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A Polymorphism with Type 2 Diabetes and Hypertension in a Chinese Population
Department of Biology, Kaohsiung Medical University (M.-S.T.), Department of Nursing, Fooyin University of Technology (S.-Y.C.), and Department of Clinical Research, Pingtung Christian Hospital (D.-M.C., J.C.-R.T., Y.-J.L.), Pingtung 90000, Taiwan
Address all correspondence and requests for reprints to: Dr. Yau-Jiunn Lee, Department of Clinical Research, Ping-Tung Christian Hospital, No. 60 Da-Lien Road, Ping-Tung 90000, Taiwan. E-mail: t3275{at}ms25.hinet.net.
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Abstract |
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A polymorphism was determined by PCR. Type 2 diabetes subjects had a lower frequency of resistin gene 3'UTR +62A allele (GG:GA/AA, 83.5%:16.5%) than the controls (GG:GA/AA, 75.1%:24.9%; odds ratio, 1.524; 95% confidence interval, 1.268–1.831; P < 0.001). Unexpectedly, diabetic patients with the GG genotype had a higher prevalence of hypertension (GG:GA/AA, 49.8%:36.2%; odds ratio, 1.375; 95% confidence interval, 1.116–1.693; P = 0.001). Logistic regression analysis confirmed that the resistin gene 3'UTR +62GA polymorphism acts as an independent contributing factor to type 2 diabetes and hypertension. The mean systolic and diastolic blood pressure levels in diabetic subjects with the GG genotype (144 ± 21/87 ± 13 mm Hg) were significantly higher than those in subjects with GA/AA variants (139 ± 21/84 ± 14 mm Hg; P = 0.004 and P = 0.002, respectively). Multiple linear regression analysis showed resistin gene polymorphism to be an independent factor associated with systolic and diastolic blood pressures in type 2 diabetes patients. These findings suggest that resistin may play a role in the pathogenesis of type 2 diabetes and insulin resistance-related hypertension. |
Introduction |
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agonist rosiglitazone, an antidiabetic agent that improves insulin action, also led to a reduction in resistin concentrations (1, 2). These findings suggested that resistin could play a part in the pathogenesis of insulin resistance and that resistin is thought to be a link between obesity and diabetes. Therefore, resistin may be a potential candidate gene associated with type 2 diabetes and the insulin resistance-related metabolic derangements, although the pathophysiological role of resistin in human subjects remains controversial (3, 4, 5, 6). The clustering of type 2 diabetes, hypertension, abdominal obesity, and dyslipidemia, known as the metabolic syndrome (7), is associated with a substantially increased cardiovascular risk (8). Although it has been argued that insulin resistance may be the primary abnormality of metabolic syndrome (7). Cao and Hegele (9) have reported two noncoding, single nucleotide polymorphisms (SNPs) in the resistin gene, one of which was located in the 3'-untranslated region (UTR). It has also been reported that SNPs in 3'UTR of genes can affect gene expression (10), and it is possible that the 3'UTR variant of the resistin gene might influence resistin gene expression. Therefore, this study was designed to investigate whether the resistin gene 3'UTR polymorphism is associated with type 2 diabetes and insulin-resistant related metabolic derangements among a Chinese population.
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Subjects and Methods |
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From February 1998 to December 2001, we studied 1102 patients with type 2 diabetes who consecutively attended the diabetic clinic at Pingtung Christian Hospital. The diagnosis of type 2 diabetes was based upon World Health Organization criteria (11). Seven hundred forty-three nondiabetic subjects were recruited from an unselected population who underwent routine health check-ups and were used as the control group. The definition of nondiabetic is subjects who have fasting plasma glucose level lower than 126 mg/dl and have no family history, including parents, siblings, and children, of type 2 diabetes. Subjects who had urinary tract infection, urolithiasis, and other known renal disorders were excluded. Each received a detailed interview about personal disease history and smoking history. All study subjects were of Han Chinese origin without any known ancestors of other ethnic origins and were living in the same region at the time of study (12). This study was approved by the human research ethic committee of our hospital, and informed consent was obtained from each patient. All patients underwent complete physical examinations and routine biochemical analyses of blood and urine as well as an assessment of the presence and extent of macrovascular or microvascular diabetic complications. The anthropometrics parameters required to calculate body mass index (BMI) and waist to hip ratio (WHR) were measured. Seated blood pressure, plasma biochemical parameters, and urinary microalbumin were measured after overnight fasting. A trained nurse measured blood pressure with a digital automatic blood pressure monitor (model HEM-907, Omron, Tokyo, Japan) after these subjects had rested for 5 min. Plasma triglycerides, total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, uric acid, creatinine, and glucose were determined by standard commercial methods on a parallel multichannel analyzer (model 7170A, Hitachi, Tokyo, Japan). Urinary albumin concentrations were measured by immunoturbidmetry (Beckman, Galway, Ireland). The detection limit was 2 mg/liter, and the inter- and intraassay coefficients of variance were less than 8%.
Hypertension was defined as raised arterial pressure (160/90 mm Hg, or receiving antihypertensive treatment). Most of the hypertensive patients were untreated, and hypertensive patients who were receiving antihypertensive agents were asked to discontinue treatment for 2 wk before the study as described previously (13).
Resistin gene 3'UTR polymorphism
Genomic DNA was prepared from peripheral blood using standard techniques. For the resistin 3'-UTR, +62GA SNP was genotyped by amplification of genomic DNA using the primers F5'-AGA GTC CAC GCT CCT GTG TT-3' and R5'-CAT CTC CAG GTT TAT TTC CAG C-3' according to the method of Cao and Hegele (9). PCR amplification products were obtained using 25-µl reactions [0.2 µg genomic DNA; 20 pmol primers; 0.2 mmol/liter each of deoxy-ATP, GTP, CTP, and TTP; 0.5 U Taq DNA polymerase (Takara Taq, Takara Shuzo Co., Ltd., Otsu Shiga, Japan); 50 mmol/liter KCl; 1.5 mmol/liter MgCl2; and 10 mmol/liter Tris-HCl, pH 8.3] in a thermal cycler (Gene Amp PCR System 9700, PerkinElmer, Foster City, CA). The amplification conditions were as follows: 94 C for 5 min, followed by 30 cycles of 30 s each at 94, 58, and 72 C, and ending with a single 10-min extension step at 72 C. The resulting fragment was 249 bp in length. Digestion of the +62G allele with BseRI produced a single fragment of 249 bp, whereas digestion of the +62A allele produced two fragments, with lengths of 238 and 11 bp. These fragments were resolved after electrophoresis in 7% polyacrylamide gels. Genotyping was performed in a blinded fashion.
Statistical analysis
The data are shown as the mean ± SD. All statistical analyses were performed using the Statistical Package for Social Science (SPSS for Windows, version 7.5.1, 1996, SPSS, Inc., Chicago, IL) program. The statistical difference in genotype distribution and allele frequencies among the groups was assessed by the Pearson 
2 statistic. The odds ratios (ORs) and 95% confidence intervals (CIs; nonadjusted or adjusted for age, sex, BMI, WHR, and systolic and diastolic blood pressures) were calculated by logistic regression analysis. Due to the small number of individuals with the AA genotype, we tested whether the variable means differed between subjects with and without the A variant (GG vs. GA and AA). Comparison of variables between groups of genotypes was performed using the ANOVA or two-tailed t test. Before statistical testing, fasting plasma glucose and serum triglycerides were logarithmically transformed to achieve a normal distribution. Differences were considered statistically significant at P less than 0.05. The effect of genotype on the clinical parameters was also estimated by analysis of covariance using genotype as a factor and age, sex, and BMI as covariates. A multiple linear stepwise regression model was used to study the association of systolic and diastolic blood pressures with the influence of independent variables (i.e. sex; age; diabetes duration; fasting plasma sugar; hemoglobin A1c (HbA1c); serum total cholesterol, low and high density lipoprotein cholesterol, triglycerides, creatinine, and uric acid levels; BMI; WHR; and resistin 3'UTR +62GA genotypes).
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Results |
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A genotype distributions within the diabetic and nondiabetic groups are presented in Table 1
. In both of the studied groups, the genotype frequency distributions of this polymorphism were in Hardy-Weinberg equilibrium. Subjects with type 2 diabetes were found to have a lower frequency of resistin gene 3'UTR +62A variant (GG:GA/AA, 83.5%:16.5%) than that of control subjects (GG:GA/AA, 75.1%:24.9%; OR, 1.524; 95% CI, 1.268–1.831; P < 0.001), indicating that the resistin gene 3'UTR +62A variant is associated with reduced risk for the development of type 2 diabetes. This association is still observed after adjustment for sex, age, WHR, BMI, and systolic and diastolic blood pressures (OR, 2.138; 95% CI, 1.283–3.536; P < 0.001). Table 1 also shows the prevalence of hypertension in control and diabetic subjects with different resistin gene 3'UTR +62GA genotypes. Diabetic patients with GG genotype were found to have a higher prevalence of hypertension (GG:GA/AA, 49.8%:36.2%; OR, 1.375; 95% CI, 1.116–1.693; P = 0.001). When pooling the controls with diabetes patients, the resistin gene 3'UTR +62GA genotype could still be significantly associated with hypertension (GG:GA/AA, 40.0%:29.4%; OR, 1.464; 95% CI, 1.180–1.817; P < 0.001), suggesting that the resistin gene 3'UTR +62A variant is associated with reduced risk for the development of insulin-resistant related hypertension. This association is still observed after adjustment for sex, age, BMI, WHR, serum total cholesterol, high density lipoprotein cholesterol, uric acid, creatinine levels, known diabetic duration, and age of diabetes onset in diabetic patients (OR, 2.393; 95% CI; 1.437–3.985; P < 0.001).
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presents the clinical characteristics of the control group and the type 2 diabetic subjects. Diabetic subjects had significantly higher levels of fasting glucose, blood pressure, and serum total cholesterol, triglyceride, and uric acid levels than those of the control group. Diabetic females had significantly higher values of BMI and WHR than those of control females. The mean systolic and diastolic blood pressure levels in diabetic subjects with the resistin gene 3'UTR GG genotype (144 ± 21/87 ± 13 mm Hg) were significantly higher than those in subjects with GA/AA variants (139 ± 21/84 ± 14 mm Hg; P = 0.004 and P = 0.002, respectively). When pooling the controls with diabetes patients, subjects with the GG genotype continue to have significantly higher systolic and diastolic blood pressure levels. These associations are still observed after adjustment for sex, age, and BMI (P < 0.001 for both systolic and diastolic blood pressures). There were no statistically significant differences in the variables found among the control subjects with different resistin gene 3'UTR +62GA genotypes.
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). In addition to age, gender, and BMI, systolic blood pressure correlated significantly with WHR, serum uric acid, creatinine, low density lipoprotein cholesterol, and total cholesterol levels. Diastolic blood pressure was correlated significantly with BMI, WHR, and serum uric acid and triglycerides. Using multiple linear stepwise regression analysis, the resistin 3'UTR genotype continued to remain a significant factor for both systolic and diastolic blood pressures (systolic blood pressure: P = 0.001; 95% CI, -11.741 to -3.081; diastolic blood pressure: P < 0.001; 95% CI, -7.611 to -2.200). Thus, the resistin 3'UTR +62GA genotype was found to be an independent contributing factor for both systolic and diastolic blood pressures in diabetic population.
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Discussion |
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A variant in the 3'UTR of the resistin gene might have an influence on resistin gene expression. Our data show that the 3'UTR polymorphism of the resistin gene is associated with type 2 diabetes and hypertension and may have some implications for resistin gene functions in human pathophysiology. Recently, investigators systematically searched for the resistin gene polymorphism and reported that they could find no evidence for the association of resistin gene polymorphism with type 2 diabetes in Caucasian and Japanese populations (16, 17, 18). Wang et al. (19) screened 44 subjects with type 2 diabetes and 20 nondiabetic family members and found 8 noncoding SNPs and 1 GAT microsatellite repeat of the resistin gene. No SNP was associated with type 2 diabetes, but the SNP in the promoter region was a significant determinant of the insulin sensitivity index. They concluded that noncoding SNPs in the resistin gene might influence insulin sensitivity in interaction with obesity. More recently, Pizzuti et al. (20) reported that an ATG repeat in the 3'UTR of the resistin gene is associated with a decreased risk of insulin resistance in Caucasians. These facts give further evidence to suggest that SNPs or 3'UTR polymorphisms in the resistin gene have some functional reference to insulin resistance and insulin-resistance related metabolic derangements.
There have been reports providing evidence that insulin resistance plays some part in blood pressure regulation (21, 22). Although a number of underlying principles have been proposed, the cause behind the association between insulin resistance and hypertension remains controversial. There is evidence that insulin-mediated vasodilatation is impaired in an insulin-resistant state (21, 22, 23), and it is possible that insulin resistance not only affects glucose metabolism, but also vasodilatation, eventually leading to hypertension. Various thiazolidinediones have been shown to have antihypertensive properties in experimental rats (24, 25, 26) and in human subjects (23, 27, 28). Nolan et al. (27) demonstrated that troglitazone resulted in a significant reduction in both systolic and diastolic blood pressures, as assessed by 24-h ambulatory monitoring, in normotensive insulin-resistant human subjects accompanied by improved glucose tolerance and enhanced insulin sensitivity.
Disordered fat storage, mobilization, and failure of fat cell proliferation may result in ectopic fat storage, insulin resistance, and type 2 diabetes (29, 30). As resistin plays a role in adipocyte differentiation (31), we proposed that the 3'UTR +62GA polymorphism might affect resistin gene expression and influence adipocyte differentiation and maturation, and thus contribute to the pathogenesis of insulin resistance and type 2 diabetes. The association of hypertension with the resistin gene polymorphism was unexpected, whereas it is well known that there are strong relationships between obesity, insulin resistance and hypertension (32, 33). Our findings that the resistin gene 3'UTR +62GA polymorphism was associated with blood pressure levels in patients with type 2 diabetes only and that the association was not observed in control subjects give further support to the suggestion that insulin resistance plays a role in the development of hypertension. The pathogenesis of obesity and insulin resistance-related hypertension is a complex process in which adipocyte-derived cytokine and insulin resistance-related endothelial dysfunction and an activated sympathetic nervous system possibly play some role (32). Our results provide a strong link between insulin resistance and hypertension, suggesting that either insulin resistance or an adipocyte-related cytokine might play a role in the pathogenesis of hypertension. Further experiments should be conduced to clarify the pathophysiological mechanism.
As resistin gene expression is regulated by thiazolidinediones, and resistin has been shown to play some part in insulin resistance (1), we conclude that resistin may have significant clinical implications for the pathogenesis of type 2 diabetes and insulin resistant-related hypertension. Because we only screened for the 3'UTR +62GA polymorphism, we cannot exclude the role of other resistin variant or variants in nearby genes, possibly in linkage disequilibrium with the +62GA variant. Some problems exist in this case-control study, such as difficulty in defining comprehensive controls and lack of reproducibility by examining multiple samples. Further studies, including functional analyses, family-based researches, and large population sample investigations, will be required to fully clarify the role of the resistin gene in type 2 diabetes and hypertension.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMI, Body mass index; CI, confidence interval; HbA1c, hemoglobin A1c; OR, odds ratio; SNP, single nucleotide polymorphism; UTR, untranslated region; WHR, waist to hip ratio.
Received September 16, 2002.
Accepted December 11, 2002.
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