Peptide profiling of casein peptide reveals its anti-diabetic effects via DPP-IV inhibition and IRS1/PI3K/AKT signaling.

Type 2 diabetes mellitus (T2DM) is a growing global health burden, increasing demand for safe food-derived strategies for glycemic control. Casein-derived peptides have potential metabolic benefits, but their antidiabetic mechanisms remain unclear. In this study, casein peptide (CNP), enriched in low-molecular-weight peptides (<3 kDa), was investigated as the principal bioactive intervention for T2DM regulation. LC-MS/MS identified 880 peptides; among the 50 most abundant, all were predicted to inhibit DPP-IV, 26 to inhibit α-glucosidase, and one to inhibit α-amylase. CNP inhibited DPP-IV in vitro in a concentration-dependent manner (IC50 = 5.06 ± 0.41 mg mL-1) and crossed Caco-2 monolayers partly through endocytosis and PepT1-mediated transport. In HFD/STZ-induced T2DM mice, CNP improved glucose tolerance and insulin sensitivity, ameliorated dyslipidemia, attenuated tissue injury and systemic inflammation, and restored GLP-1/DPP-IV balance and IRS1/PI3K/AKT signaling. These findings identify CNP as a key food-derived bioactive peptide system that modulates T2DM through complementary effects on intestinal transport, incretin regulation, glucose-lipid homeostasis, inflammation, and insulin signaling, supporting its potential for development as a functional food ingredient for T2DM management.