Lactisole interacts with the transmembrane domains of human T1R3 to inhibit sweet taste

The detection of sweet-tasting compounds is mediated in large part by a heterodimeric receptor comprised of T1R2+T1R3. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial sweeteners. Lactisole's inhibitory effect is specific to humans and other primates; lactisole does not affect responses to sweet compounds in rodents. By heterologously expressing interspecies combinations of T1R2+T1R3, we have determined that the target for lactisole's action is human T1R3. From studies with mouse/human chimeras of T1R3, we determined that the molecular basis for sensitivity to lactisole depends on only a few residues within the transmembrane region of human T1R3. Alanine substitution of residues in the transmembrane region of human T1R3 revealed 4 key residues required for sensitivity to lactisole. In our model of T1R3's seven transmembrane helices, lactisole is predicted to dock to a binding pocket within the transmembrane region that includes these 4 key residues.     

Sugar taste reception in mammals

This review evaluates behavioral and electrophysiological investigationsin an attempt to show correlation between the taste responsesof humans and other mammals to various sugar sweeteners. Methodologiesinclude whole nerve, single neuron and single cell studies.In addition, to enhance understanding of the mechanism of sweettaste reception, attention is focused on inhibition studies,mixture experiments, pH effects, analyses of concentration –response curves and structure–activity comparisons. Resultsshow that sucrose is the most potent naturally occurring disaccharidestimulant, while chloro-sucrose is the most potent syntheticdisaccharide. Among the monosaccharides, fructose is the mostpotent stimulant. Mixture experiments with sucrose and othersugars suggest that these compounds are interacting at a singlereceptor site whereas mixtures of sucrose and saccharin suggestdifferent receptor sites. Inhibitor experiments suggest thatthere are at least two major sucrose receptor sites. Singleneuron and single cell studies suggest a number of other sugarreceptors as well as a saccharin receptor. Concentration–responsecurve analyses show sigmoidally shaped curves throughout, andpH studies indicate that sucrose and saccharin are interactingwith their respective receptors by a different mechanism. Fromstructure–activity comparisons, we conclude that the bindingmechanism for sugars in humans and other mammals is very similar.     

Functional decline of sweet taste sensitivity of colobine monkeys

For many primates, sweet taste is palatable and is an indicator that the food contains carbohydrates, such as sugars and starches, as energy sources. However, we have found that Asian colobine monkeys (lutungs and langurs) have low sensitivity to various natural sugars. Sweet tastes are recognized when compounds bind to the sweet taste receptor TAS1R2/TAS1R3 in the oral cavity; accordingly, we conducted a functional assay using a heterologous expression system to evaluate the responses of Javan lutung (Trachypithecus auratus) TAS1R2/TAS1R3 to various natural sugars. We found that Javan lutung TAS1R2/TAS1R3 did not respond to natural sugars such as sucrose and maltose. We also conducted a behavioral experiment using the silvery lutung (Trachypithecus cristatus) and Hanuman langur (Semnopithecus entellus) by measuring the consumption of sugar-flavored jellies. Consistent with the functional assay results for TAS1R2/TAS1R3, these Asian colobine monkeys showed no preference for sucrose or maltose jellies. These results demonstrate that sweet taste sensitivity to natural sugars is low in Asian colobine monkeys, and this may be related to the specific feeding habits of colobine monkeys.     

The mouse taste cell response to five sugar stimuli

Intracellular recordings of mouse taste cell responses were made using glass microelectrodes filled with procion yellow dye solution. Only responses recorded from taste buds with fluorescent cells, as observed in subsequent histological preparations, were used in this study. The mouse taste cell depolarizes when stimulated with sucrose and is accompanied by either a resistance increase or no change. On the other hand, a NaCl stimulus produces a depolarization, hyperpolarization or null response and is accompanied by either a membrane resistance decrease or no change. Four sugars other than sucrose (maltose, fructose, glucose and lactose) produced the depolarization or null responses and were accompanied by an increase or no change in membrane resistance. From the above observations, it is suggested that each taste cell produces its own characteristic response profiles and membrane resistance changes for the five sugars and NaCl tested.     

Allelic variation of the Tas1r3 taste receptor gene selectively affects taste responses to sweeteners: evidence from 129.B6-Tas1r3 congenic mice.

The Tas1r3 gene encodes the T1R3 receptor protein, which is involved in sweet taste transduction. To characterize ligand specificity of the T1R3 receptor and the genetic architecture of sweet taste responsiveness, we analyzed taste responses of 129.B6-Tas1r3 congenic mice to a variety of chemically diverse sweeteners and glucose polymers with three different measures: consumption in 48-h two-bottle preference tests, initial licking responses, and responses of the chorda tympani nerve. The results were generally consistent across the three measures. Allelic variation of the Tas1r3 gene influenced taste responsiveness to nonnutritive sweeteners (saccharin, acesulfame-K, sucralose, SC-45647), sugars (sucrose, maltose, glucose, fructose), sugar alcohols (erythritol, sorbitol), and some amino acids (D-tryptophan, D-phenylalanine, L-proline). Tas1r3 genotype did not affect taste responses to several sweet-tasting amino acids (L-glutamine, L-threonine, L-alanine, glycine), glucose polymers (Polycose, maltooligosaccharide), and nonsweet NaCl, HCl, quinine, monosodium glutamate, and inosine 5'-monophosphate. Thus Tas1r3 polymorphisms affect taste responses to many nutritive and nonnutritive sweeteners (all of which must interact with a taste receptor involving T1R3), but not to all carbohydrates and amino acids. In addition, we found that the genetic architecture of sweet taste responsiveness changes depending on the measure of taste response and the intensity of the sweet taste stimulus. Variation in the T1R3 receptor influenced peripheral taste responsiveness over a wide range of sweetener concentrations, but behavioral responses to higher concentrations of some sweeteners increasingly depended on mechanisms that could override input from the peripheral taste system.     

The sweet taste quality is linked to a cluster of taste fibers in primates: lactisole diminishes preference and responses to sweet in S fibers (sweet best) chorda tympani fibers of M. fascicularis monkey

Background

Psychophysically, sweet and bitter have long been considered separate taste qualities, evident already to the newborn human. The identification of different receptors for sweet and bitter located on separate cells of the taste buds substantiated this separation. However, this finding leads to the next question: is bitter and sweet also kept separated in the next link from the taste buds, the fibers of the taste nerves? Previous studies in non-human primates, P. troglodytes, C. aethiops, M. mulatta, M. fascicularis and C. jacchus, suggest that the sweet and bitter taste qualities are linked to specific groups of fibers called S and Q fibers. In this study we apply a new sweet taste modifier, lactisole, commercially available as a suppressor of the sweetness of sugars on the human tongue, to test our hypothesis that sweet taste is conveyed in S fibers.

Results

We first ascertained that lactisole exerted similar suppression of sweetness in M. fascicularis, as reported in humans, by recording their preference of sweeteners and non- sweeteners with and without lactisole in two-bottle tests. The addition of lactisole significantly diminished the preference for all sweeteners but had no effect on the intake of non-sweet compounds or the intake of water. We then recorded the response to the same taste stimuli in 40 single chorda tympani nerve fibers. Comparison between single fiber nerve responses to stimuli with and without lactisole showed that lactisole only suppressed the responses to sweeteners in S fibers. It had no effect on the responses to any other stimuli in all other taste fibers.

Conclusion

In M. fascicularis, lactisole diminishes the attractiveness of compounds, which taste sweet to humans. This behavior is linked to activity of fibers in the S-cluster. Assuming that lactisole blocks the T1R3 monomer of the sweet taste receptor T1R2/R3, these results present further support for the hypothesis that S fibers convey taste from T1R2/R3 receptors, while the impulse activity in non-S fibers originates from other kinds of receptors. The absence of the effect of lactisole on the faint responses in some S fibers to other stimuli as well as the responses to sweet and non-sweet stimuli in non-S fibers suggest that these responses originate from other taste receptors.     

Drosophila melanogaster prefers compounds perceived sweet by humans

To understand the functional similarities of fly and mammalian taste receptors, we used a top-down approach that first established the fly sweetener-response profile. We employed the fruit fly Drosophila melanogaster, an omnivorous human commensal, and determined its sensitivity to an extended set of stimuli that humans find sweet. Flies were tested with all sweeteners in 2 assays that measured their taste reactivity (proboscis extension assay) and their ingestive preferences (free roaming ingestion choice test). A total of 21 sweeteners, comprised of 11 high-potency sweeteners, 2 amino acids, 5 sugars, 2 sugar alcohols, and a sweet salt (PbCl2), were tested in both assays. We found that wild-type Drosophila responded appetitively to most high-potency sweeteners preferred by humans, even those not considered sweet by rodents or new world monkeys. The similarities in taste preferences for sweeteners suggest that frugivorous/omnivorous apes and flies have evolved promiscuous carbohydrate taste detectors with similar affinities for myriad high-potency sweeteners. Whether these perceptual parallels are the result of convergent evolution of saccharide receptor-binding mechanisms remains to be determined.     

Use of intensity-time studies as an aid to interpreting sweet taste chemoreception

The effects of temparature on the sweetness intensity and persistenceof some polyols are described. Also, the relative sweetnessof sucrose and D-glucose in isomole fractional sloutions isinvestigated and the effects of increasing concentration andvolume on the sweetness of these sugars is elucidated. Usingintensity–time profils, the apparent K_m values of 11 sweetenershave been deternined. These reflect the apparent binding affinitiesof receptors to sweeteners and correlate well with the thresholdconcentrations.     

Inhibitory effect of the extract from Zizyphus jujuba leaves on sweet taste responses of the chorda tympani in the rat and hamster

1. One of the fractions obtained from the extract of Zizyphus jujuba leaves suppressed the response of the chorda tympani to sucrose, both in the rat and hamster. 2. In the rat and man, suppressive effect was found to be significant in responses to various sugars and artificial sweeteners but not in some sweet amino acids.     

Sweet taste receptor agonists alter ovarian functions and ovarian cycles in aged mice

Saccharine sodium and rebaudioside A are low-calorie sweeteners, and the biologic effects of these sweeteners in rat ovaries are related to the activity of sweet taste receptors. Data on the impact and regulatory mechanisms underlying such sweeteners on the reproduction of aged animals are currently lacking. In the present study we assessed how the consumption of sweeteners affects the ovarian cycle, ovulation, biochemical indices, and other biologic functions. Thirty-six 1-year-old mice were randomly divided into 3 groups: a control (C) group receiving regular water, a saccharin sodium group receiving a 7.5 mM solution, and the rebaudioside A group receiving a 2.5 mM solution for 30 days. We observed no significant changes in body weights in any group. However, uterine weight in the rebaudioside A group significantly increased in diestrus, and we recorded a significant increase in the percentage of abnormal estrous cycles and the number of corpora lutea in the treatment groups. TUNEL staining and Immunoreactivity for the apoptosis-inducing factor (AIF) confirmed apoptosis in granulosa cells, oocyte, and corpus luteum. Serum glucose increased significantly in both treatment groups and there was a significant increase in cholesterol in the rebaudioside A group. Furthermore, the saccharin sodium-treated group exhibited elevated serum progesterone levels compared with the other groups. In conclusion, sweeteners manifested deleterious effects on reproductive indices in aged mice.     

TIME-INTENSITY PROPERTIES OF SWEET AND BITTER STIMULI: IMPLICATIONS FOR SWEET AND BITTER TASTE CHEMORECEPTION

Interindividual differences in sweet and bitter taste sensitivity were investigated using time-intensity (TI) measurements and multivariate statistics. TI profiles were obtained in triplicate from 25 subjects for 23 sweet and/or bitter stimuli first matched to be approximately equi-intense to 200 mM NaCl. Sweet stimuli, except for the larger sweeteners, were less persistent, and required less time to reach maximum intensity than bitter stimuli. The results of principal component (PCA) and cluster (CA) analyses of the stimuli X subjects matrices for maximum intensity (Imax), time to maximum intensity (Tmax), total duration (Tdur), and area under the curve (Area) suggest that sweet and bitter stimuli do not share common receptors; and that there are at least two receptor mechanisms each for sweet taste (one for sugars and other small compounds, and the other for large sweeteners) and bitter taste (one for PTC/PROP and one for other bitter compounds).     

Cross-adapted sugar responses in the mouse taste cell

1. Intracellular recordings of mouse taste cell responses were made using a glass micro-electrode filled with Procion yellow dye solution. 2. Six sugars (sucrose, maltose, lactose, glucose, galactose and fructose) produced the depolarization responses. 3. Gustatory cross adaptation between sugars was determined. When the taste cell was pre-adapted with one of the six sugars, the other five sugars, cross adapted, produced depolarization, hyperpolarization or null responses. 4. From these observations, it is suggested that there are multiple sugar receptor sites on the receptor membrane of the mouse taste cell.     

Modulation of taste sensitivity by GLP-1 signaling

In many sensory systems, stimulus sensitivity is dynamically modulated through mechanisms of peripheral adaptation, efferent input, or hormonal action. In this way, responses to sensory stimuli can be optimized in the context of both the environment and the physiological state of the animal. Although the gustatory system critically influences food preference, food intake and metabolic homeostasis, the mechanisms for modulating taste sensitivity are poorly understood. In this study, we report that glucagon-like peptide-1 (GLP-1) signaling in taste buds modulates taste sensitivity in behaving mice. We find that GLP-1 is produced in two distinct subsets of mammalian taste cells, while the GLP-1 receptor is expressed on adjacent intragemmal afferent nerve fibers. GLP-1 receptor knockout mice show dramatically reduced taste responses to sweeteners in behavioral assays, indicating that GLP-1 signaling normally acts to maintain or enhance sweet taste sensitivity. A modest increase in citric acid taste sensitivity in these knockout mice suggests GLP-1 signaling may modulate sour taste, as well. Together, these findings suggest a novel paracrine mechanism for the regulation of taste function.     

Monosodium glutamate and sweet taste: generalization of conditioned taste aversion between glutamate and sweet stimuli in rats

Even though monosodium glutamate (MSG) is a prototypical umami substance, previous studies reported that a conditioned taste aversion (CTA) to MSG, mixed with amiloride to block the taste of sodium, generalizes to sucrose. These findings suggest that the taste of glutamate mimics the taste of sucrose and raise the question of whether glutamate has a broadly tuned sweet taste component. To test this hypothesis, CTA experiments were conducted to test for generalization between MSG and several sweet stimuli: sucrose, glucose, maltose, saccharin and SC-45647. Strong bidirectional generalization was seen between MSG mixed with amiloride and sucrose, glucose, saccharin and SC-45647. Weak generalization was seen between MSG and maltose, and sucrose and maltose. None of the CTAs generalized to NMDA. These findings support the hypothesis that the taste of MSG has broadly tuned, sweet-like characteristics, possibly due to the convergence of afferent signals for MSG, natural sugars and artificial sweeteners.     

The cysteine-rich region of T1R3 determines responses to intensely sweet proteins

A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose, fructose, sucrose, and sugar alcohols, small molecule artificial sweeteners such as saccharin and acesulfame K, and proteins such as monellin and thaumatin. Brazzein, like monellin and thaumatin, is a naturally occurring plant protein that humans, apes, and Old World monkeys perceive as tasting sweet but that is not perceived as sweet by other species including New World monkeys, mouse, and rat. It has been shown that heterologous expression of T1R2 plus T1R3 together yields a receptor responsive to many of the above-mentioned sweet tasting ligands. We have determined that the molecular basis for species-specific sensitivity to brazzein sweetness depends on a site within the cysteine-rich region of human T1R3. Other mutations in this region of T1R3 affected receptor activity toward monellin, and in some cases, overall efficacy to multiple sweet compounds, implicating this region as a previously unrecognized important determinant of sweet receptor function.     

Photoactive ligands probing the sweet taste receptor. Design and synthesis of highly potent diazirinyl d-phenylalanine derivatives

Some d-Amino acids such as d-tryptophan and d-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive d-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl d-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using l-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.     

Sweet taste involves several distinct receptor mechanisms

Measures of human sensitivities to various sweet compounds conductedat threshold (91 subjects, 7 sweeteners) and at suprathresholdlevels (9 subjects, 12 sweeteners) show interindividual differences.Multidimensional analysis indicates that sweet taste can berepresented in a tridimensional continuum if 12 compounds areconsidered. Results are speculatively interpreted as indirectevidence for the existence of several receptor sites cooperatingin sweet taste chemoreception.     

Genetic dimorphism in the taste sensitivity to trehalose inDrosophila melanogaster

Summary A quantitative behavioral assay was developed for the measurement of taste responses to sugars inDrosophila. The amount of the intake of a sugar solution was measured colorimetrically after homogenization of flies which had consumed sugar solutions mixed with a food-dye. A two-choice method was utilized to determine the taste sensitivity to sugars. Two kinds of sugar solutions were marked with either blue or red food-dye and placed alternately in the wells of a micro test plate. Flies were allowed to choose between the two sugar solutions. By classifying and counting the coloured flies, the relative taste sensitivity could be determined. Employing these methods, a genetic dimorphism in the taste sensitivity to trehalose was found among some laboratory strains ofDrosophila melanogaster. No difference in the taste sensitivity to glucose, fructose and sucrose was found between the trehalose high-sensitivity (T-1) and the low-sensitivity (Oregon-R) strains. Trehalose concentration equivalent to 2 mmol/1 sucrose, in terms of stimulating activity, was 57 mmol/1 inOregon-R and was 10 mmol/1 inT-1. Genetic analysis showed that theTre gene, whose locus is closely linked tocx (13.6) on theX chromosome, is responsible for the difference in the taste sensitivity to trehalose.     

Characterization of the modes of binding between human sweet taste receptor and low-molecular-weight sweet compounds

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.     

Sweetener preference of C57BL/6ByJ and 129P3/J mice

Previous studies have shown large differences in taste responses to several sweeteners between mice of the C57BL/6ByJ (B6) and 129P3/J (129) inbred strains. The goal of this study was to compare behavioral responses of B6 and 129 mice to a wider variety of sweeteners. Seventeen sweeteners were tested using two-bottle preference tests with water. Three main patterns of strain differences were evident. First, sucrose, maltose, saccharin, acesulfame-K, sucralose and SC-45647 were preferred by both strains, but the B6 mice had lower preference thresholds and higher solution intakes. Second, the amino acids D-phenylalanine, D-tryptophan, L-proline and glycine were highly preferred by B6 mice, but not by 129 mice. Third, glycyrrhizic acid, neohesperidin dihydrochalcone, thaumatin and cyclamate did not evoke strong preferences in either strain. Aspartame was neutral to all 129 and some B6 mice, but other B6 mice strongly preferred it. Thus, compared with the 129 mice the B6 mice had higher preferences for sugars, sweet tasting amino acids and several but not all non-caloric sweeteners. Glycyrrhizic acid, neohesperidin, thaumatin and cyclamate are not palatable to B6 or 129 mice.