Cellular mechanisms of incretin hormone secretion.

Enteroendocrine cells located along the gastrointestinal epithelium sense different nutrients/luminal contents that trigger the secretion of a variety of gut hormones with different roles in glucose homeostasis and appetite regulation. The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are involved in the regulation of insulin secretion, appetite, food intake and body weight after their nutrient-induced secretion from the gut. GLP-1 mimetics have been developed and used in the treatment of type 2 diabetes mellitus and obesity. Modulating the release of endogenous intestinal hormones may be a promising approach for the treatment of obesity and type 2 diabetes without surgery. For that reason, current understanding of the cellular mechanisms underlying intestinal hormone secretion will be the focus of this review. The mechanisms controlling hormone secretion depend on the nature of the stimulus, involving a variety of signalling pathways including ion channels, nutrient transporters and G-protein-coupled receptors.

A Shared Perspective on in Vitro and in Vivo Models to Assay Intestinal Transepithelial Transport of Food Compounds.

Assessing nutrient bioavailability is complex, as the process involves multiple digestion steps, several cellular environments, and regulatory-metabolic mechanisms. Several in vitro models of different physiological relevance are used to study nutrient absorption, providing significant challenges in data evaluation. However, such in vitro models are needed for mechanistic studies as well as to screen for biological functionality of the food structures designed. This collaborative work aims to put into perspective the wide-range of models to assay the permeability of food compounds considering the particular nature of the different molecules, and, where possible, in vivo data are provided for comparison.

Coupling in vitro food digestion with in vitro epithelial absorption; recommendations for biocompatibility.

As food transits the gastrointestinal tract, food structures are disrupted and nutrients are absorbed across the gut barrier. In the past decade, great efforts have focused on the creation of a consensus gastrointestinal digestion protocol (i.e., INFOGEST method) to mimic digestion in the upper gut. However, to better determine the fate of food components, it is also critical to mimic food absorption in vitro. This is usually performed by treating polarized epithelial cells (i.e., differentiated Caco-2 monolayers) with food digesta. This food digesta contains digestive enzymes and bile salts, and if following the INFOGEST protocol, at concentrations that although physiologically relevant are harmful to cells. The lack of a harmonized protocol on how to prepare the food digesta samples for downstream Caco-2 studies creates challenges in comparing inter laboratory results. This article aims to critically review the current detoxification practices, highlight potential routes and their limitations, and recommend common approaches to ensure food digesta is biocompatible with Caco-2 monolayers. Our ultimate aim is to agree a harmonized consensus protocol or framework for in vitro studies focused on the absorption of food components across the intestinal barrier.

Food peptides as inducers of CCK and GLP-1 secretion and GPCRs involved in enteroendocrine cell signalling.

The strong effect of protein digestion products on gastrointestinal-released hormones is recognised. However, little is known about the specific peptide sequences able to induce gastrointestinal hormone secretion and the receptors involved. Our objective was to identify peptides able to induce the secretion of cholecystokinin (CCK) and glucagon like peptide-1 (GLP-1) in the enteroendocrine cell line STC-1, and to evaluate the involvement of the calcium-sensing receptor and G-protein coupled receptor-93 in this cell signalling. The key role of the amino acidic sequence on CCK and GLP-1 secretion is demonstrated. Removing Ser from the N-terminus of κ-casein 33SRYPS37, or the N-terminal Trp-Ile in lysozyme 123WIRGCRL129 decreased the secretion of both hormones. However, substituting Tyr by Ala in peptide αs1-CN 90RYLG93 enhanced the CCK secretion levels but not the GLP-1 ones. In addition, the involvement of CaSR and GPR93 was evidenced, but our results pointed to the contribution of additional receptors or transporters.

In vitro digestion of milk proteins including intestinal brush border membrane peptidases. Transepithelial transport of resistant casein domains.

The use of enzymes from the brush border membrane (BBM) in simulated gastrointestinal digestion of milk proteins has been evaluated. With this purpose, the resistant sequences from casein and milk whey proteins after INFOGEST in vitro digestion with and without BBM have been analyzed by tandem mass spectrometry. The use of BBM revealed additional cleavages to those found with pancreatic enzymes, although the number of total identified peptides decreased due to the reduction of the peptide size. These new cleavages were mainly attributed to the activity of amino- and carboxy-peptidases, which was also reflected in the higher concentration of free amino acids found in the gastrointestinal digests with BBM. The peptidome of the simulated gastrointestinal digests was compared with that previously obtained in digests aspirated from human jejunum after oral administration of the same substrates. The addition of BBM did not change significantly the peptide profile, although it allowed the identification of peptides found in human digests. However, none of the models was able to reproduce the large variety of peptides found in vivo. In addition, in vitro transepithelial transport of six ß-casein derived peptides resistant to gastrointestinal digestion, including the opioid ß-casomorphin-7, was also evaluated. The results point to the importance of the nature of the N- and C-terminal end for the transport rate through the Caco-2 cell monolayer. Therefore, the use of BBM as a supplementary step after simulated pancreatic digestion can be considered in bioavailability studies since the final sequence can determine the absorption of peptides.

In vitro dipeptidyl peptidase IV inhibitory activity and in situ insulinotropic activity of milk and egg white protein digests.

Dietary proteins are involved in the regulation of glucose homeostasis by different mechanisms. Food protein digestion products are reported to inhibit dipeptidyl peptidase IV (DPP-IV), induce incretin secretion or directly exert an insulinotropic effect in pancreatic ß-cells. This study illustrates the DPP-IV inhibitory activity of gastric and intestinal digests of casein, whey and egg white proteins determined in vitro, using Gly-Pro-AMC, and in situ using non-differentiated Caco-2 cells. Comparable trends in the DPP-IV inhibitory profiles were obtained by these two methods although the extent of inhibition in situ was consistently lower than the inhibition observed in vitro. Casein intestinal digests and whey protein gastric and intestinal digests showed potent DPP-IV inhibitory activities in Caco-2 cells with IC50 values ranging from 0.8 to 1.2 mg mL-1. The absorbed fraction of the intestinal digests from whey and egg white protein induced insulin secretion in BRIN-BD11 cells when determined using a two-tiered cellular model (Caco-2 and BRIN-BD11). However, the gastric digests from the same substrates showed no insulin secretion. This may be related to limited trans-epithelial transport through the Caco-2 monolayer of the gastric digestion products. However, both, gastric and intestinal digests were able to induce insulin secretion in BRIN-BD11 cells when the monolayer was composed of a co-culture of STC-1 and Caco-2 cells. This result may be attributed to the activation of STC-1 cells and subsequent incretin secretion, induced by the gastric digest, as shown by an enhanced intracellular calcium uptake.

Compared digestibility of plant protein isolates by using the INFOGEST digestion protocol.

The use of ingredients based on plant protein isolates is being promoted due to sustainability and health reasons. However, it is necessary to explore the behaviour of plant protein isolates during gastrointestinal digestion including the profile of released free amino acids and the characterization of resistant domains to gastrointestinal digestion. The aim of the present study was to monitor protein degradation of four legume protein isolates: garden pea, grass pea, soybean and lentil, using the harmonized Infogest in vitro digestion protocol. In vitro digests were characterized regarding protein, peptide and free amino acid content. Soybean was the protein isolate with the highest percentage of insoluble nitrogen at the end of the digestion (12%), being this fraction rich in hydrophobic amino acids. Free amino acids were mainly released during the intestinal digestion, comprising 21-24% of the total nitrogen content, while the percentage of nitrogen corresponding to peptides ranged from 66 to 76%. Legume globulins were resistant to gastric digestion whereas they were hydrolysed into peptides and amino acids during the intestinal phase. However, the molecular weight (MW) distribution demonstrated that all intestinal digests, except those from soybean, contained peptides with MW > 4 kDa at the end of gastrointestinal digestion. The profile of free amino acids released during digestion supports legume protein isolates as an excellent source of essential amino acids to be used in protein-rich food products. Peptides released during digestion matched with previously reported epitopes from the same plant species or others, explaining the ability to induce allergic reactions and cross-linked reactivity.

Peptidomic data of egg white gastrointestinal digests prepared using the Infogest Harmonized Protocol.

These data are related to the research article entitled "Induction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion". In this article, the peptide and free amino acid profile of egg white gastrointestinal in vitro digests is shown. Egg white proteins were digested following the INFOGEST gastrointestinal digestion protocol. Different time points of gastric and intestinal digestion were characterized regarding protein, peptide and amino acid content. Protein degradation was followed by SDS-PAGE where some electrophoretic bands were identified by MALDI-TOF/TOF after tryptic digestion. Moreover, the molecular weight distribution of egg white peptides found at different times of gastrointestinal digestion was performed using MALDI-TOF. Peptides identified from the most abundant egg white proteins by tandem mass spectrometry were represented using a peptide profile tool and raw data are given in table format. These results reveal the protein regions resistant to digestion and illustrate the free amino acid profile of egg white protein at the end of the digestion process. These data can be used for nutritional purposes and to identify allergen epitopes or bioactive sequences.

Induction of CCK and GLP-1 release in enteroendocrine cells by egg white peptides generated during gastrointestinal digestion.

The effect of dietary protein on the induction of intestinal hormones is recognised. However, little is known about the nature of the digestion products involved in this intestinal signalling. Our aim was to characterise egg white protein digestion products and study their ability to induce CCK and GLP-1 release in enteroendocrine STC-1 cells. Intestinal digests triggered GLP-1 release at a higher rate than gastric digests. Peptides, but not free amino acids, showed a potent GLP-1 secretagogue effect, while proteins only had a modest effect. CCK was released in response to peptides and free amino acids but not proteins. Two hydrophobic negatively charged peptides triggered CCK release, while the highest GLP-1 response was found with a hydrophobic positively charged peptide, pointing to the involvement of different receptors or active sites. Identifying peptide sequences and receptors involved in hormonal secretion could open up new ways to control food intake and glucose metabolism.

Stimulation of CCK and GLP-1 secretion and expression in STC-1 cells by human jejunal contents and in vitro gastrointestinal digests from casein and whey proteins.

The present study evaluates casein and whey protein gastrointestinal digests as inducers of CCK and GLP-1 secretion and expression in STC-1 cells. In vitro digests were characterized regarding protein, peptide and free amino acid content. Digests from the intestinal phase containing small size peptides and free amino acids behaved as more potent CCK inducers than digests from the gastric phase. However, GLP-1 release was maximized with casein gastric digests and whey protein intestinal digests. Human jejunal digests from the same substrates showed a comparable response, except for casein jejunal digests which exerted a higher effect than in vitro casein gastrointestinal digests. The gene expression experiments also showed increased CCK and GLP-1 mRNA levels but the differences between the gastric and gastrointestinal phases were not as pronounced as observed by quantifying the secreted hormone. Our results demonstrate that the degree of protein hydrolysis during digestion plays an important role in CCK and GLP-1 secretion.

Intestinal Signaling of Proteins and Digestion-Derived Products Relevant to Satiety.

Luminal nutrients stimulate enteroendocrine cells through the activation of specific receptors to release hormones that inhibit appetite and promote glucose homeostasis. While food protein is the macronutrient with the highest effect on satiety, the signaling on the protein digestion products at the gut is poorly understood. This perspective aims to highlight the existing gaps in the study of protein digestion products as signaling molecules in gastrointestinal enteroendocrine cells. Because dietary protein digestion can be modulated by the technological processes applied to food, it is possible to target gut receptors to control food intake by formulating specific food ingredients or protein preloads.