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.