This study was reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University (Protocol No. 202407037).

Forty male Sprague–Dawley rats, aged 6–8 weeks, were selected as experimental subjects. The animals were obtained from the Jiangsu Experimental Animal Centre of Nanjing Medical University (production licence number: SYXK [Jiangsu] 2023–0081). They were housed in groups of four per cage under controlled environmental conditions: room temperature 20 °C–26 °C, relative humidity 40%–70%, a 12 h light-dark cycle, and ad libitum access to food and water. After a 1-week acclimatisation period on standard chow (Synergy: 1,010,088), an 8 week obesity induction phase was initiated. Rats (aged 7–9 weeks) were randomly divided into two groups: control (C, n = 10) and high-fat diet (HFD, n = 30). The control group continued to receive standard chow, whereas the HFD group was fed a high-fat diet (Synergy: XTHF45-1) containing 47% of calories from fat, 20% from protein, and 33% from carbohydrates. Following the 8 week obesity induction, rats in the HFD group with a Lee’s index 20% higher than the mean value of the control group and a Lee’s index ≥310 were classified as obese (Lee, 1929). Twenty-four obese rats (aged 15–17 weeks) meeting these criteria were randomly assigned into three subgroups: high-fat diet (H, n = 8), high-fat diet + aerobic training (HMT, n = 8), and high-fat diet + high-intensity intermittent training (HHT, n = 8). All subgroups continued their respective diets (standard or high-fat) until the conclusion of the experiment. Clean the animal room and cages daily, and replace the drinking water. Record and calculate the difference between the initial food supply and the remaining amount the next day for each cage of rats to obtain the food intake. Replace the bedding once every 2 days.

Following a 1-week adaptive training period, rats in the HMT and HHT groups underwent an 8 week training intervention (six sessions/week) beginning in Week 10, whereas no training intervention was applied to the C and H groups. Body weight and food intake were recorded daily throughout the experiment. Twenty-four hours after the final training session. Following induction with 3%–4% isoflurane anesthesia via inhalation (maintained at 2%–2.5%), the experimental rat was placed in a supine position and secured. Blood was collected via femoral artery puncture. Subsequently, the chest was rapidly opened to expose the heart. The pericardium was incised using scissors, and a perfusion cannula was inserted into the left ventricle. Perfusion with ice-cold PBS was performed rapidly until the liver blanched and the effluent became clear. After perfusion, the absence of spontaneous respiration and reflex activity confirmed that the animal was non-responsive. Tissue samples were then harvested. A portion of the tissue was reserved for detection of target gene/protein expression, while another portion was prepared for histological staining.

At the start of the training intervention, an incremental-load treadmill test was performed to determine the rats’ maximal running speed. The treadmill incline was set to 0°, and the initial speed was 10 m/min, which was increased by 2 m/min at each stage until the rats reached exhaustion and could not continue running despite re-peated encouragements. The speed of the penultimate stage was recorded as the maximal running speed.

For the HMT group, each session comprised a 10 min warm-up phase (treadmill speed: 10 m/min), a 40 min moderate-intensity aerobic phase (60%–70% of maximal running speed), and a 10 min recovery phase (treadmill speed: 10 m/min).

For the HHT group, each session comprised a 10 min warm-up phase and a 10 min recovery phase (both at 10 m/min) at the beginning and end, respectively. The intermediate 40 min interval comprised alternating treadmill exercise: 3 min at 50% of maximal running speed followed by 3 min at 90%, repeated throughout the 40 min phase (Høydal et al., 2007).

Assay kits for triglyceride (TG), total cholesterol (T-CHO), free fatty acid (FFA), cytochrome c oxidase (COX), and succinate dehydrogenase (SDH) activities were sourced from Nanjing Jiancheng Bioengineering Institute. IL-27 enzyme-linked im-munosorbent assay kits were obtained from Suzhou Jianglai Biotechnology Co., Ltd.

The tissue samples were fixed, embedded in paraffin, and sectioned. After routine dewaxing and hydration, they were stained with hematoxylin for 6 min, differentiated with acid alcohol (1% HCl in 70% ethanol) for 3 s, blued in running water, counterstained with eosin for 2 min, and finally dehydrated, cleared, and mounted with neutral gum.

Total RNA was extracted from iWAT and PRAT using TRIzol (Ambion, Texas, United States). RNA concentration was measured using an ultraviolet spectrophotometer, and cDNA was synthesised using the ReverTra Ace™ qPCR RT Kit (TOYOBO Co., Ltd., Osaka, Japan). qPCR was performed using the FastStart Universal SYBR Green Master Mix (Rox) kit (Roche Diagnostics Corporation, Indiana, United States), and relative gene ex-pression was calculated using the 2^−ΔΔCT method. The primer sequences for the target genes are shown in Table 1.

Protein samples were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. The polyvinylidene difluoride membrane was activated, equili-brated, and placed in a rotary folder using the standard sandwich configuration for 10–15 min. The transferred proteins were detected by immunoblotting, which involved rinsing and blocking, followed by sequential incubation with a primary antibody against UCP1 (1:1,000, ABclonal) and an HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:10,000, ABclonal). Signals were visualised by enhanced electro-chemiluminescence, and images were captured in greyscale.

Data were analysed using GraphPad Prism software (version 10.2) and are pre-sented as mean ± standard error of the mean (SEM). Comparisons between the C and H groups were performed using a two-tailed t-test and one-sample t-test. Differences among the H, HMT, and HHT groups were assessed by one-way analysis of variance followed by Tukey’s post hoc test. A p-value <0.05 was considered statistically significant.

After 8 weeks of HFD feeding, rats in the H group exhibited significantly higher body weight than those in the C group (Figure 1A). During the 8 week training intervention, a significant difference in food intake was observed between the exercise groups and the sedentary group. However, no significant difference was found between the HMT and HHT groups. (Figure 1B), whereas exercise training reduced overall body weight (Figure 1C). Following the 8 week intervention, rats in the HMT and HHT groups displayed lower body weight compared with the H group (Figure 1D). HFD feeding significantly increased plasma T-CHO, TG, and FFA, while exercise training enhanced lipid metabolic utilisation (Figures 1E,F). Notably, the HHT group showed greater reductions in plasma TG levels than the HMT group, an effect that may result from improved fat oxidation capacity following high-intensity intermittent training.

After dissection, iWAT and PRAT were isolated and weighed. Results showed that obesity led to significant adipose accumulation, manifesting as marked increases in iWAT (Figure 2A) and PRAT (Figure 2C) weights, which were attenuated by exercise training. Similarly, adipocyte size in iWAT (Figure 2B) and PRAT (Figure 2D) increased in obese rats and decreased following exercise. No significant differences were observed between HIIT and MAIT in reducing adipose tissue weight or cell size, consistent with previous findings (Khalafi et al., 2020).

Analysis of cellular respiratory enzyme activity levels allows assessment of mitochondrial functional status. Complex II (or succinate dehydrogenase, SDH), encoded by nuclear DNA, is typically unaffected by mitochondrial DNA damage; increased SDH activity may indicate mitochondrial biogenesis (Edgar et al., 2009; Ross et al., 2010). Complex IV (or cytochrome c oxidase, COX) is essential for mitochondrial respiration (Ross, 2011). In this study, enzymatic activity assays of iWAT and PRAT revealed that obesity suppressed SDH activity in iWAT (Figure 3A), an effect reversed by training. Training also enhanced COX activity in iWAT (Figure 3C). SDH activity in PRAT (Figure 3B) followed the trend observed in iWAT. Obesity suppressed COX activity in PRAT (Figure 3D) but was restored with training. Collectively, training improved adipose mitochondrial function by enhancing SDH and COX activity, with greater improvements in iWAT than in PRAT. MAIT had stronger effects than HIIT in improving SDH and COX activity in iWAT and PRAT, potentially because MAIT primarily relies on fat oxidation as its main energy substrate.

UCP1 is a marker of adipocyte thermogenic capacity, and its expression is used to distinguish BAT from WAT (Sidossis and Kajimura, 2015). Results showed that obesity reduced UCP1 expression in iWAT and PRAT, indicating that obesity impairs adipose thermogenic capacity, whereas training restored UCP1 expression (Figures 4A,B). Training also enhanced adipose thermogenic capacity, with HMT exhibiting superior effects. The elevated expression of Cidea, Cited, and Tbx1, genes enriched in brown or beige adipose tissue, suggests that training promotes the conversion of WAT to beige or BAT.

At the protein level, UCP1 abundance in iWAT and PRAT decreased with obesity but increased after exercise training (Figures 4C–E). HHT was more effective than HMT in elevating UCP1 protein abundance in iWAT.

To investigate the effects of obesity and long-term exercise training on IL-27 expression, we measured plasma IL-27 levels and the mRNA expression of IL-27 and related pathway components in adipose tissue. Plasma IL-27 levels were significantly reduced in obese rats (Figure 5A). Similarly, mRNA expression of IL-27 and associated signalling molecules was markedly reduced in iWAT and PRAT (Figures 5B,C). Exercise training reversed these effects, increasing plasma IL-27 levels and upregulating IL-27 and its regulatory pathway in adipose tissue. Compared with MAIT, HIIT was more effective in elevating plasma IL-27 levels and promoting mRNA expression of IL-27, p38 MAPK, and PGC-1α in adipose tissue. Mechanistically, IL-27 facilitated p38 MAPK phosphorylation, which subsequently enhanced PGC-1α activity. PGC-1α, a key regulator of UCP1-mediated thermogenesis, was thus identified as a critical downstream effector of this pathway (Ricquier and Bouillaud, 2000; Stine et al., 2013; Norheim, 2014).