The Influence of Adiponectin Gene Polymorphism on the Rosiglitazone Response in Patients With Type 2 Diabetes

A total of 166 patients were treated with rosiglitazone (4 mg/day) during a 12-week treatment course without changing previous medications. Type 2 diabetic patients with a HbA_1c values of 7.5–11.5% and fasting plasma glucose (FPG) levels of 7.8–14.0 mmol/l; (140–252 mg/dl) were enrolled in this study. The inclusion criteria were 1) age 35–80 years, 2) BMI 18.5–30 kg/m², 3) no history of PPAR agonist use, 4) no medication changes in the previous 3 months, and 5) for women, postmenopausal or using appropriate contraceptive methods. Patients with type 1 diabetes, any history of ketoacidosis, ischemic heart disease, or congestive heart failure (New York Heart Association II–IV) or who were receiving insulin therapy and pregnant or lactating women were excluded from this study. The patients were advised to consume a fixed-calorie diet and maintain a constant level of physical activity throughout the study. The Institutional Review Board of Yonsei University College of Medicine approved the study protocol. All subjects were provided adequate information about this study and gave their informed consent.

Blood samples were collected after an overnight fast. The FPG and total cholesterol and triglyceride levels were determined using an enzymatic colorimetric assay. The HDL cholesterol concentration was measured using lipoprotein electrophoresis. The LDL cholesterol level was calculated using the Friedewald formula (12). The insulin concentration was measured using a radioimmunoassay kit (DAINABOT, Tokyo, Japan). The homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by the formula: (fasting insulin [microunits per milliliter] × fasting glucose [millimoles per liter])/22.5. The HbA_1c value was determined using a high-performance liquid chromatography method (Varient II; GREENCROSS, Seoul, Korea). The serum adiponectin concentration was measured using a commercial radioimmunoassay kit (Linco Research, St. Charles, MO).

SNP45 and -276 were chosen because they are common (frequency >20%) in Korean type 2 diabetic patients and have been reported to be associated with type 2 diabetes in other Asian populations (10,11). SNP-11377 was also genotyped. The genomic DNA was extracted from leukocytes in the whole-blood samples using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA). PCR amplification was performed in a total volume of 20 μl containing the PCR buffer (10 × Optiperform Buffer III of 40 mmol/l KCl, 10 mmol/l Tris-HCl, 1.5 mmol/l MgCl₂), 45 ng/μl genomic DNA, 1 unit of Taq DNA polymerase, 250 μM concentrations each dNTPs, and 10 pmol of the sense and antisense primers. The PCR conditions and primer sequences used for ACDC PPRE, SNP45 and SNP276 are shown in the online appendix (available at http://care.diabetesjournals.org). The PCR products were genotyped by sequencing with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Data are shown as means ± SD. All calculations and statistical analyses were performed using the SPSS for Windows software (version 12.0; SPSS, Chicago, IL). A paired t test was used to evaluate the effects of rosiglitazone on the metabolic parameters. Comparisons of the continuous variables among the genotypes were assessed using an ANOVA test, and a post hoc Bonferroni correction was made to control the effects of multiple comparisons. The statistical analysis of triglyceride, HDL cholesterol, fasting insulin, and HOMA-IR levels was performed using log-transformed values because the distribution was not normal. P values <0.05 were considered significant. The allelic distribution of each SNP was verified using Hardy-Weinberg equilibrium. The haplotypes at locus 45 and locus 276 for each individual were inferred by a maximum likelihood estimation method with the Haplotyper program (http://www.people.fas.harvard.edu/∼junliu/Haplo).

Table 1 shows the allele, genotype, and haplotype distribution of ACDC. No significant deviation from Hardy-Weinberg equilibrium was observed for either locus. Table 2 shows the clinical characteristics of the patients before and after rosiglitazone treatment. The FPG levels and HbA_1c values were significantly lower after 12 weeks of treatment compared with the baseline. The serum adiponectin concentration was significantly higher after the rosiglitazone treatment (5.30 ± 4.79 μg/ml vs. 9.92 ± 6.81 μg/ml, P < 0.001). No mutation was observed at the PPRE of the adiponectin gene promoter region in any of 166 patients. Tables 3 and 4 show the clinical and biochemical characteristics of the patients according to the SNP45 and SNP276 genotypes. There were no significant differences in terms of age, duration of diabetes, and BMI according to the SNP45, SNP276, and SNP-11377 genotypes. The FPG levels, HbA_1c values, and the plasma lipid profiles were not significantly different among the genotypes of SNP45, SNP276, and SNP-11377 at baseline (Tables 3 and 4 and online appendix Table A-3, respectively).

Regarding SNP45, there was a significant difference in the decrease in the FPG level between the GG genotype and the other genotypes (TT + TG, 1.68 ± 2.40 mmol/l; GG, 0.25 ± 2.95 mmol/l; P = 0.031) (Table 5). The degree of the reduction in the HbA_1c value was smaller for the GG genotype than for the other genotypes (TT + TG, 0.85 ± 1.05%; GG, 0.05 ± 1.43%; P = 0.013) (Table 5). In addition, the degree of the increase in the serum adiponectin concentration was significantly smaller in the subjects with the GG genotype than in the subjects with the other genotypes (TT + TG, 4.81 ± 5.07 μg/ml; GG, 1.67 ± 4.45 μg/ml; P = 0.003) (Table 5).

Regarding SNP276, the degree of the decrease in the FPG level was significantly smaller in those patients with the GG genotype than in those with the other genotypes (GG, 0.86 ± 2.58 mmol/l; GT + TT, 2.23 ± 2.27 mmol/l; P = 0.001) (Table 5).

There were no significant differences in terms of insulin resistance, decrease in the FPG, decrease in the HbA_1c, and increase in the serum adiponectin levels according to SNP-11377 genotype (data are shown in the online appendix Table A-3).

In the haplotype analysis, the degree of the decrease in the FPG level was smaller in those with the GG homozygote haplotype than in those with the other haplotypes (GG homozygote haplotype, 0.03 ± 3.15 mmol/l; other haplotypes, 1.69 ± 2.40 mmol/l; P = 0.001) (Table 5). The patients with the GG homozygote haplotype had a smaller decrease in the HbA_1c level than those with the other haplotypes (GG homozygote haplotype, 0.18 ± 1.23%; other haplotypes, 0.85 ± 1.05%; P = 0.001) (Table 5). In addition, the degree of the increase in the serum adiponectin level after the rosiglitazone treatment was less in those patients with the GG homozygote haplotype than the subjects with the other haplotype (GG homozygote haplotype, 0.82 ± 3.62 μg/ml; other haplotypes, 4.81 ± 5.07 μg/ml; P = 0.003) (Table 5).

Recently, Iwaki et al. (13) characterized the PPAR-γ binding site, PPRE, in the human adiponectin gene promoter. Although a great deal of effort has been made to identify the mutation in this region, this study could not detect any mutation in the 166 type 2 diabetic patients examined.

Previous studies have shown that thiazolidinediones increase the serum adiponectin concentration by increasing the level of adiponectin transcription (14,15). Also, the association between an adiponectin gene polymorphism and the risk of type 2 diabetes has been examined in many studies (9–11,16). SNP45 and SNP276 were reported to be associated with type 2 diabetes in a Japanese population (10), whereas it was reported that SNP-11391 and SNP-11377 were related to type 2 diabetes in a French population (16). Therefore, the SNPs of ACDC for type 2 diabetes and insulin resistance appear to be different among different populations. This prospective intervention study was performed to evaluate how rosiglitazone response varies with adiponectin gene polymorphisms.

Our data suggest that there is no association between SNP-11377 genotype and rosiglitazone response, but patients with the GG genotype at SNP45 and/or the GG genotype at SNP276 are unlikely to respond to rosiglitazone. In addition, a smaller increase in the serum adiponectin concentration was observed in patients with the SNP45 GG genotype than for those with the other genotypes. It was also observed that the degree of the decrease in the FPG level and HbA_1c value was smaller in the patients with the GG homozygote haplotype than in those with the other haplotypes. In addition, patients with the GG homozygote haplotype had a smaller degree of increase in the serum adiponectin levels than in those with the other haplotypes. Patients with the GG homozygote haplotype are unlikely to respond to rosiglitazone.

The ACDC SNP45 is located in exon 2 and is a silent mutation for Gly15 (GGT to GGG). However, it might inactivate the gene or affect the adiponectin concentration by influencing the pre-mRNA splicing or the stability of the mRNA (17). Alternatively, it may be related to another functional locus via a linkage disequilibrium not yet identified. SNP276 is located in intron two of ACDC. The intronic SNP can affect the expression level of the gene via an unknown mechanism. There may be a specific linkage structure or gene-environmental interaction.

Although baseline and posttreatment FPG, HbA_1c, and serum adiponectin levels were not significantly different between the GG homozygote haplotype and the other haplotypes, there were significant differences in the changes of the FPG, HbA_1c, and serum adiponectin levels. The difference in either the adiponectin transcription activity or the adiponectin mRNA stability according to the ACDC genotypes could be responsible for the reduced serum adiponectin concentration in the GG homozygote haplotype compared with the other haplotypes. A low adiponectin level in patients with the GG homozygote haplotype may result in increased insulin resistance, which in turn contributes to a smaller decrease in the FPG levels and HbA_1c values by several mechanisms (18,19). Multiple regression tests were performed and revealed that age, sex, and BMI were not found to be major confounding factors of serum adiponectin levels according to the ACDC genotypes (data shown in online appendix).

Our findings are in contrast to those reported by Yang et al. (20), who found that subjects with the TT genotype at SNP45 are associated with insulin resistance. This discrepancy could be due to the different study population. Yang et al. (20) examined nondiabetic subjects, which is in contrast to the diabetic patients in this study. Moreover, they used HOMA-IR as an index of insulin resistance, which is not believed to be an accurate indicator for insulin resistance in patients with a low BMI and insulin secretory defect (21). Menzaghi et al. (22) reported that subjects with the TT genotype at SNP276 had a higher serum adiponectin level than the other genotypes. However, no difference was observed in the serum adiponectin level at the baseline among the SNP276 genotypes in this study. This discordance could be explained by differences in the ethnicity or specific study population.

In conclusion, this study suggests that patients with G allele homozygosity at locus 45 and locus 276 are unlikely to respond to rosiglitazone. It was found that variations in the adiponectin gene could affect the rosiglitazone treatment response to the serum adiponectin level and blood glucose control. These findings may be clinically relevant in the prediction of patients who will best respond to rosiglitazone treatment. However, further investigations will be needed to elucidate the functional mechanism of these polymorphisms.

This study was supported by the Brain Korea 21 Project for Medical Science, Yonsei University, and by Grant 03-PJ10-PG13-GD01-0002 from the Korea Health 21 R&D Project, Ministry of Health and Welfare.