Extended Abstract
Background and Aim: Overweight and obesity have become major global public health concerns, largely driven by sedentary lifestyles, reduced physical activity, and unhealthy dietary habits. These conditions are strongly associated with metabolic disorders, including type 2 diabetes mellitus, dyslipidemia, and cardiovascular diseases. An imbalance between energy intake and energy expenditure is considered the primary cause of obesity, highlighting the importance of lifestyle interventions for its prevention and management. Regular physical exercise is an effective non-pharmacological strategy for weight management, glycemic control, and the improvement of lipid metabolism; however, its physiological benefits depend on the type, intensity, and duration of exercise.. Brown adipose tissue (BAT) plays a critical role in energy expenditure through adaptive thermogenesis. Slit guidance ligand 2 (SLIT-2), a protein reduced in obese individuals, and its C-terminal fragment (SLIT2-C) activate thermogenic pathways via protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP), enhancing lipolysis, mitochondrial density, and uncoupling protein 1 (UCP-1) expression, which promotes browning of white fat. While studies show both resistance and endurance training may upregulate UCP-1, conflicting results exist, and the comparative efficacy of resistance training (RT), continuous endurance training (CET), and high-intensity interval training (HIIT) on SLIT-2 and UCP-1 gene expression in obese male Wistar rats remains unresolved.
Materials and Methods: This experimental laboratory study was conducted on 36 adult male Wistar rats (8 weeks old; mean body weight: 225 ± 40 g) obtained from the Razi Serum Research Institute, Mashhad, Iran. The animals were housed under controlled laboratory conditions at a temperature of 22 ± 5 °C, relative humidity of approximately 45%, and a 12:12-hour light–dark cycle, with free access to standard chow and water. Obesity, defined as a body weight exceeding 300 g at the time of group allocation, was used as the inclusion criterion. Following a one-week familiarization period on a motorized treadmill and a 36-rung rat climbing ladder, the animals were randomly assigned to four groups (n=9 per group): continuous endurance training (CET), resistance training (RT), high-intensity interval training (HIIT), and control. Maximal running speed was determined using an incremental exercise test before the intervention and repeated after four weeks to adjust the training intensity. The CET protocol consisted of a warm-up followed by 30 minutes of continuous running at progressively increasing intensities (60%–70% of maximal running speed) over the eight-week intervention, followed by a cool-down period. The RT protocol involved climbing a weighted ladder for three sets of five repetitions per session, with the load progressively increased from 50% to 120% of body weight throughout the intervention. The HIIT protocol consisted of repeated two-minute running bouts performed at 85%–90% of maximal running speed, interspersed with active recovery intervals, with the number of bouts gradually increasing from six to twelve over the eight-week training period.
Total ribonucleic acid (RNA) was extracted from tissue samples and reverse-transcribed into complementary DNA (cDNA) using a thermocycler-based polymerase chain reaction (PCR) system, with gene expression quantified via real-time PCR using SYBR Green dye and the threshold cycle (Ct) recorded for each sample. After confirming normal data distribution, one-way analysis of variance (ANOVA) was used to examine between-group differences in SLIT-2 and UCP-1 gene expression, followed by Tukey’s post hoc test for pairwise comparisons, with all statistical analyses performed using the Statistical Package for the Social Sciences (SPSS), version 22, at a significance level of p≤0.05.
Findings: One-way ANOVA revealed that eight weeks of CET, RT, and HIIT significantly increased SLIT-2 and UCP-1 gene expression in male Wistar rats compared with control (p≤0.05). Given this significant overall effect, Tukey’s post hoc test was subsequently performed to identify the specific source of between-group differences. For SLIT-2 gene expression, Tukey’s test showed that the CET, RT, and HIIT groups each differed significantly from the control group (p=0.001 for all three comparisons); in contrast, no significant differences were observed among the three exercise groups themselves, whether comparing RT with CET (p=0.82), RT with HIIT (p=0.99), or CET with HIIT (p=0.99).
A similar pattern was observed for UCP-1 gene expression: the CET, RT, and HIIT groups again differed significantly from control (p=0.001 for each comparison), while pairwise comparisons among the exercise groups showed no significant differences (RT vs. CET, p=0.99; RT vs. HIIT, p=0.99; CET vs. HIIT, p=0.99). Both SLIT-2 and UCP-1 gene expression increased markedly following CET, RT, and HIIT relative to the control group, with mean fold-change values ranging from approximately 3.1 to 3.3 across all three exercise groups for both genes, compared with a baseline value of roughly 1.0 in the control group, corresponding to an approximately three-fold increase in relative expression of both genes following each of the three training protocols. Moreover, these findings demonstrate a consistent and statistically robust effect of exercise training on the expression of both genes, regardless of training modality. Therefore, all three training protocols produced comparable increases in SLIT-2 and UCP-1 expression, none of the exercise groups differed significantly from one another in either gene, and this pattern, observed identically across both target genes, suggests that CET, RT, and HIIT were equally effective in upregulating SLIT-2 and UCP-1 expression in this model, with no evidence that any single training modality conferred superior efficacy over the others. The magnitude and direction of change were highly consistent between the two genes, further supporting the reliability of the observed training-induced upregulation across all three exercise protocols in this obese Wistar rat model.
Conclusion: Based on the results, it clear that the mechanisms underlying these adaptations remain incompletely understood, particularly for SLIT-2, whose exercise-related signaling pathways have not previously been characterized. The SLIT-2 is implicated in angiogenesis, anti-inflammatory processes, and lipolysis, and its C-terminal fragment promotes thermogenesis via the protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) pathway. In this way, the SLIT-2 expression appears to be driven by PRDM16, whose downstream activation may explain the training-induced increases observed here, consistent with prior reports demonstrating that PRDM16 induces SLIT-2 expression. Furthermore, SLIT-2 enhances lipolysis, mitochondrial biogenesis, and PI3K-mediated immune regulation, thereby potentially mitigating obesity-related metabolic dysfunction.
For UCP-1, exercise-induced upregulation is thought to be mediated through multiple mechanisms, including irisin release and MAPK/p38 signaling, PGC-1α-dependent mitochondrial biogenesis, sympathetic activation via β3-adrenergic receptors, and thyroid hormone–dependent deiodinase activity. UCP-1 facilitates proton leakage across the inner mitochondrial membrane, thereby uncoupling oxidative phosphorylation from ATP synthesis and dissipating energy as heat. The discrepancies observed among previous studies regarding UCP-1 responsiveness may be attributable to differences in training duration, dietary conditions, and the anatomical site of adipose tissue sampling.
Compliance with Ethical Guidelines: All experimental procedures were conducted in accordance with ethical guidelines for animal research and were approved by the Ethics Committee of Bojnourd Azad University (Approval Code: IR.IAU.BOJNOURD.REC.1402.017). All procedures were performed minimizing pain and distress to the animals.
Funding: This research is based on a doctoral dissertation from a student at the Islamic Azad University, Bojnourd Branch, and was conducted without any financial support.
Conflicts of interest: There are no conflicts of interest regarding this article.