8-week Combined Exercise Protocol Promote Health and Cardiovascular Benefits in Obese Women by Epigenetic Modifications: a Prospective Study

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This study explored the effects of an 8-week integrated strength and aerobic training protocol on the DNA methylation patterns in obese women. Methods: 13 women with a BMI of 33±2 kg/m², aged 34±5 years, underwent evaluation of body composition, waist circumference, physical performance (VO 2 max), and peripheral blood collection for DNA methylation analysis (EPIC Beadchip Illumina) before and after eight weeks of combined physical training intervention (3 times/week, 55 minutes/session, 70-90% of max HR). Results: The intervention yielded noteworthy clinical enhancements, encompassing diminished waist circumference, fat mass, and increased VO2 max (p<0.05). In DNA methylation, we identified 16 Differentially Methylated Regions (DMRs) encompassing 103 CpG sites. Most of these regions were mainly located in promoters’ regions (TSS1500 and TSS200). Among the identified DMRs, 8 exhibited hypomethylation, while the remaining 8 showed hypermethylation after exercise protocol. Enrichment analysis unveiled pathways linked to cardiac, immune, and thermogenic functions. Conclusion: Combined physical training can promote modifications in the DNA methylation profile at CpG sites in genes involved in energy metabolism pathways, increase physical performance, and reduce body fat and waist circumference. Regardless of weight loss, the exercise protocol may promote protection against cardiovascular diseases through epigenetic modifications. Trial registration: ClinicalTrials.gov (NTC 03119350). Physical exercise Obesity DNA methylation energy expenditure waist circumference fat mass Figures Figure 1 Background Obesity, a disease with a multifactorial etiopathology attributed to genetic, epigenetic, and environmental factors [ 1 ], is considered a pandemic. In Brazil, obesity increased by 60% between 2006 and 2016 [ 2 ]. It is known that obesity can be affected by genetic alterations, but little is known about the epigenetic markers related to health promotion interventions [ 3 ]. Among these interventions, physical exercise can condition the body to the point of imbalance between weight maintenance and energy expenditure, consequently reducing body weight and improving cardiorespiratory performance [ 4 ]. Epigenetics studies hereditary or non-hereditary chemical modifications in the chromatin structure without implications for DNA sequence, with DNA methylation being the most investigated process [ 5 ]. Due to its role in regulating gene expression, the DNA methylation pattern is a mechanism involved in susceptibility to several diseases, such as obesity [ 6 ]. On the other hand, although obesity has been associated with changes in the DNA methylation pattern, little is known about epigenetic markers in responses to weight loss interventions. Previous studies evidenced changes in the methylation profile of inflammatory genes after different treatments for obesity, including diet and bariatric surgery [ 7 , 8 ]. Interestingly, a study that evaluated the effect of combined exercise in epigenetic profile of women who underwent bariatric surgery also found changes in genes related to inflammatory pathways [ 9 ]. It is already known that physical exercise benefits cardiovascular, metabolic, muscular, and skeletal health through several mechanisms [ 10 ], including epigenetics modifications [ 11 ]. In this context, researchers have explored how physical exercise can modulate DNA methylation profile in different populations, including elderly individuals [ 12 ] or healthy young individuals [ 13 ]. However, there is still a gap to be filled about exercise-induced epigenetic effects in obese women. Among the training approaches discussed in the literature and the epigenetic changes induced by exercise, strength training [ 13 ] or aerobic training [ 14 ] tend to present various positive changes. Combining strength and aerobic training in the same session, known as combined training, has been emerging with excellent findings, such as epigenetic rejuvenation after eight weeks of training [ 15 ] or the remodeling of genes related to pre-diabetes after 14 weeks of training [ 16 ]. This study aims to improve conventional understandings of obesity management by analyzing the DNA methylation effects of a combined training protocol in obese women. Going beyond traditional assessments, we seek to unravel the differentially methylated regions (DMRs) and identify motifs that could unveil therapeutic opportunities in this challenging context. The innovative approach and focus on personalized interventions offer insights that could reshape current paradigms in obesity research and potentially lead to novel, tailored therapeutic strategies grounded in epigenetics. METHODS Study design and Participants For the development of this prospective and longitudinal study, women with a body mass index (BMI) between 30 and 40 kg/m², aged 20 to 40 years, sedentary, with regular menstrual cycles, and of miscegenated ethnicity from the northern region of the state of São Paulo were included (convenience sample). Exclusion criteria included medication use, smoking, alcohol consumption, associated comorbidities (diabetes mellitus, dyslipidemia, metabolic syndrome, and cardiovascular diseases), physical impairment for exercise, bariatric surgery, or weight loss treatment in the last six months. Also, participants with more than 20% absences from the intervention were excluded. This study was approved by the Hospital das Clinics Ethics Committee, Faculty of Medicine of Ribeirão Preto/USP (approval number 1.387.040) and registered in ClinicalTrials.gov (NTC 03119350). All study participants was informed and gave free written consent. After advertising on social media, approximately 100 obese women expressed interest in participating in the study. Of them, only twenty women were eligible, were included in the study and started the intervention protocol. This study lasted 12 weeks, with two weeks of pre-intervention assessments and adaptation of combined physical training, eight weeks of intervention with combined physical training and two weeks of post-intervention assessments. During the 8 weeks, 3 women dropped out, 3 were excluded due to illness, 6 were excluded due to training absences (> 20% absences), and one woman was excluded due to problems with the DNA sample. Then, thirteen women completed entire protocol study. Training Protocol Patients underwent intervention with combined physical training for 8 weeks, three times a week, with each session lasting 55 minutes (100% supervised). The training session consisted of 15 strength exercises performed in a circuit: 30 seconds of strength exercise (at least 15 repetitions) and 30 seconds of running/walking. Training intensity was individualized, between 75 and 90% of the maximum heart rate based on the results of physical tests [ 17 , 18 ]. Physical Assessments All participants were assessed before and after 8 weeks of intervention. Body weight and height were measured using Filizola® Eletrônica ID 1500 (São Paulo, Brazil). Body composition was assessed using the deuterium oxide dilution method [ 19 ], with each volunteer receiving a dose of 1 mL/k 7% deuterium oxide (Cambridge Isotope). Urine samples were collected before and three hours after dose intake. Deuterium enrichment in urine samples was determined by mass spectrometry (Europa Scientific Hydra System™). The waist circumference was measured with an inextensible tape at the largest circumference, between the last rib and the iliac crest [ 20 ]. Aerobic performance was evaluated by adapting the Shuttle Walking Test incremental test, and afterward, we estimated maximal oxygen consumption (VO2max) [ 18 ]. DNA Methylation For DNA methylation analysis, we collected peripheral blood samples in EDTA tubes (pre and post-intervention). DNA extraction was performed using an automated extractor with the Maxwell kit (Promega, WI, EUA). After extraction, bisulfite conversion was carried out using the EZ DNA Methylation-Gold kit (ZymoResearch, CA, EUA). DNA methylation analysis based on an array was conducted using the Infinium Human Methylation EPIC Beadchip (Illumina, San Diego, CA) according to manufacturer instructions [ 21 ]. Bioinformatic Analysis The whole data analysis pipeline, where done using the ChAMP package for RStudio (version 4.2.1) 18 . We extracted raw data (IDATS), performed data normalization, and corrected the cell type proportions using the myRefBase function. To identify differentially methylated regions (DMRs) we used the bumphunter method. This method stands out for defining CpG groups of probes located around the same genomic region [ 21 ]. After identifying DMRs, we used our CpG table list to perform pathway enrichment through the EWAS Toolkit [ 22 ]. After enrichment, we used ShinyGo v0.77 to identify possible motifs within the list of differentially methylated regions [ 23 ]. Statistical Analysis Data are presented as mean and standard deviation. The normality of data was checked using the Shapiro-Wilk test; all our phenotypic data showed normal distribution, adopting the Student’s t-test to compare pre and post-intervention moments. The significance level was set at p < 0.05. Analysis was performed using Graph Pad Prism 9.1 (Graph Pad Software Inc., USA). RESULTS As expected, 8-weeks combined training exercise protocol improved health and promote metabolic benefits including reduction in waist circumference (p = 0.006) and fat mass (p = 0.043), and increase in VO 2 max (p = 0.020) (Table 1 ). Table 1 Anthropometric measures, body composition and physical fitness pre- and post-8 weeks of physical training Variables Pre Post P-value Age (years) 34.4 ± 4.7 34.4 ± 4.7 - BMI (kg/m²) 33.0 ± 2.0 32.2 ± 2.0 0.959 Weight (kg) 85.6 ± 7.5 86.2 ± 7.6 0.410 WC (cm) 93.8 ± 6.1 91.1 ± 5.3 0.006 * FM (kg) 39.6 ± 3.6 38.9 ± 5.8 0.043 * FFM (kg) 45.7 ± 4.9 47.4 ± 4.2 0.329 FM (%) 47.1 ± 3.0 44.8 ± 3.0 0.810 FFM (%) 52.1 ± 3.8 55.2 ± 3.9 0.083 VO₂ max (mL/kg/min) 18.9 ± 3.2 23.4 ± 3.9 0.020 * Note - BMI: body mass index; WC: waist circumference; FM: fat mass; FFM: fat-free mass; VO₂ max: maximum oxygen consumption. Also, our analysis identified 16 Differentially Methylated Regions (DMRs) after exercise training protocol, that encompass a cumulative total of 103 CpG sites, as shown in Table 2 . Most of these regions were mainly located in promoter’s regions of specific genes (TSS1500 and TSS200). Among the identified DMRs, 8 were hypomethylated, while the remaining 8 were hypermethylated after exercise training protocol. Two DMRs of particular significance were SPON1 (DMR_13), characterized as the most hypermethylated region (Δβ 8%, FDR 0.020), and GATA5 (DMR_14), identified as the most hypomethylated region (Δβ 1,6%, FDR 0.019). These findings highlight the dynamic nature of methylation patterns in specific genomic regions and underscore the potential regulatory roles of SPON1 and GATA5 in the observed biological context. Table 2 Differentially Methylated Regions (DMRs) after 8 weeks of physical training DMR FDR Δβ Methylation Status CHR Enriched Region Genic Region Feature DMR_1 0.001 -0.003 Hypomethylated 5 miR-886 TSS1500; TSS200; IGR; Body Shore; island DMR_2 0.002 0.015 Hypermethylated 10 LRRC20 TSS1500 Shore DMR_3 0.002 0.008 Hypermethylated 2 KRTCAP3 Body; TSS200; 1stExon Island; shore DMR_4 0.005 -0.010 Hypomethylated 17 SLFN11 TSS200 Shore; island DMR_5 0.004 0.007 Hypermethylated 10 LOC441666 Body; TSS200 Shore; island DMR_6 0.005 -0.006 Hypomethylated 17 PTRH2; TMEM49 5'UTR; TSS200 Shore; island DMR_7 0.006 -0.011 Hypomethylated 6 DDX43 1stExon Island; shore DMR_8 0.007 0.012 Hypermethylated 22 C22orf26 Body; 1stExon Shore; island DMR_9 0.007 -0.010 Hypomethylated 4 DCK Body; 1stExon Island DMR_10 0.009 0.006 Hypermethylated 1 PM20D1 TSS200; TSS1500; 5'UTR; 1stExon Shore; island DMR_11 0.011 -0.005 Hypomethylated 14 LTB4R2 Body Island DMR_12 0.014 0.007 Hypermethylated 19 AURKC TSS200; 5'UTR Island DMR_13 0.020 0.081 Hypermethylated 11 SPON1 TSS1500; TSS200 Shore DMR_14 0.019 -0.016 Hypomethylated 20 GATA5 5'UTR; TSS200; 1stExon Island DMR_15 0.029 0.001 Hypermethylated 19 ISOC2 TSS200; TSS1500 Island DMR_16 0.030 -0.007 Hypomethylated 5 BDP1 TSS200; TSS1500 Island; shore Note - DMR: Differentially Methylated Region; FDR: false discovery rate; Δβ: delta beta ; CHR: chromosome; TSS200 or 1500: Transcription Start Site 200 or 1500; IGR: intergenic region; 5’UTR: 5’ untranslated region. Figure 1 showcases the primary enriched pathways associated with the 16 differentially methylated regions (DMRs). Noteworthy among these pathways are those linked to the potential therapeutic effects of the exercise. Specifically, we highlight the Notch pathway, which plays a role in heart induction (GO:0035481, GO:0035480, GO:0003137), as well as the regulation of oxidative phosphorylation uncoupler activity (GO:2000275). These findings shed light on compelling avenues for understanding the molecular mechanisms underlying the beneficial impacts of the exercise regimen. Table 3 lists the transcription factors that can bind to the enriched Motifs. The C2H2 transcription factor (C2H2 ZF), a minor structural protein motif characterized by zinc ion coordination, was consistently the most prevalent in this analysis. Table 3 Motifs identified with the genes of the 16 DMRs after 8 weeks of physical training. Enriched motif in promoter TF TF family P value FDR Score ACGT GMEB1 SAND 2.7E-04 1.1E-01 1.6E + 01 TGCGGG ZBTB1 C2H2 ZF 2.3E-03 2.2E-01 2.2E + 01 GGCGCC E2F2 E2F 2.9E-03 2.2E-01 3.6E + 01 GGGCGGGAA E2F6 E2F 3.4E-03 2.2E-01 4.6E + 01 GGGGGGGGGTGGTTTGGGG RREB1 C2H2 ZF 3.7E-03 2.2E-01 6.0E + 01 GGGGGCGGGGC SP2 C2H2 ZF 3.9E-03 2.2E-01 6.5E + 01 GGGCGGGGC KLF5 C2H2 ZF 3.9E-03 2.2E-01 4.4E + 01 GGGGGGGGGCC PATZ1 C2H2 ZF 5.0E-03 2.2E-01 4.3E + 01 GCGGGGGCGGGG EGR1 C2H2 ZF 5.4E-03 2.2E-01 5.5E + 01 GGGGCCCAAGGGGG PLAG1 C2H2 ZF 6.3E-03 2.2E-01 5.6E + 01 GGGGGTGG ZNF281 C2H2 ZF 6.5E-03 2.2E-01 3.3E + 01 GGCGGGAA E2F4 E2F 6.5E-03 2.2E-01 4.4E + 01 Note - TF: transcription factor. FDR: false discovery rate; Upstream 300bp as promoter. DISCUSSION In our study, an eight-week combined training program resulted in significant clinical improvements, including reductions in waist circumference and fat mass, and an increase in VO₂ max. Notably, our unique training protocol, combining strength and aerobic exercises in the same session, deviates from existing literature and demonstrates multiple benefits for obese women [ 24 ]. Furthermore, a meaningful increase in participants' VO₂ max was observed, a crucial factor in reducing the risk of cardiovascular disease mortality, regardless of weight loss. The combination of aerobic and strength exercises in the same session positively influenced arteriovenous oxygen difference, impacting VO₂ max through increased catecholamine production and enhanced nitric oxide bioavailability [ 25 ]. The enrichment of DMRs included several pathways: energy metabolism, thermogenic enzymes, brown adipose tissue enrichment, regulation of body weight and adiposity, autophagy process, signaling modulation, apoptosis, transcription and gene expression, and association with heart diseases. These results reenforces the important role of physical exercise in promote weight loss and induce metabolic adaptations. In fact, these metabolic effects were mediated by changes in DNA methylation profile [ 26 ]. For example, these changes in DNA methylation allow increased insulin sensitivity and expression of genes involved in energy metabolism, myogenesis, contractile properties, and oxidative stress [ 27 ]. More specifically, we found changes in DNA methylation levels of some important genes that should be highlighted. The KRTCAP3 hypermethylated in our study has been considered a candidate gene for adiposity correlated with decreased fat mass [ 28 ]. Like this, the SPON1 hypermethylated gene can influence bone mineral density, affect the balance of bone remodeling, and lead to the occurrence and development of osteoporosis [ 29 ]. While the GATA5 had hypomethylation, this gene is involved in the lipid and nitric oxide pathway and has recently been identified as a regulator of blood pressure [ 30 ]. That can promote protection against cardiovascular diseases. This hypermethylation and hypomethylation, respectively, corroborates the clinical findings of our study, in which body fat and waist circumference were reduced and cardiorespiratory performance increased. Furthermore, the investigation into cardiogenesis revealed the intricate developmental process involving cell fate determination, proliferation, differentiation, and morphogenesis. The Notch signaling pathway emerged as a pivotal orchestrator in this process, influencing cell fate decisions and exhibiting significant crosstalk with other pathways during the differentiation and patterning of cardiac tissues [ 31 ]. Our study contributes to this understanding by identifying the Notch pathway as a potential player, particularly in heart induction (GO:0035481, GO:0035480, GO:0003137) [ 32 ]. The regulation of oxidative phosphorylation uncoupler activity (GO:2000275) was also one of the exciting pathways that could be related to the metabolic and clinical changes observed in the present study. It is well established that mitochondrial energy metabolism has thermogenic effects, and these processes can be modulated during tissue remodeling and its implications for human metabolic diseases [ 33 ]. Moreover, the icosanoid and leukotrienes (GO:0004953, GO:0001632) pathways that appeared in the enrichment analysis are potential explanations for the metabolic crosstalk between the change in the substrate preference after exercise, evidenced by the increased lipid oxidation as well as the increase of the resting metabolic rate and the decrease in the absolute fat mass [ 34 ]. These findings suggest that exercise has an immunomodulatory effect. Icosanoids are lipid metabolites that influence inflammation; leukotrienes B4 are one of the key metabolites of the unsaturated fatty acid roles in obesity and subclinical inflammation, which is worsened with diets high in glucose and saturated fatty acids. Leukotriene B4 (LTB4), a proinflammatory lipid mediator, plays a pivotal role in sustaining inflammation in obesity and insulin resistance, and strategies to mitigate the LTB4-LTB4R1 axis are explored to address the inflammatory consequences of high-fat diet-induced obesity [ 34 ]. Additionally, after the 8-week training, we identified motifs within the differentially methylated regions (DMRs) genes, with particular attention to Cys2-His2 zinc finger proteins (C2H2-ZF). As the largest class of potential human transcription factors, C2H2-ZF proteins represent a relatively underexplored regulatory system [ 35 ]. The extent of their involvement in adaptive gene regulation programs, particularly in physical exercise, has just been systematically explored. Notably, Rodrigues et al.[ 12 ] identified C2H2-ZF motifs in a study related to training, mirroring the attention garnered by this TF family in our research. Collectively, our findings contribute to a growing body of evidence emphasizing the potential significance of C2H2-ZF proteins in the molecular responses to physical exercise, and the therapeutic nature of this target should be investigated in further studies. As with any study, there are limitations to consider. The sample size in our exercise intervention may limit generalizability, and individual variations in response to the training program were not extensively explored. Additionally, the scope of our analysis of Notch signaling in cardiogenesis may warrant further investigation. However, it is a longitudinal study in which the training intervention was supervised, an essential tool for evaluating clinical results. In the current context of genomics, tools that allow evaluating associations between the genome, disease, and environment have increased. The data produced by this study can elucidate the effects of physical exercise as a treatment on human health. Thus, providing a better understanding of epigenetic regulation before and after physical exercise may assist in new strategies for treating obesity. Strengths of our study include the comprehensive assessment of clinical and molecular outcomes following the unique exercise intervention. CONCLUSION In conclusion, our study provides insights into the clinical and molecular effects of an 8-week combined training program for obese women. We found a reduction in body fat and waist circumference and increased cardiorespiratory performance, an essential protector against other cardiometabolic diseases. It was also possible to observe changes in the DNA methylation profile, mainly in CpG sites involved in energy metabolism pathways. We also demonstrate that DNA methylation analysis is a viable approach to further our understanding of the complexity of metabolism and obesity. The observed effects, particularly on the Notch and mitochondrial energy metabolism pathways, suggest therapeutic avenues to address obesity-related health problems. Abbreviations BMI Body mass index. DMRs:Differentially Methylated Regions. VO 2max :maximum oxygen consumption Declarations Ethical approval and consent to participate The Research Ethics Committee of the Clinical Hospital from the Ribeirao Preto Medical School, University of São Paulo, approved the study (protocol #1.387.040), and the study is in accordance with the standards of ethics outlined in the Declaration of Helsinki. All subjects gave free written consent. All study participants were informed and gave free written consent to participate. Consent for publication Not applicable Availability of data and material All data may be made available from the corresponding author upon reasonable request. Competing interests The authors declare no conflict of interest. Funding This work was supported by Minas Gerais State Research Support Foundation – FAPEMIG (APQ-02169-21; APQ-02960-22; APQ-03029-23; APQ-03316-23), Researcher UEMG Productivity – PQ/UEMG, CNPq (National Council of Scientific and Technological Development – process 154169/2018-8 and 302231/2022-6). Authors’ Contributions JSM, CBN and CFCB conceived and designed the study. CFCB, FGC, LMD and MVMJF performed data collection. CFN, NYN and GSR measured DNA methylation data. CFCB, MVMJF and ECF analyzed clinic data. NYN and CPA performed the bioinformatics and statistical analysis. CFCB, GSR, CFN and NYN drafted the manuscript. JSM, CBN and ECF reviewed the manuscript. All authors approved the final version of the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4119029","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282165888,"identity":"b64d06f6-8110-4e35-90f1-541d26f8068b","order_by":0,"name":"Camila Fernanda Cunha Brandão","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYJCCAw8YGBJADAkGBhuwCDNBLQkILWnEaWFA0nKYsBb+9tOJBxJqGPLM288evPGj4nxiv9jxC8yFe3BrkTiTu+FAwjGGYpkzecmWPWduJ86cnVPAPOMZbi0GDCAtbAyJMxhyzCR4224nbridk8DMcwCPFv63QC3/gFr435hJ/m07l7ifoBYJoC2JbUAtEjlm0rxtBxI3SKcfwKtF4gbQlsQ+iWIJiTfG1jJnko1n3M5hODwDjxb+/tzNHz58s8mT4M8xvPmmwk62f3b6w8cFeLTALEPm8BgQ1oAG2B+QqmMUjIJRMAqGNwAARWJY1R7b55AAAAAASUVORK5CYII=","orcid":"","institution":"University of São Paulo","correspondingAuthor":true,"prefix":"","firstName":"Camila","middleName":"Fernanda Cunha","lastName":"Brandão","suffix":""},{"id":282165889,"identity":"1435f1e8-7e23-459c-a495-fe84fea78cbe","order_by":1,"name":"Guilherme Silva Rodrigues","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Guilherme","middleName":"Silva","lastName":"Rodrigues","suffix":""},{"id":282165893,"identity":"a676f3aa-741d-441f-a9e2-bca6523c0f5a","order_by":2,"name":"Natália Yumi Noronha","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Natália","middleName":"Yumi","lastName":"Noronha","suffix":""},{"id":282165894,"identity":"4da8b4af-b190-4d14-aee5-43e1c75fc23e","order_by":3,"name":"Carolina F Nicoletti","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Carolina","middleName":"F","lastName":"Nicoletti","suffix":""},{"id":282165895,"identity":"30d783cd-a04a-4d81-9ea6-09fd8d2d2435","order_by":4,"name":"Marcia VM Junqueira-Franco","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Marcia","middleName":"VM","lastName":"Junqueira-Franco","suffix":""},{"id":282165896,"identity":"6715476a-9625-4c43-850a-4e07de34b4dd","order_by":5,"name":"Cleidson Padua Alves","email":"","orcid":"","institution":"University of Cologne","correspondingAuthor":false,"prefix":"","firstName":"Cleidson","middleName":"Padua","lastName":"Alves","suffix":""},{"id":282165897,"identity":"c485ab6b-110e-47be-a3f9-2d8eae34ef53","order_by":6,"name":"Luísa Maria Diani","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Luísa","middleName":"Maria","lastName":"Diani","suffix":""},{"id":282165898,"identity":"09a4d83a-59cd-42d2-a22c-a08dfd60b977","order_by":7,"name":"Flavia G Carvalho","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Flavia","middleName":"G","lastName":"Carvalho","suffix":""},{"id":282165899,"identity":"6c222c74-4a6c-4d42-8276-f196963d282d","order_by":8,"name":"Ellen C Freitas","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Ellen","middleName":"C","lastName":"Freitas","suffix":""},{"id":282165900,"identity":"2c67d4af-6e0c-4913-8021-d3b03370e207","order_by":9,"name":"Carla B Nonino","email":"","orcid":"","institution":"Ribeirão Preto Medical School","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"B","lastName":"Nonino","suffix":""},{"id":282165901,"identity":"3ccaf998-5fac-4f3d-9b96-186a409e6877","order_by":10,"name":"Julio S Marchini","email":"","orcid":"","institution":"University of São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Julio","middleName":"S","lastName":"Marchini","suffix":""}],"badges":[],"createdAt":"2024-03-17 23:59:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4119029/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4119029/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53364240,"identity":"4d1c4a00-7f1c-4f20-8b14-22976f2cf9d1","added_by":"auto","created_at":"2024-03-25 05:45:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":293081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene Ontology (GO) enrichment of probes that are differentially methylated after the 8 weeks of combined exercise training.\u003c/strong\u003e Number of Genes Differentially Methylated in the respective pathways, the size of the circle represents if the enrichment retrieved 1 or 2 genes. The -log10(p) represents the magnitude of the FDR as more red is more significant in the post-test.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4119029/v1/306cddf9ecc9de1e097dca8a.png"},{"id":54494027,"identity":"7799ccdc-0900-453f-950f-4eaf3e80f3ce","added_by":"auto","created_at":"2024-04-11 11:14:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":652890,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4119029/v1/be443274-e51a-46d7-aa75-f9dd58b91bfb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e8-week Combined Exercise Protocol Promote Health and Cardiovascular Benefits in Obese Women by Epigenetic Modifications: a Prospective Study\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eObesity, a disease with a multifactorial etiopathology attributed to genetic, epigenetic, and environmental factors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], is considered a pandemic. In Brazil, obesity increased by 60% between 2006 and 2016 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is known that obesity can be affected by genetic alterations, but little is known about the epigenetic markers related to health promotion interventions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among these interventions, physical exercise can condition the body to the point of imbalance between weight maintenance and energy expenditure, consequently reducing body weight and improving cardiorespiratory performance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEpigenetics studies hereditary or non-hereditary chemical modifications in the chromatin structure without implications for DNA sequence, with DNA methylation being the most investigated process [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to its role in regulating gene expression, the DNA methylation pattern is a mechanism involved in susceptibility to several diseases, such as obesity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. On the other hand, although obesity has been associated with changes in the DNA methylation pattern, little is known about epigenetic markers in responses to weight loss interventions. Previous studies evidenced changes in the methylation profile of inflammatory genes after different treatments for obesity, including diet and bariatric surgery [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Interestingly, a study that evaluated the effect of combined exercise in epigenetic profile of women who underwent bariatric surgery also found changes in genes related to inflammatory pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is already known that physical exercise benefits cardiovascular, metabolic, muscular, and skeletal health through several mechanisms [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], including epigenetics modifications [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this context, researchers have explored how physical exercise can modulate DNA methylation profile in different populations, including elderly individuals [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] or healthy young individuals [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, there is still a gap to be filled about exercise-induced epigenetic effects in obese women.\u003c/p\u003e \u003cp\u003eAmong the training approaches discussed in the literature and the epigenetic changes induced by exercise, strength training [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] or aerobic training [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] tend to present various positive changes. Combining strength and aerobic training in the same session, known as combined training, has been emerging with excellent findings, such as epigenetic rejuvenation after eight weeks of training [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] or the remodeling of genes related to pre-diabetes after 14 weeks of training [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aims to improve conventional understandings of obesity management by analyzing the DNA methylation effects of a combined training protocol in obese women. Going beyond traditional assessments, we seek to unravel the differentially methylated regions (DMRs) and identify motifs that could unveil therapeutic opportunities in this challenging context. The innovative approach and focus on personalized interventions offer insights that could reshape current paradigms in obesity research and potentially lead to novel, tailored therapeutic strategies grounded in epigenetics.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and Participants\u003c/h2\u003e \u003cp\u003eFor the development of this prospective and longitudinal study, women with a body mass index (BMI) between 30 and 40 kg/m\u0026sup2;, aged 20 to 40 years, sedentary, with regular menstrual cycles, and of miscegenated ethnicity from the northern region of the state of S\u0026atilde;o Paulo were included (convenience sample). Exclusion criteria included medication use, smoking, alcohol consumption, associated comorbidities (diabetes mellitus, dyslipidemia, metabolic syndrome, and cardiovascular diseases), physical impairment for exercise, bariatric surgery, or weight loss treatment in the last six months. Also, participants with more than 20% absences from the intervention were excluded. This study was approved by the Hospital das Clinics Ethics Committee, Faculty of Medicine of Ribeir\u0026atilde;o Preto/USP (approval number 1.387.040) and registered in ClinicalTrials.gov (NTC 03119350). All study participants was informed and gave free written consent.\u003c/p\u003e \u003cp\u003eAfter advertising on social media, approximately 100 obese women expressed interest in participating in the study. Of them, only twenty women were eligible, were included in the study and started the intervention protocol. This study lasted 12 weeks, with two weeks of pre-intervention assessments and adaptation of combined physical training, eight weeks of intervention with combined physical training and two weeks of post-intervention assessments.\u003c/p\u003e \u003cp\u003eDuring the 8 weeks, 3 women dropped out, 3 were excluded due to illness, 6 were excluded due to training absences (\u0026gt;\u0026thinsp;20% absences), and one woman was excluded due to problems with the DNA sample. Then, thirteen women completed entire protocol study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTraining Protocol\u003c/h2\u003e \u003cp\u003ePatients underwent intervention with combined physical training for 8 weeks, three times a week, with each session lasting 55 minutes (100% supervised). The training session consisted of 15 strength exercises performed in a circuit: 30 seconds of strength exercise (at least 15 repetitions) and 30 seconds of running/walking. Training intensity was individualized, between 75 and 90% of the maximum heart rate based on the results of physical tests [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhysical Assessments\u003c/h2\u003e \u003cp\u003eAll participants were assessed before and after 8 weeks of intervention. Body weight and height were measured using Filizola\u0026reg; Eletr\u0026ocirc;nica ID 1500 (S\u0026atilde;o Paulo, Brazil). Body composition was assessed using the deuterium oxide dilution method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], with each volunteer receiving a dose of 1 mL/k 7% deuterium oxide (Cambridge Isotope). Urine samples were collected before and three hours after dose intake. Deuterium enrichment in urine samples was determined by mass spectrometry (Europa Scientific Hydra System\u0026trade;). The waist circumference was measured with an inextensible tape at the largest circumference, between the last rib and the iliac crest [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAerobic performance was evaluated by adapting the Shuttle Walking Test incremental test, and afterward, we estimated maximal oxygen consumption (VO2max) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDNA Methylation\u003c/h2\u003e \u003cp\u003eFor DNA methylation analysis, we collected peripheral blood samples in EDTA tubes (pre and post-intervention). DNA extraction was performed using an automated extractor with the Maxwell kit (Promega, WI, EUA). After extraction, bisulfite conversion was carried out using the EZ DNA Methylation-Gold kit (ZymoResearch, CA, EUA). DNA methylation analysis based on an array was conducted using the Infinium Human Methylation EPIC Beadchip (Illumina, San Diego, CA) according to manufacturer instructions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic Analysis\u003c/h2\u003e \u003cp\u003eThe whole data analysis pipeline, where done using the ChAMP package for RStudio (version 4.2.1)\u003csup\u003e18\u003c/sup\u003e. We extracted raw data (IDATS), performed data normalization, and corrected the cell type proportions using the myRefBase function. To identify differentially methylated regions (DMRs) we used the bumphunter method. This method stands out for defining CpG groups of probes located around the same genomic region [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. After identifying DMRs, we used our CpG table list to perform pathway enrichment through the EWAS Toolkit [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. After enrichment, we used ShinyGo v0.77 to identify possible motifs within the list of differentially methylated regions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean and standard deviation. The normality of data was checked using the Shapiro-Wilk test; all our phenotypic data showed normal distribution, adopting the Student\u0026rsquo;s t-test to compare pre and post-intervention moments. The significance level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Analysis was performed using Graph Pad Prism 9.1 (Graph Pad Software Inc., USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAs expected, 8-weeks combined training exercise protocol improved health and promote metabolic benefits including reduction in waist circumference (p\u0026thinsp;=\u0026thinsp;0.006) and fat mass (p\u0026thinsp;=\u0026thinsp;0.043), and increase in VO\u003csub\u003e2\u003c/sub\u003emax (p\u0026thinsp;=\u0026thinsp;0.020) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnthropometric measures, body composition and physical fitness pre- and post-8 weeks of physical training\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariables\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePre\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 34.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBMI (kg/m\u0026sup2;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.959\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.6\u0026thinsp;\u0026plusmn;\u0026thinsp;7.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.410\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWC (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.006\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFM (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.043\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFFM (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.329\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.810\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFFM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e55.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVO₂ max (mL/kg/min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.020\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNote\u003c/b\u003e - BMI: body mass index; WC: waist circumference; FM: fat mass; FFM: fat-free mass; VO₂ max: maximum oxygen consumption.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAlso, our analysis identified 16 Differentially Methylated Regions (DMRs) after exercise training protocol, that encompass a cumulative total of 103 CpG sites, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Most of these regions were mainly located in promoter\u0026rsquo;s regions of specific genes (TSS1500 and TSS200). Among the identified DMRs, 8 were hypomethylated, while the remaining 8 were hypermethylated after exercise training protocol. Two DMRs of particular significance were SPON1 (DMR_13), characterized as the most hypermethylated region (Δβ 8%, FDR 0.020), and GATA5 (DMR_14), identified as the most hypomethylated region (Δβ 1,6%, FDR 0.019). These findings highlight the dynamic nature of methylation patterns in specific genomic regions and underscore the potential regulatory roles of SPON1 and GATA5 in the observed biological context.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDifferentially Methylated Regions (DMRs) after 8 weeks of physical training\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFDR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔβ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMethylation Status\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCHR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEnriched\u003c/p\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGenic\u003c/p\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFeature\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003emiR-886\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS1500; TSS200; IGR; Body\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore; island\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLRRC20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eKRTCAP3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBody; TSS200; 1stExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland; shore\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSLFN11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore; island\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLOC441666\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBody; TSS200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore; island\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePTRH2; TMEM49\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5'UTR; TSS200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore; island\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eDDX43\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1stExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland; shore\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eC22orf26\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBody; 1stExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore; island\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eDCK\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBody; 1stExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ePM20D1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS200; TSS1500; 5'UTR; 1stExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore; island\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eLTB4R2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBody\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eAURKC\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS200; 5'UTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSPON1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS1500; TSS200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eShore\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eGATA5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5'UTR; TSS200; 1stExon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypermethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eISOC2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS200; TSS1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMR_16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypomethylated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eBDP1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTSS200; TSS1500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIsland; shore\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNote -\u003c/b\u003e DMR: Differentially Methylated Region; FDR: false discovery rate; Δβ: delta beta ; CHR: chromosome; TSS200 or 1500: Transcription Start Site 200 or 1500; IGR: intergenic region; 5\u0026rsquo;UTR: 5\u0026rsquo; untranslated region.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showcases the primary enriched pathways associated with the 16 differentially methylated regions (DMRs). Noteworthy among these pathways are those linked to the potential therapeutic effects of the exercise. Specifically, we highlight the Notch pathway, which plays a role in heart induction (GO:0035481, GO:0035480, GO:0003137), as well as the regulation of oxidative phosphorylation uncoupler activity (GO:2000275). These findings shed light on compelling avenues for understanding the molecular mechanisms underlying the beneficial impacts of the exercise regimen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e lists the transcription factors that can bind to the enriched Motifs. The C2H2 transcription factor (C2H2 ZF), a minor structural protein motif characterized by zinc ion coordination, was consistently the most prevalent in this analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMotifs identified with the genes of the 16 DMRs after 8 weeks of physical training.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnriched motif in promoter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTF family\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFDR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eGMEB1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSAND\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.7E-04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.1E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.6E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTGCGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eZBTB1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.3E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.2E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGCGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE2F2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE2F\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.9E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.6E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGGCGGGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE2F6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE2F\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.4E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.6E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGGGGGGGGTGGTTTGGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eRREB1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.7E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.0E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGGGGCGGGGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSP2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.5E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGGCGGGGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKLF5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.4E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGGGGGGGGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePATZ1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.3E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGCGGGGGCGGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eEGR1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.4E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.5E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGGGCCCAAGGGGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePLAG1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.3E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.6E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGGGGTGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eZNF281\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eC2H2 ZF\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.5E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.3E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGGCGGGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE2F4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE2F\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.5E-03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.2E-01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.4E\u0026thinsp;+\u0026thinsp;01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNote -\u003c/b\u003e TF: transcription factor. FDR: false discovery rate; Upstream 300bp as promoter.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn our study, an eight-week combined training program resulted in significant clinical improvements, including reductions in waist circumference and fat mass, and an increase in VO₂ max. Notably, our unique training protocol, combining strength and aerobic exercises in the same session, deviates from existing literature and demonstrates multiple benefits for obese women [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, a meaningful increase in participants' VO₂ max was observed, a crucial factor in reducing the risk of cardiovascular disease mortality, regardless of weight loss. The combination of aerobic and strength exercises in the same session positively influenced arteriovenous oxygen difference, impacting VO₂ max through increased catecholamine production and enhanced nitric oxide bioavailability [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe enrichment of DMRs included several pathways: energy metabolism, thermogenic enzymes, brown adipose tissue enrichment, regulation of body weight and adiposity, autophagy process, signaling modulation, apoptosis, transcription and gene expression, and association with heart diseases. These results reenforces the important role of physical exercise in promote weight loss and induce metabolic adaptations. In fact, these metabolic effects were mediated by changes in DNA methylation profile [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For example, these changes in DNA methylation allow increased insulin sensitivity and expression of genes involved in energy metabolism, myogenesis, contractile properties, and oxidative stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMore specifically, we found changes in DNA methylation levels of some important genes that should be highlighted. The KRTCAP3 hypermethylated in our study has been considered a candidate gene for adiposity correlated with decreased fat mass [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Like this, the SPON1 hypermethylated gene can influence bone mineral density, affect the balance of bone remodeling, and lead to the occurrence and development of osteoporosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. While the GATA5 had hypomethylation, this gene is involved in the lipid and nitric oxide pathway and has recently been identified as a regulator of blood pressure [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. That can promote protection against cardiovascular diseases. This hypermethylation and hypomethylation, respectively, corroborates the clinical findings of our study, in which body fat and waist circumference were reduced and cardiorespiratory performance increased.\u003c/p\u003e \u003cp\u003eFurthermore, the investigation into cardiogenesis revealed the intricate developmental process involving cell fate determination, proliferation, differentiation, and morphogenesis. The Notch signaling pathway emerged as a pivotal orchestrator in this process, influencing cell fate decisions and exhibiting significant crosstalk with other pathways during the differentiation and patterning of cardiac tissues [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our study contributes to this understanding by identifying the Notch pathway as a potential player, particularly in heart induction (GO:0035481, GO:0035480, GO:0003137) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe regulation of oxidative phosphorylation uncoupler activity (GO:2000275) was also one of the exciting pathways that could be related to the metabolic and clinical changes observed in the present study. It is well established that mitochondrial energy metabolism has thermogenic effects, and these processes can be modulated during tissue remodeling and its implications for human metabolic diseases [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, the icosanoid and leukotrienes (GO:0004953, GO:0001632) pathways that appeared in the enrichment analysis are potential explanations for the metabolic crosstalk between the change in the substrate preference after exercise, evidenced by the increased lipid oxidation as well as the increase of the resting metabolic rate and the decrease in the absolute fat mass [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings suggest that exercise has an immunomodulatory effect. Icosanoids are lipid metabolites that influence inflammation; leukotrienes B4 are one of the key metabolites of the unsaturated fatty acid roles in obesity and subclinical inflammation, which is worsened with diets high in glucose and saturated fatty acids. Leukotriene B4 (LTB4), a proinflammatory lipid mediator, plays a pivotal role in sustaining inflammation in obesity and insulin resistance, and strategies to mitigate the LTB4-LTB4R1 axis are explored to address the inflammatory consequences of high-fat diet-induced obesity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, after the 8-week training, we identified motifs within the differentially methylated regions (DMRs) genes, with particular attention to Cys2-His2 zinc finger proteins (C2H2-ZF). As the largest class of potential human transcription factors, C2H2-ZF proteins represent a relatively underexplored regulatory system [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The extent of their involvement in adaptive gene regulation programs, particularly in physical exercise, has just been systematically explored. Notably, Rodrigues et al.[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] identified C2H2-ZF motifs in a study related to training, mirroring the attention garnered by this TF family in our research. Collectively, our findings contribute to a growing body of evidence emphasizing the potential significance of C2H2-ZF proteins in the molecular responses to physical exercise, and the therapeutic nature of this target should be investigated in further studies.\u003c/p\u003e \u003cp\u003eAs with any study, there are limitations to consider. The sample size in our exercise intervention may limit generalizability, and individual variations in response to the training program were not extensively explored. Additionally, the scope of our analysis of Notch signaling in cardiogenesis may warrant further investigation. However, it is a longitudinal study in which the training intervention was supervised, an essential tool for evaluating clinical results. In the current context of genomics, tools that allow evaluating associations between the genome, disease, and environment have increased. The data produced by this study can elucidate the effects of physical exercise as a treatment on human health. Thus, providing a better understanding of epigenetic regulation before and after physical exercise may assist in new strategies for treating obesity. Strengths of our study include the comprehensive assessment of clinical and molecular outcomes following the unique exercise intervention.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, our study provides insights into the clinical and molecular effects of an 8-week combined training program for obese women. We found a reduction in body fat and waist circumference and increased cardiorespiratory performance, an essential protector against other cardiometabolic diseases. It was also possible to observe changes in the DNA methylation profile, mainly in CpG sites involved in energy metabolism pathways.\u003c/p\u003e \u003cp\u003eWe also demonstrate that DNA methylation analysis is a viable approach to further our understanding of the complexity of metabolism and obesity. The observed effects, particularly on the Notch and mitochondrial energy metabolism pathways, suggest therapeutic avenues to address obesity-related health problems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBody mass index. DMRs:Differentially Methylated Regions. VO\u003csub\u003e2max\u003c/sub\u003e:maximum oxygen consumption\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Research Ethics Committee of the Clinical Hospital from the Ribeirao Preto Medical School, University of São Paulo, approved the study (protocol #1.387.040), and the study is in accordance with the standards of ethics outlined in the Declaration of Helsinki. All subjects gave free written consent. All study participants were informed and gave free written consent to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data may be made available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Minas Gerais State Research Support Foundation – FAPEMIG (APQ-02169-21; APQ-02960-22; APQ-03029-23; APQ-03316-23), Researcher UEMG Productivity – PQ/UEMG, CNPq (National Council of Scientific and Technological Development – process 154169/2018-8 and 302231/2022-6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJSM, CBN and CFCB conceived and designed the study. CFCB, FGC, LMD and MVMJF performed data collection. CFN, NYN and GSR measured DNA methylation data. \u0026nbsp;CFCB, MVMJF and ECF analyzed clinic data. \u0026nbsp;NYN and CPA performed the bioinformatics and statistical analysis. CFCB, GSR, CFN and NYN drafted the manuscript. JSM, CBN and ECF reviewed the manuscript. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CFCB would like to University of the State of Minas Gerais for the Productivity scholarship – PQ/UEMG.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLin X, Li H. Obesity: Epidemiology, Pathophysiology, and Therapeutics. Front Endocrinol. 2021; 6(12):706978. \u003c/li\u003e\n\u003cli\u003eDantas LF, Marchesi JF, Peres IT, Hamacher S, Bozza FA, Quintano Neira RA. Public hospitalizations for stroke in Brazil from 2009 to 2016. Dal Pizzol F, editor. 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Cell. 2014;158(6):1431\u0026ndash;43. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Physical exercise, Obesity, DNA methylation, energy expenditure, waist circumference, fat mass","lastPublishedDoi":"10.21203/rs.3.rs-4119029/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4119029/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e It is known that obesity can be affected by genetic and epigenetic alterations, however little is known about the DNA methylation markers related to health promotion interventions aiming weigh loss. This study explored the effects of an 8-week integrated strength and aerobic training protocol on the DNA methylation patterns in obese women.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e 13 women with a BMI of 33±2 kg/m², aged 34±5 years, underwent evaluation of body composition, waist circumference, physical performance (VO\u003csub\u003e2\u003c/sub\u003emax), and peripheral blood collection for DNA methylation analysis (EPIC Beadchip Illumina) before and after eight weeks of combined physical training intervention (3 times/week, 55 minutes/session, 70-90% of max HR).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The intervention yielded noteworthy clinical enhancements, encompassing diminished waist circumference, fat mass, and increased VO2 max (p\u0026lt;0.05). In DNA methylation, we identified 16 Differentially Methylated Regions (DMRs) encompassing 103 CpG sites. Most of these regions were mainly located in promoters’ regions (TSS1500 and TSS200). Among the identified DMRs, 8 exhibited hypomethylation, while the remaining 8 showed hypermethylation after exercise protocol. Enrichment analysis unveiled pathways linked to cardiac, immune, and thermogenic functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Combined physical training can promote modifications in the DNA methylation profile at CpG sites in genes involved in energy metabolism pathways, increase physical performance, and reduce body fat and waist circumference. Regardless of weight loss, the exercise protocol may promote protection against cardiovascular diseases through epigenetic modifications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration:\u003c/strong\u003e ClinicalTrials.gov (NTC 03119350).\u003c/p\u003e","manuscriptTitle":"8-week Combined Exercise Protocol Promote Health and Cardiovascular Benefits in Obese Women by Epigenetic Modifications: a Prospective Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 05:44:55","doi":"10.21203/rs.3.rs-4119029/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5457330-cb9b-4df4-9768-501100265a43","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-11T11:05:54+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-25 05:44:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4119029","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4119029","identity":"rs-4119029","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}