Background: DNA methylation has been evidenced as a potential epigenetic mechanism related to various candi- date genes to development of obesity. Therefore, the objective of this study was to evaluate the DNA methylation levels of the ADRB3 gene by body mass index (BMI) in a representative adult population, besides characterizing this population as to the lipid profile, oxidative stress and food intake. Methods: This was a cross-sectional population-based study, involving 262 adults aged 20–59 years, of both genders, representative of the East and West regions of the municipality of João Pessoa, Paraíba state, Brazil, in that were evalu- ated lifestyle variables and performed nutritional, biochemical evaluation and DNA methylation levels of the ADRB3 gene using high resolution melting method. The relationship between the study variables was performed using analyses of variance and multiple regression models. All results were obtained using the software R, 3.3.2. Results: From the stratification of categories BMI, was observed a difference in the average variables values of age, waist-to-height ratio, waist-to-hip ratio, waist circumference, triglycerides and intake of trans fat, which occurred more frequently between the categories “eutrophic” and “obesity”. From the multiple regression analysis in the group of eutrophic adults, it was observed a negative relationship between methylation levels of the ADRB3 gene with serum levels of folic acid. However, no significant relation was observed among lipid profile, oxidative stress and food intake in individuals distributed in the three categories of BMI. Conclusions: A negative relationship was demonstrated between methylation levels of the ADRB3 gene in eutrophic adults individuals with serum levels of folic acid, as well as with the independent gender of BMI, however, was not observed relation with lipid profile, oxidative stress and variables of food intake. Regarding the absence of relationship with methylation levels of the ADRB3 gene in the categories of overweight, mild and moderate obesity, the answer probably lies in the insufficient amount of body fat to initiate inflammatory processes and oxidative stress with a direct impact on methylation levels, what is differently is found most of the times in exacerbated levels in severe obesity. Keywords: ADRB3, DNA methylation, Body mass index, Folic acid *Correspondence: firstname.lastname@example.org Health Sciences Center (Centro de Ciências da Saúde), Departament of Nutrition, Federal University of Paraíba (Universidade Federal da Paraíba), João Pessoa, Brazil Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. de Oliveira et al. J Transl Med (2018) 16:152 Page 2 of 9 which provides free fatty acids for thermogenesis, indi- Background cating the participation of the ADRB3 gene in the regula- Obesity is considered a serious public health problem, tion of body weight in humans . However, the role of affecting 39% of the world population . The epidemic the DNA methylation of the ADRB3 gene related to the projection is that by 2025, more than 2 billion adults are increase of susceptibility to visceral obesity and alter the overweight or obese . body fat distribution have not been fully elucidated yet. Multiple biological, behavioural, genetic and epigenetic In this context, the present study aims to evaluate the determinants are contributing to the multifactorial aetiol- methylation levels of the ADRB3 gene by BMI in a repre- ogy of obesity, for example, sleep disturbances, potential sentative adult population, in addition to characterizing traits of obesogenic behavior, characterized by excess cal- this population as to the lipid profile, oxidative stress and orie intake and sedentary lifestyle, peripheral and central food intake. regulation of energy balance, adipose tissue and skeletal muscle biology, in addition to changes from the gut micro- Methods biota, hormone signalling, reproductive factors, drugs, and Study design intrauterine and epigenetic intergenerational effects [3 ]. This is a cross-sectional epidemiological study linked to Increasing evidence indicates that epigenetic modifica - the project titled: “II Cycle of Diagnosis and Intervention tions in adipose tissue may be involved in pathogenesis of of Food and Nutritional Status and the Most Prevalent metabolic disorders, fat storage, cell remodeling and adi- Non-Communicable Diseases in the Population of the pogenesis, emphasizing the involvement of DNA meth- Municipality of João Pessoa/PB” (II Ciclo de Diagnóstico ylation in differentiation of adipocytes and its effect on e Intervenção da Situação Alimentar, Nutricional e das expression of genes related to obesity . Doenças não Transmissíveis mais Prevalentes da Popu- DNA methylation is the most frequent and well- lação do Município de João Pessoa/PB, II DISANDNT/PB), characterized epigenetic mark, for playing a key role on which was conducted between May 2015 and May 2016. regulating gene expression and at molecular phenotype, without any change in the sequence of DNA, it can be Ethical issues altered by environmental expositions and genetic influ - The research protocol of the project mentioned above, ences . In addition, folate is a water-soluble B vitamin to which the present study is linked, was submitted which plays an important role as donor of methyl groups and approved by the Research Ethics Committee of the required for proper epigenetic regulation . Health Sciences Center (Centro de Ciências da Saúde, Another complexity encountered in studying obesity is CCS) of the Federal University of Paraíba (Universidade the marked heterogeneity of obese individuals, through Federal da Paraíba, UFPB), under the Protocol Number the notable differences in body configuration and in the 0559/2013, in accordance with the ethical standards for regional accumulation of body fat in adiposity levels, research involving human beings included in Resolution such as subcutaneous obesity, in which excess subcuta- 466 of the December 12, 2012, meeting of the National neous fat is found around the hip and thigh areas, and Health Council/National Research Ethics Committee. visceral obesity, in which fat is mainly concentrated in After identifying the residences in randomly selected the abdominal region, mainly mesenteric adipose tissue. city blocks located in the East and West areas of the city Visceral obesity, also known as the excess of visceral adi- of João Pessoa, the researchers introduced themselves pose tissue (VAT), tends to be more pernicious in health to the residents, explaining the purpose of the study and terms, being a potential factor for cardiovascular risk . requesting their participation. To respect ethical guide- Recently, it was demonstrated the modification in lines for research involving humans, the participants methylation levels of candidate genes to development residing in the selected households were included in the of obesity exhibited a relation excellent in the discrimi- study only when they had consented to participation by nation of obesity from non-obesity status, thus suggest- signing the Informed Consent form. ing that DNA methylation levels in blood leukocytes has great potential for to characterize the obesity phenotype Sampling by to identify robust and biologically relevant epigenetic To conduct this population-based study, representative of variation related to body mass index (BMI), using an eas- the East and West regions of the municipality of João Pes- ily accessible and minimally invasive biological material soa, a representative sample of the adult group was cal- instead of adipose tissue . culated using information provided by the city hall, such Interestingly, the beta-3 adrenergic receptor (ADRB3) as the city map, number of blocks per neighborhood, is located on chromosome 8, which contains two exons and data from the Brazilian Institute of Geography and and 2.252 base pairs, and is known to play a key role in the Statistics (Instituto Brasileiro de Geografia e Estatística, regulation of lipolysis in white and brown adipose tissues, de Oliveira et al. J Transl Med (2018) 16:152 Page 3 of 9 IBGE) . In the present study, two regions (East and BMI was then calculated using the body weight (kg) West) with similar socioeconomic, epidemiological and divided by the squared body height (meters), and the lifestyle characteristics in relation to the other regions cut-off points recommended in adults aged 20–59 years were selected in order to represent the four regions of the old by the World Health Organization (WHO) were used municipality. . For the sample calculation, a single multi-level sampling Waist circumference (WC) was used to determine procedure was used. Due to the presence of heterogeneity abdominal obesity, proceeding the American Heart in income and the relationship between income, disease Association (AHA) cut-off points of ≥ 88 cm in women prevalence and nutrition , stratified sampling was used and ≥ 102 cm in men . The waist-hip ratio (WHR)  with the city blocks at the first level. In this level, the was used to define the abdominal obesity status, defined neighborhoods/city blocks of the East and West zones of the as a WHR > 0.90 in men and > 0.85 in women . municipality were ranked by income class into four strata The waist-to-height ratio (WHtR) was based on the according to information obtained from the IBGE . quotient of the waist and height measurements in cen- After stratification, the sample size, or number of rep - timeters. The cut-off point used was 0.5, as supported resentative city blocks per zone, was calculated. Next, the by studies conducted around the world in diverse popu- weight of each stratum was calculated as the number of lation as an evaluation tool to identify health risks and city blocks per zone according by strata, according to the related morbidities . formula defined by Silva, Moraes and Costa . Considering that the average income of adults in the Dietary assessment East and West areas was R$2213.26 [data obtained from To evaluate the regular food intake of the individuals, “The first cycle of diagnoses and intervention of the food three 24-h dietary recalls (24HR) were performed, con- and nutritional situation, and of the most prevalent com- templating one at the weekend, with a 15-day interval municable diseases of the municipal population of João from the beginning of the data collection, according to a Pessoa/PB” (07/2008-01/2010)], with a standard devia- study previously published . tion of R$2601.93 and a margin of error of R$3320.00 in income, the minimum sample of adults in João Pessoa Biochemical analysis necessary to be statistically representative of the east- The blood samples were collected from the individu - ern and western areas (with a confidence level of 95%) als after a 12-h fasting at home by an experienced nurse, was 236 adults. Thus, a sample of 262 adults distributed and the analysis of biochemical variables were performed across the study area was selected. according to a study previously published . The This study had the following inclusion criteria: individu - analysis of alpha-1 acid glycoprotein (AGP) was meas- als aged 20–59 years; different socioeconomic conditions, ured using the immunoturbidimetry technique and tests and medication users and non-users, with the exception were performed in an automated analyzer (LabMax 240, of the drugs described below. Exclusion criteria included Labtest, Lagoa Santa, MG, Brazil) using standardized kit the following: individuals with neuropsychiatric disorders; following the instructions provided by the manufacturer multivitamin, mineral, anorexigen, and anabolic supple- (Labtest, Lagoa Santa, MG, Brazil). ment users; and pregnant and lactating women. The Access Folate assay is a paramagnetic particle, chemiluminescent immunoassay for the quantitative Data collection determination of folic acid levels in human serum and The household visits and application of the questionnaires plasma (heparin) or red blood cells (RBC) using the were conducted by graduate students of the Nutrition Access Immunoassay Systems. Folate levels in serum Programs. These are Master’s and doctoral students in the and plasma or RBC are used to assess folate status. The Post Graduate Program in Nutrition Sciences (Programa serum folate level is an indicator of recent folate intake, de Pós Graduação em Ciências da Nutrição, PPGCN) at while that a low RBC folate value can indicate a pro- UFPB who were trained before initiating data collection longed folate deficiency. Thus, serum levels of folate after the pilot study was conducted. Questionnaires were were estimated using commercial kit (Access Folate applied for demographic, socioeconomic, and epidemio- Kit [A98032]; Beckman Coulter, Fullerton, CA, USA). logical characterization, lifestyle, nutritional and food Folate levels were determined by a corpuscle immune intake assessment, and also biochemical evaluation. chemiluminescence assay (Access 2 Immunoassay Sys- tem; Beckman Coulter, Fullerton, CA, USA). The assay Nutritional assessment was performed in accordance with the manufacturers’ Weight and height measurements were taken in tripli- protocol. Folate deficiency was defined as < 3.10 ng/ml cate, and the average of the three values was used. The with analytical sensitivity of 0.5 ng/ml. de Oliveira et al. J Transl Med (2018) 16:152 Page 4 of 9 Analysis of DNA methylation levels To identify the existence of statistically significant rela - The leukocyte DNA was isolated, quantified and trans - tionships between DNA methylation levels of the ADRB3 formed with sodium bisulfite according to the condi - gene and variables of this study, the following multiple tions described in a previously published study . linear regression models were used: Model 1: Methylation levels = β0 + β1 ∗ Total cholesterol + β2 ∗ LDL + β3 ∗ HDL + β4 ∗ Triglycerides + β5 ∗ Homocysteine + β6 ∗ TAC + β7 ∗ MDA + β8 ∗ Alpha-1 acid glycoprotein + β9 ∗ Folic acid + β10 ∗ Vitamin B12. Model 2: Methylation levels = β0 + β1 ∗ Calories + β2 ∗ Total fat + β3 ∗ Folate + β4 ∗ Vitamin B12 + β5 ∗ Monounsaturated fat + β6 ∗ Oleic acid + β7 ∗ Omega 3 + β8 ∗ Omega 6 + β9 ∗ Saturated fat + β10 ∗ Cholesterol + β11 ∗ Polyunsaturated fat + β12 ∗ Trans fat. The analysis of methylation levels in genomic DNA Considering 5% as significance. from blood was performed by High Resolution Melting (HRM) Real Time PCR method in a applied biosystems Results 7500 fast system. PCR was performed in a total volume 2+ The sample comprised 262 adults individuals, being 175 of 20 μl containing: 1× buffer, 4 mM Mg , 200 μM of female (66.79%) and 87 males (33.21%). each dNTPs (Qiagen), 250 nM of each primer, 5 μM ® In Table 1, it was performed the multiple comparison SYTO (Invitrogen), 1 U HotstarTaq DNA Polymerase testing of means for each variable, through the ANOVA (Qiagen) and 1 μl of bisulfite-modified DNA. Primers test, it was observed a difference in mean variables values were designed from the genome sequence deposited in described below, in relation to nutritional status: aver- the UCSC genome browser http://genom e-euro.ucsc. age age, waist-to-height ratio (WHtR), waist-to-hip ratio edu/(chr8:37,962,991-37,966,965), F:5′-TAG GTG ATT (WHR), waist circumference (WC), triglycerides and TGG GAG ATT TTT TTT -3′ and R:5′-CCC CTA ACA intake of trans fat. It is relevant to mention that methyla- ACC CAC TAA TAT TAA C-3′. The PCR program con - tion levels did not differ significantly by nutritional status. sisted of an initial enzymatic activation at 95 °C for To know which of BMI categories there was a significant 10 min, followed by 50 cycles of 45 s at 95 °C, 45 s at difference, Tukey’s test was performed, which occurred 60 °C and 45 s at 72 °C and the final extension at 72 °C more frequently between the categories “eutrophic” and for 10 min. The melting curves were normalized by cal - “obesity”. culation of the ‘line of best fit’ in between two normali - No significant association was observed among the vari - zation regions before and after the major fluorescence ables methylation levels of ADRB3 gene with lipid profile decrease representing the melting of the PCR product and oxidative stress in nutritional status of eutrophic, over- using the software provided with the HRM Software weight or obesity. However, it was observed that methyla- v2.0, provided by 7500 fast system. tion levels of ADRB3 gene were negatively associated with serum levels of folic acid in the eutrophic adults group; when folic acid values increased by 1 ng/ml, methylation Statistical analysis levels decreased in mean, 2.27% (p-value = 0.0327 < 0.05), All statistical analyses were performed with R software, according showed in Table 2. When applied multiple 3.3.2 . Initially it was performed the characterization regression based on model 1 including age and gender fac- of the sample by descriptive statistics represented by a tors in the three categories of BMI observed that there was single frequency, using position measurements, such as only relationship for methylation levels and male gender central tendency and dispersion. The data were tested in eutrophics (Additional file 1). This result means that, on for normality using Lilliefors test, which is a derivative average, eutrophics males have 0.1527 more methylation of the Kolmogorov–Smirnov test. Moreover, analysis of levels than females (p-value = 0.0070 < 0.05). variance (ANOVA) was used to verify if there was a mean Regarding the results of multiple regression based on difference between the BMI in which subjects were clas - model 2 described previously ont food intake, was not sified, for all quantitative variables of the study. de Oliveira et al. J Transl Med (2018) 16:152 Page 5 of 9 Table 1 Characteristics of adults population by nutritional status of the municipality of João Pessoa/PB/Brazil Parameter Eutrophic (43.51%) Overweight (36.26%) Obesity (20.23%) p-value Methylation levels (%) 43.29 ± 19.14 39.32 ± 17.57 42.74 ± 17.29 0.6020 Mean ± SD ab b a Average age 35.07 ± 11.83 41.78 ± 12.52 42.23 ± 11.58 0.0002* Mean ± SD ab bc ac WHtR (cm/cm) 0.40 ± 0.16 0.55 ± 0.06 0.61 ± 0.15 > 0.0000* Mean ± SD ab b a WHR (cm/cm) 0.79 ± 0.08 0.87 ± 0.09 0.92 ± 0.09 > 0.0000* Mean ± SD ab bc ac 89.48 ± 8.67 101.52 ± 13.90 > 0.0000* WC (cm) 74.45 ± 9.45 Mean ± SD Total cholesterol (mg/dl) 175.15 ± 35.63 195.73 ± 45.19 199.40 ± 43.48 0.9460 Mean ± SD LDL (mg/dl) 97.74 ± 47.71 112.03 ± 56.16 112.54 ± 62.30 0.1050 Mean ± SD HDL (mg/dl) 45.84 ± 11.70 41.45 ± 10.46 42.87 ± 10.31 0.2740 Mean ± SD a a Triglycerides (mg/dl) 120.39 ± 63.78 158.10 ± 93.39 158.33 ± 73.90 0.0241* Mean ± SD TAC (%) 41 ± 15 41 ± 14 42 ± 15 0.6880 Mean ± SD MDA 2.75 ± 0.99 2.98 ± 0.89 2.93 ± 0.75 0.6330 Mean ± SD Alpha-1 acid glycoprotein (mg/dl) 63.96 ± 20.86 64.66 ± 17.67 68.02 ± 16.45 0.2020 Mean ± SD Homocysteine (micromol/l) 11.42 ± 12.40 10.31 ± 6.43 10.69 ± 6.25 0.3540 Mean ± SD Serum folate (ng/ml) 14.23 ± 5.47 13.67 ± 4.87 14.70 ± 5.74 0.5660 Mean ± SD Vitamin B12 (pg/ml) 277.67 ± 136.74 261.83 ± 113.45 289.23 ± 126.91 0.0936 Mean ± SD Total fat (g) 55.12 ± 29.94 58.85 ± 52.14 58.47 ± 46.68 0.8810 Mean ± SD Calories (Kcal) 1805.16 ± 695.07 1829.67 ± 1012.35 1954.71 ± 1815.64 0.7160 Mean ± SD Dietary folate (mcg) 170.58 ± 174.83 139.58 ± 111.68 126.53 ± 89.34 0.5640 Mean ± SD Vitamin B12 (mcg) 2.10 ± 1.53 1.86 ± 1.59 2.01 ± 1.64 0.1370 Mean ± SD Monounsaturated fat (g) 14.50 ± 8.46 16.43 ± 13.80 15.07 ± 11.68 0.9240 Mean ± SD Oleic acid (g) 9.59 ± 7.33 12.03 ± 12.19 11.09 ± 10.75 0.7780 Mean ± SD Omega 3 (g) 0.64 ± 0.95 0.61 ± 0.63 0.68 ± 0.89 0.4100 Mean ± SD Omega 6 (g) 5.81 ± 4.96 6.81 ± 5.64 8.44 ± 14.17 0.2660 Mean ± SD Saturated fat (g) 17.33 ± 10.69 18.42 ± 17.72 16.85 ± 13.36 0.6750 Mean ± SD Cholesterol (mg) 264.93 ± 264.93 258.18 ± 224.64 226.09 ± 171.47 0.9460 Mean ± SD a a Trans fat (g) 0.75 ± 1.60 0.61 ± 0.54 0.76 ± 0.91 0.0172* Mean ± SD Significant difference in mean values of the variable between obese and eutrophic individuals (p-value Tukey’s test < 0.05) Significant difference in mean values of the variable between overweight and eutrophic individuals (p-value Tukey’s test < 0.05) Significant difference in mean values of the variable between obese and overweight individuals (p-value Tukey’s test < 0.05) de Oliveira et al. J Transl Med (2018) 16:152 Page 6 of 9 Table 2 Multiple regression analysis of methylation levels Discussion of the ADRB3 gene with lipid profile and oxidative stress In the present study, it was observed that methylation of adult individuals by nutritional status levels of ADRB3 gene were negatively associated with serum levels of folic acid in adult eutrophic individuals. Coefficient CI 95% Statistics t p-value Noting that as serum levels of folic acid in eutrophic indi- Total cholesterol (mg/dl) viduals increases, methylation levels decreases. However, Eutrophic* 0.23 (− 0.10 ± 0.56) 0.69 0.4999 no significant relationship was observed among lipid pro - Overweight 0.06 (− 0.04 ± 0.15) 0.61 0.5442 file, oxidative stress and food intake in individuals dis - Obesity − 0.13 (− 0.24 ± − 0.01) − 1.06 0.3000 tributed in the three categories of BMI. LDL (mg/dl) Although the mechanism responsible have not been Eutrophic* − 0.47 (− 0.82 ± − 0.11) − 1.31 0.2051 elucidated yet, higher serum folic acid concentrations Overweight − 0.02 (− 0.09 ± 0.04) − 0.38 0.7087 were associated with lower DNA methylation levels of Obesity 0.12 (0.03 ± 0.21) 1.35 0.1910 ADRB3 gene in eutrophic individuals, which may repre- HDL (mg/dl) sent a protective effect for obesity. This finding in accord - Eutrophic* 0.41 (− 0.16 ± 0.97) 0.72 0.4808 ance with the results of a study that demonstrated that Overweight 0.27 (− 0.02 ± 0.56) 0.92 0.3639 higher serum folic acid concentrations is associated Obesity 0.06 (− 0.49 ± 0.60) 0.12 0.9160 with lower levels of DNA methylation in leukocytes in Triglycerides (mg/dl) women with normal weight, and with a distinctive epi- Eutrophic* 0.08 (− 0.06 ± 0.22) 0.54 0.5941 genetic response in women with obesity, providing novel Overweight 0.02 (− 0.01 ± 0.05) 0.57 0.5742 evidences that the adequate folate metabolism may be Obesity 0.02 (− 0.04 ± 0.08) 0.36 0.720 affected by obesity, due probably to the volumetric dilu - TAC (%) tion, resulting in a redistribution of the vitamin from cir- Eutrophic* 27.64 (− 8.54 ± 63.83) 0.76 0.4543 culation into tissue . Overweight 11.30 (− 8.87 ± 31.48) 0.56 0.5789 Considering the difference between the forms and bio - Obesity 17.42 (− 7.63 ± 42.47) 0.70 0.4940 availability of folates, the folate family of compounds MDA include folic acid and its derivatives which include 5- Eutrophic* − 4.55 (− 1.37 ± 10.48) 0.77 0.4516 methyltetrahydrofolate (5-MTHF), 5-formyltetrahy- Overweight − 8.07 (− 12.60 ± − 3.53) − 1.78 0.0839 drofolate (5-FTHF or folinic acid), 10-formyl-THF, Obesity − 1.54 (− 6.81 ± 3.72) − 0.29 0.7720 5,10-methylene-THF and unsubstituted THF. Folic acid, a Alpha-1 acid glycoprotein (mg/dl) synthetic oxidized form of folate, is used in supplements Eutrophic* 0.23 (0.02 ± 0.44) 1.07 0.2964 and added to food because of its high stability and bio- Overweight 0.00 (− 0.18 ± 0.19) 0.02 0.9880 availability. Folinic acid is a 5-formyl derivative of THF. Obesity 0.08 (− 0.16 ± 0.32) 0.33 0.7420 Unlike the synthetic folate, folinic acid is naturally found Homocysteine (micromol/l) in food. It is readily converted to THF without requir- Eutrophic* − 1.74 (− 3.50 ± 0.01) − 0.99 0.3339 ing the action of the enzyme dihydrofolate reductase Overweight − 0.45 (− 0.82 ± − 0.08) − 1.20 0.2368 (DHFR). Therefore its function as a vitamin is unaffected Obesity − 0.54 (− 1.02 ± − 0.06) − 1.12 0.2750 by drugs inhibiting this enzyme, such as methotrexate. Serum folate (ng/ml) 5-MTHF is a biologically active form of folate and is the Eutrophic* − 2.27 (− 3.26 ± − 1.28) − 2.30 0.0327* most abundant form found in plasma, representing > 90% Overweight − 0.79 (− 1.53 ± − 0.05) − 1.06 0.2956 of folate and is the predominant active metabolite of Obesity − 0.44 (− 1.13 ± 0.25) − 0.63 0.5330 ingested folic acid . Vitamin B (pg/ml) Given the paucity of research on the different BMI cat - Eutrophic* 6.65 (2.72 ± 10.58) 1.69 0.1067 egories relating methylation levels in leukocytes in adults Overweight 3.67 (1.21 ± 6.14) 1.49 0.1452 of the same population, this study represents the first Obesity − 0.32 (− 2.80 ± 2.16) − 0.13 0.8970 effort on searching for differences in methylation levels of the ADRB3 gene in varying degrees of obesity, which may * Eutrophic: after adjustment for sex and age significant relationships remained for methylation levels and male gender help to understand of relations with different categories of the BMI variable. While it is well-known that central to the pathophysiol- observed significative relationship between methylation ogy of obesity is an excess amount of adiposity, on BMI levels of ADRB3 gene and food intake in the three catego- (which does not directly estimate body fat) still prevails ries of BMI (Additional file 1 ). as the most used indicator of adiposity worldwide, for practical reasons, comparability and ease to measure de Oliveira et al. J Transl Med (2018) 16:152 Page 7 of 9 weight and height . To refine risk assessment, BMI it is suggested that probably other factors have influenced could be complemented by also measuring WC to dis- the results such as sample size and non-stratification criminate between subcutaneous obesity and visceral by gender. It is also important to mention that differ - obesity, the ratio of WHR circumferences and the WHtR, ent blood cell populations may present different meth - this latter being a clinical indicator supporting of health ylation profiles, as already shown for genes implicated in risk, revealing that the accumulation of VAT and abdom- immune-related disorders , however we do not know inal subcutaneous fat is strongly associated with cardio- if this is applicable to the ADRB3 gene. metabolic risk . Nevertheless, when the gender was analyzed isolated Obesity is a heterogeneous disease with many differ - in relation to the methylation profile of ADRB3 gene, ent subtypes and DNA methylation might contribute to it was observed that males showed higher methyla- these differences . Although few researchers have tion levels than female (46.40 ± 18.81 and 39.42 ± 17.54 investigated the relationship of the ADRB3 gene and its respectively, p = 0.0037; Mann–Whitney). On the other role in susceptibility to obesity, only three studies on hand, when it evaluated the difference between levels methylation were found in the literature consulted, being of methylation by age groups no significant results were the first performed with methylation of ADRB3 gene in found (20–39 years old: 42.04 ± 19.12, 40–59 years old: severely obese men , the second with methylation in 41.40 ± 17.27; p = 0.9310; Mann–Whitney); (20–30 years VAT in obese and slim individuals , and the third with old: 39.95 ± 18.63, 31–40 years old: 45.70 ± 19.60, methylation in overweight and obese adult women . 41–50 years old: 44.39 ± 17.28, 51–59 years old: In this context, Guay et al.  demonstrated that 39.10 ± 17.04; p = 0.1338; Kruskal–Wallis). Indeed, the higher DNA methylation levels of ADRB3 gene in blood literature shows that in spite of external factors such as were significantly associated with a lower WHR and diet , smoking , alcohol consumption  and air lower LDL cholesterol levels in severely obese men. pollution , inner factors such as age and gender have Besides that, ADRB3 g.-843C>T and p.W64R polymor- also influence on the DNA methylation profile. Differ - phisms were found to be strongly associated with ADRB3 ences in the profile of DNA methylation between males DNA methylation, reinforcing the impact of both, muta- and female have already been observed in skin for miR- tions as well as DNA methylation at the ADRB3 gene 137 gene  and in buccal cells for MMP9 and TIMP- promoter. 1 genes . It is noteworthy that sex hormones may It has also been postulated that the Trp64Arg ADRB3 regulate DNA methylation differentially in the context of genetic variant is associated with 3.9 increase in BMI, different tissues, developmental stages, and pathological and these associations were observed mainly in East conditions . Asian populations. In contrast, lower gene expression of On the other hand, it is relevant to mention that it was the ADRB3 gene in Caucasians and its relationship with not detectable a relation between the methylation levels higher propensity to obesity and related comorbidities of the ADRB3 gene with overweight and obesity, since are still controversial . some studies have shown that these relations are more It was reported that the decrease in expression of prominent in severe obesity (grade III e IV) [9, 23]. Sug- ADRB3 gene in VAT is associated with obesity, but not gesting that, in overweight, mild and moderate obesity with the hypermethylation, demonstrating that the aver- (grade I e II), the state in which starts triggers the inflam - age levels of methylation of this gene in VAT was simi- matory cascade and the impacts of oxidative stress, the lar in slim individuals and with obesity from grade III amount of adiposity is still insufficient to influence the , corroborating with the present study regarding the epigenetic marks and result in alterations in phenotype. absence of difference of the methylation levels of the Interestingly, DNA methylation, especially in pro- ADRB3 gene in leukocytes for different categories of moter regions, is a well-characterized epigenetic marker BMI. u Th s, the identification in blood such as biomarker related to gene expression regulation in eukaryotes. Usu- easily accessible of the DNA methylation instead of adi- ally, methylation levels in the promoter are inversely cor- pose tissue is a promising alternative in the epigenetics related with the expression levels of the corresponding field, but still challenging. genes. However, new studies have evaluated the role of However, these findings do not apply to the present DNA methylation in the first exon, revealing that exon study, un precedented regarding methylation levels of the methylation facilitates the transcriptional process, and ADRB3 gene in leukocytes in a representative adult pop- impacts directly in genes expression . Thus, it is ulation of both genders by BMI categories. As it does not understood that other regions of the gene, spite of pro- exist in literature searching studies classifying the popu- moter region, might act in the expression control with lation based in the three categories of BMI and methyla- different methylation patterns. tion levels of the ADRB3 gene for comparative purposes, de Oliveira et al. J Transl Med (2018) 16:152 Page 8 of 9 In this context, some issues on the molecular level mild and moderate obesity, the answer probably lies in should be considered. Firstly, methylation may not be the insufficient amount of body fat to initiate inflamma - the chief mechanism involved in the regulation of activ- tory processes and oxidative stress with a direct impact ity of that gene in blood. Secondly, the analyzed regions, on methylation levels, what differently is found most although carefully chosen, may not be crucial for the times in exacerbated levels in severe obesity. regulation of gene expression . Thus, the impact of Additional file the methylation levels on ADRB3 gene and its relation- ship with lipid profile variables and oxidative stress might Additional file 1. Additional tables. be better elucidated through further studies of gene expression. In summary the beta-adrenergic receptor 3 encoded by Abbreviations the ADRB3 gene is involved in the regulation of lipoly- 5-FTHF: 5-formyltetrahydrofolate; 5-MTHF: 5-methyltetrahydrofolate; II DIS- ANDNT/JP: Second Cycle of Diagnosis and Intervention on the Diet, Nutrition sis and thermogenesis mainly in adipose tissue which and Most Prevalent Noncommunicable Diseases among the Population of provides free fatty acids for thermogenesis, indicating João Pessoa, Paraiba; ADRB3: gene encoding beta-3 adrenergic receptor; AHA: the participation of the ADRB3 gene in the regulation of American Heart Association; BMI: body mass index; DHFR: dihydrofolate reduc- tase; HDL: high density lipoprotein; HRM: high resolution melting; LDL: low body weight in humans. In the present study we analysed density lipoprotein; MDA: malondialdehyde; PCR: polymerase chain reaction; blood tissue and it is important to mention that blood PPGCN: Post Graduate Program in Nutrition Sciences; R24h: 24-hour dietary is metabolically active tissue, with an important role in recall; RBC: red blood cells; TAC : total antioxidant capacity; TC: total cholesterol; WC: waist circumference; WHO: World Health Organization; WHR: waist-to-hip the adverse inflammatory and vascular consequences of ratio; WHtR: waist-to-height ratio; UFPB: Federal University of Paraíba; VAT: adiposity, and is widely used for clinical diagnostic pur- visceral adipose tissue. poses . It has been suggested that polymorphisms in Authors’ contributions beta 3 adrenoreceptor signal transduction, binding, or YdeO contributed to data acquisition and interpretation, and writing of the regulatory mechanism may result in diminished lipolytic manuscript. RPAL coordinated the DNA methylation analyses, contributed response . In relation to association to ADRB3 meth- to data to data management and DNA methylation tests. RCPL, MGAM, CSOdaS and KQFL contributed to data management. NFPdeO was involved ylation profile and body weight the studies are still scarce in the interpretation and critical review of the manuscript. RPdeTV, FELdeLF and this relation needs to be further studied. and MdaCRG contributed to the analysis of food consumption. ASdaS and Some limitations of the present study are relevant. RAFdoN contributed to the biochemical analyses. ASD was involved in the development of the manuscript or in the critical review of relevant intellectual Regarding the absence of relationship with lipid pro- content. AHAeS, ATCA and RMdeM contributed to the study design and file, oxidative stress and food intake, probably did not statistical analysis. MJdeCC contributed to the study design, statistical analysis, occured due to the higher prevalence of eutrophic indi- data interpretation, and writing of the manuscript; she coordinated data collection and was involved in the development of the manuscript or in the viduals, lower prevalence of obesity from the degree III, critical review of relevant intellectual content. All authors read and approved and the status of all variables studied situated around 90% the final manuscript. in reference values in the total sample. And as the obeses Author details group was composed mostly by individuals with mild and Health Sciences Center (Centro de Ciências da Saúde), Departament moderate obesity (grade I e II), there was no representa- of Nutrition, Federal University of Paraíba (Universidade Federal da Paraíba), tive group of severe obesity (grade III and IV) (n = 5) in João Pessoa, Brazil. Center of Exact Sciences and Nature (Centro de Ciências Exatas e da Natureza), Federal University of Paraíba (Universidade Federal da present study. In addition, a stratification by nutritional Paraíba), João Pessoa, Brazil. Departament of Statistics, Center of Exact Sci- status according with gender was not performed, reflect - ences and Nature (Centro de Ciências Exatas e da Natureza), Federal University ing the methylation levels of the ADRB3 gene in leuko- of Paraíba (Universidade Federal da Paraíba), João Pessoa, Brazil. Departa- ment of Molecular Biology, Center of Exact Sciences and Nature (Centro de cytes based in a representative adult population of both Ciências Exatas e da Natureza), Federal University of Paraíba (Universidade genders. 5 Federal da Paraíba), João Pessoa, Brazil. Department of Economics, Center for Applied Social Sciences (Centro de Ciências Sociais Aplicadas), Federal University of Paraíba (Universidade Federal da Paraíba), João Pessoa, Brazil. Departament of Nutrition, Health Sciences Center (Centro de Ciências da Conclusions Saúde), Federal University of Pernambuco (Universidade Federal de Pernam- In conclusion, it was demonstrated a negative relation- buco), Recife, Brazil. ship between methylation levels of the ADRB3 gene in Acknowledgements individuals eutrophic adults with serum levels of folic We would like to acknowledge the professor Ph.D. José Luiz de Brito Alves acid, as well as with the independent gender of BMI, (Federal University of Paraíba), who was involved in the critical review of the however, it was not observed relationship between lipid manuscript. The funding agencies that enabled the project (National Council of Scientific and Technological Development (CNPq), Ministry of Health and profile, oxidative stress and food intake in individuals dis - Research Support Foundation of the State of Paraíba (FAPESQ, PB, Brazil); and tributed in the three categories of BMI. research collaborators (Post Graduate Program in Nutrition Sciences, Federal Regarding the absence of relationship with methylation University of Paraíba). levels of the ADRB3 gene in the categories of overweight, de Oliveira et al. J Transl Med (2018) 16:152 Page 9 of 9 Competing interests em. http://www.de.ufpb.br/~ronei /AnaHe rmini a2009 Escol a_Amost The authors declare that they have no competing interests.ragem 2009.pdf. Accessed 20 Mar 2016. 14. World Health Organization. Obesity: preventing and managing the global Availability of data and materials epidemic. Report of a WHO consultation, vol. 894. Geneva: World Health The data generated in this study are coordinated by Ph.D. Maria José de Organization; 2000. p. 11–253. Carvalho Costa. 15. Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin BA, et al. Diagnosis and management of the metabolic syndrome: an Ameri- Consent for publication can Heart Association/National Heart, Lung, and Blood Institute scientific Not applicable. statement: executive Summary. Crit Pathw Cardiol. 2005;4(4):198–203. 16. World Health Organization. Definition, diagnostics and classification of Ethics approval and consent to participate diabetes mellitus and its complications. Reports of a WHO consultation. The study was approved by the Research Ethics Committee of the Center of Geneva: World Health Organization; 1999. Health Sciences (CCS), Federal University of Paraíba, under the Protocol Num- 17. Ashwell M, Gibson S. Waist-to-height ratio as an indicator of ‘early health ber 0559/13. All subjects gave their written informed consent. risk’: simpler and more predictive than using a ‘matrix’ based on BMI and waist circumference. BMJ Open. 2016;6:1–7. Funding 18. Lima RPA, do Nascimento RAF, Luna RCP, Persuhn DC, da Silva AS, da Con- The funding agencies that enabled the project (National Council of Scientific ceição M, et al. Eec ff t of a diet containing folate and hazelnut oil capsule and Technological Development (CNPq), Ministry of Health and Research Sup- on the methylation level of the ADRB3 gene, lipid profile and oxidative port Foundation of the State of Paraíba (FAPESQ, PB, Brazil) under the Number stress in overweight or obese women. Clin Epigenetics. 2017;9:110. EFP_00008187. 19. R Development Team. R: A language and environment for statistical com- puting. R foundation for statistical computing. 2009. http://www.r-proje ct.org. Accessed 16 Oct 2017. Publisher’s Note 20. Scaglione F, Panzavolta G. 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Published: Jun 5, 2018
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