Denatonium Benzoate-Induces Oxidative Stress in the Heart and Kidney of Chinese Fast Yellow Chickens by Regulating Apoptosis, Autophagy, Antioxidative Activities and Bitter Taste Receptor Gene Expressions.
ABSTRACT: The sense of taste which tells us which prospective foods are nutritious, poisonous and harmful is essential for the life of the organisms. Denatonium benzoate (DB) is a bitter taste agonist known for its activation of bitter taste receptors in different cells. The aim of the current study was to investigate the mRNA expressions of bitter taste, downstream signaling effectors, apoptosis-, autophagy- and antioxidant-related genes and effector signaling pathways in the heart/kidney of chickens after DB dietary exposure. We randomly assigned 240, 1-day-old Chinese Fast Yellow chicks into four groups with five replicates of 12 chicks and studied them for 28 consecutive days. The dietary treatments consisted of basal diet and feed containing DB (5, 20 and 100 mg/kg). The results revealed that dietary DB impaired (p < 0.05) the growth performance of the chickens. Haemotoxylin and eosin staining and TUNEL assays confirmed that medium and high doses of DB damaged the epithelial cells of heart/kidney and induced apoptosis and autophagy. Remarkably, the results of RT-PCR and qRT-PCR indicated that different doses of DB gradually increased (p < 0.05) mRNA expressions of bitter taste, signaling effectors, apoptosis-, autophagy- and antioxidant- related genes on day 7 in a dose-response manner, while, these expressions were decreased (p < 0.05) subsequently by day-28 but exceptional higher (P < 0.05) expressions were observed in the high-dose DB groups of chickens. In conclusion, DB exerts adverse effects on the heart/kidney of chickens in a dose-response manner via damaging the epithelium of the heart/kidney by inducing apoptosis, autophagy associated with bitter taste and effector gene expressions. Correlation analyses for apoptosis/autophagy showed agonistic relationships. Our data provide a novel perspective for understanding the interaction of bitter taste, apoptosis, autophagy and antioxidative genes with bitter taste strong activators in the heart/kidney of chicken. These insights might help the feed industries and pave the way toward innovative directions in chicken husbandry.
Project description:Chickens have been reported to have a low taste bud count and thus low taste acuity. However, more recent studies indicate that the earlier reported count of chicken taste buds may have been significantly underestimated. To answer the question of whether the taste sensing system in broiler chickens evolved during the breeding selection over the past decades, we compared the taste sensitivity to bitter and taste buds between a meat-type control strain - the 1955 Athens Canadian Random Bred (ACRB), and a modern high-yielding broiler strain - the 2012 Cobb 500. The behavioral tests showed that the ACRB did not avoid bitter taste solutions of quinine hydrochloride (QHCl) at the examined concentrations (0.5, 1, 2, and 4 mM) (P > 0.05), while the Cobb 500 significantly avoided both the 2 mM and 4 mM QHCl solutions (P < 0.01). The labeling of chicken taste buds using the molecular marker Vimentin revealed that Cobb 500 chickens had a slightly higher number (P < 0.1), but lower density of taste bud clusters in the palate (P < 0.01) and the base of the oral cavity (P < 0.05) compared to the ACRB. We also found that a single amino acid change occurred in the bitter taste receptor T2R7. However, the functional analyses using HEK293T cells transiently expressing T2R7 revealed that the functions of T2R7 were comparable between the two strains. Taken together, our results demonstrated that taste sensitivities could be affected by the selection of the broiler chickens. The modern high-yielding broilers, which have massive feed intake and appetite, had a higher sensitivity to bitter taste stimuli than the meat-type chicken strain which was established decades ago. This evolvement of taste sensitivities may be associated with the alterations of an upper level of taste system, rather than the peripheral taste system, including distribution of taste buds and functions of taste receptors.
Project description:AIMS/HYPOTHESIS:This study was designed to ascertain whether human enteroendocrine cells express bitter taste receptors, and whether activation of these receptors with bitter-tasting ligands induces secretion of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). METHODS:We used human enteroendocrine NCI-H716 cells, isolated duodenal segments from mice, and whole mice as our experimental systems for investigating stimuli and mechanisms underlying GLP-1- and PYY-stimulated release. We measured hormone levels by ELISA and determined bitter taste receptor expression by real-time quantitative PCR. We adopted a pharmacological approach using inhibitors and enhancers of downstream signalling pathways known to be involved in bitter taste transduction in taste bud cells to investigate these pathways in NCI-H716 cells. RESULTS:Using a pharmacological approach, we identified signalling pathways triggered by the denatonium benzoate (DB)-activated bitter receptors. This involved activation of ?-gustducin (G?gust)-the specific G-protein subunit that is also present in taste bud cells-reduction of intracellular cAMP levels and enhancement of phospholipase C (PLC) activity, which ultimately led to increased intracellular calcium concentrations and hormone release. Gavage of DB, followed by gavage of glucose, to db/db mice stimulated GLP-1 and subsequent insulin secretion, leading to lower blood glucose levels. CONCLUSIONS/INTERPRETATION:Our study demonstrates that activation of gut-expressed bitter taste receptors stimulates GLP-1 secretion in a PLC-dependent manner. In diabetic mice, DB (a ligand of bitter taste receptor cells), when given via gavage, lowers blood glucose levels in diabetic mice after oral glucose administration, through increased secretion of GLP-1.
Project description:The present study was conducted to investigate the responsiveness expressions of ggTas2Rs against denatonium benzoate (DB) and genistein (GEN) in several organs of the Chinese Fast Yellow Chicken. A total of 300 one-day-old chicks that weighed an average of 32 g were randomly allocated into five groups with five replicates for 56 consecutive days. The dietary treatments consisted of basal diet, denatonium benzoate (5 mg/kg, 20 mg/kg, and 100 mg/kg), and genistein 25 mg/kg. The results of qRT-PCR indicated significantly (p < 0.05) high-level expressions in the heart, spleen, and lungs in the starter and grower stages except for in bursa Fabricius. The responsiveness expressions of ggTas2Rs against DB 100 mg/kg and GEN 25 mg/kg were highly dose-dependent in the heart, spleen, lungs, and kidneys in the starter and grower stages, but dose-independent in the bursa Fabricius in the finisher stage. The ggTas2Rs were highly expressed in lungs and the spleen, but lower in the bursa Fabricius among the organs. However, the organ growth performance significantly (p < 0.05) increased in the groups administered DB 5 mg/kg and GEN 25 mg/kg; meanwhile, the DB 20 mg/kg and DB 100 mg/kg treatments significantly reduced the growth of all the organs, respectively. These findings indicate that responsiveness expressions are dose-dependent, and bitterness sensitivity consequently decreases in aged chickens. Therefore, these findings may improve the production of new feedstuffs for chickens according to their growing stages.
Project description:Bitter taste elicits an aversive reaction, and is believed to protect against consuming poisons. Bitter molecules are detected by the Tas2r family of G-protein-coupled receptors, with a species-dependent number of subtypes. Chickens demonstrate bitter taste sensitivity despite having only three bitter taste receptors-ggTas2r1, ggTas2r2 and ggTas2r7. This minimalistic bitter taste system in chickens was used to determine relationships between in-vitro (measured in heterologous systems) and in-vivo (behavioral) detection thresholds. ggTas2r-selective ligands, nicotine (ggTas2r1), caffeine (ggTas2r2), erythromycin and (+)-catechin (ggTas2r7), and the Tas2r-promiscuous ligand quinine (all three ggTas2rs) were studied. Ligands of the same receptor had different in-vivo:in-vitro ratios, and the ggTas2r-promiscuous ligand did not exhibit lower in-vivo:in-vitro ratios than ggTas2r-selective ligands. In-vivo thresholds were similar or up to two orders of magnitude higher than the in-vitro ones.
Project description:The objective of this study was to investigate the effects of dietary 3 kinds of sweeteners supplementation on growth performance, serum biochemicals, and jejunal physiological functions of broiler chickens for 21 D. A total of one hundred ninety-two 1-day-old male Ross 308 broiler chicks were randomly divided into 4 treatments with 6 replicates for each treatment. The treatments were basal diet (CON), a basal diet supplemented with 250 mg/kg stevioside (STE), a basal diet supplemented with 100 mg/kg sucralose (SUC), and a basal diet supplemented with 600 mg/kg saccharin sodium (SAC). All birds were housed in 3-level battery cages. The results showed that dietary STE supplementation increased (P < 0.05) growth performance, serum total protein, serum albumin, and jejunal antioxidant capacity of broiler chickens. Both SUC and SAC supplementation decreased (P < 0.05) serum total protein and albumin. Dietary SAC supplementation impaired the intestinal integrity, permeability, and mucus layer of the jejunum in broiler chickens. In addition, SAC supplementation elevated (P < 0.05) the transcription expression level of jejunal bitter taste receptors and induced excessive jejunal apoptosis. Our data suggest that STE could be potentially applied as a growth-promoting and antioxidant feed additive in broiler chickens. Whereas, dietary supplementation with high level SAC has side-effects on the jejunal physiological functions of broiler chickens.
Project description:Bitter taste is one of the basic taste modalities, warning against consuming potential poisons. Bitter compounds activate members of the bitter taste receptor (Tas2r) subfamily of G protein-coupled receptors (GPCRs). The number of functional Tas2rs is species-dependent. Chickens represent an intriguing minimalistic model, because they detect the bitter taste of structurally different molecules with merely three bitter taste receptor subtypes. We investigated the binding modes of several known agonists of a representative chicken bitter taste receptor, ggTas2r1. Because of low sequence similarity between ggTas2r1 and crystallized GPCRs (~10% identity, ~30% similarity at most), the combination of computational approaches with site-directed mutagenesis was used to characterize the agonist-bound conformation of ggTas2r1 binding site between TMs 3, 5, 6 and 7. We found that the ligand interactions with N93 in TM3 and/or N247 in TM5, combined with hydrophobic contacts, are typically involved in agonist recognition. Next, the ggTas2r1 structural model was successfully used to identify three quinine analogues (epiquinidine, ethylhydrocupreine, quinidine) as new ggTas2r1 agonists. The integrated approach validated here may be applicable to additional cases where the sequence identity of the GPCR of interest and the existing experimental structures is low.
Project description:Chickens sense the bitter taste of structurally different molecules with merely three bitter taste receptors (Gallus gallus taste 2 receptors, ggTas2rs), representing a minimal case of bitter perception. Some bitter compounds like quinine, diphenidol and chlorpheniramine, activate all three ggTas2rs, while others selectively activate one or two of the receptors. We focus on bitter compounds with different selectivity profiles toward the three receptors, to shed light on the molecular recognition complexity in bitter taste. Using homology modeling and induced-fit docking simulations, we investigated the binding modes of ggTas2r agonists. Interestingly, promiscuous compounds are predicted to establish polar interactions with position 6.51 and hydrophobic interactions with positions 3.32 and 5.42 in all ggTas2rs; whereas certain residues are responsible for receptor selectivity. Lys3.29 and Asn3.36 are suggested as ggTas2r1-specificity-conferring residues; Gln6.55 as ggTas2r2-specificity-conferring residue; Ser5.38 and Gln7.42 as ggTas2r7-specificity conferring residues. The selectivity profile of quinine analogs, quinidine, epiquinidine and ethylhydrocupreine, was then characterized by combining calcium-imaging experiments and in silico approaches. ggTas2r models were used to virtually screen BitterDB compounds. ~50% of compounds known to be bitter to human are likely to be bitter to chicken, with 25, 20, 37% predicted to be ggTas2r1, ggTas2r2, ggTas2r7 agonists, respectively. Predicted ggTas2rs agonists can be tested with in vitro and in vivo experiments, contributing to our understanding of bitter taste in chicken and, consequently, to the improvement of chicken feed.
Project description:Bitterness is one of the five basic tastes, and sensitivity to bitterness is important in that it enables animals to avoid harmful and toxic substances. In humans, taste sensitivity decreases with age, although the extent of loss varies depending on the taste quality. In chickens (Gallus gallus domesticus), baby chicks have been found to be more sensitive to salt and sour taste qualities than adults. In this study, therefore, we investigated the growth-associated changes in bitter taste sensitivity in chicks. We examined the behavioral perceptions toward the bitter compounds chloramphenicol and andrographolide in chicks at three different growth stages. Then, we measured the relative expression of the functional bitter taste receptors in the chick palate. In behavioral drinking tests, the 0-1-week-old chicks consumed a significantly lower amount of bitter solutions than water, whereas the 8-9-week-old chicks showed lower avoidance of the bitter solutions than the 0-1-week-old and 4-5-week-old chicks. Real-time PCR assay showed that the 0-1-week-old chicks had significantly higher expression of one of the functional bitter taste receptors in the palate than that in the older chicks. These results suggest that baby chicks are more sensitive to bitterness than older chicks. These findings may be useful in the production of new feedstuff for chicks according to their growth stages.
Project description:Bitter taste receptors (TAS2Rs) are present in extra-oral tissues, including gut endocrine cells. This study explored the presence and mechanism of action of TAS2R agonists on gut smooth muscle in vitro and investigated functional effects of intra-gastric administration of TAS2R agonists on gastric motility and satiation. TAS2Rs and taste signalling elements were expressed in smooth muscle tissue along the mouse gut and in human gastric smooth muscle cells (hGSMC). Bitter tastants induced concentration and region-dependent contractility changes in mouse intestinal muscle strips. Contractions induced by denatonium benzoate (DB) in gastric fundus were mediated via increases in intracellular Ca(2+) release and extracellular Ca(2+)-influx, partially masked by a hyperpolarizing K(+)-efflux. Intra-gastric administration of DB in mice induced a TAS2R-dependent delay in gastric emptying. In hGSMC, bitter compounds evoked Ca(2+)-rises and increased ERK-phosphorylation. Healthy volunteers showed an impaired fundic relaxation in response to nutrient infusion and a decreased nutrient volume tolerance and increased satiation during an oral nutrient challenge test after intra-gastric DB administration. These findings suggest a potential role for intestinal TAS2Rs as therapeutic targets to alter gastrointestinal motility and hence to interfere with hunger signalling.
Project description:The characterization of molecular mechanisms underlying the taste-sensing system of chickens will add to our understanding of their feeding behaviors in poultry farming. In the mammalian taste system, the heterodimer of taste receptor type 1 members 1/3 (T1R1/T1R3) functions as an umami (amino acid) taste receptor. Here, we analyzed the expression patterns of T1R1 and T1R3 in the taste cells of chickens, labeled by the molecular markers for chicken taste buds (vimentin and α-gustducin). We observed that α-gustducin was expressed in some of the chicken T1R3-positive taste bud cells but rarely expressed in the T1R1-positive and T2R7-positive taste bud cells. These results raise the possibility that there is another second messenger signaling system in chicken taste sensory cells. We also observed that T1R3 and α-gustducin were expressed mostly in the vimentin-positive taste bud cells, whereas T1R1 and bitter taste receptor (i.e., taste receptor type 2 member 7, T2R7) were expressed largely in the vimentin-negative taste bud cells in chickens. In addition, we observed that T1R1 and T1R3 were co-expressed in about 5% of chickens' taste bud cells, which express T1R1 or T1R3. These results suggest that the heterodimer of T1R1 and T1R3 is rarely formed in chickens' taste bud cells, and they provide comparative insights into the expressional regulation of taste receptors in the taste bud cells of vertebrates.