Association of Angiotensin-Converting Enzyme and Angiotensin II Type I Receptor Gene Polymorphisms with Extreme Obesity in Polish Individuals

Abstract

There is strong evidence for the presence of a functional renin–angiotensin system in human adipose tissue. The aim of our study was to investigate the association of polymorphic variants of angiotensin-converting enzyme gene (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 extreme obesity. However, extremely obese patients with T2DM showed an increased frequency of ACE II genotype compared with controls (p<0.05) and with non-diabetic extremely obese patients (p<0.01). The results suggest that 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 II and AGTR1 AC genotypes were most frequently observed in patients with extreme obesity and T2DM. On the basis of our results, we suggest that ACE II homozygosity may be a significant predictor of extreme obesity and T2DM and that the interaction between ACE and AGTR1 genes may be considered a predisposing factor for extreme obesity and extreme obesity-associated T2DM development.

Introduction

I n recent years, the number of subjects with obesity as well as severe obesity has increased rapidly and is described as an epidemic (Bray and Bellanger, 2006). Obesity is an important clinical and public health challenge in both developed and developing countries, and it is related to the risk for hypertension, cardiovascular disease, renal impairment, 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 interactions between environmental and genetic factors that influence an individual's susceptibility to obesity (Qi and Cho, 2008). Polymorphisms in several obesity candidate genes have been the subject of intensive research, but a limited number of studies have investigated a possible link between obesity and renin–angiotensin system (RAS), an important regulator of blood pressure (BP) and electrolyte and homeostasis. In addition to the classical RAS, all of its components are expressed in different tissues, including adipose tissue and other metabolically active tissues, such as skeletal muscle and liver. This permits local production and action of angiotensin II (Ang II) in adipose tissue (Goossens et al., 2003; Yvan-Charvet and Quignard-Boulangé, 2011). The overproduction of Ang II is related to hypertension and several diseases, including cardiovascular and kidney diseases, dyslipidemia, and glucose intolerance (Yvan-Charvet and Quignard-Boulangé, 2011). Ang II, the product of the action of angiotensin-converting enzyme (ACE), may play a role in adipocyte growth and differentiation and possibly in body fat accumulation and glucose metabolism, consequently contributing to obesity and insulin resistance in Ang II-responsive tissues (Karlsson et al., 1998; Goossens et al., 2003; Yvan-Charvet and Quignard-Boulangé, 2011).

The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme gene (ACE) is characterized by the presence (I) or absence (D) of a 287-bp Alu repeat sequence in intron 16 (Sayed-Tabatabaei et al., 2006). Patients homozygous for 287-bp deletion (genotype DD) have higher plasma or tissue activity of ACE compared with ID heterozygotes or II homozygotes (Rigat et al., 1990).

The angiotensin II type I receptor gene (AGTR1) A1166C polymorphism consists of A/C nucleotide transversion and has been located at the 1166 position in the 3′ untranslated region of the AGTR1 gene. Thus, in the human population, three possible genotypes exist: homozygotes—AA, CC, and heterozygote—AC (Bonnardeaux et al., 1994).

Specific polymorphic variants of ACE and AGTR1 genes affect the activity of protein products of these genes (Rigat et al., 1990; Thekkumkara and Linas, 2003). Thus, they are considered as playing a significant role in the pathogenesis of obesity, including extreme obesity, and they can also play a role in the pathogenesis of T2DM that is associated with obesity (Karlsson et al., 1998; Engeli et al., 1999; Yvan-Charvet and Quignard-Boulangé, 2011).

Several studies have been recently published with regard to the association of the ACE I/D polymorphism (Cooper et 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 A1166C polymorphism (Strazzullo et al., 2003; Abdollahi et al., 2005; Akasaka et al., 2006; Mehri et al., 2010) with the development of obesity and T2DM in humans.

The objective of the present study was to analyze the distribution of the ACE gene and AGTR1 gene polymorphic variants in extremely obese Polish patients. We also aimed at investigating the possible interaction between ACE and AGTR1 genotype in extreme obesity. Moreover, we sought to explore the impact of the I/D polymorphism of the ACE gene and A1166C polymorphism of the AGTR1 gene on T2DM development in the group of extremely obese patients.

Most of the studies investigated the polymorphism of a single gene in relation to overweight or obesity, and their results were often conflicting. Therefore, we focused on the synergistic effects of genetic variants of ACE and AGT1R genes in morbidly obese patients. In world literature, no reports have been published related to the role of the RAS gene polymorphisms and their interaction in the development of extreme obesity; thus, our study may be considered pioneering in this field of knowledge.

Materials and Methods

Study subjects

A total of 461 subjects: 173 men and 288 women participated in this study. The study group consisted of 276 patients (101 men and 175 women, mean age 54.24±11.98 years) with extreme obesity (BMI [body mass index] ≥40 kg/m²). Basing on the earlier diagnosed T2DM in the group of patients with extreme obesity, two subgroups were distinguished: 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 (72 men and 113 women, mean age 47.88±16.93 years), who had never been obese.

Both the control and the study groups were members of the Polish population, and they were recruited from the Spa Clinic of Balneology and Metabolic Disorders in Ciechocinek, Poland (from November 2010 to July 2012). Exclusion criteria were the following: secondary form of obesity; type 1 diabetes mellitus; renal, hematologic, hepatic, and thyroid diseases; evidence of other metabolic diseases; and corticosteroid therapy. The majority of patients took antihypertensive and lipid-lowering drugs, which was not considered exclusion criteria.

Study protocol

The study protocol was approved by the Bioethical Committee of the Medical University in Lodz (RNN/656/10/KB from 16 November, 2010). Informed written consent was obtained from each participant after full explanation of the purpose of investigation and the nature of all procedures used.

Measurements and biochemical analyses

All subjects underwent anthropometric measurements (weight and height) in the fasting state and lightweight clothes using standard anthropometric techniques. BMI (kg/m²) was calculated. Waist and hip circumferences were measured, and waist-to-hip ratio was determined. BP readings were taken in sitting position after resting for at least 15 min using a standard sphygmomanometer on the left arm. The mean BP value was calculated from two to six measurements. All biochemical and anthropometric measurements were taken during a 3-week stay at the inpatient ward of the Spa Clinic of Balneology and Metabolic Disorders in Ciechocinek.

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 glucose tolerance test was also performed in all extremely obese patients according to clinical recommendations. Data on age, gender, smoking habit, and family history of obesity and diabetes were obtained during a baseline examination. Peripheral venous blood samples were obtained from each patient for molecular testing using ethylenediaminetetraacetic acid-containing tubes.

Genotyping

Genomic DNA was isolated from 200 μL of peripheral blood leucocytes with the use of DNA extraction kit (GeneJET™ Genomic DNA Purification Kit; Fermentas, Vilnius, Lithuania), according to the manufacturer's protocol.

Polymerase chain reaction (PCR) was performed to determine ACE I/D polymorphism of the ACE gene according to the method described by Rigat et al. (1990) with modification. PCR was performed with thermal cycler and thermostable Taq polymerase (Fermentas) using primers that flank the I/D region in intron 16. of the ACE gene. The sequences of the oligonucleotide primers were as follows: forward [sense] F 5′-CTG GAG ACC ACT CCC ATC CTT TCT-3′ and reverse [antisense] R 5′-GAT GTG GCC ATC ACA TTC GTC AGA T-3′. Each DD sample was subjected to the second independent amplification with a primer pair that 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 CCA TGC CCA TAA-3′). PCR was performed under the same conditions except the annealing temperature (Lindpaintner et al., 1995).

The AGTR1 A1166C polymorphism was determined using polymerase chain reaction-restriction fragment length polymorphism method with primers: forward [sense] F 5′-GCA GCA CTT CAC TAC CAA ATG GGC-3′ and reverse [antisense] R 5′-CAG GAC AAA AGC AGG CTA GGG AGA-3′ (Bonnardeaux et al., 1994). The 255 bp PCR products were digested with the restriction enzyme BsuRI (Fermentas).

Statistical analysis

Basic statistics such as mean and standard deviation (SD) of anthropometric and biochemical measures were calculated. Chi-square test was used to compare discrete variables between the groups and to evaluate statistical differences of genotype distributions and allele frequencies of the ACE and AGTR1 genotypes between patients with extreme obesity and the control group. Hardy–Weinberg equilibrium was evaluated 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 normal distribution. A p value of 0.05 or less was considered significant. To find out the effect of genotypes on the extremely obese and on T2DM status, the odds ratio (OR) was calculated with their 95% designed research project. One-way ANOVA test was used to evaluate differences in quantitative variables according to genotype. STATISTICA 10.0 PL was used for all calculations.

Results

The clinical characteristics and biochemical parameters in patients with extreme obesity and in the lean control subjects are summarized in Table 1. The control and study groups were well matched for gender, but the mean (±SD) age of the extremely obese subjects was higher than that of the controls. The values of systolic and diastolic blood pressure, mean fasting glucose, TG, and HDL-C levels differed between extremely obese patients and control subjects (Table 1).

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

Allele and genotype frequencies of ACE and AGTR1 genes in the studied population are shown in Table 2. Genotype frequencies of the ACE and AGTR1 genes in all groups were in accordance with the Hardy–Weinberg equilibrium (p>0.1), showing that the study groups excluded selection pressure for the investigated genotypes.

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	27.88
ID	106	57.30	139	50.36	66	59.46	73	44.24
II	31	16.76	61	22.10	15	13.51	46	27.88
p-Value		0.260^a		0.757^b		0.019^c		0.010^d
OR^e			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
OR^e 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	50.00
I	168	45.41	261	47.28	96	43.24	165	50.00
p-Value		0.575^a		0.608^b		0.224^c		0.119^d
AGTR1 genotypes	N	%	n	%	n	%	n	%
AA	97	52.43	147	53.26	58	52.25	89	53.94
AC	80	43.24	109	39.49	46	41.44	63	38.18
CC	8	4.32	20	7.25	7	6.31	13	7.88
p-Value		0.377^a		0.744^b		0.298^c		0.802^d
OR^f			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
OR^f for T2DM							1.07 (0.71–1.61) P=0.783
AGTR1 alleles	N	%	n	%	n	%	n	%
A	274	74.05	403	73.01	162	72.97	241	73.03
C	96	25.95	149	26.99	60	27.03	89	26.97
p-Value		0.724^a		0.773^b		0.759^c		0.988^d

Controls versus all extremely obese cases.

Controls versus extremely obese cases without T2DM.

Controls versus extremely obese cases with T2DM.

Extremely obese cases without T2DM versus extremely obese cases with T2DM.

II versus DD+ID.

AA versus AC+CC.

OR, odds ratio.

ACE gene I/D polymorphism

The frequencies of DD, ID, and II genotypes of ACE gene in extremely obese patients did not differ significantly from those in controls (p>0.1). The results showed that the frequency of the II genotype was found more often in extremely obese 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 distribution of ACE genotypes between extremely obese patients with T2DM and controls (p<0.05). The frequency of the II genotype in extremely obese patients with T2DM was significantly higher than in the control group (p<0.05) and compared with the non-diabetic extemely obese patients (p=0.01, Table 2).

AGTR1 gene A1166C polymorphism

No statistically significant differences were found in the distribution of AA, AC, and CC genotypes of AGTR1 gene A1166C polymorphism between the control group and the study group of patients with extreme obesity (p>0.1) (Table 2). The CC genotype was more frequently detected in the whole group of patients with extreme obesity compared with the controls and with non-diabetic extremely obese subjects, although these differences were not statistically significant (p>0.1).

Clinical and biochemical characteristics according to ACE and AGTR1 genotype distributions

No differences were detected in most of the clinical and biochemical parameters according to ACE I/D polymorphism genotypes distribution. All obese patients with the DD genotype of the ACE gene had a higher TG level than subjects with either ID or II genotype (p<0.05). Among all extremely obese patients, the highest frequency of T2DM was observed in those with the II genotype; whereas the lowest was observed in patients with the ID genotype (p<0.01). The control subjects with the II genotype had a higher BMI than those with the DD or ID genotype (p<0.05). Mean BMI±SD was 23.52±2.32 kg/m² for DD, 23.81±2.20 kg/m² for DI, and 24.79±2.48 kg/m² for II genotype (data not shown). The remaining variables were similar among the three ACE genotypes. No difference was detected in any of the clinical and biochemical parameters according to the AGTR1 A1166C polymorphism genotype distribution.

Interaction between the ACE and AGTR1 genes

We failed to detect any statistically significant differences in the distribution of ACE/AGTR1 combined genotypes between 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 genotypes in the extremely obese subjects with that observed in the lean control subjects, to study the specific effects of the AGTR1 A1166C genotypes. The frequency of the A allele in all extremely obese patients with the II genotype was higher than in controls with the II genotype (p<0.05). The difference in allele A frequency between II genotype in extremely obese patients with T2DM and control subjects was more considerable (p<0.001). There was also a significantly higher prevalence of the II genotype in extremely obese patients with T2DM who had AA genotype than among AA homozygotes in the control group (p<0.01) (Table 3).

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	34.90
ID	74	50.34	56	51.38	9	45.00	204	50.62	74	49.66
II	42	28.57	15	13.76	4	20.00	99	24.57	23	15.44
OR 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	36.67
ID	36	62.07	26	56.52	4	57.14	98	60.49	34	56.66
II	12	20.69	2	4.35	1	14.29	26	16.05	4	6.67
OR 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	33.71
ID	38	42.79	30	47.62	5	38.46	106	43.98	40	44.94
II	12	33.70	13	20.63	3	23.08	73	30.29	19	21.35
OR 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	33.33
ID	62	63.92	42	52.50	2	25.00	166	60.58	46	47.92
II	16	16.49	12	15.00	3	37.50	44	16.06	18	18.75
p Value	0.061^a	0.787^b	0.934^a	0.179^b	0.524^a	0.405^b	0.013^a	1.000^b	0.794^a	0.106^b
	0.007^c	0.068^d	0.666^c	0.051^d	0.729^c	0.720^d	<0.001^c	0.001^d	0.884^c	0.048^d
OR for T2DM II/AC	4.66 (1.03–21.06) p=0.019

Controls versus all cases.

Controls versus cases without T2DM.

Controls versus cases with T2DM.

Cases without T2DM versus cases with T2DM.

Then, we analyzed the joint effect of the two polymorphisms of ACE and AGTR1 genes on the risk of extreme obesity and T2DM in extremely obese patients. The values of ORs indicated that individuals with II/AA combined genotype 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 in the 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 polymorphic genotypes differed only between non-diabetic extremely obese patients and patients with extreme obesity with T2DM (p<0.05). The prevalence of II genotype was higher in obese patients with T2DM who had AC genotype than in non-diabetic extremely obese patients with AC genotype, although the difference was on the borderline of statistical significance (p=0.05).

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

Discussion

ACE gene I/D polymorphism

In our study, no statistically significant difference was observed in the distribution of ACE I/D polymorphism genotypes and allele frequencies between subjects with normal body weight (control group) and all patients with extreme obesity (p>0.1). The mean value of BMI did not differ in patients with extreme obesity dependent on the genotype of ACE gene (p>0.1). These results suggested that the ACE I/D polymorphism was not associated with the development of extreme obesity.

However, in our study, we observed a higher, although statistically insignificant (p>0.1), frequency of genotype II in the group of patients with extreme obesity compared with the control group. The risk for the development of extreme obesity was not elevated in the investigated group of genotype II carriers (p>0.1). Strazzullo et al. (2003) observed, in a prospective study, that the prevalence of overweight and central obesity is higher among men aged ≥54 years with DD genotype compared with the carriers of insertion (I) allele. In our study, the investigated group included only patients with extreme obesity (mean BMI=45 kg/m²). We expected that in extreme obesity, the contribution of genetic factors in pathogenesis of excessive fat accumulation can be substantially higher than in the lower class of obesity. A similar assumption was made by Bell et al. (2007), whose research was carried out with the participation of patients with extreme obesity. However, they did not observe the association of genetic variation in locus of the ACE gene with the risk for extreme obesity in the examined population (Bell et al., 2007). With regard to our observation that II genotype was more prevalent among the extreme obese patients, we strongly suggest that the correlation between ACE gene I/D polymorphism and extreme obesity requires further support in a much larger number of participants with a morbid form of obesity.

Observations made in recent years have pointed to a strong association between abdominal obesity and insulin resistance. In subjects genetically predisposed to T2DM, obesity induced by high-calorie diet and low physical activity can lead to the development of tissue resistance to insulin (Kahn and Flier, 2000). The gathered data indicate complex correlations between adipose tissue metabolism and insulin action. These data also enable us to suppose that insulin resistance and compensatory hyperinsulinemia associated with it not only result from obesity but also contribute to it (Kahn and Flier, 2000; Yvan-Charvet and Quignard-Boulangé, 2011). Thus, in our study, we also assessed the possible effect of I/D polymorphism of ACE gene on the development of obesity in patients with T2DM and on the development of T2DM in patients with extreme obesity. In our analysis, a significantly higher frequency of the II genotype was observed in extremely obese patients with T2DM compared with the control group (p<0.05) and compared with non-diabetic extremely obese patients (p<0.01). The calculated OR for II genotype indicated almost a twice higher risk for extreme obesity related to concomitant T2DM (p<0.05). Based on OR, the relative risk for T2DM associated with II genotype with coexisting extreme obesity was nearly 2.5-fold higher in relation to other genotypes (p<0.01). The results of our study point to the possible association of ACE gene II genotype with the development of T2DM in subjects with extreme obesity. The results obtained in our study are in agreement with the results of research studies published by other authors (Ryan et al., 2001; Thomas et al., 2001). In the study of Thomas et al. (2001) carried out among the population of Chinese patients with metabolic syndrome, no significant correlation was found between I/D polymorphism of ACE gene and the development of obesity or metabolic syndrome. However, a statistically lower frequency of D allele (p<0.05) was noted in each subgroup of the examined patients with diagnosed T2DM. Mean fasting plasma glucose level was the highest in patients with II genotype, but the difference was not statistically significant (p=0.081) (Thomas et al., 2001). Ryan et al. (2001) observed that overweight and obese sedentary women with the II genotype had greater insulin resistance and potential risk for T2DM than women with the DD genotype (Ryan et al., 2001). Some other studies reported that the ACE DD genotype was associated with an increased susceptibility to T2DM (Feng et al., 2002; Yang et al., 2006; Akin et al., 2010; Mehri et al., 2010).

The RAS is inappropriately activated in extreme obesity and diabetes, including the mechanism by which Ang II promotes adipocyte growth and differentiation and induces insulin resistance (Goossens et al., 2003; Luther and Brown, 2011). Recent studies suggest that drugs which decrease the formation and action of Ang II (e.g., ACE inhibitors and AT₁ receptor blockers) may also improve the insulin sensitivity and reduce an incidence of diabetes (Luther and Brown, 2011). On the basis of the protective effect of pharmacological blockade of the RAS on the development of T2DM (Luther and Brown, 2011), it should be thought that subjects with II genotype and genetically determined lower ACE levels would have lower risk for the development of T2DM. This correlation has neither been confirmed in our study nor has it been confirmed in the studies of other authors (Ryan et al., 2001; Thomas et al., 2001). However, in recent years, the results of experimental studies have been gathered, with regard to the role of RAS in the metabolism of adipocytes, which could partly explain the possible effect of II genotype observed in our study on the development of extreme obesity and T2DM. It has been shown that Ang II via AT₁ receptor inhibits adipogenic differentiation of human preadipocytes to mature adipocytes in vitro, and this impairs the fat cells ability to store fat. This, in turn, results in the shunting of fats to the liver, skeletal muscle, and pancreas, which worsens insulin resistance. Furthermore, adipocytes are able to inhibit preadipocytes differentiation, suggesting a paracrine negative-feedback loop that inhibits further recruitment of preadipocytes by maturing adipocytes (Janke et al., 2002). Another study showed that Ang II leads to a distinct reduction in insulin-induced differentiation of preadipocytes. Therefore, Ang II could be a protective factor against uncontrolled expansion of adipose tissue in contact with high insulin levels, and increased activity of ACE (DD genotype) could play a protective role against obesity and associated T2DM (Schling and Löffler, 2001). The results obtained in our study suggest that ACE gene I/D polymorphisms may influence the development of extreme obesity and T2DM. Our findings also confirm the suggestion that the etiology of obesity and diabetes may have a common factor(s) and they also provide clues to high incidence of T2DM in patients with obesity.

AGTR1 gene A1166C polymorphism

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

Interaction between the ACE and AGTR1 genes

Most studies on the genetic determinants of obesity and T2DM focused on a single gene. The fact that the effects attributable to a single gene are often very small may explain conflicting results. Therefore, we explored gene–gene interactions between ACE and AGTR1 genes to assess the possible risk for extreme obesity and related T2DM. We observed a significant interaction between the A allele or AA genotype of the AGTR1 gene and three genotypes of the ACE gene. We found that the development of extreme obesity correlated with the II/AA combination of genotypes with significantly increased OR in extreme obesity (p<0.05), which was particularly elevated in extreme obesity with T2DM (p<0.01). Among all extremely obese patients, a nearly twofold higher percentage of II/AA combined genotype carriers was found than in the group of lean subjects (15.22% vs. 8.56%), but the difference was not statistically significant (p>0.1). The relative risk (OR) for extreme obesity associated with II/AA combined genotype was elevated in relation to all the remaining double genotypes (p<0.05). We also observed similar and insignificant over-representation of homozygous A allele carriers with the II genotype among cases with T2DM (18.18%) compared with the normal weight control group (p>0.1). The relative risk (OR) for extreme obesity development associated with T2DM was in our study nearly 2.5-fold higher in II/AA double homozygotes than in all remaining combined genotypes (p<0.01). Our study also demonstrated that the II/AC combined genotype was associated with greater than a fourfold higher risk for T2DM in patients with extreme obesity (p<0.05). Our results emphasize the hypothesis that functional allelic variants of ACE and AGTR1 genes, individually not contributing to extreme obesity, can interact significantly in a combined analysis. The observed interaction may result from a synergistic but independent effect of each genetic factor. On the other hand, functional considerations support the hypothesis that there is a true interaction between both alleles. Ang II, the level of which is affected by the ACE gene I/D polymorphism (Rigat et al., 1990), activates AT₁ receptors, whose signaling properties depend on the AGTR1 gene A1166C polymorphism (Thekkumkara and Linas, 2003; Sethupathy et al., 2007). It is possible that the AGTR1 gene polymorphism may influence post-transcriptional receptor modification, which alters cell signaling (Thekkumkara et al., 1998; Thekkumkara and Linas, 2003). In addition, it has been shown that the 1166C allele may lead to elevation of AT₁ receptor levels (Sethupathy et al., 2007). Thus, it can be supposed that due to an interaction of ACE and AGTR1 genes, a smaller amount of Ang II (II genotype) acts through AT₁ receptors that have lower response to ligand binding (AA genotype). In this study, the presence of combined genotypes showed an increased risk for extreme obesity and T2DM. The results of studies published by Mehri et al. (2010) enable us to suppose that there is an important interaction among the two RAS gene polymorphisms investigated in our study and extreme obesity and T2DM, as all of them are a part of the same metabolic pathway.

Some potential limitations should be considered in our study. Given the limited number of patients, though representative of our population, further studies with larger samples and different populations are necessary in order to confirm our findings. An interaction between lifestyle, physical activity, and a familial history of extreme obesity could be a confounding factor, and these were not investigated. The present study has to be interpreted within the context of its limitations.

Conclusions

To conclude, the results of our study suggest that ACE gene I/D polymorphisms may influence the development of extreme obesity and T2DM. Our study has shown for the first time that the development of extreme obesity correlated with II/AA combined genotype regardless of the presence of diabetes and that II/AC combined genotype was associated with the risk for the development of type 2 diabetes in patients with extreme obesity.

Footnotes

Acknowledgments

The authors wish to thank Beata Błaszkiewicz for assistance in data collection for this study and for technical assistance, and Jadwiga Kacprzak for laboratory assistance.

Disclosure Statement

No competing financial interests exist.

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