A Lysozyme-Derived Peptide Induces GLP-1 Secretion in Mouse Jejunal Organoids and Modulates Glycemia in Rats

Abstract

Protein digestion products promote the release of incretins, such as GLP-1, which regulate glucose homeostasis. Our previous studies demonstrated that the egg white peptide fraction stimulates GLP-1 secretion in STC-1 cells. Here, the GLP-1 secretion induced by the lysozyme-derived peptide ¹²³WIRGCRL¹²⁹ and six alanine-substituted analogues was evaluated in mouse jejunal organoids, alongside egg white digest and the amino acid phenylalanine (Phe). Phe induced a faster GLP-1 response, but the peptide ¹²³WIRGCRL¹²⁹ elicited a similar GLP-1 response, although tested at a 20-fold lower concentration. The GLP-1 release in organoids elicited by the peptide was 57.8 ± 5 pM, while Phe reached 43.7 ± 1 pM at 60 min. In STC-1 cells, the peptide induced 667.7 ± 24.2 pM of GLP-1 compared to 416.6 ± 40.1 pM induced by Phe. Results obtained in STC-1 suggested the involvement of ERK- and AMPK-mediated pathways in the GLP-1 secretion induced by the peptide. Oral glucose tolerance tests in Wistar rats after oral administration of ¹²³WIRGCRL¹²⁹ showed a reduction in glucose levels, while no changes were observed in the group receiving the amino acid mixture at equimolar concentration. These findings suggest the potential therapeutic application of some food peptides against type 2 diabetes.

Keywords: GLP-1; egg white peptides; enteroendocrine cells; mouse jejunal organoids; type 2 diabetes.

1

GLP-1 release at 10, 30, and 60 min after exposure to EWD, ¹²³WIRGCRL¹²⁹ (peptide), or Phe in mouse intestinal organoids (A) and relative proglucagon gene expression at 10 min (B), 30 min (C), and 60 min (D). EWD was assayed at 2 mg/mL, Phe at 20 mM, and synthetic peptide at 1 mM. Experiments were performed in triplicate, followed by technical duplicates. Error bars indicate SEM (n = 3). Statistical significance compared with control in GLP-1 release (one-way ANOVA with Tukey’s post hoc test) is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical significance (p < 0.05) in the comparison between different samples at the same time is indicated by different letters (a, b). Unpaired t test was used in gene expression and statistical significance compared with control is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2

GLP-1 release at 10, 30, and 60 min after exposure to EWD, ¹²³WIRGCRL¹²⁹ (peptide), or Phe to STC-1 cells (A) and relative proglucagon gene expression at 10 min (B), 30 min (C), and 60 min (D). EWD was assayed at 2 mg/mL, Phe at 20 mM, and synthetic peptide at 1 mM. Experiments were performed in triplicate, followed by technical duplicates. Error bars indicate SEM (n = 3). Statistical significance compared with control (one-way ANOVA with Tukey’s post hoc test) is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical significance (p < 0.05) in the comparison between different samples at the same dose is indicated by different letters (a, b, c). Unpaired t test was used in gene expression and statistical significance compared with control is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3

GLP-1 release after 1 h incubation of mouse intestinal organoids with 1 mM of synthetic peptides. Experiments were performed in triplicate, followed by technical duplicates. Error bars indicate SEM (n = 3). Statistical significance compared with control (one-way ANOVA with Tukey’s post hoc test) is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical significance (p < 0.05) in the comparison between different samples at the same dose is indicated by different letters (a, b).

4

p-IRS1, IRS1, p-AMPK, AMPK, p-ERK, ERK, GAPDH, and HSP90 presence were analyzed at 5, 15, 30, and 60 min after peptide ¹²³WIRGCRL¹²⁹ (1 mM) or EWD (2 mg/mL) or buffer addition in STC-1 cells (A). Relative protein expression of p-IRS/IRS (B), p-AMPK/AMPK (C), and p-ERK/ERK (D). Error bars indicate SEM (n = 3). Statistical significance compared with control (one-way ANOVA with Tukey’s post hoc test) is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical significance (p < 0.05) in the comparison between different samples at the same time is indicated by different letters (a, b).

5

Blood glucose levels in mg/dL 45 min before the 3 mg/kg of glucose overload administration and after 15, 30, 60, and 120 min (A). GIP (B) and GLP-1 (C) plasma levels in mg/mL 15 min after glucose overload in basal, overload, peptide (¹²³WIRGCRL¹²⁹), and AA mixture groups. Statistical significance compared with control (one-way ANOVA with Tukey’s post hoc test) is indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Statistical significance (p < 0.05) in the comparison between different samples at the same dose is indicated by different letters (a, b).