Functional and genomic comparative study of the bitter taste receptor family TAS2R: Insight into the role of human TAS2R5.

Bitterness is perceived in humans by 25 subtypes of bitter taste receptors (hTAS2R) that range from broadly tuned to more narrowly tuned receptors. hTAS2R5 is one of the most narrowly tuned bitter taste receptors in humans. In this study, we review the literature on this receptor and show there is no consensus about its role. We then compare the possible role of hTAS2R5 with that of the proteins of the TAS2R family in rat, mouse, and pig. A phylogenetic tree of all mammalian TAS2R domain-containing proteins showed that human hTAS2R5 has no ortholog in pig, mouse, or rat genomes. By comparing the agonists that are common to hTAS2R5 and other members of the family, we observed that hTAS2R39 is the receptor that shares most agonists with hTAS2R5. In mouse, some of these agonists activate mTas2r105 and mTas2r144, which are distant paralogs of hTAS2R5. mTas2r144 seems to be the receptor that is most similar to hTAS2R5 because they are both activated by the same agonists and have affinities in the same range of values. Then, we can conclude that hTAS2R5 has a unique functional specificity in humans as it is activated by selective agonists and that its closest functional homolog in mouse is the phylogenetically distant mTas2r144.

GSPE Pre-Treatment Exerts Long-Lasting Preventive Effects against Aging-Induced Changes in the Colonic Enterohormone Profile of Female Rats.

The impact that healthy aging can have on society has raised great interest in understanding aging mechanisms. However, the effects this biological process may have on the gastrointestinal tract (GIT) have not yet been fully described. Results in relation to changes observed in the enteroendocrine system along the GIT are controversial. Grape seed proanthocyanidin extracts (GSPE) have been shown to protect against several pathologies associated with aging. Based on previous results, we hypothesized that a GSPE pre-treatment could prevent the aging processes that affect the enteroendocrine system. To test this hypothesis, we treated 21-month-old female rats with GSPE for 10 days. Eleven weeks after the treatment, we analyzed the effects of GSPE by comparing these aged animals with young animals. Aging induced a greater endocrine response to stimulation in the upper GIT segments (cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1)), a decrease in the mRNA abundance of GLP-1, peptide YY (PYY) and chromogranin A (ChgA) in the colon, and an increase in colonic butyrate. GSPE-treated rats were protected against a decrease in enterohormone expression in the colon. This effect is not directly related to the abundance of microbiome or short-chain fatty acids (SCFA) at this location. GSPE may therefore be effective in preventing a decrease in the colonic abundance of enterohormone expression induced by aging.

Protective properties of grape-seed proanthocyanidins in human ex vivo acute colonic dysfunction induced by dextran sodium sulfate.

PURPOSE: Anti-inflammatory and barrier-protective properties have been attributed to proanthocyanidins in the context of intestinal dysfunction, however little information is available about the impact of these phytochemicals on intestinal barrier integrity and immune response in the human. Here we assessed the putative protective properties of a grape-seed proanthocyanidin extract (GSPE) against dextran sodium sulfate (DSS)-induced acute dysfunction of the human colon in an Ussing chamber system. METHODS: Human proximal and distal colon tissues from colectomized patients were submitted ex vivo for a 30-min preventive GSPE treatment (50 or 200 µg mL-1) followed by 1-h incubation with DSS (12% w v-1). Transepithelial electrical resistance (TEER), permeation of a fluorescently-labeled dextran (FD4) and proinflammatory cytokine release [tumor necrosis factor (TNF)-α and interleukin (IL)-1ß] of colonic tissues were determined. RESULTS: DSS reduced TEER (45-52%) in both the proximal and distal colon; however, significant increments in FD4 permeation (fourfold) and TNF-α release (61%) were observed only in the proximal colon. The preventive GSPE treatment decreased DSS-induced TEER loss (20-32%), FD4 permeation (66-73%) and TNF-α release (22-33%) of the proximal colon dose-dependently. The distal colon was not responsive to the preventive treatment but showed a reduction in IL-1ß release below basal levels with the highest GSPE concentration. CONCLUSIONS: Our results demonstrate potential preventive effects of GSPE on human colon dysfunction. Further studies are required to test whether administering GSPE could be a complementary therapeutic approach in colonic dysfunction associated with metabolic disorders and inflammatory bowel disease.

Glucagon-like peptide-1 regulation by food proteins and protein hydrolysates.

Glucagon-like peptide-1 (GLP-1) is an enterohormone with a key role in several processes controlling body homeostasis, including glucose homeostasis and food intake regulation. It is secreted by the intestinal cells in response to nutrients, such as glucose, fat and amino acids. In the present review, we analyse the effect of protein on GLP-1 secretion and clearance. We review the literature on the GLP-1 secretory effects of protein and protein hydrolysates, and the mechanisms through which they exert these effects. We also review the studies on protein from different sources that has inhibitory effects on dipeptidyl peptidase-4 (DPP4), the enzyme responsible for GLP-1 inactivation, with particular emphasis on specific sources and treatments, and the gaps there still are in knowledge. There is evidence that the protein source and the hydrolytic processing applied to them can influence the effects on GLP-1 signalling. The gastrointestinal digestion of proteins, for example, significantly changes their effectiveness at modulating this enterohormone secretion in both in vivo and in vitro studies. Nevertheless, little information is available regarding human studies and more research is required to understand their potential as regulators of glucose homeostasis.

A specific dose of grape seed-derived proanthocyanidins to inhibit body weight gain limits food intake and increases energy expenditure in rats.

PURPOSE: Several studies have suggested that flavanols may have antiobesity effects; however, those effects clearly depend on the experimental conditions. In a previous study, we found that a single acute dose of grape seed proanthocyanidin extract (GSPE) has satiating effects. We therefore hypothesise that satiating doses of GSPE could be used to reduce body weight gain, and our present objective was to define the most effective dose. METHODS: We assayed two GSPE doses in aged male Wistar rats. First we performed a subchronic (8-day) treatment by intragastric administration, which was repeated after a washout period. We measured body weight, energy intake and faeces composition; we performed indirect calorimetry; and we analysed the mRNA expression of genes involved in lipid metabolism to determine the target tissue for the GSPE. RESULTS: We observed that 0.5 g GSPE/kg BW significantly reduced food intake and thus the amount of energy absorbed. This dosage also increased lipid oxidation in subcutaneous adipose tissue, thus causing a higher total energy expenditure. These combined effects caused a decrease in body weight. Conversely, 1 g GSPE/kg BW, which also reduced energy absorption after the first treatment, had a rebound effect on body weight gain which resulted in a lower response to the proanthocyanidin extract. That is, after the second treatment, the GSPE did not reduce the energy absorbed or modify energy expenditure and body weight. CONCLUSION: GSPE at a dose of 0.5 g/kg can reduce body weight by limiting food intake and activating energy expenditure in subcutaneous adipose tissue.

Assessing the impact of insect protein sources on intestinal health and disease: insights from human ex vivo and rat in vivo models.

The exploration of edible insects, specifically Alphitobius diaperinus and Tenebrio molitor, as sustainable sources of protein for human consumption is an emerging field. However, research into their effects on intestinal health, especially in relation to inflammation and permeability, remains limited. Using ex vivo and in vivo models of intestinal health and disease, in this study we assess the impact of the above insects on intestinal function by focusing on inflammation, barrier dysfunction and morphological changes. Initially, human intestinal explants were exposed to in vitro-digested extracts of these insects, almond and beef. Immune secretome analysis showed that the inflammatory response to insect-treated samples was comparatively lower than it was for samples exposed to almond and beef. Animal studies using yellow mealworm (Tenebrio molitor) and buffalo (Alphitobius diaperinus) flours were then used to evaluate their safety in healthy rats and LPS-induced intestinal dysfunction rats. Chronic administration of these insect-derived flours showed no adverse effects on behavior, metabolism, intestinal morphology or immune response (such as inflammation or allergy markers) in healthy Wistar rats. Notably, in rats subjected to proinflammatory LPS-induced intestinal dysfunction, T. molitor consumption did not exacerbate symptoms, nor did it increase allergic responses. These findings validate the safety of these edible insects under healthy conditions, demonstrate their innocuity in a model of intestinal dysfunction, and underscore their promise as sustainable and nutritionally valuable dietary protein sources.

Bitter taste receptors along the gastrointestinal tract: comparison between humans and rodents.

For decades bitter taste receptors (TAS2R) were thought to be located only in the mouth and to serve as sensors for nutrients and harmful substances. However, in recent years Tas2r have also been reported in extraoral tissues such as the skin, the lungs, and the intestine, where their function is still uncertain. To better understand the physiological role of these receptors, in this paper we focused on the intestine, an organ in which their activation may be similar to the receptors found in the mouth. We compare the relative presence of these receptors along the gastrointestinal tract in three main species of biomedical research (mice, rats and humans) using sequence homology. Current data from studies of rodents are scarce and while more data are available in humans, they are still deficient. Our results indicate, unexpectedly, that the reported expression profiles do not always coincide between species even if the receptors are orthologs. This may be due not only to evolutionary divergence of the species but also to their adaptation to different dietary patterns. Further studies are needed in order to develop an integrated vision of these receptors and their physiological functionality along the gastrointestinal tract.

Effect of an Acute Insect Preload vs. an Almond Preload on Energy Intake, Subjective Food Consumption and Intestinal Health in Healthy Young Adults.

Protein is considered the most satiating macronutrient, and its effect on satiety and food intake is source-dependent. For the first time, we compared the effect of the administration of an insect or almond preload, both containing 20 g of protein, on appetite and food intake in human subjects. Participants consumed both foods and a vehicle as a liquid preload on three separate days. They were then offered a breakfast and lunch buffet meal at which food intake was measured. Visual analogue scale (VAS) questionnaires were completed following the three preloads to assess appetite and other sensations. At breakfast, reduced energy intake was observed for both preloads compared with vehicle. At lunch, food intake only differed in the insect group, which consumed more than the vehicle. Insect preload increased the total amount of protein ingested with a slight increase in total energy consumed, differently than almond, which significantly increased total protein and energy consumed. There was no correlation between indigestion-sensation ratings and food intake. Moreover, the insect preload resulted in lower sleepiness and tiredness ratings compared with the almond preload. Thus, insect-derived protein may be suitable as a safe ingredient for snacks intended for elderly or infirm patients who require increased protein intake.

Development of a coculture system to evaluate the bioactivity of plant extracts on pancreatic ß-cells.

Natural plant extracts are candidates for the development of new functional foods. Most of them are usually complex mixtures of molecules of uncertain bioavailability that are often partially metabolized before they finally reach the target cells IN VIVO. IN VITRO studies of the bioactivity of these extracts suggest that their direct application to some cell cultures might be a long way from becoming a reality. To overcome this limitation, we seeded Caco-2 cells onto culture inserts and after 21 days, cocultured these with INS-1E on the base of the well. After 24 hours of coculture, TEER (transepithelium electrical resistance) measurements indicated no changes in the permeability of the Caco-2 barrier. We also found no changes in either the ability of Caco-2 cells to metabolize the flavan-3-ol component of a grape-seed procyanidin-rich extract, or in the flavanols' ability to pass through the barrier. However, the expression of the Caco-2 SGLT-1 gene increased due to the coculture. GSIS (glucose stimulated insulin secretion) was maintained in the INS-1E cells with higher levels of insulin secretion despite the fact that the insulin gene expression was unmodified by the cocultivation. Furthermore, we found that in some of the assays requiring several medium changes there was a tendency to lose ß-cells. Neutral red assay showed that seeded cells should only be cocultured for a short time to obtain a higher consistency. In conclusion, four hours coculture with Caco-2 cells and INS-1E is a suitable method for checking the bioactivity of natural plant extracts of unknown bioavailability on ß-cells.

Health-Promoting Properties of Proanthocyanidins for Intestinal Dysfunction.

The intestinal barrier is constantly exposed to potentially harmful environmental factors, including food components and bacterial endotoxins. When intestinal barrier function and immune homeostasis are compromised (intestinal dysfunction), inflammatory conditions may develop and impact overall health. Evidence from experimental animal and cell culture studies suggests that exposure of intestinal mucosa to proanthocyanidin (PAC)-rich plant products, such as grape seeds, may contribute to maintaining the barrier function and to ameliorating the pathological inflammation present in diet-induced obesity and inflammatory bowel disease. In this review, we aim to update the current knowledge on the bioactivity of PACs in experimental models of intestinal dysfunction and in humans, and to provide insights into the underlying biochemical and molecular mechanisms.

Gastrointestinally Digested Protein from the Insect Alphitobius diaperinus Stimulates a Different Intestinal Secretome than Beef or Almond, Producing a Differential Response in Food Intake in Rats.

In this study we compare the interaction of three protein sources-insect, beef, and almond-with the gastrointestinal tract. We measured the enterohormone secretion ex vivo in human and pig intestine treated with in vitro digestions of these foods. Insect and beef were the most effective in inducing the secretion of CCK, while almond was the most effective in inducing PYY in pig duodenum. In the human colon, almond was also the most effective in inducing PYY, and GLP-1 levels were increased by insect and beef. The three digested proteins reduced ghrelin secretion in pig duodenum, while only insect reduced ghrelin secretion in human colon. We also found that food intake in rats increased in groups fed a raw insect pre-load and decreased when fed raw almond. In conclusion, the insect Alphitobius diaperinus modulates duodenal and colonic enterohormone release and increases food intake in rats. These effects differ from beef and almond.

Effects of Flavanols on Enteroendocrine Secretion.

Some beneficial effects of grape seed proanthocyanidin extract (GSPE) can be explained by the modulation of enterohormone secretion. As GSPE comprises a combination of different molecules, the pure compounds that cause these effects need to be elucidated. The enterohormones and chemoreceptors present in the gastrointestinal tract differ between species, so if humans are to gain beneficial effects, species closer to humans-and humans themselves-must be used. We demonstrate that 100 mg/L of GSPE stimulates peptide YY (PYY) release, but not glucagon-like peptide 1 (GLP-1) release in the human colon. We used a pig ex vivo system that differentiates between apical and basolateral intestinal sides to analyse how apical stimulation with GSPE and its pure compounds affects the gastrointestinal tract. In pigs, apical GSPE treatment stimulates the basolateral release of PYY in the duodenum and colon and that of GLP-1 in the ascending, but not the descending colon. In the duodenum, luminal stimulation with procyanidin dimer B2 increased PYY secretion, but not CCK secretion, while catechin monomers (catechin/epicatechin) significantly increased CCK release, but not PYY release. The differential effects of GSPE and its pure compounds on enterohormone release at the same intestinal segment suggest that they act through chemosensors located apically and unevenly distributed along the gastrointestinal tract.

Modulation of Food Intake by Differential TAS2R Stimulation in Rat.

Metabolic surgery modulates the enterohormone profile, which leads, among other effects, to changes in food intake. Bitter taste receptors (TAS2Rs) have been identified in the gastrointestinal tract and specific stimulation of these has been linked to the control of ghrelin secretion. We hypothesize that optimal stimulation of TAS2Rs could help to modulate enteroendocrine secretions and thus regulate food intake. To determine this, we have assayed the response to specific agonists for hTAS2R5, hTAS2R14 and hTAS2R39 on enteroendocrine secretions from intestinal segments and food intake in rats. We found that hTAS2R5 agonists stimulate glucagon-like peptide 1 (GLP-1) and cholecystokinin (CCK), and reduce food intake. hTAS2R14 agonists induce GLP1, while hTASR39 agonists tend to increase peptide YY (PYY) but fail to reduce food intake. The effect of simultaneously activating several receptors is heterogeneous depending on the relative affinity of the agonists for each receptor. Although detailed mechanisms are not clear, bitter compounds can stimulate differentially enteroendocrine secretions that modulate food intake in rats.

Grape-Seed Proanthocyanidins are Able to Reverse Intestinal Dysfunction and Metabolic Endotoxemia Induced by a Cafeteria Diet in Wistar Rats.

We evaluated the effectiveness of pharmacological doses of grape-seed proanthocyanidin extract (GSPE) in reversing intestinal barrier alterations and local inflammation in female Wistar rats fed a long-term obesogenic diet. Animals were fed a 17-week cafeteria diet (CAF diet), supplemented with daily GSPE doses (100 or 500 mg kg-1 body weight) during the final two weeks. CAF diet enhanced the intestinal permeation of an orally administered marker (ovalbumin, OVA) and increased the plasma levels of tumor necrosis factor-α (TNF-α) and lipopolysaccharides (LPS) in 2-3-fold. Ex vivo Ussing chamber assays showed a 55-70% reduction in transepithelial electrical resistance (TEER) and increased the TNF-α secretions in both small and large intestinal sections with a 25-fold increment in the ileum. Ileal tissues also presented a 4-fold increase of myeloperoxidase (MPO) activity. Both GSPE-treatments were able to restitute TEER values in the ileum and colon and to reduce plasma LPS to basal levels without a dose-dependent effect. However, effects on the OVA permeation and TNF-α secretion were dose and section-specific. GSPE also reduced ileal MPO activity and upregulated claudin 1 gene expression. This study provides evidence of the efficacy of GSPE-supplementation ameliorating diet-induced intestinal dysfunction and metabolic endotoxemia when administered at the end of a long-term obesogenic diet.

Proanthocyanidins Limit Adipose Accrual Induced by a Cafeteria Diet, Several Weeks after the End of the Treatment.

A dose of proanthocyanidins with satiating properties proved to be able to limit body weight increase several weeks after administration under exposure to a cafeteria diet. Here we describe some of the molecular targets and the duration of the effects. We treated rats with 500 mg grape seed proanthocyanidin extract (GSPE)/kg BW for ten days. Seven or seventeen weeks after the last GSPE dose, while animals were on a cafeteria diet, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA of the key energy metabolism enzymes from the liver, adipose depots and muscle. We found that a reduction in the expression of adipose Lpl might explain the lower amount of adipose tissue in rats seven weeks after the last GSPE dose. The liver showed increased expression of Cpt1a and Hmgs2 together with a reduction in Fasn and Dgat2. In addition, muscle showed a higher fatty oxidation (Oxct1 and Cpt1b mRNA). However, after seventeen weeks, there was a completely different gene expression pattern. At the conclusion of the study, seven weeks after the last GSPE administration there was a limitation in adipose accrual that might be mediated by an inhibition of the gene expression of the adipose tissue Lpl. Concomitantly there was an increase in fatty acid oxidation in liver and muscle.

Long-Lasting Effects of GSPE on Ileal GLP-1R Gene Expression Are Associated with a Hypomethylation of the GLP-1R Promoter in Female Wistar Rats.

Flavonoids have been shown to modulate GLP-1 in obesity. GLP-1 induces some of its effects through the intestinal GLP-1 receptor (GLP-1R), though no data exist on how flavonoids affect this receptor. Here, we examine how a dose of grape seed proanthocyanidin extract (GSPE) with anti-obesity activity affects intestinal GLP-1R and analyze whether epigenetics play a role in the long-lasting effects of GSPE. We found that 10-day GSPE administration prior to the cafeteria diet upregulated GLP-1R mRNA in the ileum 17 weeks after the GSPE treatment. This was associated with a hypomethylation of the GLP-1R promoter near the region where the SP1 transcription factor binds. In the colon, the cafeteria diet upregulated GLP-1R without showing any GSPE effect. In conclusion, we have identified long-lasting GSPE effects on GLP-1R gene expression in the ileum that are partly mediated by hypomethylation at the gene promoter and may affect the SP1 binding factor.

Grape seed proanthocyanidins influence gut microbiota and enteroendocrine secretions in female rats.

Grape seed proanthocyanidin extract (GSPE) modulates several parameters involved in metabolic syndrome. GSPE is a mixture of compounds, some which are rapidly absorbed, while others remain in the lumen where they might have effects that are translated to the whole organism. Our aim was to decipher if the 8-day treatment of GSPE, previously shown to reduce food intake, induces changes in the microbiota and enterohormone secretion. The ratio of Firmicutes : Bacteroidetes was lower in the microbiota of GSPE-treated rats compared to controls, and differences in several taxonomic families and genera were observed. Such modulation led to a reduction in cecal butyrate content. GSPE also increased plasma glucagon-like-peptide-1 (GLP-1). Gallic acid did not induce major changes in the microbiota profile nor in GLP-1 secretion. Correlations between several microbiota taxa and plasma triacylglycerol, adiposity, and enterohormones were observed. Modulation of microbiota may be one of the mechanism by which GSPE impacts metabolic health.

Acute selective bioactivity of grape seed proanthocyanidins on enteroendocrine secretions in the gastrointestinal tract.

Background: Enteroendocrine cells respond to food components by secreting an array of hormones that regulate several functions. We have previously shown that grape seed proanthocyanidins (GSPE) modulate GLP-1 levels. Objective: To deepen on the knowledge of the mechanisms used by GSPE to increase GLP-1, and extend it to its role at modulation of other enterohormones. Design: We used an ex vivo system to test direct modulation of enterohormones; STC-1 cells to test pure phenolic compounds; and rats to test the effects at different gastrointestinal segments. Results: GSPE compounds act at several locations along the gastrointestinal tract modulating enterohormone secretion depending on the feeding condition. GSPE directly promotes GLP-1 secretion in the ileum, while unabsorbed/metabolized forms do so in the colon. Such stimulation requires the presence of glucose. GSPE enhanced GIP and reduced CCK secretion; gallic acid could be partly responsible for this effect. Conclusions: The activity of GSPE modulating enterohormone secretion may help to explain its effects on metabolism. GSPE acts through several mechanisms; its compounds and their metabolites are GLP-1 secretagogues in ileum and colon, respectively. In vivo GLP-1 secretion might also be mediated by indirect pathways involving modulation of other enterohormones that in turn regulate GLP-1 release.

Acutely administered grape-seed proanthocyanidin extract acts as a satiating agent.

Grape-seed proanthocyanidins' role as stimulators of active GLP-1 in rats suggests that they could be effective as satiating agents. Wistar rats were used to study the effects of proanthocyanidins on food intake with different doses, administration times and proanthocyanidin extract compositions. A dose of 423 mg of phenolics per kg body weight (BW) of grape-seed proanthocyanidin extract (GSPE) was necessary to decrease the 12-hour cumulative food intake by 18.7 ± 3.4%. Proanthocyanidins were effective when delivered directly into the gastrointestinal tract one hour before, or simultaneously at the start of the feeding period. Proanthocyanidins without galloyl forms, such as those from cocoa extract, were not as effective as grape-seed derived forms. GSPE increased the portal levels of active GLP-1 and total ghrelin and decreased the CCK levels, simultaneously with a decrease in gastric emptying. In conclusion, grape-seed proanthocyanidins could be useful as a satiating agent under the conditions defined in this study.

Effect of the co-occurring olive oil and thyme extracts on the phenolic bioaccessibility and bioavailability assessed by in vitro digestion and cell models.

Olive oils flavoured with edible herbs have grown in popularity because of their added value and potential health benefits. However, the combined presence of different phytochemicals from olive oil and herbs requires study of their possible interactions during intestinal transport and metabolism. The aim of this study was firstly to evaluate the effect on bioaccessibility of the co-occurring bioactive compounds from olive oil and thyme through an in vitro digestion model of three extracts: olive extract (OE), thyme extract (TE) and a combination of both (OTE). The bioaccessible fractions were exposed to Caco-2 and HepG-2 cell models, as well as to a co-culture of both of these. Results indicated that the bioaccessibility of hydroxytyrosol was enhanced when OTE was digested. After Caco-2 cells exposure, no significant differences were observed in hydroxytyrosol transport, whereas the main flavonoids from thyme seemed to undergo an enhanced basolateral permeation when both phenolic sources where exposed.