Soybean- and Lupin-Derived Peptides Inhibit DPP-IV Activity on In Situ Human Intestinal Caco-2 Cells and Ex Vivo Human Serum

Abstract

Recent investigations have focused on food-derived peptides as novel natural inhibitors of dipeptidyl peptidase IV (DPP-IV), a new target for diabetes. This study aimed to optimize fast, sensitive, and cost-effective DPP-IV assays in situ on human intestinal Caco-2 cells and ex vivo on human serum. Both assays were applied to investigate the inhibitory activity of soy and lupin peptides. The best conditions for in situ DPP-IV activity in Caco-2 cells were obtained using 2-day cells and 50 µM Gly-Pro-AMC. Sitagliptin, used as reference inhibitor, showed a dose-dependent response with a 50% inhibition concentration (IC₅₀) of 0.6 µM. A lower IC₅₀ (0.2 µM) was obtained for sitagliptin on human serum incubated with the substrate for 24 h. Both assays were applied to assess the activity of Lup1 (LTFPGSAED) and Soy1 (IAVPTGVA) on DPP-IV. Lup1 and Soy1 inhibited DPP-IV in situ, with IC₅₀ values of of 207.5 and 223.2 µM, respectively, and maintained their inhibitory activity ex vivo on circulating DPP-IV with a slightly lower potency. These assays can be used to characterize the DPP-IV inhibitory activity of food-derived molecules more accurately than in vitro biochemical tests. This combined approach also considers their effects on the circulating form of DPP-IV, correlated to metabolic diseases.

Keywords: anti-diabetic activity; bioactive peptides; dipeptidyl peptidase IV; glucose metabolism; sitagliptin.

Figure 1

In situ DPP-IV activity on 2-, 4-, and 6-day Caco-2 cells: (a) using 50 µM Gly-Pro-AMC as a substrate, the fluorescence signal increased as a function of time and was linear at all cell ages over the first 3 min. (b) The fluorescence signal was measured after 3 min as a function of substrate Gly-Pro-AMC concentration at all cell ages. Data are the means ± SD of three experiments performed in triplicate.

Figure 2

Inhibitory activity of sitagliptin on DPP-IV activity measured in situ on 2-day Caco-2 cells: (a) at 1 µM, sitagliptin inhibited DPP-IV activity in Caco-2 cells by over 50%; (b) sitagliptin inhibited DPP-IV activity in a dose-response manner, exhibiting an IC₅₀ of 0.6 µM. Data are the means ± SD of three experiments performed in triplicate.

Figure 3

Inhibition of in situ DPP-IV activity by Lup1 and Soy1: (a) after pre-incubation (1 h) with 100 µM Lup1 and Soy1, DPP-IV activity was inhibited by 50% by both peptides; (b) measuring inhibitory activity as a function of peptide concentration, a dose-dependent response was observed with IC₅₀ values of 207.5 µM for Lup1 and 223.2 µM for Soy1. Data are the means ± SD of three experiments performed in triplicate. *: p > 0.05; **: p > 0.01.

Figure 4

Ex vivo assay of circulating DPP-IV in human serum: (a) sitagliptin showed a dose-dependent inhibition of circulating DPP-IV with an IC₅₀ of 0.2 µM; (b) Lup1 inhibited circulating DPP-IV activity by 18.1% and 24.7% at 100 µM and 300 µM, respectively. Soy1, at the same concentrations, showed a slightly higher inhibitory activity of 27.7% and 35.0%, respectively. Data are the means ± SD of three experiments performed in triplicate. *: p > 0.05.