New Bioactive Peptides Identified from a Tilapia Byproduct Hydrolysate Exerting Effects on DPP-IV Activity and Intestinal Hormones Regulation after Canine Gastrointestinal Simulated Digestion

Abstract

Like their owners, dogs and cats are more and more affected by overweight and obesity-related problems and interest in functional pet foods is growing sharply. Through numerous studies, fish protein hydrolysates have proved their worth to prevent and manage obesity-related comorbidities like diabetes. In this work, a human in vitro static simulated gastrointestinal digestion model was adapted to the dog which allowed us to demonstrate the promising effects of a tilapia byproduct hydrolysate on the regulation of food intake and glucose metabolism. Promising effects on intestinal hormones secretion and dipeptidyl peptidase IV (DPP-IV) inhibitory activity were evidenced. We identify new bioactive peptides able to stimulate cholecystokinin (CCK) and glucagon-like peptide 1 (GLP-1) secretions, and to inhibit the DPP-IV activity after a transport study through a Caco-2 cell monolayer.

Keywords: DPP-IV inhibitory peptides; bioactive peptides; cholecystokinin; fish byproduct hydrolysate; glucagon-like peptide 1; in vitro gastrointestinal digestion.

Figure 1

Peptide profiles and peptide molecular weight (MW) distributions. (A) Peptide profiles were obtained by size exclusion chromatography-fast protein liquid chromatography (SEC-FPLC): FBPH (blue curves), FBP (green curves); oral (light colored curves), gastric (colored curves) and intestinal (dark colored curves) digests. (B) The MW distribution of peptides in the different SGID compartments, expressed in percentage of total area under the curve (AUC), was calculated from the linear regression relationship which correlates the Log of known MW standard peptides and the elution volume.

Figure 2

FBPH and FBP induction of intestinal hormones release during simulated GI digestion. The amounts of intestinal hormones released in STC-1 cells in the supernatants after 2 h of contact with the FBP and FBPH digests (2; 5 and 10 mg mL⁻¹ w/v) were determined by radioimmunoassay for CCK (A) and active GLP-1 (B). Values are means ± SD and are expressed in fold of control (buffer). Means without a common letter within the same graph are significantly different (p < 0.05) using one-way ANOVA following by Tukey post-hoc test for pairwise comparisons.

Figure 3

FBPH and FBP effects on the Caco-2 DPP-IV activity inhibition during SGID. The Caco-2 DPP-IV activity inhibition (%) obtained with FBP and FBPH digests assayed at increasing concentrations (0.5; 0.99 and 1.98 g L⁻¹, w/v). Means without a common letter within the same graph are significantly different (p < 0.05) using one-way ANOVA followed by Tukey post-hoc test for pairwise comparisons. Inset: IC₅₀ were determined by linear regression correlating the DPP-IV activity inhibition percentage and the Ln of the concentration.

Figure 4

FBPH intestinal digest SEC fractionation effects on gut hormones release in STC-1 cells. The SEC fractionation of the FBPH intestinal digest was performed using a HiLoad 16/600 Superdex prepgrade column with an isocratic gradient of 30% acetonitrile 0.1% TFA (A). The amounts of intestinal hormones in the supernatants, after 2 h of contact with the fractions and or the FBPH digest (0.5% w/v), were determined by radioimmunoassay for CCK (B) and active GLP-1 (C). Values are the means of three repeated measurements and are expressed in fold of control (buffer) ± SD. Means without a common letter within the same graph are significantly different (p < 0.05) using one-way ANOVA followed by a Tukey post-hoc test for pairwise comparisons.

Figure 5

SEC-F2 RP-HPLC fractionation effect on gut hormones release in STC-1 cells. The RP-HPLC fractionation of the FBPH intestinal digest’s SEC-F2 fraction was performed using a C18 Gemini column (150 × 10 mm, particles of 5 μm, 110 Å, Phenomenex) with an ACN gradient represented in red (A). The amounts of intestinal hormones released in the supernatants, after 2 h of contact, with the subfractions, the SEC-F2 fraction and the FBPH digest (0.5% w/v), were determined by radioimmunoassay for CCK (B) and active GLP-1 (C). Values are means of three repeated measurements and are expressed in fold of control (buffer) ± SD. Means without a common letter within the same graph are significantly different (p < 0.05) using one-way ANOVA following by Tukey post-hoc test for pairwise comparisons.

Figure 6

Mass signal 3D-map of the FE subfraction issued from the RP-HPLC separation of the FBPH intestinal digest’s SEC-F2 fraction. Peptide map showing the repartition of all peptides detected by RP-UPLC-MS/MS analysis according to their retention time during chromatography, their mass to charge ratio (m/z) and their intensity. Grey signals represent all ions detected, blue squares represent identified peptides by database confrontation (false discovery rate (FDR) < 1%) and orange squares represent peptides sequenced by de novo mode (ALC score > 80%).

Figure 7

Synthetic peptide effects on intestinal hormones release in STC-1 cells. The amounts of intestinal hormones released in the supernatants, after 2 h of contact with the peptides (1 mM), were determined by radioimmunoassay for CCK (A) and active GLP-1 (B). Values are means of three repeated measurements and are expressed in fold of control (buffer) ± SD. Means were compared to control mean using one-way ANOVA following by a Dunnett post-hoc test, **** p < 0.0001; *** p < 0.001.