Characterization of DPP-IV Inhibitory Peptides Using an In Vitro Cell Culture Model of the Intestine

Abstract

Here, we characterize the activities of two depeptidyl peptidase-IV (DPP-IV) inhibitory peptides, VLATSGPG and LDKVFER, using the Caco-2 monolayer model for the intestine. VLATSGPG and LDKVFR inhibited the DPP-IV in the cells via a mixed-type inhibition mode, with in situ IC50 values of 207.3 and 148.5 μM, respectively. Furthermore, VLATSGPG and LDKVFR were transported intact across the cells, with P app values of 2.41 ± 0.16 and 4.23 ± 0.29 × 10–7 cm/s, respectively. Fragmented peptides were identified in the basolateral side of the membrane. Two of these, GPG and VLA, exhibited high inhibitory activities of 83.6 ± 3.3 and 58.5 ± 2.5%, respectively, at 100 μM concentration. Although 3 mM VLATSGPG and LDKVFR were transported across the epithelium in a concentration-dependent manner, their transport did not damage the tight junction proteins, ZO-1 and occludin. This study demonstrates that the two peptides potentially regulate DPP-IV activity in the intestine.

Transepithelial Transport of the Bifunctional Peptide IPYWTY Indirectly Induced Insulin Release Mediated by Active GLP-1

“…The P app value was used for evaluating peptide permeability in the Caco-2 cell monolayer, and the P app values of DPP-4IPs are always between 10 –8 and 10 –7 cm/s, when transported across the Caco-2 cell monolayer . For instance, the P app values of DPP-4IPs, such as IADHFL, VLATSGPG, and VPLVM, were 4.91 × 10 –8 , 2.41 × 10 –7 , and 6.96 × 10 –7 cm/s, respectively. In Figure , the content of IPYWTY significantly increased from 2.06 μM at 30 min to 7.49 μM at 120 min, and the P app value of IPYWTY was 4.65 × 10 –7 cm/s.…”

“…Although the in situ DPP-4 IC 50 of IPYWTY (80.48 μM) was 7.29 times higher than its in vitro DPP-4 IC 50 (11.04 μM), which was still lower than other DPP-4IPs, including LDKVFER, VLATSGPG, LTFPGSAED, and IAVPTGVA, with the in situ DPP-4 IC 50 values of 148.5, 207.3, 207.5, and 223.2 μM, respectively. 8,21 Various brush border membrane peptidases are secreted from Caco-2 cells, which may degrade peptides and thus decrease their DPP-4 inhibitory activity. 6 Especially, DPP-4 preferentially releases dipeptides from peptides with a proline or alanine at position 2 of the Nterminal.…”

“…Assay in Caco-2 Cell Monolayers. According to Jin et al 8 Caco-2 cell monolayers were incubated with 0.5 mL of HBSS, cytochalasin D (0.5 μg/mL), wortmannin (0.5 μM), and Gly-Sar (10 mM) on the AP side for 30 min. Subsequently, the AP and BL solutions were rinsed three times with HBSS and replaced with IPYWTY (2 mM) and HBSS, respectively.…”

“…As an illustration, the transepithelial transport of two DPP-4IPs, VLATSGPG and LDKVFER, was characterized by using a Caco-2 cell monolayer model. 8 Notably, Caco-2 cells were not applicable for evaluating the insulinotropic effects of DPP-4IPs mediated by active GLP-1 because it cannot express and secrete insulin. Accordingly, the coculture cell models are being increasingly used to precisely study the in situ insulinotropic effects of DPP-4IPs.…”

Mechanism of interactions between egg protein–derived tri‐peptides and cellular membrane by molecular dynamic simulation and isothermal titration calorimetry

“…Enzyme inhibitors and nanocarriers have enabled food‐derived bioactive peptides to resist the hydrolysis by gastrointestinal proteases and membrane peptidases (Xu et al ., 2019). Peptides obtained from the gastrointestinal digestion of dairy proteins showed resistance to brush border membrane enzymes (Jin et al ., 2021). Hen egg–derived tri‐peptides ADF (Ala‐Asp‐Phe), FGR (Phe‐Gly‐Arg) and MIR (Met‐Ile‐Arg) were obtained from the in silico gastrointestinal digestion with ACE inhibitory activities (IC 50 : 27.75 mM, 66.98 µM and 24.97 mM respectively) and membrane peptidase inhibitory activities (IC 50 : 16.83, 46.22 and 4.86 mM respectively) were also identified in our previous study (Zhao et al ., 2020).…”