Molecular Biology Reports

https://doi.org/10.1007/s11033-019-05041-w

ORIGINAL ARTICLE

Genetic association of insulin receptor substrate‑1 (IRS‑1, rs1801278)

gene with insulin resistant of type 2 diabetes mellitus in a Pakistani
population
Abdullah Abdo Albegali1 · Muhammad Shahzad1 · Saqib Mahmood2 · Muhammad Ikram Ullah3

Received: 5 May 2019 / Accepted: 20 August 2019

© Springer Nature B.V. 2019

Abstract
Insulin resistance (IR), a pathological condition of type 2 diabetes mellitus (T2DM) is characterized by an inability of body’s
tissue to respond the secreted or administered insulin, a necessary step for cellular glucose transportation. The prevalence
of insulin resistance progresses with age, especially in overweight people with central obesity. Insulin receptor substrates
(IRS) are important molecular proteins in the insulin signalling pathway, where IRS-1 plays a key function in cells insulin
sensitivity. The common mutation (rs1801278; r.2963G > A: Gly972Arg) of the IRS-1 gene occurs when residue glycine
changes to arginine at codon 972. The objective of this study was to detect the genetic association of rs1801278 polymorphism
of the IRS-1 gene with insulin resistance in type 2 diabetes from the Lahore region of Pakistan. A total of 322 subjects (161
cases and 161 healthy individuals) were included. DNA was isolated for detection of the genotype distribution and allele
frequencies by PCR–RFLP. The results showed a significant difference in the genotype distribution and allele frequency
between the T2DM cases and controls for single nucleotide polymorphism (SNP) rs1801278 (OR 17.61, 95% CI 8.06–38.4,
p < 0.001). In conclusion, association between rs1801278 polymorphism of the IRS-1 gene and insulin resistance in T2DM
has been established in a Pakistani population.

Keywords Type 2 diabetes mellitus (T2DM) · Insulin resistance · Insulin resistance substrate-1 (IRS-1) gene ·
Polymorphism · Pakistani population

Introduction Insulin resistance (IR) along with hypertension and dys-

Insulin is a well known anabolic hormone and is vital for the
appropriate tissue development, growth, and maintenance
of whole-body glucose homeostasis. Insulin’s binding to
its receptor is translated by activation of various docking
proteins like insulin receptor substrates (IRS) which play an
important role in further signalling pathway [19]. Modifica-
tion of such molecular proteins may result in impairment
of insulin and subsequent reduction in insulin sensitivity.
* Muhammad Shahzad
shahzad912@gmail.com
1
Department of Pharmacology, University of Health Sciences,
Khayaban‑e‑Jamia Punjab Street, Lahore 54600, Pakistan
2
Department of Human Genetics and Molecular Biology,
University of Health Sciences, Lahore 54600, Pakistan
3
Department of Clinical Laboratory Sciences, Jouf University,
Sakaka 2014, Kingdom of Saudi Arabia
lipidemia are regarded as the main clustering risk factors of
metabolic syndrome that are strongly related to the progress
of type 2 diabetes mellitus (T2DM) and other illnesses of
cardiovascular diseases [14, 24, 27].
The IRS protein is mostly found in cytoplasm, but its
nuclear existence may occur in some cell types and under
specific stimulating factors. IRS is an intracellular signal-
ling adaptor protein which coordinates and integrates several
biologically key extracellular signalling cascades within the
cell. The IRS-1 was first recognized as a signalling interme-
diate of insulin receptor [8].
The IRS-1 gene is located at chromosome 2q36 and
it encodes a member of the IRS protein substrate family.
IRS-1 is an endogenous substrate of the insulin receptor,
plays a crucial role in the insulin signalling pathway, and
is expressed in insulin-sensitive tissues. Several mecha-
nisms have currently revealed as a potential explanation for
IR states. One of which include variations in IRS-1 either
because of genetic mutations or serine phosphorylation of

13
Vol.:(0123456789)

IRS protein that would minimize their ability to attract PI
3-kinase, and hence reducing its activation [26].
Previously, some studies described the genetic analysis
of the IRS-1 gene and have revealed that nucleotide varia-
tions like rs1801278 (r.2963G > A: Gly972Arg) considerably
impaired the function of IRS-1 due to IR, lipid abnormali-
ties [13, 27], polycystic ovarian syndrome [11] and T2DM
pathologies [4, 16], Zaman [21, 28]. The association of this
common variation Gly972Arg with a significant risk of
T2DM has also been described in different ethnic groups like
European, Caucasian and Saudi population [1, 13]. Though,
another meta-analysis study has been conducted on a large
scale but did not associate the Gly972Arg polymorphism to
the risk of T2DM [18].
In the present study, we determined the association of
a genetic variant (Gly972Arg) of IRS-1 gene with insulin
resistant T2DM in a Pakistani population for the first time
according to the published data.
Materials and methods
Ethical approval for this research was obtained from the
Advance Studies and Research Board (AS&RB), Univer-
sity of Health Sciences, Lahore, Pakistan for the inclusion
of human subjects according to the Helsinki guidelines
(2008). Written informed consent was taken from all the
participants.
Subjects
A total of 322 participants were recruited in this investi-
gation and grouped equally into cases (161) and controls
(161). The healthy subjects were enrolled from the Lahore
region of Pakistan and the cases were recruited from differ-
ent teaching hospitals of Lahore. The cases were diagnosed
with T2DM and were not responding to insulin or other oral
hypoglycemic agents, but their glucose levels improved with
insulin-sensitising agents like metformin were included. The
blood samples were collected from each participant for bio-
chemical and molecular studies.
Biochemical evaluation for insulin resistance
Anthropometric measurements including, height, weight,
and waist were taken using standard techniques. The body
mass index (BMI) of each subject was calculated as the
weight (Kg) divided by the square of height (in m) at the
time of blood collection. Biochemical analysis was carried
out for fasting plasma glucose (FPG) [Randox, Cat. No:
GL1021], lipid profiles (Randox, HDL-Choles, Cat. No:
CH3811; Tot. Cholest, Cat No. CH200; TG, TR212) and
HbA1c (Randox, Cat No. HA3830) levels were performed
13
Molecular Biology Reports
according to standard protocols and calibrated methods.
Insulin resistance (IR) was confirmed using the homeostasis
model assessment for insulin resistance (HOMA-IR) equa-
tion ([FPG (mmol/L) × FIRI (mU/mL)]/405) according to
the previous guidelines [17]. The cut off value for HOMA-
IR was considered ≥ 1.6 according to International Diabetes
Federation [10].
Genetic studies of IRS‑1 polymorphism
Total genomic DNA extraction was performed on the whole
blood by standard phenol–chloroform method. Single
nucleotide polymorphism (SNP) was carried out by PCR
amplification and restriction fragment length polymorphism
(RFLP) of PCR products was performed by a site specific
restriction enzyme. Amplification of unique oligonucleo-
tide sequence of primers F-5′-CTT​TCC​ACA​GCT​CAC​CTT​
C-3′ and R-5′-GTT​AGG​CCT​GCA​AAT​GTC​TA-3′ of IRS-
1 gene was done, and genotyping was done as previously
described [22]. Concisely, after amplification, the digestion
of PCR products was performed with 2 μl of SmaI restriction
enzyme (BioLabs, New England, USA) and then digested
products were resolved on 2.0% agarose gel visualized by
UV illumination after ethidium bromide staining for sized
fragments. The size of the bands indicated the interpretation
of the fragments for genotype and allele frequency.
Statistical analysis
The data analysis was performed by SPSS 21.0 for the quan-
titative variables by applying Student’s t test for continuous
values and by the Chi square (χ2) test for nominal variables.
The genotype distributions of the control group, which were
anticipated to be with Hardy–Weinberg equilibrium, were
established using a goodness-of-fit Chi square test. The dif-
ferences in variables and genotype frequency distributions
between the patients and controls were tested using a two-
sided Chi square test. The association between the IRS-1
rs1801278 (Gly972Arg) polymorphism and IR in T2DM
was designated by calculating odds ratios and 95% CIs
through a univariate logistic regression analysis. Statistical
significance was considered if p ≤ 0.05.
Results
Demographic and biochemical features
The demographic parameters of all participants are shown in
Table 1. The participant’s range of age was 45 to 65 years.
The IR group comprised of 82 (50.9%) male patients and 79
(49.1%) female patients. There was observed no significant
difference in gender (p = 1.00) between cases and controls.
Molecular Biology Reports

Table 1  Demographic and clinical characteristics in study groups

Parameter Controls n (%) Cases n (%) χ2 (df) p-value

Age
≤ 50 years 70 (43.5) 111 (68.9) 21.209 (1) < 0.001*
> 50 years 91 (56.5) 50 (31.1)
Body mass index
Under weight 06 (3.7) 00 (0.0) 123.953 (3) < 0.001*
Normal 153 (95.0) 69 (42.9) Fig. 1  PCR product size for IRS-1 (rs1801278; Gly972Arg) polymor-
weight phism. DNA ladder 100 bp, amplified products sizes of 221 bp
Overweight 02 (1.2) 91 (56.5)
Obese 00 (0.0) 01 (0.3)
Association between the IRS‑1 rs1801278
Gender
polymorphism and insulin resistance
Males 82 (50.9) 82 (50.9) 0.000 (1) 1.00
Females 79 (49.1) 79 (49.1)
The genotype frequency distribution of the IRS-1
*Significant p-value < 0.05 (Gly972Arg) polymorphism was 46.0% (GG), 7.5% (GA),
and 46.6% (AA) in cases and was 86.3% (GG), 8.7% (GA),
and 5.0% (AA) in controls as presented in Table 3. After
The frequency of BMI in cases revealed 91 (56.5%) patients adjustment for Age, Gender, BMI, HOMA-IR, and lipid
overweight, and 01 (0.3%) patients were obese. The clinical profile parameters, the association between the IRS-1
parameters including waist circumference, HOMA-R, lipid (Gly972Arg) polymorphism and IR in T2DM patients
profile were found to be significantly different in cases as was analyzed. In a co-dominant model, the homozygous
compared to controls (p < 0.001) which describe the meta- AA genotype of IRS-1 Gly972Arg polymorphism was
bolic abnormalities among IR in T2DM patients (Table 2). contributed to 46.6% occurrence of IR (OR 17.61, 95%
CI 8.06–38.4, p < 0.001). In dominant model, the hete-
Genotype distribution of IRS‑1 alleles rozygous GA/AA genotypes of Gly972Arg polymorphism
contributed to 54.0% occurrence of insulin resistance (OR
The amplification products of IRS-1 rs1801278 (Gly972Arg) 7.43, 95% CI 4.30–12.83, p < 0.001) and after adjustment
polymorphism was found to be a band size of 221 bp as (OR 10.64, 95% CI 3.11–36.45, p < 0.001). Concerning
shown in Fig. 1. The RFLP products showed single (221 bp), allele frequency, the A allele was more frequent 87/161
double (190 bp, 31 bp) and triple (221, 190, and 31 bp) (54.1%) in T2DM cases as compared to the controls
bands for GG (wild), GA (heterozygous) and AA (homozy- 22/161 (13.7%) showed significant difference (p < 0.001).
gous) genotypes respectively as shown in Fig. 2.

Table 2  Comparison Parameter Controls Cases t/U* p-value

(Mean ± SD) of biochemical
parameters in patients (T2D) Waist circumference (cm) 77.43 ± 8.7 104.22 ± 8.7 27.479 < 0.001**
and controls
FPG (mg/dL) 73.53 ± 9.1 180.13 ± 16.3 72.453 < 0.001**
HbA1c (mmol/mol) 45 ± 3.8 55 ± 6.6 95.66 < 0.001**
FPI (uU/mL) 6.29 ± 1.1 37.38 ± 3.8 60.610 < 0.001**
HOMA-IR 1.16 ± 0.3 32.00 ± 19.9 63.166 < 0.001**
TC (mg/dL) 171.30 ± 8.7 230.84 ± 60.5 4304.00 < 0.001**
TG (mg/dL) 122.09 ± 12.1 203.12 ± 50.0 825.00 < 0.001**
HDL (mg/dL) 63.32 ± 5.1 38.70 ± 3.3 105.50 < 0.001**
LDL (mg/dL) 210.21 ± 10.9 151.51 ± 61.2 5017.50 < 0.001**
VLDL (mg/dL) 24.41 ± 2.4 40.63 ± 9.6 820.50 < 0.001**

*t-test used for normal distributed data while Mann–Whitney; U test is used for not-normally distributed
data, FPG fasting plasma glucose, FPI fasting plasma insulin
**p-value < 0.05 is significant

13
 Molecular Biology Reports

risk of IR development in T2DM patients. In accordance

Fig. 2  Restriction fragments result for IRS-1 (rs1801278; Gly972Arg)
polymorphism. Lane M shows 100 bp DNA Ladder, and Lanes 1, 2,
and 3 show single fragment (221 bp). Lane 4 and 5 show the double
fragments (190 bp and 31 bp) and Lane 6 shows the triple fragments
(221 bp, 190 bp, and 31 bp)
Discussion
The association between insulin resistance of T2DM and
IRS-1 Gly972Argpolymorphism was analyzed in the pre-
sent case–control study. The IRS-1 gene has been consid-
ered to be a candidate gene for metabolic diseases such
as T2DM and obesity. The presence of genetic variation
of the IRS-1 gene has been reported to be associated with
the development of IR [15]. The IRS-1 gene is located at
chromosome 2q36 encodes a member of the IRS protein
substrate family. IRS-1 is an endogenous substrate of the
insulin receptor, plays a crucial role in the insulin signal-
ling pathway, and is expressed in insulin-sensitive tissues
[2].
In our study, a significant association between the IRS-1
Gly972Arg polymorphism and IR was observed in patients
of T2DM (Table 3). The minor AA and combined GA/
AA genotypes of IRS-1 Gly972Arg established the high
of published data, it is the first investigation to search the
association role of IRS-1 genetic polymorphism in insulin
resistance of T2DM patients from the Pakistani popula-
tion. Previously, many studies for the putative associa-
tion between the known IRS-1 polymorphism and T2DM
have been carried out by different researchers in different
populations.
A study on Indian and Finnish subjects failed to reveal
any link of the Arg972 variant with T2DM, however, a com-
bined assessment of individuals from French and Danish
ethnic groups revealed an elevated Arg972 polymorphism
prevalence in patients with T2DM [12]. In a study from
South Indian population indicated that the IRS-1 Gly972Arg
variant was not associated with T2DM among Indian sub-
jects in which no difference was observed for ‘‘A’’ allele
frequency between the control and diabetic subjects. The
frequency of minor homozygous genotype AA was found
to be very low in type 2 diabetes (0.1%) subject while the
frequency of combined GA & AA genotypes was not sig-
nificantly different between the control and type 2 diabe-
tes groups [5]. In another study from Saudi population, the
results showed that the variant allele of Arg972 of the IRS-1
gene was significantly associated with T2DM [1]. Also in
Mexican population, the significant role of Arg972 has been
established with higher allele frequency in T2DM patients
as compared to the controls [6].
The effects of IRS-1 variants might be different because of
ethnicity or less sample size or even degree of obesity. More-
over, vast regional differences in the prevalence of Arg972
polymorphism have also been described [7, 25] which fur-
ther complicate the mechanism of the association studies
of this variant. In this regard, replication studies within the
same ethnicity are a more strong choice, particularly among

Table 3  Allele and genotype distribution of IRS-1 (Gly972Arg; rs1801278) in cases and controls

Genotypes/models Controls Cases p -value OR crude p-value adjusted* OR ­adjusteda

n (%) n (%) [95% CI] [95% CI]

Genotypes
Co-dominant model
GG (wild-type) 139 (86.3) 74 (46.0) < 0.001 1.00 < 0.001 1.00
GA (heterozygous) 14 (8.7) 12 (7.5) 1.61 (0.71–3.66) 0.91 (0.10-8.61)
AA (homozygous) 08 (5.0) 75 (46.6) 17.61 (8.06–38.48) –
A-carrier 22 (13.7) 87 (54.1)
Dominant model
GG 139 (86.3) 74 (46.0) < 0.001 1.00 < 0.001 1.00
GA & AA 22 (13.7) 87 (54.0) 7.43 (4.30–12.83) 10.64 (3.11–36.45)
GG&GA 153 (95.0) 86 (53.4) < 0.001 1.00 < 0.001 1.00
AA 08 (5.0) 75 (46.6) 16.68 (7.68–36.21) --

*p-value < 0.05 is significant

a
Adjusted for: Age, Gender, BMI, HOMA, TC, TG, HDL, LDL, and VLDL

13
Molecular Biology Reports
Asians, where they do have higher IR, greater susceptibility
to T2DM, and a powerful genetic background [23]. There
was lack of association between the IRS-1 Gly972Arg poly-
morphism and T2DM among Eastern Saudis [3] and this
common polymorphism was not associated with insulin
resistance and risk of T2DM in non-obese Turkish popula-
tion [4]. In a study of South Indian population, the dominant
model was considered to associate the Gly972Arg variant
among T2DM and normal subjects with no association
(p = 0.25) between minor allele and T2DM [5]. On the other
hand, some other studies conducted in different populations
did not show any association between IRS-1 Gly972Arg and
T2DM [9, 20, 29].
Conclusion
In conclusion, this study presented the significant difference
in genotype distribution of IRS-1 Gly972Arg polymorphism
between controls and T2DM patients. Further continuous
scientific contribution is categorising the distribution of gen-
otypes from diverse ethnic groups would enhance our under-
standing of the molecular mechanism of how the genetic risk
of T2DM is disseminated.
Acknowledgements We would like to thank Mr. Ali Amar Research
fellow and Miss. Osheen Sajjad Lecturer of Department of Human
Genetics for their assistance. We wish to thank Mrs. Amara Lab Man-
ager of the department of Pharmacology for continuous support.
Author contributions MS and SM: conceived and designed the study:
AAA: collected the samples from subjects and performed laboratory
experiments, AAA, MIU and MR: data integration and statistical analy-
ses and its interpretation; AAA and MIU: draft the manuscript, MS,
SM and MR: edit and approve the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
References
1. Alharbi KK et al (2014) Association of the genetic variants of
insulin receptor substrate 1 (IRS-1) with type 2 diabetes mellitus
in a Saudi population. Endocrine 47:472–477
2. Almind K et al (1996) A common amino acid polymorphism in
insulin receptor substrate-1 causes impaired insulin signalling.
Evidence from transfection studies. J Clin Invest 97:2569–2575
3. Alsalman HA, Kaabi YA (2015) Lack of association between the
insulin receptor substrates-1 Gly972Arg polymorphism and type-2
diabetes mellitus among Saudis from Eastern Saudi Arabia. Saudi
Med J 36:1420–1424
4. Arikoglu H et al (2014) IRS1 gene polymorphisms Gly972Arg
and Ala513Pro are not associated with insulin resistance and
type 2 diabetes risk in non-obese Turkish population. Meta Gene
24:579–585
5. Bodhini D, Radha V, Mohan V (2011) Association study of
IRS1 gene polymorphisms with type 2 diabetes in south Indians.
Diabetes Technol Ther 13:767–772
6. Burguete-Garcia AI et al (2010) Association of Gly972Arg pol-
ymorphism of IRS1 gene with type 2 diabetes mellitus in lean
participants of a national health survey in Mexico: a candidate
gene study. Metabolism 59:38–45
7. Clausen JO et al (1995) Insulin resistance: interactions between
obesity and a common variant of insulin receptor substrate-1.
Lancet 346:397–402
8. Dearth RK et al (2007) Oncogenic transformation by the sig-
nalling adaptor proteins insulin receptor substrate (IRS)-1 and
IRS-2. Cell Cycle 6:705–713
9. Florez JC et al (2004) Association testing in 9,000 people
fails to confirm the association of the insulin receptor sub-
strate-1 G972R polymorphism with type 2 diabetes. Diabetes
53:3313–3318
10. Gayoso-Diz P et al (2013) Insulin resistance (HOMA-IR) cut-off
values and the metabolic syndrome in a general adult population:
effect of gender and age: EPIRCE cross-sectional study. BMC
Endocr Disord 13:47
11. Giandalia A et al (2018) Influence of peroxisome proliferator-
activated receptor-γ exon 2 and exon 6 and insulin receptor sub-
strate (IRS)-1 Gly972Arg polymorphisms on insulin resistance
and beta-cell function in southern mediterranean women with
polycystic ovary syndrome. J Clin Transl Endocrinol 13:1–8
12. Hitman GA et al (1995) Insulin receptor substrate-1 gene muta-
tions in NIDDM; implications for the study of polygenic disease.
Diabetologia 38:481–486
13. Jellema A et al (2003) Gly972Arg variant in the insulin receptor
substrate-1 gene and association with Type 2 diabetes: a meta-
analysis of 27 studies. Diabetologia 46:990–995
14. Mahmutovic L et al (2019) Association of IRS1 genetic variants
with glucose control and insulin resistance in type 2 diabetic
patients from Bosnia and Herzegovina. Drug Metab Pers Ther.
https​://doi.org/10.1515/dmpt-2018-0031
15. Marín C et al (2011) The insulin sensitivity response is determined
by the interaction between the G972R polymorphism of the insu-
lin receptor substrate 1 gene and dietary fat. Mol Nutr Food Res
55:328–335
16. Martínez-Gómez LE et al (2011) A replication study of the IRS1,
CAPN10, TCF7L2, and PPARG gene polymorphisms associated
with type 2 diabetes in two different populations of Mexico. Ann
Hum Genet 75:612–620
17. Matthews DR et al (1985) Homeostasis model assessment: insulin
resistance and beta-cell function from fasting plasma glucose and
insulin concentrations in man. Diabetologia 28:412–419
18. Morini E et al (2009) IRS1 G972R polymorphism and type 2 dia-
betes: a paradigm for the difficult ascertainment of the contribu-
tion to disease susceptibility of ‘low-frequency-low-risk’ variants.
Diabetologia 52:1852–1857
19. Mousavinasab F et al (2005) Common polymorphisms in the
PPARgamma2 and IRS-1 genes and their interaction influence
serum adiponectin concentration in young Finnish men. Mol
Genet Metab 84:344–348
20. Suer FEO, Mergen H, Bolu E, Ozata M (2005) Molecular scan-
ning for mutations in the insulin receptor substrate-1 (IRS-1) gene
in Turkish with type 2 diabetes mellitus. Endocr J 52:593–598
21. Owen K, Ayres S, Corbett S, Hattersley A (2002) Increased risk
of diabetes in first-degree relatives of young-onset type 2 diabetic
patients compared with relatives of those diagnosed later. Diabetes
Care 25:636–637
22. Pappalardo MA et al (2010) Very high frequency of the polymor-
phism for the insulin receptor substrate 1 (IRS-1) at codon 972
(glycine972arginine) in Southern Italian women with polycystic
ovary syndrome. Horm Metab Res 42:575–584
13

23. Radha V, Mohan V (2007) Genetic predisposition to type 2 dia-
betes among Asian Indians. Indian J Med Res 125:259–274
24. Roberts CK, Hevener AL, Barnard RJ (2013) Metabolic syndrome
and insulin resistance: underlying causes and modification by
exercise training. Compr Physiol 3:1–58
25. Sesti G et al (2001) Defects of the insulin receptor substrate (IRS)
system in human metabolic disorders. FASEB J 15:2099–2111
26. Yamauchi T et al (1996) Insulin signalling and insulin actions in
the muscles and livers of insulin-resistant, insulin receptor sub-
strate 1-deficient mice. Mol Cell Biol 16:3074–3084
27. Yousef AA et al (2018) IRS-1 genetic polymorphism
(r.2963G > A) in type 2 diabetes mellitus patients associated with
insulin resistance. Dovepress 11:99–106
13
Molecular Biology Reports
28. Huri HZ et al (2012) Optimisation of glycaemic control during
episodes of severe/acute hyperglycaemia in patients with type 2
diabetes mellitus. Int J Clin Pharm 34:863–870
29. Zeggini E et al (2004) Association studies of insulin receptor sub-
strate 1 gene (IRS1) variants in type 2 diabetes samples enriched
for family history and early age of onset. Diabetes 53:3319–3322
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.