Association of β 2-adrenergic receptor and insulin receptor substrate-1 polymorphisms with obesity in a Northern Indian population

researchers showed association of such a large range of factors together. Obesity being a multifactorial disorder, in the present study we tried to associate various demographic and hormonal factors with obesity. Further, the study was extended to conÞ rm the existence of genetic factors with obesity. In this study, we want to explore the relationship between all these demographic, hormonal, and genetic factors in obese north Indians.


Introduction
Obesity has become a global pandemic and long standing obesity is often among one of the risk factors for metabolic syndrome.Though studies have been performed on causation of obesity but none of the which is proportional to degree of obesity.There is conß icting information on interaction of growth hormone and its relation to cardiovascular risk and obesity. [9]pertension is being observed in obese subjects with high leptin levels. [10]Recent studies have shown increased expression and activation of the circulating vasoconstrictor enzymes in adipose tissue, elevating the blood pressure in obese individuals. [11]Thyroid hormones play an important role in regulating energy homeostasis by stimulating expression of adrenergic receptor by enhancing responsiveness of catecholamines and thus regulating obesity. [12]art from these factors, role of genetic polymorphisms has also been known to affect obesity phenotype.
Kentaro et al. [13] were the Þ rst to identify two promoter polymorphisms (T to C) at -47 and -20 in 5' leader cistron of β 2 -AR gene and observed the high frequency of variant allele (-47 C) in obese as compared with nonobese subjects.After this report, known to our best, no study has been published so far showing the association of these promoter polymorphisms with obesity or other conditions.IRS-1, principal substrate for insulin and insulin like growth factor (IGF-1) receptors is involved in glucose clearance [14,15] and is an attractive candidate gene to harbor genetic variation that might inß uence insulin resistance and obesity.Glycine to arginine amino acid substitution at 972 codon of its gene product leads to reduced activity.
Although, several reports on obesity has been published but none of them studied such a wide range of factors together which are involved in causation of obesity.In the present study, we aimed to Þ nd out the association of demographic, hormonal and genetic factors like β 2 -AR (-47 and -20 T/C) and IRS-1 Gly972Arg gene polymorphisms with obesity in north Indian population.

Subjects
A total of 534 subjects were enrolled initially from the out patients department of Chatrapati Shahuji Maharaj Medical University, Lucknow and volunteers from general population of Lucknow (North India).Out of these, only 111 obese and 89 non-obese individual were selected beÞ tting the strict inclusion criteria.All subjects were asked for detailed clinical history and required measurements were done for height, weight, body mass index (BMI) and waist-to-hip ratio.Subjects were considered as normal if they fall in normal ranges of various parameters.For example, growth hormone (GH) = 0-14 IU/ml, leptin=2-11 mg/ml, glucagon=50-150 pg/ml, serum insulin=0-30 µU/ml.Only non-smoker, non-diabetic, normotensive subjects who did not have history of coronary artery disease, neoplasia, congenital and mental disorders, and endocrine disorders like Myxoedema and Cushing syndrome were included.Study was approved from the ethical committee of the institute.

Sample collection
After an informed consent, overnight fasting blood samples (5 ml) were taken from all subjects.Two ml blood was taken in EDTA for analysis of DNA.
The genomic DNA was extracted from peripheral blood leucocytes pellet using the standard salting out method. [16]Remaining 3 ml blood was used for serum/ plasma isolation.

Hormonal assays
Assays for glucagon and leptin hormones were done in serum/plasma by radio-immunoassay using RIA kit (Linco Research, USA). [17]Insulin hormone was assayed using RIA Kit [BARC, India].GH assay was done using hCG [ 125 I] IRMA kit (RK-5CT) [IZOTOP, Budapest]. [18]Blood sugar was assayed by Glucose oxidase-Peroxidase (GOD-POD) method. [19]notyping for β2-AR (-20 and -47 C/T) Polymorphism A fragment of 353 bp in promoter of the β 2 -AR gene was amplified by polymerase chain reaction (PCR) using primers forward 5'-GAA TGA GGC TTC CAG GCG TC-3' and reverse 5'-GGC CCA TGA CCA GAT CAG CA-3'. [13]Each ampliÞ cation was performed using 200ng of genomic DNA in a volume of 50 µl using 25 pmol of each primer, 200 µM each dNTPs, 15 mM MgCl 2 , 100 mM Tris and 1.5 units of Taq polymerase (Bangalore Genei, Bangalore).DNA templates were initially denatured at 95°C for three minutes, followed by 30 cycles with denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec and, extension at 72°C for 45 sec and Þ nally, an extension at 72°C for Þ ve minutes.T to C variation at promoter sites -47 and -20 create a restriction site for MspA1I and HphI (NEB) restriction enzymes respectively.The PCR products were subjected to digestion with restriction endonucleases at 37°C for overnight.Digested products were run on 15% polyacrylamide gel.

Genotyping for IRS-1 Gly 972 Arg polymorphism
The primers used to analyze the IRS-1 gene were as follows: forward 5'-GCA GCC TGG CAG GAG AG-3' and reverse 5'-CTC ACC TCC TCT GCA GC-3'. [20]The PCR products were initially denatured at 95°C for Þ ve min, cycling conditions were 95°C for one minute, 58°C for one minute, 70°C for one minute ( 30

Results
The demographic and clinical proÞ les are shown in Table 1.A total of 200 individuals (89 non-obese and 111 obese) with BMI (25.69±5.27kg/m 2 ) waist to hip ratio (WHR) (0.87±0.08) and mean age 31.38±11.70years were included in the present study.

Association of BMI with demographic and hormonal profi le
Subjects were categorized in 2 groups according to BMI 14-24.99 (non-obese) and BMI 25-51 (obese).

Frequency distribution of IRS-1 Gly972Arg (G/A), β 2 -AR -20 (MspA1) and ß2-AR -47 (Hph1) (T/C) gene polymorphism in non-obese and obese
In Table 2, we present genotypic data for the studied polymorphisms.The polymorphism distribution was not signiÞ cantly different for case and control subjects, with the TT genotype of β 2 -AR -20 (MspA1) gene, found in  The frequency of variant allele was very low.We found only one variant genotype (CC) so we include this in the heterozygous CT genotype.There was no signiÞ cant difference in the frequencies of the TT genotype and CT genotype of β 2 -AR -47 (Hph1) gene polymorphism between the non-obese and obese subjects.
The frequency of GG, GA genotype of IRS-1 Gly972Arg gene polymorphism was also not signiÞ cantly different in non-obese and obese subjects.Here also, no variant AA was found in non-obese and obese subjects.
Arg genotype) for demographic and hormonal proÞ le in non-obese and obese subjects.There was no inß uence of IRS-1 gene polymorphism on demographic (P>0.05) and hormonal proÞ le (P>0.05)[Table 5].

Discussion
The combined effects of genes, environment, and lifestyle are responsible for development of obesity.In this study, BMI was taken as major criteria of obesity and correlation of BMI with different clinical and genetic parameter was seen like waist to hip ratio and hormonal levels (insulin, leptin, glucagon, and growth hormone).
A statistically significant (P≤0.001)association of increased leptin levels (20.4±5.5 vs. 4.7±3.3)was observed in subjects with high BMI.The leptin level was signiÞ cantly higher in obese females.These results are similar to the Þ ndings of Considine et al. [1] where leptin levels were 31.3±24.1 ng/ml in obese subjects and 7.5± 9.3 ng/ml in normal weight subjects (P<0.001).Richard et al. [21] also found increased leptin levels in obese group (17.1± 4.8 vs. 5.8± 1.42 ng/ml, P=<0.001) than normal subjects.Recent studies also revealed a correlation of BMI with increased leptin in obese women. [2]Subjects with higher BMI and WHR showed higher leptin levels in present study.This was in agreement (P<0.001) with the Þ nding of previous study conducted by Paul et al. [22] Richard et al. [21] reported negative correlation between waist to hip ratio and leptin levels, which was not statistically signiÞ cant (P=0.99).
GH level showed a signiÞ cant fall with obesity in our study and these Þ ndings are in conformity with the observations of a recent study. [9]Savastano et al. [23] observed a negative correlation with age, BMI, waist circumference and fat mass which also favors this study.
In this study, we investigated 2 genetic variants of the β 2 AR gene and 1 genetic variant of IRS-1 gene as candidates to predispose obesity.Blood pressure as well as fat metabolism are regulated by the β 2 AR, so we tested the β 2 AR polymorphisms for association with hypertension obesity.Genetic variance in the IRS-1 is thought to play a key role in the insulin resistance that characterizes type 2 diabetes. [24], we wanted to look for association of the polymorphisms in IRS-1 and β 2 -AR genes.Our results showed that Gly to Arg variation in IRS-1 gene was observed in 7 (3.5%)subjects.Clausen et al. [25] found the increased insulin resistance with IRS-1 Gly 972 Arg polymorphism in heterozygous state in obese patients.
Caucasian study performed in two cohorts found the increased insulin resistance in obese children. [26]qal et al. [27] reported that Gly to Arg polymorphism predisposes to NIDDM only in the presence of excess of body weight.
Insulin promotes adipocyte triglyceride stores by a number of mechanisms stimulating tri-glyceride synthesis (lipogenesis).In adipocytes of obese human IRS-1 protein expression is down regulated, resulting in decreased IRS-1 associated phosphoinisitide 3 kinase (PI3K) activity which comes down stream in the insulin metabolism pathway and has antilipolytic action which is preserved in diabetic obese despite of low insulin levels resulting in maintenance or expansion of fat stores.
Studies have shown that β 2 -adrenergic receptor (ADRB2) controls energy balance and storage of fat.This receptor is down regulated in white adipose tissue in obesity.Its gene is known to be highly polymorphic. [28]1][32] The most common polymorphism Arg16Gly was found to be associated with risk for obesity. [33]A study from western world showed that Arg16Gly polymorphism was associated with weight gain from childhood to young adulthood in males. [34]However, some contradictory observations have been seen in some of the studies. [35]Earlier study published from Japan [13] stated the importance of promoter polymorphisms in β 2 -AR gene.Therefore, to Þ nd out if any association is present in Indian population, we performed the present study.
However, this study also did not reveal any signiÞ cant cycles) followed by Þ nal extension at 72°C for 10 min.The GGG to AGG substitution at codon 972 creates a restriction site for BstNI (NEB) restriction enzyme.PCR product of 221 bp was digested with restriction enzyme (10 U) at 37°C for overnight and digested products were run on 15% polyacrylamide gel at 300V.Gels were stained with ethidium bromide and visualized under ultraviolet light.All PCR reactions were performed in a Thermal Cycler (MJ Research Inc, Waltham MA).Gel documentation was done by Alphaimager TM 1220, Alpha Innotech Corporation, USA.Statistical analysis Statistical analysis was performed by SPSS (version 11.5) software.All continuous variables were expressed as mean ± SD and tested by ANOVA test.Comparisons of categorical variables were assessed using χ 2 tests or Fisher's exact test.P-value <0.05 was considered as signiÞ cant.
association of β 2 -AR -20 and -47 T/C polymorphisms with obesity.In conclusion, the present study revealed signiÞ cant association of demographic and hormonal factors with obesity in north Indians.No signiÞ cant association of any obesity related factor could be established with Srivastava, et al.: β2-AR and IRS-1gene polymorphism and obesity β 2 -AR promotor polymorphisms and IRS-1 Gly 972 Arg polymorphism.The limitations of this study is low sample size and the subjects consider as obese have BMI ≤25 rather then BMI ≤30.Moreover, frequencies of variant alleles in these polymorphisms were very low, and large sample size may be required to achieve deÞ nitive results of the association.