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Bioinformatics Analysis: The datasets GSE205891 and GSE230002 from the GEO database were mined, and differential expression analysis was performed on each dataset using the online tool GEO2R to screen for differentially expressed genes (DEGs). The criteria for identifying DEGs were a fold change of more than 2 or less than 0.5, with P < 0.05. The lists of DEGs from both datasets were loaded into R, and the intersecting genes were identified. GO and KEGG enrichment analyses were conducted on these intersecting genes. The protein-protein interaction (PPI) network of the intersecting genes was constructed using the STRING database and Cytoscape software, and the top 10 key genes were calculated. The interactions between the proteins encoded by these genes were analyzed to identify those at critical nodes.
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Experimental Animals and Grouping: Thirty-six 8-week-old male db/db mice (diabetic model) and 12 age-matched db/m mice (normal control) were purchased and housed in the SPF-level animal facility at Wuhan Sports University. After one week of acclimatization, random blood glucose levels were measured to ensure that all db/db mice had random blood glucose levels ≥ 16.6 mmol/L, meeting the diabetic model criteria. Subsequently, the db/db mice were randomly divided into three groups: DC group (diabetic control), MICT group, and HIIT group, with 12 mice in each group. The 12 db/m mice served as the NC group (normal control).
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Exercise Intervention: Prior to formal exercise, the MICT and HIIT groups underwent one week of treadmill adaptation training, followed by a maximum exercise capacity test to determine the maximum running speed (Vmax) for each mouse. The formal intervention lasted for 10 weeks, with exercises conducted 5 days per week, once daily. The HIIT group exercised at 90% Vmax, while the MICT group exercised at 70% Vmax. The specific protocols were as follows: The HIIT group started with a 5-minute warm-up at 50% Vmax, followed by 10 repetitions of 2-minute runs at 90% Vmax, with 2-minute rest intervals between repetitions, and ended with a 5-minute cool-down at 50% Vmax. The MICT group began with a 5-minute warm-up at 50% Vmax, followed by continuous treadmill exercise at 70% Vmax, covering the same total distance as the HIIT group, and concluded with a 5-minute cool-down at 50% Vmax. Maximum exercise capacity tests were performed every two weeks to adjust the exercise protocol based on changes in Vmax.
Measurements: Body weight and random blood glucose levels were measured weekly. Pathological examination of gastrocnemius muscle tissue (HE staining, PAS staining) and transmission electron microscopy to observe mitochondrial ultrastructure were performed. Enzyme activity assays for L-LDH and G6PDH were conducted using commercial kits. Western blotting was used to detect the protein expression levels of GLUT4, MSTN, AMPK, P-AMPK, PGC-1α, NRF2, TFAM, OPA1, MFN2, DRP1, PINK1, and PARKIN. Real-time quantitative PCR was used to measure mitochondrial DNA copy number.
