The interaction between dietary Non-Enzymatic Antioxidant Capacity (NEAC) and variant of Cocaine and Amphetamine-Regulated Transcript Prepropeptide (CARTPT) 5q13-14 - rs2239670 locus on hypothalamic-pituitary axis hormones and metabolic risk factors in obese individuals

Epidemiological studies have illustrated that polymorphisms of Cocaine and Amphetamine-Regulated Transcript Prepropeptide gene (CARTPT) may affect obesity outcomes. However, no study has investigated the CARTPT gene-diet interactions in term of antioxidant potential of diet. This study aimed to evaluate the interactions between CARTPT gene and dietary non-enzymatic antioxidant capacity on hypothalamic- pituitary axis hormones and cardio-metabolic risk factors. In the present cross-sectional study, 288 apparently healthy obese adults aged 20–50 years with a body mass index (BMI) of 30–40 kg/m 2 completed the study. Dietary assessment was performed by food frequency questionnaire (FFQ) and antioxidant capacity of diet was estimated by calculating the oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), total radical trapping antioxidant parameter (TRAP) and Trolox equivalent antioxidant capacity (TEAC). The CARTPT rs2239670 polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) technique.


Abstract Background
Epidemiological studies have illustrated that polymorphisms of Cocaine and Amphetamine-Regulated Transcript Prepropeptide gene (CARTPT) may affect obesity outcomes. However, no study has investigated the CARTPT gene-diet interactions in term of antioxidant potential of diet. This study aimed to evaluate the interactions between CARTPT gene and dietary non-enzymatic antioxidant capacity on hypothalamic-pituitary axis hormones and cardio-metabolic risk factors.

Method and Material
In the present cross-sectional study, 288 apparently healthy obese adults aged 20-50 years with a body mass index (BMI) of 30-40 kg/m 2 completed the study. Dietary assessment was performed by food frequency questionnaire (FFQ) and antioxidant capacity of diet was estimated by calculating the oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), total radical trapping antioxidant parameter (TRAP) and Trolox equivalent antioxidant capacity (TEAC). The CARTPT rs2239670 polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique.

Results
There was a signi cant interaction between CARTPT rs2239670 and dietary ORAC on BMI (P interaction = 0.048) and fat mass percent (FM%) (P interaction = 0.008); in A allele carriers, higher adherence to the dietary ORAC was related to lower level of BMI and FM%. As well as, the signi cant interactions were observed between FRAP index and rs2239670 in relation to HOMA (P interaction = 0.049) and QUICKI (P interaction = 0.048). Moreover, there were signi cant interactions of rs2239670 with TRAP (P interaction = 0.029) and TEAC (P interaction = 0.034) on the serum glucose level; individuals with AG genotype were more respondent to higher intake of TRAP.

Conclusion
The present study indicated the interactions between CARTPT genotypes and dietary NEAC on obesity and its-related metabolic parameters. Further studies are warranted to con rm our ndings.

Background
Obesity, as a worldwide pandemic, has become a global public health problem and its prevalence is rising at an alarming rate in many countries [1]. The worldwide prevalence of overweight and obesity has doubled since 1980 in all age-sex groups regardless of race, ethnicity and socioeconomic status [2].
Obesity is a complex and preventable disease which substantially increases risk of multiple comorbidities such as: diabetes mellitus, cardiovascular diseases (CVDs), hypertension, fatty liver disease, several types of cancers and stroke.. Thereby it can affect quality of life, life expectancy, work productivity and healthcare costs [1]. There is scienti c evidence that obesity plays an important role in the initiation and progression of these pathological conditions through oxidative stress which is characterized by elevated reactive oxygen species (ROS) and/or reduced antioxidant capacity [3]. Oxidative stress damages or alterations in antioxidant defenses have been implicated in the pathogenesis and development of obesity-related consequences such as CVDs and cancer [3][4][5]. Emerging evidence suggest that the development of obesity, as a multifactorial disorder, and its-related co-morbidities is determined by interactions between genetic and environmental variables particularly diet [6]. Accumulating evidence suggests that intake of dietary antioxidants (e.g. selenium, vitamin C and vitamin E) through nonenzymatic defense mechanisms can protect against cell damage caused by oxidative stress and also related in ammatory outcome [7]. In this regard, the epidemiologic studies have reported an inverse association between intakes of fruits and vegetables, which are rich source of antioxidants, and the risk of several chronic conditions such as CVDs and cancers [8,9]. Since various antioxidants are combined in foods and they act in a cumulative and synergistic way in complex matrixes, assessment of single speci c antioxidant may not re ect the potential overall antioxidant effect of the diet. Thus, investigation of the dietary total antioxidant capacity (TAC) can provide a better tool to examine the bene cial effects of dietary antioxidants on chronic diseases prevention [10]. Non-enzymatic antioxidant capacity (NEAC), also recognized as TAC, is a new approach to capture synergistic effects of antioxidants in diet and generally can be estimated through four different methods including: Trolox equivalent antioxidant capacity (TEAC), ferric reducing-antioxidant power (FRAP), total radical-trapping antioxidant parameter (TRAP) and oxygen radical absorbance capacity (ORAC) [11]. Recently, observational studies have shown that high dietary NEAC is inversely related to the risk of stroke, diabetes and different cancers [12][13][14].
Since obesity is highly heritable, genetic factors substantially contribute to development of obesity and its serious consequences [15]. Cocaine-and amphetamine-regulated transcript prepropeptide gene (CARTPT), which is located at the chromosome 5q13-14 and expresses the cocaine-and amphetamineregulated transcript (CART) protein, has been recognized to be a susceptibility locus for obesity ( Fig. 1).
CART protein, as one of the various neuropeptides in the arcuate nucleus (ARC) of the hypothalamus, is involved in the regulation of food intake and body weight [16]. In this regard, previous evidence have shown that, CART peptides which are co-expressed with other neurotransmitters such as α-melanocytestimulating-hormone (α-MSH) inhibit food intake [17]. Recent studies have indicated that polymorphisms in the CARTPT gene might in uence obesity, metabolic syndromes (MetS) and its components [18]. However, the results of studies regarding these associations are controversial [19]. These heterogeneous associations may be due to the complex pathogenesis and etiology of obesity which involve interaction between genetic and environmental factors especially diet. A positive association between CARTPT rs2239670 variant and alcoholism has been reported in the Korean population and as obesity [20] and substance abuse have a common neurobiological basis, investigation of the association between this polymorphism and obesity and metabolic factors is interested. Therefore, since the better understanding of the gene-diet interactions can provide more e cient strategies for personalized treatment, and also to our knowledge, no studies have assessed the modi cation effects of diet in term of dietary antioxidant capacity on the association of CARTPT gene, we aimed to with cardio-metabolic risk factors and hypothalamic hormones, we aimed to evaluate the potential interactions between CARTPT rs2239670 and dietary NEAC on cardio-metabolic risk factors and hypothalamic hormones among obese population.

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/m 2 ). Detailed illustration of recruitment of research participants has been provided elsewhere [21]. 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).

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) [22]. 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. .

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 highdensity 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.

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 [27].Then, daily nutrient consumption was analyzed by Iranian Food Composition Table (FCT) [28]. Information missing from this FCT was completed with the United States Department of Agriculture FCT [29].
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 reducingantioxidant power (FRAP) [30] which measures the ability of dietary antioxidants to reduce ferric to ferrous ions, total radical-trapping antioxidant parameter (TRAP) [31] that evaluates the chain-breaking antioxidant potential for scavenging peroxyl radicals, oxygen radical absorbance capacity (ORAC) [32] which measures the antioxidant capacity against peroxyl radicals using an area-under-curve (AUC) technique and Trolox equivalent antioxidant capacity (TEAC) [31] 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 [33] and also its still unknown whether these products are e ciently absorbed and exert an antioxidant effect in vivo [34], 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.

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

Results
The present cross-sectional study was conducted on 288 individuals (51.1% men and 48.9% women).    As elucidated in Fig. 2, after adjusting for sex and age, there was a signi cant interaction between CARTPT rs2239670 variant and dietary ORAC on BMI (P interaction = 0.048) and fat mass percent (FM%) (P interaction = 0.008); in A allele carriers, higher adherence to the dietary ORAC was related to lower level of BMI and FM%. Moreover, the signi cant interactions were observed between FRAP indicator and CARTPT rs2239670 polymorphism in relation to HOMA (P interaction = 0.049) and QUICKI (P interaction = 0.048). Such that, adherence to the dietary FRAP modi ed positively the association of the CARTPT rs2239670 variant with HOMA and QUICKI in G allele carriers. It seemed that dietary NEAC couldn't modulate detrimental effects of CARTPT rs2239670 polymorphism in participants carrying AA genotype.
Additionally, the signi cant interactions were observed for CARTPT rs2239670 variant with FRAP (0.029) and TEAC (0.034) in relation to serum glucose level. The highest serum glucose concentration was observed in the AA genotype with moderate compliance with FRAP and TEAC; while high compliance with these indices couldn't effect on genetic predisposition to blood sugar abnormalities.

Discussion
To the best of our knowledge, the current research is the rst attempt to study the interaction between Korean (17%) [20] and Malaysian (30%) [19]. This discrepancy in the minor allele frequency reported might be explained by differences in study sample size, demographic characteristics of population like age, ethnicity, and gender and also various lifestyles. Previous evidence has revealed that polymorphisms in the CARTPT gene are linked to human obesity. In this regard, leu34Phe missense mutation in CART gene was detected in Italian subjects with early-onset obesity [16]. Likewise, it was reported that the A-156G polymorphism in the promoter region of CARTPT was related to obesity among Japanese subjects [35]. Moreover, the studies have shown that genetic variation in the CART locus might in uence susceptibility to MetS and its components such as dyslipidemia, high blood pressure and hyperglycemia [18]. It should be taken into account that all variants in CARTPT gene are not associated with obesity phenotypes and nding in this regard is con icting [36]. For example, in accordance with the results of Walder study, C1442G variant of CARTPT gene was not associated with obesity in Pima Indians [36]. The previous studies which have speci cally examined the association between the CARTPT rs2239670 polymorphism and addictive behaviors such as alcohol dependence have reported a positive association [20]. However, the studies which have evaluated this variant in relation to obesity are scarce. To the best of our knowledge, only one study have investigated the CARTPT rs2239670-obesity relation and showed no association between this variant and obesity among the Malaysian subjects [19]. All of these heterogeneities in nding warrants further research efforts among different populations. Since the rs2239670 variant located in the intron 1 of CARTPT gene and the strong effects on obesity and related metabolic factors may be removed during mRNA splicing encoding CART proteins, effects of this variant on CART function is still unknown [19]. However, genetic variation in the CART locus may effect on the expression of the CART peptide, which is related to hypothalamic anorectic and orexigenic neuropeptides [37]. Totally, it seems that CART peptides have an anorexigenic effect, although the mechanism of this function is not completely known [38].
As far as we are aware, no previous research has investigated the gene-diet interactions of CARTPT with adherence to the dietary NEAC on metabolic pro le in obesity to compare our nding. However, there have been numerous researches investigating the interaction between genetic variation and diet or dietary ingredients on obesity and its-related complications [39]. For example, Mirzababaei and et al. examined the interaction of the rs1333048 variant on 9p21 genetic region with TAC on the risk of MetS and they revealed that high ORAC intake may modify the increased risk of MetS in homozygous subjects for the minor allele (AA genotype) [39]. Accordingly, in other our recent study, we documented the signi cant interactions between dietary quality indices and CARTPT rs2239670 genotypes affecting metabolic parameters [40]; higher adherence to diet quality index-international (DQI-I) reduced the metabolic risk in obese individuals with the homozygous minor allele genotype (AA).
Noticeably, the main nding of our study was the association between the CARTPT rs2239670 polymorphism and cardio-metabolic factors depended on the dietary antioxidant intakes; a good compliance with NEAC blunted the association of the CARTPT gene with metabolic factors. While the underlying mechanisms behind this interactions is not still clari ed, these favorable effects of the total antioxidant capacity may be mediated by vitamin C, vitamin E and its isomers, selenium, carotenoids, iso avones, avonoids, and proanthocyanidins [41,42]. In this regard, there are numerous studies which have indicated the bene cial effects of antioxidant-rich foods (e.g. fruits and vegetables and tea) on the obesity, insulin resistance, glucose homeostasis and lipid pro les [43,44]. These favorable effects of these antioxidants on the improvement of metabolic pro le may be partly attributed to other activities of antioxidants such as regulation of brown adipose tissue metabolism and enhancement of thermogenesis, suppression of adipogenesis and induction of catabolism in adipose tissue [45].
The strengths of the present study is that according to our current knowledge, it is the rst study to examine the interaction between CARTPT rs2239670 genotypes and dietary NEAC on the metabolic factors in obese subjects and identifying these gene-diet interactions may provide the best personalized dietary advice for high-risk participants according to their genetic makeup to reduce the burden of obesity and its-related chronic diseases.
However, the present study has several potential limitations that should be considered. Firstly, since the present study is a cross-sectional study, ascertained causality cannot be argued but it helps to generate hypotheses that can be examined by prospective cohort or other studies. Secondly, a rather small sample size of our study may not have su cient statistical power to detect the interaction effect. So, the results of our study should be interpreted with caution and require replication and con rmation in larger and different populations. Thirdly, the present study was limited to the assessment of only one variant from a single gene, while multiple genes are known to be involved in the pathogenesis of obesity and its-related consequences. Fourthly, because dietary intakes and other lifestyle factors in Tabriz may differ from those in other parts of the country with different cultures, our results may not necessarily be extrapolated to the general population. Fifthly, despite adjustment for several confounders in the analyses, residual confounding by other unmeasured factors was inevitable. Lastly, under-reporting of dietary intakes particularly in obese individuals is a source of measurements error which may cause underestimation of the true effect [46]. Thus, we excluded the extreme-energy reporters from analysis.

Conclusion
In conclusion, our nding for the rst time showed a statistically signi cant gene-diet interaction of the CARTPT rs2239670 with adherence to dietary NEAC, as a novel measurement of antioxidant intake, in relation to obesity and related-metabolic phenotypes; high intake of NEAC by minor-allele-carriers attenuated genetic association with BMI and FM%, however high compliance with these indices couldn't effect on genetic predisposition to blood sugar abnormalities. Further studies are warranted to con rm our ndings, which may have important implications for public health.

Declarations
Ethical approval and consent to participate Each participant was completely informed about the study protocol and provided a written informed consent form before taking part in the study. The study protocol was approved and registered by the ethics committee of Tabriz University of Medical Sciences (Ethics number: IR.TBZMED.REC.1397.266).

Consent to publish
Not applicable.

Availability of data and materials
All of the data are available with reasonable request from the corresponding author Competing interest Figure 1 Genomic organization of the CARTPT rs2239670 polymorphism region P-for interaction between different NEAC tertiles according to CARTPT genotype.