J Life Sci Res 2011; 9:1-5Ⓒ 2011 Osaka Prefecture University

Journal of Life Science Research
Online ISSN 2186-5809

Original

Gender-Dependent Effect of AGT M235T Polymorphisms on Glycated Hemoglobin in
Japanese Females with Type 2 Diabetes
Saeko IMAI,†1 Mikuko MATSUDA,2 Michiaki FUKUI,3 Goji HASEGAWA,3 Neiko OZASA,4 Motoko MIYAKE,1 Tatsushi
KOMATSU,5 and Shizuo KAJIYAMA2,3
1

Department of Clinical Nutrition, Faculty of Comprehensive Rehabilitation, Osaka Prefecture University, 3-7-30, Habikino, Habikino,
Osaka 583-8555, Japan; 2Kajiyama Clinic, Kyoto, Japan; 3Department of Endocrinology and Metabolism, Kyoto Prefectural
University of Medicine, Graduate School of Medical Science, Kyoto, Japan; 4Department of Cardiovascular Medicine, Kyoto
University, Graduate School of Medicine, Kyoto, Japan; 5Faculty of Human Life and Science, Doshisha Women’s College of Liberal
Arts, Kyoto 602-0893, Kyoto, Japan.
Received 4 August 2011; accepted 26 October 2011

We investigated to determine how AGT (M235T), β3AR (Trp64Arg), and UCP1 (-3826 A/G)
polymorphisms effect on blood pressure, body mass index (BMI), glycemic control and serum lipids levels
in Japanese patients with type 2 diabetes mellitus (T2DM). A total of 157 patients were screened for thevariants of SNPs. The AGT (M235T), β3AR (Trp64Arg), and UCP1 (-3826A/G) polymorphisms wereanalyzed by PCR-RFLP, and BMI, blood pressure, HbA1c, and serum lipids were examined. The patients
who carry either or both β3AR TrpArg/ArgArg and UCP1 AG/GG genotypes demonstrated the higher BMI
than patients with β3AR TrpTrp and UCP1 AA genotypes. Male patients with β3AR TrpArg/ArgArg
and/or UCP1 AG/GG genotypes had more frequently had high systolic blood pressure than those with
β3AR TrpTrp (wild) and UCP1 AA (wild) genotypes (133 ± 2 vs. 122 ± 3 mmHg, p < 0.01). Age and theexistence of β3AR TrpArg/ArgArg and/or UCP1 AG/GG genotypes were independent factors significantly
associated with systolic blood pressure by multivariate analysis in male patients with T2DM. Bloodpressure did not show AGT genotype-specific difference. However, female patients with AGT TT genotype
showed higher mean HbA1c levels than females with AGT MM/MT genotypes (8.6 ± 1.8 vs. 7.6 ± 1.4%, p< 0.001). The AGT TT genotype was an independent factor significantly associated with HbA1c levels infemale patients by multivariate analysis. Thus, gender is a very important factor in the effects of the AGT(M235T) polymorphism on HbA1c levels in Japanese patients with T2DM.
Key words: diabetes; SNPs; AGT; β3AR; UCP1; HbA1c

1 Introduction
Obesity and metabolic disorders have been associated with mutations called the single nucleotide polymorphism (SNPs) in the thrifty genotypes, such as β3adrenergic receptor (β3AR) Trp64Arg or uncoupling
protein 1 (UCP1) -3826 A/G polymorphisms. 1 The
condition is brought on by larger visceral fat mass accompanied by insulin resistance. It has been suggested thatthese SNPs were associated with an increased tendency togain weight and a low basal metabolic rate, and as a result
causes with type 2 diabetes mellitus (T2DM) or hyperlipidemia.2, 3 One of brown adipose tissue's specific innermitochondrial components is UCP1 which varies respiration coupling and dissipates oxidation energy as heat. An†

Corresponding author. E-mail: poooch@rehab.osakafu-u.ac.jp

association between the polymorphic Bcl I site (A-G
polymorphism at position -3826 bp in the 5' flanking
domain) of the UCP1 gene and fat gain has beenreported.4 Its dysfunction also leads to obesity and insulin
resistance. There are several reports in which thefrequency of the β3AR (Trp64Arg) or the UCP1 (-3826
A/G) SNPs in obese people, especially Japanese women,
was found to be approximately two times higher thanlevels found in Finns or Canadians.5, 6
The angiotensinogen gene (AGT) Met235Thr(M235T) polymorphism has been shown to be associatedwith essential hypertension in Caucasian populations.7
This polymorphism was found to be in the tight linkage
disequilibrium with a molecular variant of the genepromoter, which was shown to affect the transcription rateof the gene.8 Some researchers demonstrated a positive

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association between the AGT T235 allele and hypertension. 9 However, the reports are controversial. 10 The
factors such as age, sex, and race explain the different
results. Particularly, ethnic differences in the frequenciesof these genotypes have been reported.11
In this study we investigated to determine how AGT(M235T), β3AR (Trp64Arg), and UCP1 (-3826 A/G)
polymorphisms effect on blood pressure, glycemic control
and body mass index (BMI) in Japanese patients with
T2DM.
2 Subjects and methods
2.1 Subjects
Patients diagnosed with T2DM who fulfilled the
WHO criteria for diabetes were recruited from outpatients
visiting Kajiyama Clinic, specializes in diabetes treatment(Kyoto city, Japan), between 2008 and 2009. Patientswere excluded if they had any significant diseases thatwere likely to affect the outcome or compliance with thisstudy. The exclusion criteria were as follows: heart
failure, hepatic dysfunction, renal failure, or serious
physical or mental conditions. The study protocol wasapproved by the Ethics Committee of the School ofComprehensive Rehabilitation at Osaka Prefecture
University. Informed consent was obtained from all thesubjects before enrollment in the study. A total of 157
adults with T2DM were assigned to the study group.
2.2 Methods
Laboratory data, body weight and body mass index(BMI) were collected of all participants. BMI was
calculated as weight in kilograms divided by height inmeters squared. Fasting blood samples were collected in
the morning after an overnight fast from all participants.
HbA1c levels (JDS) were determined by a latex cohesion
method (JCA-BM2250, KYOWA MEDEX, Co. Ltd.,Tokyo, Japan). The value for HbA1c (%) was estimatedas a National Glycohemoglobin Standardization Program(NGSP) equivalent value (%) calculated by the formulaHbA1c (%) = HbA1c (JDS)(%) + 0.4%, considering therelational expression of HbA1c (JDS)(%) measured by
the previous Japanese standard substance and measurement methods and HbA1c (NGSP).12 Total cholesterol
and triglyceride levels were determined by enzyme assay.
High density lipoprotein cholesterol (HDL-cholesterol)levels were determined by a direct method (Labospect
008K, Bio Majesty JCA-BM 8060, JEOL, Ltd., Tokyo,
Japan) and low density lipoprotein cholesterol (LDLcholesterol) levels by an enzymatic method (Bio Majesty
JCA-BM 8060, JEOL, Ltd., Tokyo, Japan). Blood pressure
was measured from the right arm using a mercury sphygmomanometer in a sitting position after 5 minutes of rest.

A total of 157 patients were screened for the variantsof β3AR (Trp64Arg), UCP1 (-3826 A/G), and AGT(M235T) SNPs. Genomic DNA was extracted fromperipheral blood leukocytes of the subjects with DNA
Extractor WB Kit (Wako. Pure Chem., Osaka, Japan).
The variants of the genes were detected using a polymerase chain reaction-restriction fragment length polymor phism (PCR-RFLP) method. 13 The amplification
protocol and method of measurement were the same aspreviously reported for β3AR (Trp64Arg),14 UCP1 (-3826
A/G),5 and AGT (M235T).15
All continuous variables are reported as mean ± SD
unless otherwise indicated. Comparisons of clinical andlaboratory characteristics between the patients with SNPs
and without SNPs were done by unpaired Student's t-testor chi-square test as appropriate using SPSS version 15.0
for Windows (SPSS Inc, Chicago, IL). Multivariate
analysis was conducted using a stepwise method by JMP
version 9.0 for Windows (SAS Inc. Cary, NC). SNPs,
Age, BMI, and duration of diabetes were incorporated as
independent variables for analysis of HbA1c, systolicblood pressure levels. A two-tailed value of p < 0.05 wasconsidered significant.
3 Results
The characteristics of the patients are shown in Table 1.
The baseline biophysical parameters show no genderdifferences in age, duration of T2DM, BMI, HbA1c,
blood pressure, and LDL-cholesterol. The frequencies ofthe β3AR (Trp64Arg) genotypes in the 157 subjects with
T2DM were TrpTrp (wild) type, 69%; TrpArg type, 27%;
and ArgArg type, 4%. The frequencies of the UCP1(-3826 A/G) AA (wild), AG, and GG genotypes were 28,
47, and 25%. The patients who carry both or either
genotype of β3AR TrpArg/ArgArg or UCP1 AG/GG

Table 1

Clinical characteristics of patients with T2DM.

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were 79% in the subjects. The patients who carrygenotype of β3AR TrpArg/ArgArg showed higher BMI
than those with β3AR wild genotypes (24.7 vs. 23.1
kg/m 2, p < 0.05), although there were no differences
between UCP1 wild and AG/GG genotypes with respectof BMI, blood pressure, HbA1c, or serum lipids
concentrations. However, the patients who carry both oreither β3AR TrpArg/ArgArg and/or UCP1 AG/GG
genotypes demonstrated the higher BMI than subjects
with β3AR wild and UCP1 wild genotypes (Fig. 1). Malepatients with β3AR TrpArg/ArgArg and/or UCP1
AG/GG genotypes showed significant higher systolicblood pressure than males with β3AR wild and UCP1
wild genotypes when analyzed separately in gender(133 ± 2 vs. 122 ± 3 mmHg, p < 0.01, Fig. 1). As shownin Table 2, age and β3AR TrpArg/ArgArg and/or UCP1

Fig. 1Comparison of (A) BMI or (B) systolic blood pressure
with or without thrifty SNPs β3AR (Trp64Arg) and/or
UCP1 -3826 A/G polymorphisms. Values are mean ±SD. Differences between with and without thrifty
SNPs were examined by Student’s t-test. *p < 0.05,**p < 0.01. Significant differences in each genotype.
BMI; body mass index, SBP; systolic blood pressure.

AG/GG genotypes were independent factors significantly
associated with increased systolic blood pressure levels
by multivariate analysis in male patients with T2DM. No
difference was observed in HbA1c or serum lipids inpatients with or without β3AR or UCP1 SNPs.
The frequencies of the AGT (M235T) MM (wild),MT, and TT genotypes were 3, 35, and 61%. Because of
the low frequency of the MM genotype (n = 5), we
combined the patient groups with the MM/MT genotypes

Table 2Multivariate analysis of systolic blood pressure inmale patients with T2DM.

Fig. 2 Comparison of (A) BMI or (B) HbA1c with or without
SNPs of AGT M235T genotypes. Values are mean ±SD. Differences between with and without SNPs were
examined by Student’s t-test. *p < 0.05, **p < 0.001.
Significant differences in each genotype.

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Table 3

Multivariate analysis of of HbA1c levels in female
patients with T2DM.

for comparison with patients who carry the AGT TT
genotype. No association was found in hypertension
between the AGT TT and MM/MT genotypes (132 ± 15
vs.133 ± 18 mmHg, p = 0.686). However, the femaleswith AGT TT genotype demonstrated significant higher
HbA1c values than females with the MT/MM genotypes(8.6 ± 1.8 vs. 7.6 ± 1.4%, p < 0.001, Fig. 2), whereas thisassociation was not found in male patients. By multivariate analysis AGT TT genotype was independently
associated with increased HbA1c levels in female patientswith T2DM (Table 3). No difference was observed inblood pressure or serum lipids in patients with or without
AGT SNPs.
4 Discussion
In this study the genotype frequencies of β3AR
(Trp64Arg), UCP1 (-3826A/G), or AGT (M235T)
polymorphisms did not differ the reports of Japanese
subjects in the previous reports. 5, 6, 10, 14, 16 The β3AR
(Trp64Arg) or UCP1 (-3826A/G) gene did not have an
independent effect on the blood pressure, but its existencewith β3AR TrpArg/ArgArg and/or UCP1 AG/GG
genotypes and age resulted in higher systolic bloodpressure in Japanese male patients with T2DM.
The AGT TT genotype is found in low frequency in
the Caucasian,17 in contrast to its high prevalence in east
Asians,13, 18 and African-American populations.11, 19 It is
generally believed that AGT M235T polymorphism itself
does not cause hypertension but it is rather a marker ofthe DNA segment that actually is responsible forsusceptibility to hypertension.20 However, the reports arecontroversial.10, 21-23 The inconsistency among the previous
studies might be due to differences in the criteria forsubject selection. Especially, differences in age andethnic background play an important role. The
informative value of this AGT (M235T) polymorphism
may be sensitive to phylogenetic distance and the heterogeneity of the genetic background. Iwai et al. indicatedthat the TT genotype of the angiotensinogen gene was a
predictor of blood pressure in a subpopulation less than
50 years of age of Japanese subjects.24 In contrast, BMI
was reported to be a strong predictor of blood pressure ina subpopulation more than 50 years of age.
In our study the AGT TT genotype was not associat-

ed with a hypertension, however, the female diabetic
carriers of the AGT TT genotype was associated the higher glycated hemoglobin levels, which might be related tovisceral obesity and hyperinsulinemia.25 Because AGT isregarded as one of the cytokines produced from
adipocytes and serum AGT concentrations are reported to
be positively correlated with BMI. The mean BMI offemale patients tended to be higher than that of female
patients with AGT AA/AG genotypes, although it was notstatistically significant (Fig. 2A). However, larger andfurther investigation requires more detail information.
The β3AR TrpArg/ArgArg or UCP1 AG/GG
genotypes did not have an independent effect on the bloodpressure, but with the β3AR TrpArg/ArgArg and/or
UCP1 AG/GG genotypes resulted in higher systolic bloodpressure in male patients with T2DM. The female
diabetic carriers of the AGT TT genotype might associate
the higher glycated hemoglobin levels. Thus, gender is avery important factor in the effects of AGT TT genotypes
on glycated hemoglobin levels in Japanese patients with
T2DM. Since the number of patients in this study issmall, these conclusions must be interpreted with caution.
Acknowledgements
This work was supported by a Grants-in Aid for
Scientific Research (project number 19500605), the Kao
Research Council for the Study of Healthcare Science, theJapan Dietetic Association, the St Luke's Life Science
Institution, and Osaka Prefecture University.
Conflict of interest
The authors declared no conflict of interest.
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