It was reported that gene polymorphisms in the fat-mass and obesity-associated gene (FTO) were associated with obesity and diabetes in several genome-wide association studies. A recent report indicated that FTO-knockout mice exhibited phenotypes of skinny body shape and normal metabolic profiles. Thus, FTO could be important in metabolic disorders. The aim of this study was to clarify the role of single nucleotide polymorphisms (SNPs) in FTO in metabolic disorders such as hypertension, obesity, diabetes, dyslipidemia, insulin resistance and metabolic syndrome in the Japanese general population using data from a cohort study in Hokkaido, namely the Tanno–Sobetsu study. Written informed consent for the genetic analysis was obtained from each subject participating in the study. A total of 1514 subjects were genotyped by TaqMan PCR methods for three SNPs, rs9939609, rs1121980 and rs1558902, in FTO. Association analyses between the SNPs and metabolic parameters were performed. Although two SNPs, rs9939609 and rs1558902, were not significantly associated with hypertension, obesity, metabolic syndrome or any metabolic parameters, additive and recessive models of rs1121980 were strongly associated with plasma immunoreactive insulin (IRI) level and homeostasis model assessment insulin resistance (HOMA-IR), even after adjusting for confounding factors such as age, gender and body mass index. A haplotype of three SNPs was also significantly associated with IRI and HOMA-IR. One SNP, rs1121980, and a haplotype of three SNPs in FTO that contains this SNP, might be important in the progression of insulin resistance in Japanese subjects.
Metabolic syndrome (MS) consisting of central obesity, high blood pressure, abnormal glucose tolerance or abnormal lipid profiles is considered an independent risk factor for cardiovascular diseases such as ischemic heart disease and stroke.1, 2 In Japan, central obesity based on visceral fat accumulation is an essential diagnosis criterion for MS. One of the main pathways to central obesity is considered to be an imbalance in the secretion of adipocytokines from adipose tissues and subsequent following insulin resistance.3 Genetic background influences metabolic disorders. Recently, several genome-wide association studies revealed that single nucleotide polymorphisms (SNPs) of the fat-mass and obesity-associated gene (FTO) might be predisposing factors for obesity, diabetes and MS.4, 5, 6
Recently, it was reported that FTO-knockout mice showed several characteristic phenotypes, which included skinny body shape and normal metabolic profiles.7 Thus, FTO could be important in fat accumulation and the regulation of glucose or lipid metabolism.
In previous genetic analyses, SNPs rs9939609, rs1121980 and rs1558902 in FTO were strongly associated with obesity, defined by body mass index (BMI).8, 9, 10, 11 There remain questions concerning whether these SNPs affect obesity and other metabolic disorders in Japanese subjects, who have a quite different body shape and diet from subjects in Western countries. Furthermore, the allele frequencies of FTO SNPs in Japanese subjects are quite different from those in Caucasian subjects (http://hapmap.ncbi.nlm.nih.gov/).
It was reported by Japanese investigators that rs1558902, but not rs9939609, in FTO was associated with BMI in a case (severe obesity group: BMI⩾30 kg m−2)–control (nonobesity group: BMI⩽23 kg m−2) study.9 However, there are no reports on investigations of the relationship between FTO SNPs and metabolic disorders including hypertension, obesity, DM and MS as well as other parameters such as insulin resistance in the Japanese general population. In this study, we investigate the role of FTO SNPs in metabolic disorders in a cohort study, namely, the Tanno–Sobetsu study in Hokkaido, northern Japan.
We recruited 1514 subjects (803 in Tanno town and 711 in Sobetsu town) who had undergone medical checkups in these towns in Hokkaido, Japan, in 2002. The detailed epidemiological findings have already been reported.12, 13, 14, 15, 16, 17 Subjects completed a standard questionnaire regarding their medical history, and smoking and drinking habits. We measured the systolic blood pressure (SBP), diastolic blood pressure (DBP), BMI, abdominal circumference, total cholesterol, triglyceride, high-density lipoprotein cholesterol, plasma glucose and immunoreactive insulin (IRI). Blood samples were collected during fasting in the early morning. Obesity was defined as a BMI >25 kg m−2. Dyslipidemia was defined as total cholesterol ⩾220 mg per 100 ml and/or drug treatment for hypercholesterolemia. Diabetes was defined as fasting blood sugar ⩾126 mg per 100 ml and/or drug treatment for hyperglycemia. The Japanese definition of MS 18 was used as the diagnosis for MS. Briefly, criterion 1 and two of criteria 2–4 needed to be met.
visceral fat: (male) abdominal circumference ⩾85 cm (female) abdominal circumference ⩾90 cm
lipid abnormality: treatment for dyslipidemia or triglyceride ⩾150 mg per 100 ml and/or high-density lipoprotein cholesterol <40 mg per 100 ml
blood pressure: treatment for hypertension or SBP⩾130 and/or DBP⩾85 mm Hg
hyperglycemia: treatment for diabetes or fasting blood sugar ⩾110 mg per 100 ml.
Homeostasis model assessment insulin resistance (HOMA-IR) was used to determine insulin sensitivity, and was calculated as plasma glucose (mg per 100 ml) × IRI (μU ml−1)/405.19 Blood pressure was measured twice after 5 min of rest, with the subjects seated. Hypertension was defined as SBP ⩾140, DBP ⩾90 mm Hg or the current use of antihypertensive agents. Three hundred and ninety-five subjects were taking antihypertensive agents, and these subjects were included in the study. Individuals undergoing medical treatment and receiving diet therapy or exercise therapy for diabetes mellitus (n=84) were also included. Precise information on the types of antihypertensive agents or the nature of the treatment for diabetes was not obtained. All participants gave written informed consent to participate in the genetic analyses and in all other procedures associated with the study. The ethics committee of Osaka University approved the study protocol. The final number of subjects participating in the genetic study was 1488.
Genomic DNA was extracted from 200 μl of buffy coat using a QIAamp DNA Blood Kit (Qiagen, Hilden, Germany). We selected three SNPs, rs9939609, rs1121980 and rs1558902, in FTO, which were identified as being associated with obesity and/or diabetes in previous reports.6, 9, 20, 21 These SNPs were genotyped using TaqMan PCR methods with the following probes: C__30090620_10 for rs9939609, C__2031261_10 for rs1121980 and C__8917111_10 for rs1558902 (Applied Biosystems, Foster City, CA, USA).
Hardy–Weinberg equilibrium was calculated by a χ2-test. Linkage disequilibrium was evaluated by SNP Alyze version 2.1 (DYNACOM Co., Ltd, Mohara, Japan). Associations between the polymorphisms and clinical variables were analyzed using one-way analysis of variance and analysis of covariance adjusted for confounding factors. Differences in genotype or allele distribution were examined by χ2-analysis. All numerical values are expressed as mean±s.d. Values of P<0.05 were considered to indicate statistical significance. To adjust for multiple testing of the three gene polymorphisms by Bonferroni's correction, we arbitrarily adopted P<0.017 (=0.05/3) as the level of statistical significance. Haplotype estimation was performed by the expectation-maximization algorithm. All analyses except analysis of covariance were performed with JMP statistical software (version 5; SAS Institute Inc., Cary, NC, USA), and analysis of covariance was performed with SPSSα statistical software (release 11.0.1; SPSS Inc., Chicago, IL, USA).
The total number of study subjects who were successfully genotyped for all three SNPs, rs9939609, rs1121980 and rs1558902 of FTO, was 1488. The characteristics of study subjects are shown in Table 1. In this cohort, the average BMI was much lower than that of study subjects in previous studies.5, 9, 22, 23 The prevalence of obesity defined as BMI ⩾25 kg m−2 according to Japanese criteria was 33%. DM, hypertension and MS by the Japanese definitions were exhibited by the study subjects at 7.8, 44.4 and 15.9%, respectively.
From the genotyping, the prevalences of each genotype in the three SNPs were determined to be AA/AT/TT=56/475/957 in rs9939609, AA/AG/GG=81/519/885 in rs1121980 and AA/AT/TT=59/468/959 in rs1558902. These allele frequencies are in accordance with Hardy–Weinberg equilibrium (data not shown).
The three SNPs, rs9939609, rs1121980 and rs1558902 in FTO, were tested for associations with hypertension, diabetes, dyslipidemia, obesity and MS in all the subjects using χ2-tests. As shown in Table 2, there were no significant associations with hypertension or metabolic disorders in additive, dominant or recessive models. Table 3 shows blood pressure level and various metabolic parameters compared among genotypes for the three models of three SNPs in FTO using analysis of variance.
Additive and dominant models of rs1121980 showed significant differences in levels of IRI (P=0.022, 0.01, respectively) and HOMA-IR (P=0.029, 0.008, respectively), as shown in Table 3. Table 4 shows detailed data of IRI and HOMA-IR values composed among the genotypes in rs1121980. After adjusting for confounding factors including age, gender, BMI, abdominal circumference and presence of DM, there were still significant differences in IRI and HOMA-IR among genotypes in both additive and dominant models (P=0.005, 0.001, respectively) as determined by analysis of covariance.
Because some antihypertensive drugs might affect insulin sensitivity, we investigated the genotype comparison of HOMA-IR in both additive and dominant models for rs1121980 in subjects without hypertension (n=740). Significant differences were again identified: AA (n=38): 1.82±4.04 vs. AG(223): 1.09±0.84 vs. GG(418): 1.10±0.94 in the additive model (P=0.004) and AA: 1.82±4.04 vs. AG+GG(641): 1.10±0.91 in the dominant model (P=0.0009).
There was strong linkage disequilibrium among the three SNPs in FTO. The r2 values were 0.811 between rs9939609 and rs1121980, 0.956 between rs9939609 and rs1558902 and 0.821 between rs1121980 and rs1558902. Table 5 shows the results of comparison for metabolic parameters for each haplotype of the three SNPs in additive, dominant and recessive models analyzed by analysis of variance. Low-frequency haplotypes, namely, those with frequencies below 1%, were excluded from analysis. Five haplotypes were analyzed. H2 had a strong association with insulin resistance (Table 5), although it was present at a low frequency (n=21).
This study is the first reported investigation of the association between genetic variations in FTO and detailed metabolic parameters in the Japanese general population. We selected three SNPs, rs9939609, rs1121980 and rs1558902 in FTO, that had been found to be strongly associated with obesity, defined by BMI, in genome-wide association studies in various ethnicities.22, 24, 25, 26, 27, 28, 29 In Japan, Hotta et al.9 reported that rs1558902 in FTO was most significantly associated with obesity in a case–control association study using severely obese Japanese subjects (average BMI⩾30 kg m−2).9 In this study, we investigated the association between various metabolic parameters including hypertension, DM, obesity and MS in subjects participating in the Tanno–Sobetsu cohort study, a study of a Japanese representative rural cohort in Hokkaido. The average BMI of the study subjects was 23.81±3.24 kg m−2, which is close to the national average in Japan. In this study, none of the three SNPs was associated with obesity, defined by BMI, higher abdominal circumference or prevalence of MS, defined by Japanese criteria. In addition, none of the three SNPs was associated with hypertension, dyslipidemia or prevalence of DM.
Only one SNP, rs1121980, showed a strong correlation with HOMA-IR, which is an index of insulin resistance, in additive and dominant models. Subjects with AA in rs1121980 had a much higher HOMA-IR and a higher insulin resistance than subjects without the AA genotype (P=0.008). This P-value is considered significant (P<0.017) after Bonferroni’s correction to adjust for multiple testing of the three SNPs. Subjects with the haplotype H2, which includes AA in rs1121980, had a higher HOMA-IR than other subjects (Table 5). Thus, we conclude that rs1121980 in FTO is associated with insulin resistance in the Japanese general population. Because a recent report indicates that a gain of function of FTO induces insulin resistance,7 rs1121980 located in an intron may regulate FTO gene function by affecting splicing variation. After adjusting for obesity and the prevalence of DM, rs1121980 is independently associated with insulin resistance. Therefore, rs1121980 may affect insulin resistance, directly and not only indirectly by obesity.
In this study, three SNPs in FTO were not associated with obesity. Several reasons for this are considered. One is the difference between Caucasian and Japanese general populations in the severity of obesity. In fact, SNPs in FTO were associated with obesity in a study using Japanese subjects with severe obesity (average BMI⩾30 kg m−2).9 Another reason is the differences in allele frequency among FTO SNPs. In the cases of rs9939609, allele frequency information obtained from HapMap database (http://hapmap.ncbi.nlm.nih.gov/) shows significant differences between Caucasian (AA/AT/TT=0.117/0.667/0.217) and Japanese populations (0.067/0.200/0.733).
In summary, an SNP located in an intron, rs1121980, and a haplotype of three SNPs in FTO that includes this SNP, may be important in the progression of insulin resistance in Japanese subjects. This SNP may be an independent risk factor for future MS, hypertension and DM in Japanese subjects. However, this study has limitations because of its cross-sectional design. Prospective studies investigating the relationship between these SNPs and the development of MS, hypertension and DM over a long time scale are necessary.
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We thank Ms Kazuko Iwasa for continuous support of this investigations. This study was supported by research grant from the Japanese Ministry of Health, Labor, and Welfare and Osaka Heart Club.
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Shimaoka, I., Kamide, K., Ohishi, M. et al. Association of gene polymorphism of the fat-mass and obesity-associated gene with insulin resistance in Japanese. Hypertens Res 33, 214–218 (2010) doi:10.1038/hr.2009.215
- insulin resistance
- metabolic syndrome
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