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REGULATION OF TASTE SIGNALING MOLECULES BY HIGH PROTEIN DIET IN THE PIG PYLORUS Vallorani C.*[1], Latorre R.[1], Mazzoni M.[1], Bacci M.L.[1], Monica F.[1], Giancola F.[1], Falconi M.[2], De Giorgio R.[3], Sternini C.[4], Clavenzani P.[1] [1]Department of Veterinary Medical Science, University of Bologna. ~ Bologna, [2]Department of Biomedical and Neuromotorial Sciences, University of Bologna ~ Bologna, [3]Department of Medical and Surgical Sciences, University of Bologna ~ Bologna, [4]CURE/DDRC, Division of Digestive Diseases, Department of Medicine, UCLA, Los Angeles, CA, USA ~ Los Angeles Taste receptors (TRs) and their signaling molecules are widely expressed in extra‐oral sites, including the gastrointestinal (GI) tract mucosa(1). Fasting and refeeding have been shown to modify the expression of α‐ transducin / α‐gustducin in enteroendocrine cells of the pig GI tract(2), however, the effects of individual nutrients on TR‐related molecules remain unknown. The gustatory system is fundamental for detecting dietary nutrients and evoking appropriate functional responses leading to digestion and absorption. Thus, the aim of this study was to test whether a short‐ and long‐term high protein diet (3 and 30 days, respectively) affected the enteroendocrine profile of the TRs signalling molecules α‐transducin / α‐ gustducin expressing cells in the pig gastric mucosa. Twelve hybrid (LW x D) female pigs (weighing 30‐40 Kg) were subdivided in three experimental groups (n= 4 each group): A) fed standard diet (controls, CTR, 14,5% protein); B) fed high protein diet for 3 days (HP‐3, 33% protein); and C) fed high protein diet for 30 days (HP‐30, 33% protein). The protein used to supplement the diet was fish and potatoes protein. Mucosal samples from pylorus were harvested, fixed and processed for single and double labelling immunofluorescence with a mixture of the following primary antisera to Gα‐transducin (Gαtran), Gα‐gustducin (Gαgust) and 5‐hydroxytryptamine (5‐HT). In the pyloric mucosa, the average number of Gαtran immunoreactive (‐IR) cells were 111.5 ± 26.2 (CTR), 148.8 ± 13.2 (HP3), and 218.3 ± 28.8 (HP30) (CTR vs HP‐3 P< 0.04; CTR vs HP‐30 P< 0.002; HP‐3 vs HP‐30 P< 0.005), while Gαgust‐IR cells were 138.8 ± 45.1 8 (CTR), 177.3 ± 14 (HP3), 211.5 ± 29.1 (HP30) (CTR vs HP‐30 P

The gustatory system plays the critical function of distinguishing between nutrients and non‐nutrient, potentially dangerous substances (1) allowing adaptation to different habitats (2). Different tastes are detected by G protein couple receptors and their signalling molecules,including the heterotrimeric G proteins,α‐transducin (Gαtran)and α‐gustducin(Gαgust) (3,4). Taste‐related molecules have been discovered in the gastrointestinal tract of a variety of species from fish to humans. In this study we examined the distribution and peptide content of cells expressing Gαtran‐immunoreactivity in the gastrointestinal(GI)mucosa of the sea bass. Adult European sea bass(Dicentrarchus labrax)were sampled from different tanks and euthanized by an overdose of anaesthetic. The stomach,pyloric caeca and intestine were harvested;the intestine was divided into three regions: cranial, middle and caudal. Tissue sections were processed for immunofluorescence with the following primary antisera: Gαtran, Gαgust, ghrelin(GHR), 5‐hydroxytryptamine (5‐HT), obestatin (OB), somatostatin (SOM), gastrin/cholecystokinin (GAS/CCK), glucagon‐like peptide‐1 (GLP‐1), calcitonin gene‐related peptide (CGRP) and substance P(SP). Each antibody was either used alone for single labelling or in combination with others for double labelling. Gαtran immunoreactivity was observed throughout the sea bass GI tract, but was more abundant in the stomach compared to the intestine with decreasing density of cells from the cranial to the caudal regions. Specificity of Gαtran and Gαgust immunostaining was established by Western blot analysis, which showed immunopositive bands at the expected molecular weight of ~45 and ~40 kDa, respectively, in sea bass gut tissue as well as in positive tissue (e.g. brain and eye). Staining specificity was also demonstrated by immunoblocking with the homologous peptides. Colocalization of Gαtran and Gαgust immunoreactivity was visualized in some cells in the stomach. A subpopulation of Gαtran immunoreacitve cells in the stomach also contained GHR, OB or 5‐HT immunoreactivity. Colocalization of Gαtran immumoreactivity with SOM, GAS/CCK, GLP‐1, SP, or CGRP immunistaining was not observed in any GI tract regions. Our data provide evidence that Gαtran and Gαgust are involved in chemosensory transmission in the sea bass GI enteroendocrine system. It is likely that nutrients activate taste receptors and their signalling molecules to induce the release of peptides from enteroendocrine cells, which in turn activate other cells types directly or via neural reflexes thus initiating a variety of physiological responses controlling GI functions and caloric intake. 1 Sternini C. Am J Physiol Gastrointest Liver Physiol, 2007, 292:G457‐G461. 2 Barreiro‐Iglesias et. all, Brain Behav Evol 2010,75:241‐250. 3 Mazzoni et. all, J Cell Mol Med 2013, Apr;17(4):466‐74. 4 Oike H et. all, J Neurosci, 2007, 27:5584‐5592.

Taste receptors (TR1s, TR2s) for sweet and bitter along with related G-proteins (α-transducin and α-gustducin) have been localized in the GI tract mucosa of several mammals. Taste signalling molecules are now conceived to be part of a wide chemosensing system contributing to sense luminal contents. In the pig model (resembling the human gut) we established whether feeding changes, i.e. fasting and re-feeding, wereable to affect: 1) the expression of α-transducin throughout the GI tract; 2) neuropeptide coding of the cells expressing α-transducin; and 3) the spatial relationship between α-transducin cells and nerves supplying the gut. Pig stomach-to-rectum specimens (n= 12; 45 days of age), subdivided in 3 groups, i.e. control (C), fasted for 24h (F) and re-fed (R), were fixed in 4%-paraformaldehyde, embedded either in paraffin and processed for single and double labelling immunofluorescence with antibodies to: α-transducin, chromogranin-A (CgA), gastrin/cholecystokinin (Gas/CCK), somatostatin (SOM) and PGP9.5. The highest density of α-transducin-immunoreactive (IR) cells has been shown in the pylorus (C: 191±22, F: 99±28 and R: 112±51) and in the cardial mucosa (C: 79±46, F: 29±13 and R: 57±10), whereas in the intestine the highest α-transducin cell density was observed in the duodenum of C (54±22) vs. F and R (41±20,14±6, respectively). The density of α-transducin-IR cells decreased along the large intestine. The percentage of α-transducin/CgA-IR cells was reduced in the GI tract of F and R vs. C. In the jejunum,α -transducin/CCK cells decreased in F and R vs. C. Gas/α-transducin and SOM/α -transducin colocalizations have never been detected. PGP9.5 varicose nerve fibers, running either singly or in small fascicles, throughout the lamina propria of the small bowe l mucosa were seen in close spatial relationship with α-transducin cells. In conclusion, changes to α-transducin expression in neuropeptide-containing cells following F and R provide a basis to the concept that these cells participate to luminal chemosensing in the GI tract. The identification of chemosensory molecule plasticity in response to fasting and nutrients in specialized GI cells could open novel therapeutic strategies in feeding behaviour or metabolic disorders.

SUMMARY - Transcripts for sweet and bitter taste receptors and the gustatory G proteins, alpha-gustducin and alpha-transducin, are expressed by the oral cavity and the gastrointestinal (GI) mucosa. This study evaluated: 1) distribution of alpha-transducin in the pig GI tract; 2) type of cells expressing alpha-transducin (chromogranin-A, gastrin/cholecystokinin, somatostatin, villin) 3) relationship between alpha-transducin cells and enteric fibres. The presence of alpha-transducin-IR in enteroendocrine cells supports an active chemosensory machinery in the GI mucosa and that taste signalling molecules contribute to luminal chemosensing. These mechanisms may occur via neuro-endocrine cross-talk activating digestive or protective reflex pathways (e.g. vomiting or food aversion).

No abstract available

Tachykinin (TK) peptides influence neuronal activity in the inner retina of mammals. The aim of this investigation was to determine the cellular localization of the neurokinin 1 receptor (NK1), whose preferred ligand is the TK peptide substance P (SP), in the rat retina. These studies used a polyclonal antiserum directed to the C-terminus of rat NK1. The majority of NK1-immunoreactive (IR) cells were located in the proximal inner nuclear layer (INL), and very rarely they were found in the distal INL. Some small and large NK1-IR somata were present in the ganglion cell layer. NK1-IR processes were densely distributed across the inner plexiform layer (IPL) with a maximum density over lamina 2 of the IPL. Immunoreactive processes also crossed the INL and ramified in the outer plexiform layer where they formed a sparse meshwork. NK1-IR processes were rarely observed in the optic nerve fiber layer. Double-label immunofluorescence studies with different histochemical markers for bipolar cells indicated that NK1 immunoreactivity was not present in bipolar cells. Together, these observations indicate that NK1 immunoreactivity is predominantly expressed by amacrine, displaced amacrine, interplexiform, and some ganglion cells. Double-label immunofluorescence experiments were also performed to characterize NK1-containing amacrine cells. Sixty-one percent of the γ-aminobutyric acid (GABA)-IR cells, 71% of the large tyrosine hydroxylase (TH)-IR cells, and 100% of the small TH-IR cells contained NK1 immunoreactivity. In addition, most (91%) of the NK1-IR cells had GABA immunoreactivity. In contrast, vasoactive intestinal polypeptide-, TK-, choline acetyltransferase-, and parvalbumin-IR amacrine cells did not express NK1 immunoreactivity. Overall, the present findings suggest that SP acts directly upon several cell populations, including GABA-containing amacrine cells and ganglion cells, to influence visual information processing in the inner retina. J. Comp. Neurol. 389:496–507, 1997.