2.1. Study participants
This cross-sectional study was conducted in Tabriz, the city in the north-west of Iran, from December 2017 to April 2019 among apparently healthy obese subjects using convenience sampling method. Study participants included 288 adult subjects (20-50 years old) who were obese (body mass index (BMI) ≥ 30 kg/m2). Detailed illustration of recruitment of research participants has been provided elsewhere . Individuals were excluded if they met any of the following criteria: menopause, pregnancy, lactation, a history of cardiovascular diseases, hypertension, hyperlipidemia, cancer, type 2 diabetes mellitus, renal diseases, taking any medication affecting the variables studied (hypoglycemic, lipid-lowering, antihypertensive, corticosteroids, or antidepressants). The written informed consent was obtained from all the participants at the beginning of the study. The study protocol was approved and registered by the ethical committee of Tabriz University of Medical Sciences (Ethics number: IR.TBZMED.REC.1397.266).
2.2. Demographic and anthropometric assessments
Information of physical activity and other demographic characteristics (age, sex) were collected by trained interviewer at the beginning of the study. Physical activity level was also estimated using the short form of the international physical activity questionnaire (IPAQ) . Anthropometric measurements including weight, height, waist circumference (WC), and hip circumference (HC) were conducted by a trained dietitian. Weight was measured in minimally clothed subjects using a Seca scale (Seca, Germany) to the nearest 0.01 kg. Height was measured in light clothing and without shoes by means of a tape measure with a precision of 0.1 cm. The narrowest level and the largest part were used to measure the waist and hip circumference (WC and HC), respectively, with an unscratched tape without applying any pressure to the body. Assessments of body composition were performed using bioelectrical impedance analysis (BIA) technology (Tanita, BC-418 MA, Tokyo, Japan). Waist-to-hip ratio (WHR) was calculated by dividing WC to HC. Systolic and diastolic blood pressure (SBP and DBP) were measured after at least 10 min of rest in the seated position. This measurement was carried out two times and the average of them was considered as subjects BP.
. 2.3. Biochemical measurements
Blood samples were obtained from each study participant after 10-12 hour of fasting. Plasma and serum were prepared by centrifugation (10 min at 4500 rpm, 4°C) and their aliquots were stored at − 80 °C until assay. Serum concentrations of glucose, total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) were measured by commercial kits (Pars Azmoon Inc, Tehran, Iran). As well as, serum low-density lipoprotein cholesterol level (LDL-C) was calculated using friedewald equation. Insulin, α-MSH and AgRP concentrations were analyzed by enzyme-linked immunosorbent assay kits (Bioassay Technology Laboratory, Shanghai Korean Biotech, Shanghai City, China) based on manufacturer’s instructions. Moreover, following formulas were applied to compute homeostasis model assessment insulin resistance index (HOMA-IR) and quantitative insulin sensitivity check index (QUICKI) values.
HOMA-UR = [fasting glucose (mmol/L) × fasting insulin (μIU/mL)] / 22.5 
QUICKI = 1 / [log fasting glucose (mmol/L) + log fasting insulin (μIU/mL)] 
2.4. Dietary assessments and dietary TAC calculation
Dietary intake of participants over the last year was evaluated using a 147-item semi-quantitative food frequency questionnaire (FFQ) which was validated in the previous studies [25, 26]. Interviewer asked participants to report their frequency and amount of each food item during the previous year based on a daily, weekly, monthly and yearly basis. Portion sizes of recorded food items were converted to grams by using household measures .Then, daily nutrient consumption was analyzed by Iranian Food Composition Table (FCT) . Information missing from this FCT was completed with the United States Department of Agriculture FCT .
NEAC is an indicator of diet quality which describes the ability of food antioxidants to scavenge free radicals. To estimate the dietary NEAC for each subject, four assays were used: ferric reducing-antioxidant power (FRAP)  which measures the ability of dietary antioxidants to reduce ferric to ferrous ions, total radical-trapping antioxidant parameter (TRAP)  that evaluates the chain-breaking antioxidant potential for scavenging peroxyl radicals, oxygen radical absorbance capacity (ORAC)  which measures the antioxidant capacity against peroxyl radicals using an area-under-curve (AUC) technique and Trolox equivalent antioxidant capacity (TEAC)  which is based on scavenging ability of antioxidants against a radical cation in both lipophilic and hydrophilic environments, Since maillard products of the coffee, which is produced during roasting process, are the main contributors to the high antioxidant capacity of coffee in vitro  and also its still unknown whether these products are efficiently absorbed and exert an antioxidant effect in vivo , we decided to exclude the contribution of coffee to NEAC. Dietary NEAC values for 64, 63, 59 and 65 food items in the FFQ were assigned by ORAC, FRAP TRAP and TEAC, respectively. Finally, total dietary NEAC was calculated by multiplying the NEAC values of individual foods by the amount of each food consumed and then summing up these to obtain dietary NEACE for each participant.
2.5. DNA extraction and genotyping:
The extraction of genomic DNA from blood samples was performed by phenol/chloroform extraction method. Nano Drop 2000C spectrophotometer was used to determine the concentration and purity of the DNA extracted from each sample. Genotyping the CARTPT rs2239670 variant was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) method using the following primers: forward 5′-CCTGCTGCTGATGCTACCTCT-3′ and reverse 5′-GCGCTTCGATCTGCAACACAC-3′. The cycling conditions in DNA thermocycler were as follows: an initial denaturation at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 20 s, and final extension at 72 °C for 10 min. PCR amplification was carried out in a final volume of 25 μl which included 2 µl genomic DNA, 12.5 µl distilled water, 10 µl Taq DNA Polymerase Master Mix (Ampliqon; Denmark) and 0.5 µl of each primer. PCR product was digested with
ApaI restriction enzyme (Takara, Japan) and then digested product was subjected to electrophoresis on 3% agarose gel. Fragments containing three possible genotypes of the CARTPT rs2239670 were detected: uncut homozygous AA (552 bp), cut heterozygous AG (212, 340 and 552 bp) and cut homozygous GG (340 and 212 bp). To confirm the results of PCR-RFLP, 10 percent of PCR samples were directly sequenced.
2.6. Statistical analyses
Normal distribution of data was checked by Kolmogorov-Smirnov test. The comparison of qualitative and quantitative variables was conducted by chi- square test and analysis of variance (ANOVA), respectively. Data on continuous and categorical variables were presented as the mean ± SD and the frequencies or percentages, respectively. ANCOVA multivariate interaction model was used to estimate the interactions of CARTPT rs2239670 polymorphism with dietary NEAC in relation to cardio-metabolic risk factors and hypothalamic hormones after adjusting for confounding factors. All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS, Chicago, Il, USA) version 22.0. A p. value < 0.05 was considered as statistically significant.