The association between FTO genotype and macro-nutrients’ intake in overweight adults

Background Macro-nutrients can influence on body their interactions with FTO gene polymorphisms. This study aimed to investigate the association between FTO gene rs9939609 polymorphism with macro-nutrients intake. Methods This cross-sectional study was carried out on 196 overweight adults. The rs9939609 SNP in FTO was genotyped. Dietary intake was assessed by a valid 168-item semi-quantitative food frequency questionnaire (FFQ). The association between dietary macro-nutrients and the FTO genotype were assessed using linear regression after adjustments for sex, age, physical activity, triglycerides, fasting blood sugar (FBS), and low density lipoprotein (LDL). Results The higher carbohydrates (p=0.000), fat intake (p=0.009) and calorie intake (p=0.001) were significantly associated with FTO gene risk allele (P=0.001). Carriers of the AA/AG genotype of rs9939609 had significantly higher calorie, fat, and carbohydrate intake than the carriers of the TT genotype (p=0.019, p=0.010, & p=0.000, respectively). Conclusion The dietary carbohydrate and fat intake were associated with FTO genotype. Further studies are warranted to investigation of these interactions and the underlying mechanisms.


Introduction
Prevalence of obesity dramatically increased worldwide in developed and underdeveloped countries and today obesity is a global health-related problem (1,2). More than 34.9% of adult's population of the United States are obese (3).
Obesity is associated with other diseases such as cancer, hypertension, dyslipidemia cardiovascular disease, type 2 diabetes, and psychological disorders (4). Obesity is a multifactorial disorder caused by genetics, lifestyle and environmental factors (5) and recent studies reported that genetics may exert its effects by changing lifestyle. (4-6).
An important role of some genes in obesity has been reported in many studies (7-9). One of the most important gene is fat mass and obesity gene (FTO) that is found to be strongly associated with obesity (7-8). The FTO gene is located on the chromosome region 16q12.2, and it is widely expressed in several tissues such as brain, visceral fat, liver and hypothalamus. FTO genotype had a strong association with body mass index (BMI) and obesity (8-9).
FTO gene has an important role in regulation of food intake, energy balance, appetite and basal metabolic rate (BMR) (10). Recent studies reported an interaction between FTO genotypes and dietary intake (11-12). On the other hand, FTO genotypes may influence the effects of dietary macronutrients intake on BMI, body weight, food intake, energy balance, appetite, and hormone secretion (12)(13)(14).
Dietary macronutrients including carbohydrate, fat, and protein, as the main source of energy, have key roles in regulation appetite, body weight, and BMI (14)(15). In order to understand whether the intake of dietary macronutrients is associated with FTO gene, we conducted a study to investigate the interactions between changes in the amount of dietary carbohydrate, protein and fat, with the FTO genotype in overweight adults.

Methodology
This study was a cross-sectional study was carried out from September 2016 to October 2017 on 199 randomly selected participants referred to the Shohadaye Valfajr health center, Shiraz, Iran. In brief, participants were overweight adults with BMI between 24/9 to 29/9 kg/m2 and aged 20 to 45 years. The Inclusion criteria was defined as willingness to participate in the study, not participating in a weight management programs during two past months and no recent weight loss greater than 5%. We excluded participants with alcohol or drugs addiction (n=1), smoking, certain weight-related diseases (including specific psychological or neurological disorders, insulin resistance, thyroid disease, liver disease, renal failure, infectious and other specific diseases) (n=1), and pregnant or lactating women (n=1). All participants signed a consent form before participation in the study.

Anthropometric Measures
The height of the participants was measured with a calibrated tape line fastened to a wall and without shoes with a precision of 0.5 cm. A bio impedance analysis scale (BIA) (Tanita, Japan/BC-418) was used to measure anthropometric indices such as body weight, Body Mass Index (BMI), skeletal muscle percentage (SM%), body fat (BF), skeletal muscle (SM) and body fat percentage (BF%) after entering their height, age and gender.

Genotyping:
DNA was extracted from whole peripheral blood sample using the DNA extraction kit (SinaPure DNA Kit, PR881612/EX6001/CinnaGen/Iran). DNA samples were stored at −20 °C before genotyping. After DNA extraction, the concentration of the extract material was obtained by spectrophotometer NanoDrop device (ND1000,USA). FTO gene was genotyped for rs9939609 polymorphism via tetra primer amplification refractory mutation system polymerase chain reaction(ARMS-PCR)

Macronutrients' intake
Usual Macronutrients' intakes of participants were assessed by a validated 168-item semi-quantitative FFQ (16). The FFQ consisted of 168 food items with standard portion sizes commonly consumed by Iranian people. Face-to-face interviews were administered by a trained dietitian.
Macronutrients' consumption frequencies were converted to grams per day by using household measures. Daily intakes of energy were also measured for each person by using the modified US Department of Agriculture food consumption database, which was modified for Iranian foods.

Laboratory Measurement
Serum TG, TC, HDL, LDL, glucose and insulin levels were measured after 12 hours of an overnight fasting. leptin and adiponectin serum were measured using EDTAanticoagulated tubes. Insulin, leptin and adiponectin level was determined by ELISA test (LDN, Germany).

Statistical Analysis
ANOVA test was used to compare anthropometric indices, plasma levels of hormones and lipid and sugar profile between different FTO genotypes. Tukey test was used for comparison the calorie and macronutrient intake between three genotypes. To adjust the effects of confounders, linear regression was used after that the assumptions of the linear regression model were confirmed. Statistical analyses were performed using SPSS version 23.0 (IBM SPSS Statistics for Windows, IBM Corp., Armonk, NY, USA). The results were considered statistically significant at P<0.05.

Ethics approval and consent to participate
This study has been approved by Local ethics review boards at Shiraz University of medical sciences (Code: ir.sums.rec.1395.100).
Tukey tests conducted to recognize differences between three genotypes identified for identified a significant difference between calorie, carbohydrate, fat intake and genotypes of FTO. Homozygotes for the rs9939609 risk allele A had significantly higher calorie, fat, and carbohydrate intake than the carriers of the TT genotype (p=0.019, p=0.010, & p=0.000, respectively) but not significant for heterozygotes than the carriers of the TT genotype (p=0.869, p=1.000, & p=0.966, respectively) ( Table 2).
To adjust the effects of confounders, linear association of FTO rs9939609 different genotypes with the level of macronutrients' intake (carbohydrate, Fat, Protein) was assessed ( Table 3). The level of calorie intake, carbohydrate, and fat intake were significantly different between FTO genotypes. This association remained significant for carbohydrate, calorie and fat intake after adjustment for age and sex (p= 0.000, p=0.001 and p=0.009 respectively) (model1). The results did not change after further adjustments for physical activity, TG, LDL, FBS (p= 0.001, p=0.000 and p=0.019 respectively) (model 2).

Discussion
In the present study, we evaluated the associations between rs9939609 FTO polymorphism with calorie, fat, carbohydrate and protein intake. The results identified that there was a significant difference between FTO genotype with calorie, carbohydrate, and fat intake, but not significant for protein intake. This association remained significant for calorie and macronutrients' intake after adjustments for sex, age, physical activity, LDL, HDL, and FBS. In AA carriers, dietary carbohydrate, fat, and calorie intake were higher than TT carriers. However, the results of recent studies about association between dietary macronutrients and FTO polymorphism were inconsistent. Timpson et al. reported higher energy intake and fat intake among rs9939609 AA genotype carriers. They suggest that FTO polymorphism may influence on appetite and food intake (17). Some studies reported that carriers of risk allele FTO received higher energy intake (17,18). The AA carriers was reported to intake higher fat then TT genotype.
Consistent with our study, Daya et al. reported that carriers of AT/AA genotype had higher fat intake (1.40 times) and obesity risk than TT genotype (19). Fat intake may modifiy the effect of the FTO rs9939609 polymorphism on adiposity. It was observed that carriers of the A risk allele FTO rs9939609 had no significant influence on adiposity in subjects whose dietary fat intake was below 30% of total energy but fat intake higher than 30% increased central and total adipose tissues This study found that the AA carriers had higher carbohydrate intake than TT genotype. While Sonest et al found low carbohydrate intake associated with FTO genetic variants. In homozygous for alleles A, BMI higher than TT genotype but the increase in BMI was mainly restricted to subjects who reported low physical activity (15). Carbohydrate intake (especially glucose intake) increase FTO gene expression This study found no association between protein intake and FTO genotype. While some studies indicated that protein intake was associated with FTO genotype (27-28) and leucine decreased FTO gene expression in the hypothalamus (27). However, another study reported that leucine intake increased FTO gene expression (28).
Doaei et al found that higher protein intake up-regulated the FTO gene and also indicated that only in A allele carriers, protein intake was positively associated with FTO gene expression (25).
The FTO variants was associated with intake of energy-dense foods such as fat-rich foods (29). FTO gene variants played important roles in appetite regulation, food intake, tendency to choose energy-dense food (high fat and high carbohydrate diet) (30). The carriers of A allele FTO rs9939609 had Energy-dense food choices, higher body weight, and overeating behaviors (31).
Some studies suggested that FTO play a crucial role in regulating energy homeostasis. FTO gene is expressed in brain that controls feeding and energy expenditure (32). On the other hand, Fto expression level in hypothalamus is regulated by dietary intake. Interestingly, it was reported that ahigh-fat diet can down-regulate FTO expression in short-term and up regulate in long-term (33, 34).
On the other hand, the FTO gene is related with gut hormones such as orexigenic hormone, acyl-ghrelin, satiety hormone, peptide YY that regulate food intake and appetite (35). FTO gene polymorphism (AA genotype) influence on circulating PYY3-36 and acyl-ghrelin levels that lead to increased food intake especially energydense foods and reduced satiety (36,37).In rs9939609 AA carriers, suppression of acylated ghrelin led to overeating and obesity (38). So, it is plausible that FTO gene polymorphisms could change appetite and food intake that may lead to weight gain and obesity.

Conclusion
FTO gene rs9939609 polymorphism is associated with dietary intake. The intake of calorie, carbohydrate, and fat intake were associated with FTO gene polymorphisms and this association remained significant for calorie and macronutrient intake after adjustments for sex, age, physical activity, LDL, HDL, and FBS. In AA carriers, dietary carbohydrate, fat, calorie was higher than TT carriers. Further studies are needed to increase our understanding of the underlying mechanisms of the association between FTO gene and dietary intake.

Ethics approval and consent to participate
This study has been approved by Local ethics review boards at Shiraz University of medical sciences (ir.sums.rec.1395.100).

Availability of data and material:
Not applicable

Competing interests
The authors declare that they have no competing interests

Consent for publication
Not applicable.

Authors' contributions
MM and MHE designed the study, involved in the data collection, analysis, and drafting of the manuscript.MM, MGh, SD were involved in the design of the study, analysis of the data, and critically reviewed the manuscript. All authors read and approved the final manuscript.