EFFECT OF THE Pro12Ala POLYMORPHISM OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ2 GENE ON LIPID PROFILE AND ADIPOKINES LEVELS IN OBESE SUBJECTS
Becer E1,2,*, Çırakoğlu A3
*Corresponding Author: Eda Becer, Ph.D., M.Sc., Department of Biochemistry, Faculty of Pharmacy, Near East University, Nicosia, Mersin 10, Turkey. Tel: +90-392-680-2000, Ext: 128. Fax:+90-392-680-2038. E-mail: edabecer@yahoo.com
page: 71

MATERIALS AND METHODS

Subjects. This study was performed on two groups. One group was composed of 160 obese patients having a mean age of 41.4 ± 8.19 years and BMI 34.12 ± 6.84 kg/ m2. The second group was composed of 140 non obese subjects. The mean age of subjects was 39.6 ± 9.63 years and their mean BMI was 21.56 ± 4.65 kg/m2. Obese patients were recruited from the Endocrinology Department at the Famagusta Goverment Hospital, Famagusta, Cyprus, and non obese controls from the general population. None of the participants had hypertension, liver, kidney, thyroid, cardiovascular or any active inflammatory diseases and they were also questioned as to whether they were undergoing any medical therapy that might affect the lipid and glucose metabolism. The participants neither received any medications nor participated in any dietary or exercise programs. All subjects provided written informed consent before enrollment in the study, and the study was approved by the Near East University Research Ethics Committee. Anthropometric Measurements. All the measurements were performed in the morning with the patients in a fasting state and anthropometric measurements, including weight (kg), height (m), hip circumference (cm) and waist circumference (cm) of each subject were measured barefoot and lightly clothed. Hip circumference was measured by placing a tape measure around the patient’s hips at the level of the prominence over the greater trochanter of both femurs. Waist circumference was taken midway between the lowest rib (laterally) and the iliocristale landmark by flexible tape. The BMI was calculated as body weight (kg) divided by the square of height (m2) and obesity was defined as BMI ≥30 kg/m2 [14]. Biochemical Parameters. Blood samples were obtained after an overnight fast. The levels of serum glucose, triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were measured by fully automated clinical chemistry analyzer (Abbott Architect C8000; Abbott Laboratories, Abbott Park, IL, USA). Fasting insulin concentrations were measured by electro chemiluminescence kit (Ref. 12017547) (Elecsys Corporation, Lenexa, KS, USA). Insulin resistance index were calculated using the HOMA-IR, as the product of fasting insulin (µU/mL) and fasting glucose (mmol/L) divided by 22.5 [15]. Serum leptin (ng/mL), resistin (ng/mL), chemerin (ng/mL) and adiponectin levels (µg/mL) were measured using commercially available enzyme linked immunosorbent assay (ELISA) kits (DRG International Inc., Springfield Townsip, NJ, USA for leptin and Biovendor Laboratory Inc., Brno, Czech Republic for adiponectin, chemerin and resistin) according to the manufacturers’ protocols. The PPARγ2 Gene Pro12Ala Polymorphism. In the screening phase, we assayed for the Pro12Ala (rs1801282) genomic variation of the PPARγ2 gene that exhibited associations with obesity, T2DM and insulin sensitivity in previous population studies [8-11]. The studies involving the PPARγ2 gene Pro12Ala polymorphism on adipokine levels such as leptin, adiponectin, resistin and chemerin in obese subjects was limited. For this reason, we decided to study the PPARγ2 gene Pro12Ala polymorphism. Genomic DNA was extracted from whole blood by the salting out procedure [16]. Genotyping of the PPARγ2 gene Pro12Ala polymorphism (rs1801282) was carried out using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with previously described primer pairs [6]. The PCR reactions were performed on a total volume of 50 µL using 1 µg of genomic DNA, 0.4 µM of each primer, 23.5 µL nuclease-free water (Fermentas International Inc., Burlington, ON, Canada) and 25 µL DreamTaq PCR Master Mix (Fermentas International Inc.). The PCR consisted of one cycle of initial denaturation for 5 min. at 95 °C, followed by 30 cycles denaturation for 1 min. at 95 °C, annealing for 1 min. at 56 °C and extension for 1 min. at 72 °C, and a final extension at 72 °C for 10 min. Electrophoresis was conducted to confirm the 270 bp PCR products. The PCR products were digested for 2 hours at 37 °C with BstUI restriction enzyme (Bsh 12361; Thermo Fisher Scientific, Waltham, MA, USA). Digest products were visualized on a 2.5% agarose gel stained with ethidium bromide. The expected products after digestion with BstUI were 270 bp for normal homozygotes, 227 and 43 bp for Pro12Ala homozygotes, and 270, 227, and 43 bp for heterozygotes. The quality of SNP genotyping was verified by independently replicating the genotyping using randomly selected samples. The results from quality control were 100.0% in agreement with the initial genotyping results. Statistical Analysis. Distribution of continuous variables in groups were expressed as mean ± standard deviation (SD). Differences in baseline characteristics between groups were analyzed by Student’s t-test. The χ2 analyses were used to compare the categorical variables, to compare the association between the genotypes and alleles in relation to obese and non obese subjects, and to test for deviation of the genotypic distribution from Hardy-Weinberg equilibrium (HWE). Analysis of variance (ANOVA) was used to compare means of continuous variables in the three genotype subgroups. The differences in the mean values of continuous variables in the three genotype subgroups were confirmed by the post hoc Tukey test. A p value of less than 0.05 was considered statistically significant. All statistical analyses were performed with the Statistical Package for the Social Sciences (SPSS) version 15.0 (SPSS Inc., Chicago, IL, USA).



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