Structural Characteristics of Theragra chalcogramma Milt Peptides and Their Anti-Fatigue Activity via AMPK/PGC-1αMediated Energy Metabolism Regulation in Exercised Mice

Objectives: While several physiological functions of milt peptides have been discovered, the structural characteristics of Theragra chalcogramma milt peptides (TMP) and their anti-fatigue mechanisms remain unclear. Methods: TMP was obtained by hydrolysis via flavor enzyme and alkaline protease, and its structural characteristics were analyzed. A mice model of exercise-induced fatigue was established. The anti-fatigue effect of TMP was evaluated by determining the main biochemical indices in the serum, liver, and skeletal muscle of mice. Additionally, qPCR analysis was conducted to investigate its regulatory effects on relevant energy metabolism pathways. Results: TMP contained 18.2% branched-chain amino acids, with those with molecular weights below 1000 Da accounting for 91.6%. A total of 154 characteristic peptides, such as VPFPR and LPPGR, were identified from TMP, among which 64% of the peptides contained glutamic acid, arginine, or aspartic acid. Molecular docking of potential bioactive peptides to AMP-activated protein kinase (AMPK) revealed binding energies from −9.1 to −5.5 kcal/mol. The exhaustive swimming test showed that oral administration of TMP prolonged the swimming duration. In the fatigue murine model, TMP reduced blood urea nitrogen and blood lactic acid levels while enhancing the content of muscle glycogen. Meanwhile, TMP significantly increased the activity of glutathione peroxidase and superoxide dismutase and reduced the accumulation of malondialdehyde, demonstrating antioxidant properties. Additionally, TMP significantly decreased creatine kinase and lactate dehydrogenase extravasation, thereby protecting muscle tissue, as corroborated by immunohistochemical analyses. Mechanistically, TMP upregulated AMPK and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) expression, promoting mitochondrial biogenesis via the AMPK/PGC-1α pathway. Conclusions: These findings suggest TMP has potential as a dietary supplement for alleviating physical fatigue.