.7 ± 0.77 0.01*
*Data are expressed as mean ± SD. HOMA-IR = Homeostasis model as-
sessment-insulin resistance; LDL = Low-density lipoprotein; HDL = High-
density lipoprotein.
tiple gene-environment interactions. Several potential
candidate genes have been suggested according to their
biological relevance, such as genes in systems of energy
balance, nutrient partitioning, lipid and insulin metabo-
lism, lipolysis, thermogenesis, fuel oxidation and glucose
uptake in skeletal muscle. Many of these genes have
been associated with MetS in various ethnic populations
[2]. Although many different genes have been proposed
as diabetogenes, this study focused on the IRS-1 and
IRS-2 genes. These are the major substrates participating
in insulin action in skeletal muscle. Defects in IRS-1 or
IRS-2 which are major substrates tiyrosine phosphoryla-
tion characterizes insulin resistance associated with dia-
betes [20]. To our knowledge, insulin resistance is a key
player in the pathophysiology of the MetS and has even
been postulated as its underlying cause [21]. In general,
the stigmata of the MetS are significantly associated with
insulin resistance [22]. In addition, certain genetic vari-
ants have been observed to increase or decrease the risk
of developing the entire syndrome [23]. The present
study showed that there was statistically significant asso-
ciations of IRS-1 and IRS-2 gene polymorphisms with
MetS. However, no relationships between gene poly-
morphisms and components of MetS were found.
Several polymorphisms of the IRS-1 gene have been
identified and studied with regard to their influence on
insulin action [24]. Ura et al. [25] reported that three
genetic variants of IRS-1 exist (Pro170Arg; Met209Thr;
Ser809Phe) in Japanese patients with NIDDM. In an-
other study [12], it was reported that Ala512Pro,
Ser892Gly in IRS-1 gene were rare and Gly972Arg in
IRS-1 gene was more common in Turkish population but
might not be a major determinant of genetic susceptility
to type 2 diabetes. However, Ranjith et al. [26] reported
that their findings did not support a role for any of the
polymorphic variant alleles such as IRS-I G972R, PPAR-
gamma P12A, KCNJ11 E23K, and TNF-alpha -308G/A
genes examined in the etiology of insulin resistance re-
inforce the notion of a multifactorial etiology for the
MetS. In the present study, we found 34.0% G972R gene
polymorphism in IRS-1 gene in MetS. However, none of
the controls had nucleotide substitution in IRS-1 gene.
Thus, we can conclude that G972R in IRS-1 gene is
associated with metabolic syndrome.
Insulin receptor substrate-2, like IRS-1, is thought to
be involved in insulin signaling and glucose intolerance
[27-29]. In humans, a number of polymorphisms have
been identified in the IRS2 gene, including Gly1057Asp
variant that occured with an allelic frequency of 34%
[30]. In another study, Attaoua et al. [31] suggested an
independent association of IRS-2 variant with insulin
resistance. In the present study, at the IRS-2 gene 18.0%
of the patients were heterozygous and 11.0% were ho-
mozygous for the G1057D mutation, 2.0% were het-
erozygous for the P1031P and P1033PG1057 mutations,
17.0% were heterozygous for P1033P, 3.0% were ho-
mozygous for P1033P and 5% were heterozygous for the

F. Sarac et al. / Journal of Diabetes Mellitus 3 (2013) 214-220
218
Tabl e 4. Demographic and biochemical parameters were shown according to insulin receptor substrat-1 (IRS-1) and insulin receptor
substrat-2 (IRS-2) genes in patients with metabolic syndrome.
IRS-1
polymorphism (−)
(n = 66)
IRS-1
polymorphism (+)
(n = 34)
p IRS-2 polymorphism (−)
(n = 42)
IRS-2
polymorphism (+)
(n = 58)
p
Age 49.7 ± 8.7 48.7 ± 6.1 0.8148.8 ± 8.2 50.36 ± 8.9 0.61
Body weight (kg) 93.0 ± 17.8 83.9 ± 7.7 0.3292.7 ± 17.5 92.6 ± 17.6 0.32
Body Mass Index (kg/m2) 34.9 ± 6.4 30.0 ± 3.4 0.7334.1 ± 6.1 35.1 ± 6.5 0.07
Waist circumference (cm) 105.8 ± 10.3 100.2 ± 3.3 0.20106.3 ± 10.4 105.1 ± 10.1 0.20
Hip circumference (cm) 112.2 ± 9.9 105.5 ± 5.1 0.20113.0 ± 9.9 111.2 ± 9.8 0.26
Fat mass (kg) 39.8 ± 9.2 41.1 ± 5.8 0.9041.9 ± 14.8 40.8 ± 26.1 0.70
% fat 41.4 ± 7.1 42.4 ± 4.2 0.8040.8 ± 5.0 40.1 ± 4.1 0.35
Systolic blood pressure (mmHg) 126.6 ± 19.5 127.5 ± 6.4 0.60123.7 ± 15.0 128.7 ± 21.6 0.60
Diastolic blood pressure (mmHg) 70.1 ± 15.6 66.2 ± 4.7 0.9269.0 ± 13.0 70.26 ± 16.4 0.92
Triglyceride (mg/dl) 178.2 ± 61.3 163.7 ± 23.3 0.88179.8 ± 68.8 178.0 ± 53.8 0.88
Total-cholesterol (mg/dl) 210.0 ± 48.1 198.5 ± 40.0 0.67200.5 ± 47.1 215.1 ± 44.0 0.67
LDL-cholesterol (mg/dl) 149.0 ± 36.8 150.0 ± 41.8 0.95135.5 ± 33.7 158.8 ± 36.1 0.95
HDL-cholesterol (mg/dl) 45.2 ± 7.3 45.5 ± 7.2 0.7046.2 ± 8.1 44.54 ± 6.4 0.70
Fasting glucose (mg/dl) 90.4 ± 10.7 95.5 ± 2.8 0.6491.8 ± 13.4 89.7 ± 9.0 0.64
Fasting insulin (μU/ml) 9.9 ± 3.8 10.2 ± 3.2 0.7810.1 ± 3.3 9.8 ± 4.0 0.78
HOMA-IR 2.2 ± 20.9 2.40 ± 0.7 0.492.30 ± 0.8 2.17 ± 0.9 0.49
*Data are expressed as mean ± SD. Abbreviations: HOMA-IR = Homeostasis model assessment-insulin resistance; LDL = Low-density lipoprotein; HDL =
High-density lipoprotein.
G 1067D and P1033P mutations in patients with MetS.
However, none of the controls had nucleotide substitu-
tions in IRS-2 gene.
Clinical studies [32,33] have shown that genetic vari-
ant in IRS-1 is associated with reduced insulin sensitivity.
Likewise, the IRS1 gene contains polymorphisms located
at codon 972 (BstNI) which has been associated with
type 2 diabetes mellitus and insulin resistance. Moreover,
earlier observation has indicated that the presence of a
mutated IRS-1 gene is associated with dyslipidemia [34].
Recently, it was reported that rs2289046 polymorphism
at the IRS2 gene locus might influenced insulin sensitiv-
ity by interacting with certain plasma fatty acids in MetS
subjects [35]. In another study, Thomas et al. [36] sug-
gested that an insulin receptor gene polymorphism was
associated with diastolic blood pressure in Chinese sub-
jects with components of the MetS. Similarly, it was
suggested that a putative role of IGF1R variants in indi-
vidual susceptibility to metabolic syndrome-related phe-
notypes, in particular on the risk of having insulin resis-
tance and arterial hypertension [37]. In the present study,
we didn’t find any relationships between IRS-1 and
IRS-2 gene polymorphisms and components of MetS
such as blood pressures, waist circumferences, fasting
glucose, triglyceride and HDL-Cholesterol levels.
5. CONCLUSION
In summary, insulin receptor substrate-1 and insulin
receptor substrate-2 gene polymorphisms are related to
metabolic syndrome, but not its components.
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