Sensitivity to chemical stimuli plays a fundamental role in the food preferences. Examples in the evolutionary scale:ud1. Role of the walking leg chemoreceptors in the red swamp crayfish Procambarus Clarkiiud2. PROP bitter taste sensitivity and its nutritional implications in Humans

摘要

In this thesis, we studied two examples of the sensitivity to chemical stimuli and its role in the food preferences in two models of the evolutionary scale.udThe red swamp crayfish Procambarus clarkii (Girard, 1852) (Crustacea: Decapoda) is an invasive species of freshwater habitats that has spread worldwide. In crayfish, like in other decapod crustaceans, reception of chemical cues occurs by way of peripheral chemoreceptors grouped within sensory hairs and typically located on the cuticle of cephalothoracic appendages. Antennules and pereopods (walking legs), in particular, have been reported to be olfactory organs involved in a number of behavioral responses, such as, sex recognition and localization of food sources in the environment. By way of extracellular nerve recordings coupled with behavioral bioassays, we investigated the sensitivity spectra of the walking leg chemoreceptors in the crayfish P. clarkii in response to different compounds of feeding significance and related to its omnivorous habits. Our results confirmed a marked sensitivity of the legs to trehalose, cellobiose, sucrose, maltose, glycine and leucine. Some sensitivity to glucose, fructose, asparagine (all food indicators) and taurocholic acid was also found, the sugar-sensitive chemoreceptor units resulting as broadly tuned to the carbohydrates. Responses were highly phasic to trehalose (hemolymph sugar in the body fluid of many invertebrates), phasic to glycine and leucine and phasic-tonic to the other compounds. This suggests that chemoreceptor phasicity is an additional property for better discrimination of the protein components in the diet from other stimuli. The behavioral bioassays excluded, at least under confined experimental conditions, any involvement of antennules in the detection of food-related compounds, thus emphasizing the role of the crayfish legs as the main short-distance, broad-spectrum sensors for feeding. Such information may be valuable for the identification of key chemicals aimed at the future development of strategies for crayfish population control programs.udTaste sensitivity varies greatly in humans, influencing eating behavior and therefore may play a role in body composition. PROP bitter taste sensitivity is the most studied example of the individual variability of taste sensitivity. Some studies show that PROP bitter taste sensitivity may be correlated with sensitivity to other oral stimuli, food preferences and BMI, while other studies did not confirm this association. It is known that PROP phenotype is associated with variant in bitter taste receptors TAS2R38 and with density of fungiform papillae on tongue surface. Although most of PROP phenotypic variations are explained by the allelic diversity of the bitter receptor TAS2R38, they cannot explain the PROP taster status-related differences above all that in the perception to different oral stimuli. The aim of this study was identify and characterize other factors that may contribute to differences in the genetic predisposition to taste PROP and identify confounding variables which may explain the controversial data in the literature about the relationship between PROP taste sensitivity and BMI. 1) We investigated the possible relationship between PROP bitter taste responsiveness and salivary proteins by using HPLC-ESI-MS on saliva sample before and after PROP taste stimulation. 2) We evaluated the role of proteins and free amino acids in modulating bitter taste responsiveness. Subjects rated PROP bitterness after supplementation of two salivary proteins (Ps-1 and II-2), and the free form of constituent amino acids of the two proteins sequences (L-Arg and L-Lys) whose interaction with PROP was demonstrated by 1H-NMR spectroscopy. 3) We investigate the role of polymorphism rs2274333 (A/G) in the gene that codify for the salivary trofic factor gustin protein, in PROP sensitivity and fungiform papilla density and morphology and in vitro we investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. 4) We investigated whether the endocannabinoid system, which modulates hunger/satiety and energy balance, plays a role in modulating eating behaviour influenced by a sensitivity to PROP which could explain the controversial data in literature. In particular we determined the plasma profile of the endocannabinoids 2-arachidonoylglycerol (2-AG), anandamide (AEA) and congeners in normal-weight PROP super-tasters and non-tasters, also we assessed the cognitive eating behavior disorder by the Three-Factor Eating Questionnaire.udThe results showed that: 1) Basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in non-taster unstimulated saliva, and PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. 2) Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter responsiveness. 1H-NMR results showed that the interaction between PROP and L-Arg is stonger than that involving L-Lys, and taste experiments confirmed that oral supplementation with L-Arg increase more PROP bitterness intensity than L-Lys. 3) Gustin and TAS2R38 genotypes were associated with PROP threshold, while bitterness intensity was mostly determined by TAS2R38 genotypes. Fungiform papillae densities were associated with both genotypes (with a stronger effect for gustin), but papilla morphology was a function of gustin alone. In vitro experiment, the treatment of isolated cells with saliva from individuals with AA form, and direct application of the active iso-form of gustin protein, increased cell proliferation and metabolic activity. 4) The disinhibition score of non-taster was higher than those of super-tasters. In addition, we found that the concentrationudof endocannabinoid AEA (anandamide) and 2-AG (2-arachidonoylglycerol) was lower in the plasma of non taster compared with super-tasters subjects.udIn conclusion, among the factors contributing to individual differences of PROP sensitivity, in addition to the TAS2R38 variants with its different affinity for the stimulus, we found: 1-2) the specific salivary proteins of bPRP family (Ps-1) and L-Arg that could be involved in twist and turn of the PROP molecule, thus facilitating its binding with the receptor. 3) A gustin gene polymorphism that, by modulating the protein activity, controls the growth and maintenance of taste buds and 4) the higher disinhibition behaviour in non-tasters may be compensated in part, in normal-weight subjects, by the decrease of peripheral endocannabinoids to downregulate the hunger-energy intake circuitry.