Plasma Resistin, Associated With Single Nucleotide Polymorphism −420, Is Correlated With Insulin Resistance, Lower HDL Cholesterol, and High-Sensitivity C-Reactive Protein in the Japanese General Population

RESEARCH DESIGN AND METHODS

All subjects were native to Japan. We analyzed community-dwelling subjects attending a yearly medical checkup in a rural town located in Ehime prefecture, Japan, in 2002. Of the 2,889 subjects who agreed to participate, 2,078, for whom overnight fasting plasma samples (>11 h) were available, were analyzed for plasma resistin levels. Because of the availability of plasma samples, immunoreactive insulin (IRI) and hs-CRP were measured in 2,017 and 1,875 subjects, respectively. Of the 2,078 subjects, 157 with A1C levels <5.6%, FPG levels <110 mg/dl, no history of diabetes, and no evidence of diabetes within first-degree relatives were used as nondiabetic control subjects in a previous study (9). There was no overlapping of samples between the present study and the other previous study (8). Of the 2,078 subjects, 151 were considered diabetic because they were being treated with antihyperglycemic agents or had FPG levels of ≥126 mg/dl. The association between SNP −420 and diabetes was not significant, possibly because of the lack of power using the small numbers of diabetic subjects. The plasma samples were immediately separated, frozen, and stored at −80°C. The baseline characteristics of the study subjects, such as alcohol habituation, history or symptoms of CVD, and medication, were investigated in an individual interview using a structured questionnaire. The clinical characteristics of these subjects are summarized in Table 1. All subjects were informed of the purpose of the study and their consent was obtained. The study was approved by the ethics committee of the Ehime University Graduate School of Medicine. Definitions used are as follows: obesity, BMI ≥25 kg/m²; impaired glucose tolerance, FPG ≥110 mg/dl (6.1 mmol/l) and/or under medication of antihyperglycemic agents; high blood pressure, systolic blood pressure (SBP) ≥140 mmHg and/or diastolic pressure ≥90 mmHg and/or under medication with antihypertensive agents; hypertriglyceridemia, triglyceride levels ≥150 mg/dl (1.69 mmol/l) and/or under medication with antihyperlipidemic agents; and low HDL cholesterol, HDL cholesterol <40 mg/dl (1.04 mmol/l). CVD includes stroke, myocardial infarction, and angina pectoris. Because Japanese individuals are generally leaner than Caucasians, BMI ≥25 kg/m² was used as the standard cutoff value for the diagnosis of obesity (19). Waist circumference data were not available in this study. Blood pressure was measured using an automatic cuff-oscillometric device with an appropriately sized cuff on the left arm (BP-103i; Colin, Aichi, Japan) after a resting period of at least 5 min in the sitting position.

SNP typing

SNP −420 was typed by TaqMan analysis (Applied Biosystems). The probes used were VIC 5′-CATGAAGACGGAGGCC-3′ for −420C and FAM 5′-ATGAAGAGGGAGGCC-3′ for −420G. Forward and reverse primers were 5′-CCACCTCCTGACCAGTCTCT-3′ and 5′-AGCCTTCCCACTTCCAACAG-3′, respectively. When required, PCR direct sequencing was performed as previously described (8,20).

Measurement of plasma resistin and hs-CRP levels

Plasma resistin was measured using a human resistin enzyme-linked immunosorbent assay kit (LINCO Research) following the manufacturer's protocol as described (8). The linearity was maintained <0.16 ng/ml. Inter- and intra-assay coefficients of variation (CVs) were 6.9 and 1.7% (low levels) and 7.2 and 8.1% (high levels), respectively. The kit used had a good correlation with the other kit (r = 0.978; y = 2.216x + 8.0, where y is this kit and x is BioVender's kit). Plasma hs-CRP concentration was measured using a previously validated assay system (Dade Behring) (21). Inter- and intra-assay CVs were 3.2 and 6.7%, respectively.

Statistical analysis

To examine effects of SNP −420 on plasma resistin, a multiple regression analysis involving SNP −420, age, sex, and BMI as independent variables and plasma resistin as a dependent variable was used. In this analysis, the genotypes for SNP −420, C/C, C/G, and G/G were denoted by two dummy variables (c1 and c2 [0 and 0, 1 and 0, and 0 and 1, respectively]). To examine the relation of plasma resistin with age, sex, BMI, SBP, HDL cholesterol, triglyceride levels, FPG, IRI, HOMA-IR, or hs-CRP, simple regression analysis involving plasma resistin as a dependent variable was performed. A multiple regression analysis was then performed using only the significant factors. HOMA-IR, HDL cholesterol, hs-CRP, or SBP was analyzed as a dependent variable, and plasma resistin, age, sex, and BMI were involved as independent variables. CVD was involved as a dependent variable in logistic regression analysis. ANOVA was used where indicated. All analyses were performed with SPSS version 14.0J (SPSS, Chicago, IL). Bonferroni's correction was applied to the initial analyses of the relation between plasma resistin and either categories (raw P value ×9 for ANOVA) or continuous parameters (raw P value ×10 for simple regression analysis) and the subsequent multiple and logistic regression analyses using factors selected from these results (raw P value ×5). The proportion of variance of plasma resistin explained by SNP −420 was assessed based on results of a simple regression analysis. Power was calculated based on the observed effect and sample sizes using general linear model for ANOVA (simple and multiple regression analyses with α = 0.05). Null hypotheses were rejected at P < 0.05.

RESULTS

SNP −420 was associated with plasma resistin in the Japanese general population

We first assessed plasma resistin based on each genotype of SNP −420 in 2,078 subjects (Fig. 1A). Fasting plasma resistin was highest in subjects with the G/G genotype, followed in order by those with C/G and those with C/C (F = 368.6, P < 0.0001, power = 0.999). This association was consistent when analyzed in either male (F = 150.6, P < 0.0001) or female (F = 221.3, P < 0.0001) subjects. When 50, 20, and 5% of the subjects were randomly selected and compared using the SPSS program, these P values were consistently low (P < 0.0001). Therefore, plasma resistin was associated with SNP −420 in this population.

We then examined the number of subjects in each 2.5 ng/ml range of plasma resistin concentration based on the SNP −420 genotype (Fig. 1B). The plasma resistin at the highest number of subjects with each genotype appears to be in the order of G/G > C/G > C/C. The range of plasma resistin was broadest in subjects with G/G, followed in order by C/G and C/C (1.9–52.7, 2.2–46.2, and 2.2–35.2 ng/ml, respectively), suggesting that factors other than SNP −420 genotype may affect plasma resistin.

To examine isolated effects of SNP −420 on plasma resistin, a multiple regression analysis involving SNP −420, age, sex, and BMI as independent variables was used. The SNP −420 genotype including G alleles (G/G vs. C/C, P < 0.001, power = 0.999 and C/G vs. C/C, P < 0.001, power = 0.999), higher age (P < 0.001, power = 0.999), and female sex (P = 0.001, power = 0.894), but not higher BMI (P = 0.195, power = 0.254), was positively correlated with plasma resistin. The standardized coefficient (β) of the G/G genotype compared with C/C was highest (β = 0.480), followed by that of C/G compared with C/C (β = 0.384) (age, β = 0.100; female sex, β = 0.060; and BMI, β = 0.024). Therefore, SNP −420 genotype was the strongest determinant of plasma resistin among these factors. The contribution of this genotype to the observed total variance of resistin (R²) was 26.1%.

Plasma resistin was higher in elderly individuals, female subjects, nondrinkers, and subjects with high blood pressure

We then examined mean plasma resistin in each category without considering the SNP −420 genotype. Plasma resistin was higher in elderly individuals (aged ≥65 years) (mean ± SD 12.2 ± 7.1 vs. 10.9 ± 6.1; ANOVA P < 0.001, power = 0.994), female subjects (11.9 ± 6.6 vs. 11.0 ± 6.6; P = 0.003, power = 0.852), nonhabitual alcohol drinkers (12.0 ± 6.7 vs. 10.4 ± 6.3; P < 0.001, power = 0.999), subjects with high blood pressure (11.9 ± 6.9 vs. 11.0 ± 6.2; P = 0.001, power = 0.905), those with low HDL cholesterol (13.0 ± 8.3 vs. 11.4 ± 6.5; P = 0.014, power = 0.693), and those with a history of CVD (12.5 ± 6.7 vs. 11.4 ± 6.6; P = 0.045, power = 0.516). Age, sex, alcohol drinking, and high blood pressure remained significant after Bonferroni's correction. Obesity (P = 0.613, power = 0.080), IGT (P = 0.733, power = 0.063), or hypertriglyceridemia (P = 0.497, power = 0.104) was not associated with plasma resistin.

Age, female sex, SBP, low HDL cholesterol, HOMA-IR, and hs-CRP were correlated with plasma resistin

We then examined which factors are correlated with plasma resistin (Table 2). Simple regression analysis revealed that age, female sex, SBP, low HDL cholesterol, IRI, HOMA-IR, and hs-CRP were correlated with plasma resistin. Each of these P values remains significant after Bonferroni's correction. BMI, triglyceride levels, and FPG were not correlated with plasma resistin. Therefore, with possible effects of age and sex, high plasma resistin was correlated with insulin resistance, low HDL cholesterol, high SBP, and high hs-CRP.

Plasma resistin was correlated with HOMA-IR, low HDL cholesterol, or hs-CRP, independent of age, sex, and BMI

To examine isolated effects of plasma resistin on each factor, a multiple regression analysis adjusted for age, sex, and BMI was performed (Table 3). Factors significantly associated with plasma resistin in Table 2, namely, HOMA-IR, HDL cholesterol, hs-CRP, and SBP, were individually analyzed as a dependent variable. Among these factors, only HOMA-IR, low HDL cholesterol, and hs-CRP were correlated with plasma resistin, with the caution that plasma resistin has a relatively small effect on these parameters based on the regression coefficients. Therefore, plasma resistin, associated with SNP −420, was correlated with HOMA-IR, low HDL cholesterol, and hs-CRP, independent of age, sex, and BMI.

CONCLUSIONS

Our cross-sectional study that included 2,078 subjects from the Japanese general population shows that plasma resistin was associated with SNP −420. Plasma resistin was higher in the elderly, female subjects, nondrinkers, and subjects with high blood pressure. Multiple regression analysis adjusted for age, sex, and BMI revealed that plasma resistin was an independent factor for HOMA-IR, low HDL cholesterol, and hs-CRP.

We found that SNP −420 was associated with plasma resistin in the order G/G > C/G > C/C in a large number of samples. This finding provides strong evidence for a tight correlation between a functional promoter SNP and its gene product as the final output in humans. The association is also supported by studies in which smaller numbers of samples were used, namely by Cho et al. (11) and ourselves (8). Haplotypes including SNP −420 also show this similar tendency in Japanese subjects (22). A total of four independent groups reported that the activity of the mutant RETN promoter including −420G is higher than that of the wild type including −420C in vitro (8,11,22,23). Therefore, we propose that SNP −420 is a determinant of plasma resistin. Because only SNP −420 was typed in this study, the other SNPs in RETN should be analyzed to further examine this hypothesis.

Our findings have shown that plasma resistin was positively associated with HOMA-IR, independent of age, sex, and BMI. To our knowledge, the positive correlation between circulating resistin and HOMA-IR in humans is supported in 2 of >10 previous studies, whereas the role of resistin as a factor inducing insulin resistance has been established in mice (16,24). The lower power with small numbers of subjects may account for this difference. The broader range of the assay used in this study could also be a contributing factor. It should be noted that serum resistin probably exists as a hexamer (major form) or trimer (a more biologically active form) in mice, which may also affect the assay results (25). The existence of multimers in human serum has recently been implicated (26).

We have shown that plasma resistin was inversely associated with serum HDL cholesterol, independent of age, sex, and BMI. Resistin was reported to be associated with low HDL cholesterol in a smaller numbers of subjects (27,28). Overexpression of resistin in the liver using adenovirus in mice shows enhanced insulin resistance, low serum HDL cholesterol, and high triglyceride levels, which resembles the metabolic syndrome in humans (29). Insulin is known to upregulate lipoprotein lipase, a critical factor producing HDL cholesterol through lipoprotein metabolism. Therefore, insulin resistance caused by elevated plasma resistin could result in reduced serum HDL cholesterol.

We found that plasma resistin was positively associated with hs-CRP. Shetty et al. (28) and McTernan et al. (15) reported that resistin is positively correlated with C-reactive protein (CRP) in a cross-sectional analysis of 77 subjects having diabetes or its risk and 45 type 2 diabetic subjects, respectively. Al-Daghri et al. (30) showed that resistin is associated with CRP in subjects with type 2 diabetes or coronary artery disease in the Saudi Arabian population. Reilly et al. (31) reported that plasma resistin is correlated with inflammatory markers and is predictive of coronary atherosclerosis in humans, independent of CRP. In vitro, resistin increases the expression of critical factors involved in atherosclerotic lesion, such as vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 (32,33). Resistin also enhances human aortic smooth muscle cell proliferation (34). Therefore, resistin could enhance vascular inflammation resulting in elevated serum hs-CRP, whereas an inflammatory cascade has been proposed to lead to hyperresistinemia in humans (35).

In summary, SNP −420 was associated with plasma resistin in the Japanese general population. Plasma resistin was correlated with insulin resistance, lower HDL cholesterol, and high hs-CRP. It is not clear what genetic or environmental factors other than SNP −420, age, and sex affect plasma resistin and how resistin induces insulin resistance in humans. Further studies will be required to clarify these points.