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. 2024 Jun 5;72(22):12719-12724.
doi: 10.1021/acs.jafc.4c00974. Epub 2024 May 24.

Investigating the Transepithelial Transport and Enzymatic Stability of Lactononadecapeptide (NIPPLTQTPVVVPPFLQPE), a 19-Amino Acid Casein-Derived Peptide in Caco-2 Cells

Eriko Nakatani  1 , Masaki Sasai  2 , Hidetoshi Miyazaki  2 , Shimako Tanaka  1 , Tatsuhiko Hirota  2 , Takashi Okura  1
Affiliations

Investigating the Transepithelial Transport and Enzymatic Stability of Lactononadecapeptide (NIPPLTQTPVVVPPFLQPE), a 19-Amino Acid Casein-Derived Peptide in Caco-2 Cells

Eriko Nakatani et al. J Agric Food Chem. .

Abstract

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE), a casein-derived peptide comprising 19 residues, is known for its capacity to enhance cognitive function. This study aimed to explore the transepithelial transport and stability of LNDP. Results showed that LNDP retained over 90% stability after 2 h of treatment with gastrointestinal enzymes. The stability of LNDP on Caco-2 cell monolayers ranged from 93.4% ± 0.9% to 101.1% ± 1.2% over a period of 15-60 min, with no significant differences at each time point. The permeability of LNDP across an artificial lipid membrane was very low with the effective permeability of 3.6 × 10-11 cm/s. The Caco-2 assay demonstrated that LNDP could traverse the intestinal epithelium, with an apparent permeability of 1.22 × 10-6 cm/s. Its transport was significantly inhibited to 67.9% ± 5.0% of the control by Gly-Pro, a competitor of peptide transporter 1 (PEPT1). Furthermore, PEPT1 knockdown using siRNA significantly inhibited LNDP transport by 77.6% ± 1.9% in Caco-2 cell monolayers. The LNDP uptake in PEPT1-expressing HEK293 cells was significantly higher (54.5% ± 14.6%) than that in mock cells. These findings suggest that PEPT1 plays a crucial role in LNDP transport, and LNDP exhibits good resistance to gastrointestinal enzymes.

Keywords: Caco-2 cells; beta-casein; intestinal transport; lactononadecapeptide (LNDP); peptide; peptide transporter 1 (PEPT1).

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Conflict of interest statement

The authors declare the following competing financial interest(s): This research received funding from Asahi Group Holdings, Ltd., Japan. M.S., H.M., and T.H. are employed by Asahi Quality and Innovations Ltd., which is affiliated with Asahi Group Holdings Ltd. E.N., S.T., and T.O. state that the research was conducted without any financial interests that could be perceived as conflicts of interest. The funders did not influence the study design, data collection, analysis, interpretation, manuscript writing, or the decision to publish the results.

Figures

Figure 1
Figure 1
Stability of LNDP (10 μM). All data are presented as means ± SE (n = 3). (a) Recovery rate of LNDP following exposure to digestive tract gastrointestinal enzymes. (b) Recovery rate of LNDP on the apical side of Caco-2 cell monolayers within 60 min.
Figure 2
Figure 2
Transepithelial transport of LNDP in Caco-2 cells. (a) Impact of incubation time on LNDP transport. Data are presented as means ± SE (n = 3). (b) Effects of GP, Cytochalasin D, and Wortmannin on LNDP transport. Values are means ± SE (*P < 0.05 compared to the control group, n = 18).
Figure 3
Figure 3
Influence of siRNA targeting PEPT1 on LNDP transport across Caco-2 cell monolayers. Postincubation, LNDP concentration was assessed via LC-MS/MS. Values are means ± SE (*P < 0.05 compared to the control group, n = 12).
Figure 4
Figure 4
Uptake of LNDP into hPEPT1 cells versus mock HEK293 cells. Cells were incubated at 37 °C for 5 min. LNDP concentration postincubation was determined using LC-MS/MS. All data are means ± SE (*P < 0.05 compared to mock cells, n = 6).
Figure 5
Figure 5
Schematic diagram of the transepithelial transport pathway for the 19-residue peptide LNDP across enterocyte monolayers: Active transport via PEPT1.
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