Association of Angiotensin-Converting Enzymeand Angiotensin II Type I Receptor Gene Polymorphisms

with Extreme Obesity in Polish Individuals

Marta Pacholczyk,1 Tomasz Ferenc,1 Jan Kowalski,2 Przemys1aw Adamczyk,3

Jacek Chojnowski,3 and Irena Ponikowska3

There is strong evidence for the presence of a functional renin–angiotensin system in human adipose tissue. Theaim of our study was to investigate the association of polymorphic variants of angiotensin-converting enzymegene (ACE I/D) and angiotensin II type I receptor gene (AGTR1 A1166C) with extreme obesity and obesity-associated type 2 diabetes mellitus (T2DM) and to examine their combined effect on extremely obese patients.Overall, no significant associations were detected between ACE and AGTR1 gene polymorphisms and extremeobesity. However, extremely obese patients with T2DM showed an increased frequency of ACE II genotypecompared with controls ( p < 0.05) and with non-diabetic extremely obese patients ( p < 0.01). The results suggestthat II genotype of ACE was a significant contributor to extreme obesity in AA homozygotes of AGTR1 gene,regardless of the presence of T2DM. Moreover, the analysis of genetic polymorphisms demonstrated that ACE IIand AGTR1 AC genotypes were most frequently observed in patients with extreme obesity and T2DM. On thebasis of our results, we suggest that ACE II homozygosity may be a significant predictor of extreme obesity andT2DM and that the interaction between ACE and AGTR1 genes may be considered a predisposing factor forextreme obesity and extreme obesity-associated T2DM development.

Introduction

In recent years, the number of subjects with obesity aswell as severe obesity has increased rapidly and is de-

scribed as an epidemic (Bray and Bellanger, 2006). Obesity isan important clinical and public health challenge in bothdeveloped and developing countries, and it is related to therisk for hypertension, cardiovascular disease, renal impair-ment, type 2 diabetes mellitus (T2DM), metabolic syndrome,and certain types of cancer (Hensrud and Klein, 2006;Soverini et al., 2010; Koebnick et al., 2012).

Obesity is a multifactorial condition due to complex in-teractions between environmental and genetic factors thatinfluence an individual’s susceptibility to obesity (Qi andCho, 2008). Polymorphisms in several obesity candidategenes have been the subject of intensive research, but alimited number of studies have investigated a possible linkbetween obesity and renin–angiotensin system (RAS), animportant regulator of blood pressure (BP) and electrolyteand homeostasis. In addition to the classical RAS, all of itscomponents are expressed in different tissues, includingadipose tissue and other metabolically active tissues, such as

skeletal muscle and liver. This permits local production andaction of angiotensin II (Ang II) in adipose tissue (Goossenset al., 2003; Yvan-Charvet and Quignard-Boulange, 2011).The overproduction of Ang II is related to hypertension andseveral diseases, including cardiovascular and kidney dis-eases, dyslipidemia, and glucose intolerance (Yvan-Charvetand Quignard-Boulange, 2011). Ang II, the product of theaction of angiotensin-converting enzyme (ACE), may play arole in adipocyte growth and differentiation and possibly inbody fat accumulation and glucose metabolism, conse-quently contributing to obesity and insulin resistance in AngII-responsive tissues (Karlsson et al., 1998; Goossens et al.,2003; Yvan-Charvet and Quignard-Boulange, 2011).

The insertion/deletion (I/D) polymorphism of the angio-tensin-converting enzyme gene (ACE) is characterized by thepresence (I) or absence (D) of a 287-bp Alu repeat sequence inintron 16 (Sayed-Tabatabaei et al., 2006). Patients homozy-gous for 287-bp deletion (genotype DD) have higher plasmaor tissue activity of ACE compared with ID heterozygotes orII homozygotes (Rigat et al., 1990).

The angiotensin II type I receptor gene (AGTR1) A1166Cpolymorphism consists of A/C nucleotide transversion and

Departments of 1Biology and Medical Genetics and 2Internal Diseases and Cardiological Rehabilitation, Medical University of Lodz, Lodz,Poland.

3Department of Balneology and Physical Medicine, Spa Clinic of Balneology and Metabolic Disorders in Ciechocinek, Collegium Medicumin Bydgoszcz, Ciechocinek, Poland.

DNA AND CELL BIOLOGYVolume 32, Number 8, 2013ª Mary Ann Liebert, Inc.Pp. 435–442DOI: 10.1089/dna.2013.2014

435

has been located at the 1166 position in the 3¢ untranslatedregion of the AGTR1 gene. Thus, in the human population,three possible genotypes exist: homozygotes—AA, CC, andheterozygote—AC (Bonnardeaux et al., 1994).

Specific polymorphic variants of ACE and AGTR1 genesaffect the activity of protein products of these genes (Rigatet al., 1990; Thekkumkara and Linas, 2003). Thus, they areconsidered as playing a significant role in the pathogenesis ofobesity, including extreme obesity, and they can also play arole in the pathogenesis of T2DM that is associated withobesity (Karlsson et al., 1998; Engeli et al., 1999; Yvan-Charvetand Quignard-Boulange, 2011).

Several studies have been recently published with regardto the association of the ACE I/D polymorphism (Cooperet al., 1997; Ryan et al., 2001; Thomas et al., 2001; Feng et al.,2002; Strazzullo et al., 2003; Um et al., 2003; Kramer et al.,2005; Yang et al., 2006; Bell et al., 2007; Wacker et al., 2008;Akin et al., 2010; Mehri et al., 2010) and AGTR1 A1166Cpolymorphism (Strazzullo et al., 2003; Abdollahi et al., 2005;Akasaka et al., 2006; Mehri et al., 2010) with the developmentof obesity and T2DM in humans.

The objective of the present study was to analyze thedistribution of the ACE gene and AGTR1 gene polymorphicvariants in extremely obese Polish patients. We also aimed atinvestigating the possible interaction between ACE andAGTR1 genotype in extreme obesity. Moreover, we sought toexplore the impact of the I/D polymorphism of the ACEgene and A1166C polymorphism of the AGTR1 gene onT2DM development in the group of extremely obese pa-tients.

Most of the studies investigated the polymorphism of asingle gene in relation to overweight or obesity, and theirresults were often conflicting. Therefore, we focused on thesynergistic effects of genetic variants of ACE and AGT1Rgenes in morbidly obese patients. In world literature, noreports have been published related to the role of the RASgene polymorphisms and their interaction in the develop-ment of extreme obesity; thus, our study may be consideredpioneering in this field of knowledge.

Materials and Methods

Study subjects

A total of 461 subjects: 173 men and 288 women partici-pated in this study. The study group consisted of 276 pa-tients (101 men and 175 women, mean age 54.24 – 11.98years) with extreme obesity (BMI [body mass index] ‡ 40 kg/m2). Basing on the earlier diagnosed T2DM in the group ofpatients with extreme obesity, two subgroups were distin-guished: without T2DM (n = 111) and with T2DM (n = 165).The control group included 185 normal weight (BMI£ 25 kg/m2) or overweight (BMI 25–29.5 kg/m2) subjects (72men and 113 women, mean age 47.88 – 16.93 years), who hadnever been obese.

Both the control and the study groups were members ofthe Polish population, and they were recruited from the SpaClinic of Balneology and Metabolic Disorders in Ciechoci-nek, Poland (from November 2010 to July 2012). Exclusioncriteria were the following: secondary form of obesity; type1 diabetes mellitus; renal, hematologic, hepatic, and thyroiddiseases; evidence of other metabolic diseases; and corti-costeroid therapy. The majority of patients took antihy-

pertensive and lipid-lowering drugs, which was notconsidered exclusion criteria.

Study protocol. The study protocol was approved by theBioethical Committee of the Medical University in Lodz(RNN/656/10/KB from 16 November, 2010). Informedwritten consent was obtained from each participant after fullexplanation of the purpose of investigation and the nature ofall procedures used.

Measurements and biochemical analyses

All subjects underwent anthropometric measurements(weight and height) in the fasting state and lightweightclothes using standard anthropometric techniques. BMI (kg/m2) was calculated. Waist and hip circumferences weremeasured, and waist-to-hip ratio was determined. BP read-ings were taken in sitting position after resting for at least15 min using a standard sphygmomanometer on the left arm.The mean BP value was calculated from two to six mea-surements. All biochemical and anthropometric measure-ments were taken during a 3-week stay at the inpatient wardof the Spa Clinic of Balneology and Metabolic Disorders inCiechocinek.

Basic clinical characteristics, including total cholesterol,triglycerides (TG), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein cholesterol, and glucose,were measured in all subjects in the fasting state. Oral glu-cose tolerance test was also performed in all extremely obesepatients according to clinical recommendations. Data on age,gender, smoking habit, and family history of obesity anddiabetes were obtained during a baseline examination. Per-ipheral venous blood samples were obtained from each pa-tient for molecular testing using ethylenediaminetetraaceticacid-containing tubes.

Genotyping

Genomic DNA was isolated from 200 mL of peripheralblood leucocytes with the use of DNA extraction kit (Gene-JET� Genomic DNA Purification Kit; Fermentas, Vilnius,Lithuania), according to the manufacturer’s protocol.

Polymerase chain reaction (PCR) was performed to de-termine ACE I/D polymorphism of the ACE gene accordingto the method described by Rigat et al. (1990) with modifi-cation. PCR was performed with thermal cycler and ther-mostable Taq polymerase (Fermentas) using primers thatflank the I/D region in intron 16. of the ACE gene. Thesequences of the oligonucleotide primers were as follows:forward [sense] F 5¢-CTG GAG ACC ACT CCC ATC CTTTCT-3¢ and reverse [antisense] R 5¢-GAT GTG GCC ATCACA TTC GTC AGA T-3¢. Each DD sample was subjectedto the second independent amplification with a primer pairthat recognizes an insertion-specific sequence (Forward[sense] F 5¢–TGG GAC CAC AGC GCC CGC CAC TAC-3¢and Reverse [antisense] R 5¢-TCG CCA GCC CTC CCATGC CCA TAA-3¢). PCR was performed under the sameconditions except the annealing temperature (Lindpaintneret al., 1995).

The AGTR1 A1166C polymorphism was determined us-ing polymerase chain reaction-restriction fragment lengthpolymorphism method with primers: forward [sense] F 5¢-GCA GCA CTT CAC TAC CAA ATG GGC-3¢ and reverse

436 PACHOLCZYK ET AL.

[antisense] R 5¢-CAG GAC AAA AGC AGG CTA GGGAGA-3¢ (Bonnardeaux et al., 1994). The 255 bp PCR prod-ucts were digested with the restriction enzyme BsuRI (Fer-mentas).

Statistical analysis

Basic statistics such as mean and standard deviation (SD)of anthropometric and biochemical measures were calcu-lated. Chi-square test was used to compare discrete variablesbetween the groups and to evaluate statistical differences ofgenotype distributions and allele frequencies of the ACE andAGTR1 genotypes between patients with extreme obesityand the control group. Hardy–Weinberg equilibrium wasevaluated on the basis of the expected genotype distribution.T-Student test or one-way analysis of variance (ANOVA)was used for normally distributed data, and U-Mann–Whitney test was applied when there was lack of normaldistribution. A p value of 0.05 or less was considered sig-nificant. To find out the effect of genotypes on the extremelyobese and on T2DM status, the odds ratio (OR) was calcu-lated with their 95% designed research project. One-wayANOVA test was used to evaluate differences in quantitativevariables according to genotype. STATISTICA 10.0 PL wasused for all calculations.

Results

The clinical characteristics and biochemical parameters inpatients with extreme obesity and in the lean control subjectsare summarized in Table 1. The control and study groupswere well matched for gender, but the mean ( – SD) age ofthe extremely obese subjects was higher than that of thecontrols. The values of systolic and diastolic blood pressure,mean fasting glucose, TG, and HDL-C levels differedbetween extremely obese patients and control subjects(Table 1).

Allele and genotype frequencies of ACE and AGTR1 genesin the studied population are shown in Table 2. Genotypefrequencies of the ACE and AGTR1 genes in all groups werein accordance with the Hardy–Weinberg equilibrium

( p > 0.1), showing that the study groups excluded selectionpressure for the investigated genotypes.

ACE gene I/D polymorphism

The frequencies of DD, ID, and II genotypes of ACE gene inextremely obese patients did not differ significantly fromthose in controls ( p > 0.1). The results showed that the fre-quency of the II genotype was found more often in extremelyobese patients (22.10%) compared with lean control subjects(16.76%), although it was not statistically significant ( p > 0.1).There were statistically significant differences in the distribu-tion of ACE genotypes between extremely obese patients withT2DM and controls ( p < 0.05). The frequency of the II geno-type in extremely obese patients with T2DM was significantlyhigher than in the control group ( p < 0.05) and compared withthe non-diabetic extemely obese patients ( p = 0.01, Table 2).

AGTR1 gene A1166C polymorphism

No statistically significant differences were found in thedistribution of AA, AC, and CC genotypes of AGTR1 geneA1166C polymorphism between the control group andthe study group of patients with extreme obesity ( p > 0.1)(Table 2). The CC genotype was more frequently detected inthe whole group of patients with extreme obesity comparedwith the controls and with non-diabetic extremely obesesubjects, although these differences were not statisticallysignificant ( p > 0.1).

Clinical and biochemical characteristics accordingto ACE and AGTR1 genotype distributions

No differences were detected in most of the clinical andbiochemical parameters according to ACE I/D polymorphismgenotypes distribution. All obese patients with the DD ge-notype of the ACE gene had a higher TG level than subjectswith either ID or II genotype ( p < 0.05). Among all extremelyobese patients, the highest frequency of T2DM was observedin those with the II genotype; whereas the lowest was ob-served in patients with the ID genotype ( p < 0.01). The controlsubjects with the II genotype had a higher BMI than those

Table 1. Clinical and Biochemical Characteristics of the Extremely Obese Patients and Control Subjects

Control subjects (N = 185) Extremely obese patients (N = 276) p-Value

Age (years) 47.88 – 16.93 54.24 – 11.98 < 0.001Male/female (%) 38.92/61.08 36.59/63.41 0.613BMI (kg/m2) 23.89 – 2.31 45.04 – 5.61 < 0.001WHR: Women 0.7974 – 0.0810 0.9481 – 0.0701 < 0.001WHR: Men 0.9112 – 0.0834 1.0247 – 0.0631 < 0.001Hypertension (%) 35.68 90.22 < 0.001SBP 126.87 – 13.46 123.16 – 13.64 < 0.001DBP 80.12 – 8.22 77.46 – 4.05 < 0.001Fasting glucose (mg/dL) 93.62 – 11.80 121.48 – 33.20 < 0.001T2DM% 0 59.78 < 0.001TC (mg/dL) 202.71 – 44.08 197.22 – 46.71 0.193LDL-C (mg/dL) 119.36 – 39.01 115.91 – 36.43 0.481HDL-C (mg/dL) 61.76 – 17.76 53.12 – 17.05 < 0.001TG (mg/dL) 107.89 – 58.73 148.18 – 61.10 < 0.001

Data are presented as mean – SD.BMI, body mass index; WHR, waist-to-hip ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; T2DM, type 2 diabetes mellitus; TC,

total cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglicerydes; SD, standard deviation.

RAS GENES POLYMORPHISM IN EXTREME OBESITY 437

with the DD or ID genotype ( p < 0.05). Mean BMI – SD was23.52 – 2.32 kg/m2 for DD, 23.81 – 2.20 kg/m2 for DI, and24.79 – 2.48 kg/m2 for II genotype (data not shown). The re-maining variables were similar among the three ACE geno-types. No difference was detected in any of the clinical andbiochemical parameters according to the AGTR1 A1166Cpolymorphism genotype distribution.

Interaction between the ACE and AGTR1 genes

We failed to detect any statistically significant differencesin the distribution of ACE/AGTR1 combined genotypes be-tween lean control subjects and extremely obese patients( p > 0.1) or extremely obese patients with T2DM ( p > 0.05).Subsequently, we compared the distribution of the DD, ID,and II genotypes among AGTR1 A1166C polymorphic ge-notypes in the extremely obese subjects with that observed inthe lean control subjects, to study the specific effects of theAGTR1 A1166C genotypes. The frequency of the A allele inall extremely obese patients with the II genotype was higherthan in controls with the II genotype ( p < 0.05). The differ-ence in allele A frequency between II genotype in extremelyobese patients with T2DM and control subjects was moreconsiderable ( p < 0.001). There was also a significantly higher

prevalence of the II genotype in extremely obese patientswith T2DM who had AA genotype than among AA homo-zygotes in the control group ( p < 0.01) (Table 3).

Then, we analyzed the joint effect of the two polymor-phisms of ACE and AGTR1 genes on the risk of extremeobesity and T2DM in extremely obese patients. The values ofORs indicated that individuals with II/AA combined geno-type had almost twofold increased risk for extreme obesity,OR = 1.90 (95% [confidence interval] CI 1.04–3.47) ( p < 0.05).The risk for extreme obesity was almost 2.5-fold increased inthe group of extremely obese patients with T2DM, OR = 2.34(95% CI 1.24–4.46) ( p < 0.01) (Table 3).

The occurrence of the C allele among ACE polymorphicgenotypes differed only between non-diabetic extremelyobese patients and patients with extreme obesity with T2DM( p < 0.05). The prevalence of II genotype was higher in obesepatients with T2DM who had AC genotype than in non-diabetic extremely obese patients with AC genotype, al-though the difference was on the borderline of statisticalsignificance ( p = 0.05).

A significant association was also observed between II/AC combined genotype of ACE and AGTR1 polymorphismsand the increased risk of T2DM in the group of extremelyobese patients; OR = 4.66, 95% CI (1.03–21.06), p < 0.05.

Table 2. ACE I/D and AGTR1 A1166C Genotype and Allele Distribution in Extremely Obese Patients

and Subgroups and in Control Subjects

Control subjects Extremely obese patients

(N = 185) All cases (N = 276) Cases without T2DM (N = 111) Cases with T2DM (N = 165)

ACE genotypes N % n % N % n %DD 48 25.95 76 27.54 30 27.03 46 27.88ID 106 57.30 139 50.36 66 59.46 73 44.24II 31 16.76 61 22.10 15 13.51 46 27.88p-Value 0.260a 0.757b 0.019c 0.010d

ORe 1.41 (0.88–2.26)p = 0.156

0.77 (0.40–1.50)p = 0.452

1.92 (1.16–3.17)p = 0.012

ORe for T2DM 2.47 (1.32–4.65)p = 0.004

ACE alleles N % n % n % n %D 202 54.59 291 52.72 126 56.75 165 50.00I 168 45.41 261 47.28 96 43.24 165 50.00p-Value 0.575a 0.608b 0.224c 0.119d

AGTR1 genotypes N % n % n % n %AA 97 52.43 147 53.26 58 52.25 89 53.94AC 80 43.24 109 39.49 46 41.44 63 38.18CC 8 4.32 20 7.25 7 6.31 13 7.88p-Value 0.377a 0.744b 0.298c 0.802d

ORf 1.03 (0.75–1.42)p = 0.861

0.99 (0.66–1.48)p = 0.976

1.06 (0.74–1.52)p = 0.778

ORf for T2DM 1.07 (0.71–1.61) P = 0.783AGTR1 alleles N % n % n % n %A 274 74.05 403 73.01 162 72.97 241 73.03C 96 25.95 149 26.99 60 27.03 89 26.97p-Value 0.724a 0.773b 0.759c 0.988d

aControls versus all extremely obese cases.bControls versus extremely obese cases without T2DM.cControls versus extremely obese cases with T2DM.dExtremely obese cases without T2DM versus extremely obese cases with T2DM.eII versus DD + ID.fAA versus AC + CC.OR, odds ratio.

438 PACHOLCZYK ET AL.

Discussion

ACE gene I/D polymorphism

In our study, no statistically significant difference wasobserved in the distribution of ACE I/D polymorphism ge-notypes and allele frequencies between subjects with normalbody weight (control group) and all patients with extremeobesity ( p > 0.1). The mean value of BMI did not differ inpatients with extreme obesity dependent on the genotype ofACE gene ( p > 0.1). These results suggested that the ACE I/Dpolymorphism was not associated with the development ofextreme obesity.

However, in our study, we observed a higher, althoughstatistically insignificant ( p > 0.1), frequency of genotype II inthe group of patients with extreme obesity compared withthe control group. The risk for the development of extremeobesity was not elevated in the investigated group of geno-type II carriers ( p > 0.1). Strazzullo et al. (2003) observed, in aprospective study, that the prevalence of overweight andcentral obesity is higher among men aged ‡ 54 years withDD genotype compared with the carriers of insertion (I) al-lele. In our study, the investigated group included only pa-tients with extreme obesity (mean BMI = 45 kg/m2). Weexpected that in extreme obesity, the contribution of geneticfactors in pathogenesis of excessive fat accumulation can besubstantially higher than in the lower class of obesity. Asimilar assumption was made by Bell et al. (2007), whose

research was carried out with the participation of patientswith extreme obesity. However, they did not observe theassociation of genetic variation in locus of the ACE gene withthe risk for extreme obesity in the examined population (Bellet al., 2007). With regard to our observation that II genotypewas more prevalent among the extreme obese patients, westrongly suggest that the correlation between ACE gene I/Dpolymorphism and extreme obesity requires further supportin a much larger number of participants with a morbid formof obesity.

Observations made in recent years have pointed to astrong association between abdominal obesity and insulinresistance. In subjects genetically predisposed to T2DM,obesity induced by high-calorie diet and low physical ac-tivity can lead to the development of tissue resistance toinsulin (Kahn and Flier, 2000). The gathered data indicatecomplex correlations between adipose tissue metabolism andinsulin action. These data also enable us to suppose thatinsulin resistance and compensatory hyperinsulinemia as-sociated with it not only result from obesity but also con-tribute to it (Kahn and Flier, 2000; Yvan-Charvet andQuignard-Boulange, 2011). Thus, in our study, we also as-sessed the possible effect of I/D polymorphism of ACE geneon the development of obesity in patients with T2DM and onthe development of T2DM in patients with extreme obesity.In our analysis, a significantly higher frequency of the IIgenotype was observed in extremely obese patients with

Table 3. Association Between ACE I/D and AGTR1 A1166C Genotypes in Extremely Obese Patients

and Subgroups and in Control Subjects

AGTR1 AA AGTR1 AC AGTR1 CC Allele A Allele C

All cases n % n % n % n % n %DD 31 21.09 38 34.86 7 35.00 100 24.81 52 34.90ID 74 50.34 56 51.38 9 45.00 204 50.62 74 49.66II 42 28.57 15 13.76 4 20.00 99 24.57 23 15.44OR II/AA 1.90 (1.04–3.47)

p = 0.033

Cases without T2DM n % n % n % n % n %DD 10 17.24 18 39.13 2 28.57 38 23.46 22 36.67ID 36 62.07 26 56.52 4 57.14 98 60.49 34 56.66II 12 20.69 2 4.35 1 14.29 26 16.05 4 6.67OR II/AA 1.28 (0.58–2.81)

p = 0.541

Cases with T2DM n % n % n % n % n %DD 21 23.60 20 31.75 5 38.46 62 25.73 30 33.71ID 38 42.79 30 47.62 5 38.46 106 43.98 40 44.94II 12 33.70 13 20.63 3 23.08 73 30.29 19 21.35OR II/AA 2.34 (1.24–4.46)

p = 0.008

Control subjects n % n % n % n % n %DD 19 19.59 26 32.50 3 37.50 64 23.36 32 33.33ID 62 63.92 42 52.50 2 25.00 166 60.58 46 47.92II 16 16.49 12 15.00 3 37.50 44 16.06 18 18.75p Value 0.061a 0.787b 0.934a 0.179b 0.524a 0.405b 0.013a 1.000b 0.794a 0.106b

0.007c 0.068d 0.666c 0.051d 0.729c 0.720d < 0.001c 0.001d 0.884c 0.048d

OR for T2DM II/AC 4.66 (1.03–21.06)p = 0.019

aControls versus all cases.bControls versus cases without T2DM.cControls versus cases with T2DM.dCases without T2DM versus cases with T2DM.

RAS GENES POLYMORPHISM IN EXTREME OBESITY 439

T2DM compared with the control group ( p < 0.05) andcompared with non-diabetic extremely obese patients( p < 0.01). The calculated OR for II genotype indicated almosta twice higher risk for extreme obesity related to concomitantT2DM ( p < 0.05). Based on OR, the relative risk for T2DMassociated with II genotype with coexisting extreme obesitywas nearly 2.5-fold higher in relation to other genotypes( p < 0.01). The results of our study point to the possible as-sociation of ACE gene II genotype with the development ofT2DM in subjects with extreme obesity. The results obtainedin our study are in agreement with the results of researchstudies published by other authors (Ryan et al., 2001; Thomaset al., 2001). In the study of Thomas et al. (2001) carried outamong the population of Chinese patients with metabolicsyndrome, no significant correlation was found between I/Dpolymorphism of ACE gene and the development of obesityor metabolic syndrome. However, a statistically lower fre-quency of D allele ( p < 0.05) was noted in each subgroup ofthe examined patients with diagnosed T2DM. Mean fastingplasma glucose level was the highest in patients with II ge-notype, but the difference was not statistically significant( p = 0.081) (Thomas et al., 2001). Ryan et al. (2001) observedthat overweight and obese sedentary women with the IIgenotype had greater insulin resistance and potential risk forT2DM than women with the DD genotype (Ryan et al., 2001).Some other studies reported that the ACE DD genotype wasassociated with an increased susceptibility to T2DM (Fenget al., 2002; Yang et al., 2006; Akin et al., 2010; Mehri et al.,2010).

The RAS is inappropriately activated in extreme obesityand diabetes, including the mechanism by which Ang IIpromotes adipocyte growth and differentiation and inducesinsulin resistance (Goossens et al., 2003; Luther and Brown,2011). Recent studies suggest that drugs which decrease theformation and action of Ang II (e.g., ACE inhibitors and AT1

receptor blockers) may also improve the insulin sensitivityand reduce an incidence of diabetes (Luther and Brown,2011). On the basis of the protective effect of pharmacologicalblockade of the RAS on the development of T2DM (Lutherand Brown, 2011), it should be thought that subjects with IIgenotype and genetically determined lower ACE levelswould have lower risk for the development of T2DM. Thiscorrelation has neither been confirmed in our study nor has itbeen confirmed in the studies of other authors (Ryan et al.,2001; Thomas et al., 2001). However, in recent years, the re-sults of experimental studies have been gathered, with re-gard to the role of RAS in the metabolism of adipocytes,which could partly explain the possible effect of II genotypeobserved in our study on the development of extreme obe-sity and T2DM. It has been shown that Ang II via AT1 re-ceptor inhibits adipogenic differentiation of humanpreadipocytes to mature adipocytes in vitro, and this impairsthe fat cells ability to store fat. This, in turn, results in theshunting of fats to the liver, skeletal muscle, and pancreas,which worsens insulin resistance. Furthermore, adipocytesare able to inhibit preadipocytes differentiation, suggesting aparacrine negative-feedback loop that inhibits further re-cruitment of preadipocytes by maturing adipocytes ( Jankeet al., 2002). Another study showed that Ang II leads to adistinct reduction in insulin-induced differentiation of pre-adipocytes. Therefore, Ang II could be a protective factoragainst uncontrolled expansion of adipose tissue in contact

with high insulin levels, and increased activity of ACE (DDgenotype) could play a protective role against obesity andassociated T2DM (Schling and Loffler, 2001). The resultsobtained in our study suggest that ACE gene I/D polymor-phisms may influence the development of extreme obesityand T2DM. Our findings also confirm the suggestion that theetiology of obesity and diabetes may have a common fac-tor(s) and they also provide clues to high incidence of T2DMin patients with obesity.

AGTR1 gene A1166C polymorphism

In the present study, we found no evidence of associationbetween the polymorphic variants of the AGTR1 gene andthe risk of extreme obesity and associated T2DM. However,among all extremely obese patients and in obese patientswith T2DM, a higher proportion of individuals were foundto have CC genotype as compared with lean subjects. Ourresults did not confirm the previous report (Mehri et al.,2010), demonstrating a significant association between CCgenotype or C allele of AGTR1 gene A1166C polymorphismand increased risk of T2DM ( p < 0.001). In our study, theeffect of a single polymorphism may be masked by the in-teraction with environmental and genetic factors, which re-flects differences between populations.

Interaction between the ACE and AGTR1 genes

Most studies on the genetic determinants of obesity andT2DM focused on a single gene. The fact that the effectsattributable to a single gene are often very small may explainconflicting results. Therefore, we explored gene–gene inter-actions between ACE and AGTR1 genes to assess the possiblerisk for extreme obesity and related T2DM. We observed asignificant interaction between the A allele or AA genotypeof the AGTR1 gene and three genotypes of the ACE gene. Wefound that the development of extreme obesity correlatedwith the II/AA combination of genotypes with significantlyincreased OR in extreme obesity ( p < 0.05), which was par-ticularly elevated in extreme obesity with T2DM ( p < 0.01).Among all extremely obese patients, a nearly twofold higherpercentage of II/AA combined genotype carriers was foundthan in the group of lean subjects (15.22% vs. 8.56%), but thedifference was not statistically significant ( p > 0.1). The rela-tive risk (OR) for extreme obesity associated with II/AAcombined genotype was elevated in relation to all the re-maining double genotypes ( p < 0.05). We also observedsimilar and insignificant over-representation of homozygousA allele carriers with the II genotype among cases withT2DM (18.18%) compared with the normal weight controlgroup ( p > 0.1). The relative risk (OR) for extreme obesitydevelopment associated with T2DM was in our study nearly2.5-fold higher in II/AA double homozygotes than in allremaining combined genotypes ( p < 0.01). Our study alsodemonstrated that the II/AC combined genotype was asso-ciated with greater than a fourfold higher risk for T2DM inpatients with extreme obesity ( p < 0.05). Our results empha-size the hypothesis that functional allelic variants of ACE andAGTR1 genes, individually not contributing to extremeobesity, can interact significantly in a combined analysis. Theobserved interaction may result from a synergistic but in-dependent effect of each genetic factor. On the other hand,functional considerations support the hypothesis that there is

440 PACHOLCZYK ET AL.

a true interaction between both alleles. Ang II, the level ofwhich is affected by the ACE gene I/D polymorphism (Rigatet al., 1990), activates AT1 receptors, whose signaling prop-erties depend on the AGTR1 gene A1166C polymorphism(Thekkumkara and Linas, 2003; Sethupathy et al., 2007). It ispossible that the AGTR1 gene polymorphism may influencepost-transcriptional receptor modification, which alters cellsignaling (Thekkumkara et al., 1998; Thekkumkara and Li-nas, 2003). In addition, it has been shown that the 1166Callele may lead to elevation of AT1 receptor levels (Sethu-pathy et al., 2007). Thus, it can be supposed that due to aninteraction of ACE and AGTR1 genes, a smaller amount ofAng II (II genotype) acts through AT1 receptors that havelower response to ligand binding (AA genotype). In thisstudy, the presence of combined genotypes showed an in-creased risk for extreme obesity and T2DM. The results ofstudies published by Mehri et al. (2010) enable us to supposethat there is an important interaction among the two RASgene polymorphisms investigated in our study and extremeobesity and T2DM, as all of them are a part of the samemetabolic pathway.

Some potential limitations should be considered in ourstudy. Given the limited number of patients, though repre-sentative of our population, further studies with largersamples and different populations are necessary in order toconfirm our findings. An interaction between lifestyle,physical activity, and a familial history of extreme obesitycould be a confounding factor, and these were not investi-gated. The present study has to be interpreted within thecontext of its limitations.

Conclusions

To conclude, the results of our study suggest that ACEgene I/D polymorphisms may influence the development ofextreme obesity and T2DM. Our study has shown for thefirst time that the development of extreme obesity correlatedwith II/AA combined genotype regardless of the presence ofdiabetes and that II/AC combined genotype was associatedwith the risk for the development of type 2 diabetes inpatients with extreme obesity.

Acknowledgments

The authors wish to thank Beata B1aszkiewicz for assis-tance in data collection for this study and for technicalassistance, and Jadwiga Kacprzak for laboratory assistance.

Disclosure Statement

No competing financial interests exist.

References

Abdollahi, M.R., Gaunt, T.R., Syddall, H.E., Cooper, C., Phillips,D.I., Ye, S., and Day, I.N. (2005). Angiotensin II type I receptorgene polymorphism: anthropometric and metabolic syndrometraits. J Med Genet 42, 396–401.

Akasaka, H., Katsuya, T., Saitoh, S., Sugimoto, K., Fu, Y., Takagi,S., Ohnishi, H., Rakugi, H., Ura, N., Shimamoto, K., and OgiharaT. (2006). Effects of angiotensin II type 1 receptor gene poly-morphisms on insulin resistance in a Japanese general popula-tion: the Tanno-Sobetsu study. Hypertens Res 29, 961–967.

Akin, F., Turgut, S., Bastemir, M., Turgut, G., Kursunluoglu, R.,Karasu, U., and Guclu, A. (2010). Angiotensin-converting en-zyme gene polymorphism in overweight and obese Turkishpatients with insulin resistance. DNA Cell Biol 29, 207–212.

Bell, C.G., Meyere, D., Petretto, E., Levy-Marchal, C., HercBerg,S., Charles, M.A., Boyle, C., Weill, J., Tauber, M., Mein, C.A.,Aitman, T.J., Froguel, P., and Walley, A.J. (2007). No contri-bution of angiotensin-converting enzyme (ACE) gene variantsto severe obesity: a model for comprehensive case/control andquantitative cladistic analysis of ACE in human diseases. Eur JHum Genet 15, 320–327.

Bonnardeaux, A., Davies, E., Jeunemaitre, X., Fery, I., Charru, A.,Clauser, E., Tiret, L., Cambien, F., Corvol, P., and Soubrier, F.(1994). Angiotensin II Type 1 receptor gene polymorphisms inhuman essential hypertension. Hypertension 24, 63–69.

Bray, G.A., and Bellanger, T. (2006). Epidemiology, trends, andmorbidities of obesity and the metabolic syndrome. Endocrine29, 109–117.

Cooper, R., McFarlane-Anderson, N., Bennett, FI., Wilks, R.,Puras, A., Tewksbury, D., Ward, R., and Forrester, T. (1997).ACE, angiotensinogen and obesity: a potential pathwayleading to hypertension. J Hum Hypertens 11, 107–111.

Engeli, S., Gorzelniak, K., Kreutz, R., Runkel, N., Distler, A., andSharma, A.M. (1999). Coexpression of renin-angiotensin sys-tem genes in human adipose tissue. J Hypertens 17, 555–560.

Feng, Y., Niu, T., Xu, X., Chen, C., Li, Q., Qian, R., Wang, G., andXu, X. (2002). Insertion/deletion polymorphism of the ACEgene is associated with type 2 diabetes. Diabetes 51, 1986–1988.

Goossens, G.H., Blaak, E.E., and van Baak, M.A. (2003). Possibleinvolvement of the adipose tissue renin-angiotensin system inthe patophysiology of obesity and obesity-related disorders.Obes Rev 4, 43–55.

Hensrud, D.D., and Klein, S. (2006). Extreme obesity: a newmedical crisis in the United States. Mayo Clin Proc 2006 81

(suppl. 10), S5–S10.Janke, J., Engeli, S., Gorzelniak, K., Luft, F.C., and Sharma, A.M.

(2002). Mature adipocytes inhibit in vitro differentiation ofhuman preadipocytes via angiotensin type 1 receptors. Dia-betes 51, 1699–1707.

Kahn, B.B., and Flier, J.S. (2000). Obesity and insulin resistance. JClin Invest 106, 473–481.

Karlsson, C., Lindell, K., Ottosson, M., Sjostrom, L., Carlsson, B.,and Carlsson, L.M. (1998). Human adipose tissue expressesangiotensinogen and enzymes required for its conversion toangiotensin II. J Clin Endocrinol Metab 83, 3925–3929.

Koebnick, C., Smith, N., Huang, K., Martinez, M.P., Clancy,H.A., and Kushi, L.H. (2012). The prevalence of obesity andobesity-related health conditions in a large, multiethnic cohortof young adults in California. Ann Epidemiol 22, 609–616.

Kramer, H., Wu, X., Kan, D., Luke, A., Zhu, X., Adeyemo, A.,McKenzie, C., and Cooper, R. (2005). Angiotensin-convertingenzyme gene polymorphisms and obesity: an examination ofthree black populations. Obes Res 13, 823–828.

Lindpaintner, K., Pfeffer, M.A., Kreutz, R., Stampfer, M.J.,Grodstein, F., LaMotte, F., Buring, J., and Hennekens, C.H.(1995). A prospective evaluation of an angiotensin-convertingenzyme gene polymorphism and the risk of ischemic heartdisease. N Engl J Med 332, 706–711.

Luther, J.M., and Brown, N.J. (2011). The renin-angiotensin-aldosterone system and glucose homeostasis. Trends Phar-macol Sci 32, 734–739.

Mehri, S., Koubaa, N., Hammami, S., Mahjoub, S., Chaaba, R.,Nakbi, A., Zouari, B., Abid, M., Ben Arab, S., Baudin, B., and

RAS GENES POLYMORPHISM IN EXTREME OBESITY 441

Hammami, M. (2010). Genotypic interactions of renin-angio-tensin system genes with diabetes type 2 in a Tunisian pop-ulation. Life Sci 87, 49–54.

Qi, L., and Cho, Y. (2008). Gene-environment interaction andobesity. Nutr Rev 66, 684–694.

Rigat, B., Hubert, C., Alhenc-Gelas, F., Cambien, F., Corvol, P.,and Soubrier, F. (1990). An insertion/deletion polymorphism inthe angiotensin converting enzyme gene accounting for half thevariance of serum enzyme levels. J Clin Invest 86, 1343–1346.

Ryan, A.S., Nicklas, B.J., Berman, D.M., and Ferrell, R.E. (2001).The insertion/deletion polymorphism of the ACE gene is re-lated to insulin sensitivity in overweight women. DiabetesCare 24, 1646–1652.

Sayed-Tabatabaei, F.A., Oostra, B.A., Isaacs, A., van Duijn, C.M.,and Witteman, J.C. (2006). ACE polymorphisms. Circ Res 98,

1123–1133.Schling, P., and Loffler, G. (2001). Effects of angiotensin II on

adipose conversion and expression of genes of the renin-angiotensin system in human preadipocytes. Horm Metab Res33, 189–195.

Sethupathy, P., Borel, C., Gagnebin, M., Grant, G.R., Deutsch, S.,Elton, T.S., Hatzigeorgiou, A.G., and Antonarakis, S.E. (2007).Human microRNA-155 on chromosome 21 differentially inter-acts with its polymorphic target in the AGTR1 3¢ untranslatedregion: a mechanism for functional single-nucleotide polymor-phisms related to phenotypes. Am J Hum Genet 81, 405–413.

Soverini, V., Moscatiello, S., Villanova, N., Ragni, E., Di Dom-izio, S., and Marchesini, G. (2010). Metabolic syndrome andinsulin resistance in subjects with morbid obesity. Obes Surg20, 295–301.

Strazzullo, P., Iacone, R., Iacoviello, L., Russo, O., Barba, G.,Russo, P., D’Orazio, A., Barbato, A., Cappuccio, F.P., Farinaro,E., and Siani, A. (2003). Olivetti Prospective Heart Study.Genetic variation in the renin-angiotensin system and ab-dominal adiposity in men: the Olivetti Prospective HeartStudy. Ann Intern Med 138, 17–23.

Thekkumkara, T.J., and Linas, S.L. (2003). Evidence for in-volvement of 3¢-untranslated region in determining angio-tensin II receptor coupling specificity to G-protein. Biochem J370, 631–639.

Thekkumkara, T.J., Thomas, W.G., Motel, T.J., and Baker, K.M.(1998). Functional role for the angiotensin II receptor (AT1A)3¢-untranslated region in determining cellular responses toagonist: evidence for recognition by RNA binding proteins.Biochem J 329 (Part 2), 255–264.

Thomas, G., Tomlinson, B., Chan, J.C., Sanderson, J.E., Cockram,C.S., and Critchley, J.A. (2001). Renin-angiotensin system genepolymorphisms, blood pressure, dyslipidemia and diabetes inHong Kong Chinese: a significant association of the ACEinsertion/deletion polymorphism with type 2 diabetes. Dia-betes Care 24, 356–361.

Um, J.Y., Mun, K.S., An, N.H., Kim, S.D., Song, Y.S., Lee, K.N.,Wi, D.H., You, Y.O., and Kim, H.M. (2003). Polymorphism ofangiotensin-converting enzyme gene and BMI in obese Koreanwomen. Clin Chim Acta 328, 173–178.

Wacker, M.J., Godard, M.P., McCabe, E.H., Donnelly, J.E., andKelly, J.K. (2008). Sex difference in the association of the an-giotensin converting enzyme I/D polymorphism and bodymass index. Med Sci Monit 14, CR353–CR357.

Yang, M., Qiu, C., Xu, Q., and Xiang, H.D. (2006). Association ofangiotensin converting enzyme gene I/D polymorphism withtype 2 diabetes mellitus. Biomed Environ Sci 19, 323–327.

Yvan-Charvet, L., and Quignard-Boulange, A. (2011). Role ofadipose tissue renin-angiotensin system in metabolic and in-flammatory diseases associated with obesity. Kidney Int 79,

162–168.

Address correspondence to:Marta Pacholczyk, PhD

Department of Biology and Medical GeneticsMedical University of Lodz

Haller’s Sq. 1Lodz 90-647

Poland

E-mail: marta.pacholczyk@umed.lodz.pl; marta-mp17@o2.pl

Received for publication February 14, 2013; received inrevised form May 7, 2013; accepted September May 7, 2013.

442 PACHOLCZYK ET AL.