Dipeptidyl-peptidase (DPP)-IV inhibition by penultimate N-terminus Pro-containing peptides is a promising strategy for Type 2 diabetes (T2D) management, as it prevents the degradation of incretin hormones (DPP-IV substrates) like glucagon-like peptide-1 (GLP-1), thereby prolonging their half-life. However, the stability and bio-accessibility of these peptides are crucial to their efficacy in orally administered therapeutics. We previously identified LPCL and TPFLPDE peptides from tilapia viscera by-products hydrolysates, which exhibited significant DPP-IV inhibition in vitro and in situ while effectively preserving active GLP-1 levels after 2 h treatment in STC-1 cells under basal glucose conditions. In this study, we assessed the gastrointestinal (GI) stability of these peptides using a static in vitro GI digestion model and evaluated their DPP-IV inhibition capacity after post-GI digestion and post-DPP-IV pre-incubation. Our findings revealed that both peptides retained their DPP-IV inhibition capacity after GI digestion, with RP-HPLC and mass spectrometry confirming their intact forms. At post-DPP-IV pre-incubation, peptides exhibited decreased DPP-IV inhibition potency, indicating a substrate-type inhibition mechanism, forming N-terminal dipeptide fragments. Furthermore, we extended our previous findings by demonstrating that LPCL not only inhibits DPP-IV activity but also modulates GLP-1 expression and increases PepT1 protein levels in STC-1 cells. Unlike glycyl sarcosine, a known PepT1 substrate, LPCL effectively activates the PepT1 extracellular domain (ECD), leading to increased receptor expression and receptor-ligand interactions, as confirmed by molecular docking analysis. These results provide novel insights into the functional roles of LPCL in modulating DPP-IV activity and GLP-1 expression, potentially via PepT1 ECD conformational stabilization.
