Saccharin: Artificial Sweetener, Bitter Tastant, and Sweet Taste Inhibitor

Chapter 16

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Saccharin: Artificial Sweetener, Bitter Tastant, and Sweet Taste Inhibitor 1

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Marcel Winnig , Christina Kuhn , Oliver Frank , Bernd Bufe , Maik Behrens, Thomas Hofmann, and Wolfgang Meyerhof 1

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Department of Molecular Genetics, German Institute of Human Nutrition Potsdam-Rehbruecke, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany Institute of Food Chemistry, University of Münster, Corrensstrasse 45, 48149 Münster, Germany 2

Like all other sweet tasting compounds reported to date, saccharin activates the sweet taste receptor TAS1R2/TAS1R3. Its threshold of activation is in the sub-mM range and the receptor responses saturate at 1-3 m M . In the same concentration range saccharin also activates the human bitter taste receptors TAS2R43 and TAS2R44. They likely mediate saccharin's bitter aftertaste that many subjects complain. A t concentrations above 3 m M , saccharin antagonizes activation of TAS1R2/TAS1R3 by itself and other sweeteners. Apparently, saccharin binds to two sites, a high-affinity agonist-binding site and a low-affinity allosteric site. While only the former is occupied at low agonist concentrations leading to receptor activation, the latter becomes occupied at higher agonist concentrations causing receptor inhibition. Thus, we suggest that with rising concentrations the sensory properties of saccharin are impaired by a disproportionate increase in its bitter taste at the expense of its sweet taste.

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© 2008 American Chemical Society Weerasinghe and DuBois; Sweetness and Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Introduction The taste system provides the organisms with essential information about their food. Humans, like other mammals, detect and distinguish the five basic taste modalities salty, sour, umami, sweet, and bitter. Each modality is assumed to hold a specific subtask. Whereas sweet taste monitors carbohydrate-rich food and activates attractive neural pathways that stimulate intake, bitter taste serves as a warning system that activates repulsive behaviors protecting organisms from ingesting spoiled or toxic food. Sweet taste is elicited by the class C G proteincoupled receptors TAS1R2 and TAS1R3 with typical large N-terminal "venusfly-trap modules" and short C-termini (7-5). They function as dimers, the TAS1R2/TAS1R3 heterodimer being a general sweet taste receptor for numerous natural and artificial ligands including mono- and disaccharides, D and L-amino acids, peptides, proteins, metal ions, sulfamates, sulfonyl amides, and isovannillyl compounds, and the TAS1R3 homodimer being a low affinity receptor for some saccharides (2-4). Apparently various binding sites of the sweet taste receptor allow the interactions with so many different ligands (5-8). Bitter taste is initiated through the interactions of bitter substances with members of the TAS2R family, G protein-coupled receptors with short N - and C-termini, which are encoded by -25 genes in humans(9-72). The use of high potency artificial sweeteners is constantly increasing as weight-conscious subjects and diabetics use these compounds to reduce their calorie or sugar intake (13, 14). However, the sweet taste of the two commonly used sulfonyl amide sweeteners, saccharin and acesulfame K , is accompanied by a lingering bitter after taste that increases with higher concentrations, thereby limiting their use (75, 16). We here show by functional expression of the recombinant receptors that the two sulfonyl amide sweeteners activate specific TAS2R bitter taste receptors and inhibit at high concentrations the sweet taste receptor heteromer. We conclude that both actions contribute to the off-taste of sulfonyl amides.

Materials and Methods We transfected HEK293T cells stably expressing the chimeric G protein Gal6gust44 with c D N A constructs (150 ng per well) for hTAS2R bitter taste receptors or TAS1R2/TAS1R3 sweet taste receptor. 24 h after transfections cells were stained with Fluo-4-AM (Molecular Probes). After administration of tastants we analyzed the cells for changes of fluorescence by calcium imaging experiments in an automated fluorometric system, FLIPR (Molecular Devices) or by the single cell calcium imaging technique. We established dose response

Weerasinghe and DuBois; Sweetness and Sweeteners ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

232 curves by performing quadruplicates in at least two independent experiments. Calcium signals were corrected for the response of mock transfected cells and the data normalized to the fluorescence of cells prior to the stimulus (AF/F=(FF0)/F0). We calculated concentration-response curves and E C values with SigmaPlot by nonlinear regression using the function f=((a-d)/(l+(x/EC ) )+d). 5 0

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Results and Discussion Identification of TAS2R Bitter Taste Recptors for Sulfonyl Sweeteners

Amide

We used the sulfonyl amide sweeteners, saccharin and acesulfame K , to challenge HEK293T-Gctl6Gust44 cells individually transfected with cDNAs of the 25 human TAS2R bitter taste receptors (Figure 1) tagged with the somatostatin receptor 3 plasma membrane targeting sequence at their N-termini and with the herpes simplex virus glycoprotein D epitope at the C-termini (70).

Figure 1. Heterologous expression ofhTAS2R43 andhTAS2R44. Calcium responses of cells transfected with DNA for hTAS2R43 (left) or hTAS2R44 (middle) or of mock-transfected cells (right) elicited by 10 μΜ aristolochic acid, 10 mM saccharin, 10 mM acesulfame Κ or vehicle (from top to Bottom). Arrows denote the application of compounds. Scale bars, horizontal, 100 s; vertical AF/F = ft 7.


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Cells expressing hTAS2R43 or hTAS2R44 concentration dependently responded to this treatment with elevated C a levels, while mock-transfected cells or cells transfected with any other hTAS2R D N A did not (17). hTAS2R44 showed higher affinities for saccharin and acesulfame Κ ( E C values, 1.1 ± 0.01 m M and 2.5 ± 0.02 m M , respectively) than hTAS2R43 ( E C values, 1.7 ± 0.02 m M and >10 m M , respectively). Human TAS2R44-mediated signals displayed also -twofold higher amplitudes, suggesting that it contributes stronger to the bitter taste of sulfonyl amides than hTAS2R43. Both receptors are also activated by the purely bitter compound aristolochic acid with much higher affinity ( E C values, 81 ± 0.8 n M for hTAS2R43 and 455 ± 5.3 n M for hTAS2R44), whereas various other sweeteners, bitter or umami compounds failed to activate them (Figure 2). Moreover, the inhibitor lactisole did not dimmish sulfonyl amide2+

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Figure 2. Calcium responses ofhTAS2R43 (black) or hTAS2R44 (grey) expressing cells to various taste compounds.



234 induced responses from hTAS2R43 and hTAS2R44 at concentrations known to block sweet taste perception in subjects or responses from recombinant TAS1R2/TAS1R3 (not shown). Together, these results indicate that hTAS2R43 and hTAS2R44 are true bitter taste receptors and not contributing to the sweet taste of the sulfonyl amides. The data are in line with previous psychophysical studies which suggested a common receptor mechanism for the bitter taste of saccharin and acesulfame Κ (75, 16). We performed adaptation and cross-adaptation experiments to further examine the role of hTAS2R43 and hTAS2R44 for bitter tasting o f sulfonyl amides (Figure 3). Adaptation refers to the decline in taste responses of subjects

Figure 3. Adaptation and cross-adaptation of subjects ' taste responses to various tastants. a, Adaptation to the bitterness of the WTAS2R16 agonist salicin (Sal). No cross-adaptation is seen withTAS2R43/hTAS2R44 agonists, b, Adaptation to the bitterness of the hTAS2R43/hTAS2R44 agonist aristolochic acid (AA). Cross-adaptation is observed with the other hTAS2R43/hTAS2R44 agonists, saccharin (Sac) and acesulfame Κ (AcK) but not with the KTAS2R16 agonist salicin. Figures indicate the time in seconds that tastant were kept in the mouth before intensity rating was done. The phases of the experiment were separated by 30 min.



235 seen after prolonged stimulation with a taste stimulus (18, 19). It has successfully been employed to determine whether taste stimuli elicit the same or different signaling mechanisms (10, 20, 21). We investigated adaptation and cross-adapatation behaviors of the three hTAS2R43 and hTAS2R44 agonists saccharin, acesulfame Κ and aristolochic acid and the hTAS2R16 agonist salicin (17). After 15 s subjects rated, at appropriate concentrations, all four compounds as equally intense bitter on an arbitrary scale of 0 to 5. However, the bitterness of all four compounds declined over a time period from 15 to 90 s from 5 to ~1. When subjects with declined responses to the hTAS2R16 agonist salicin as first stimulus tasted any of the hTAS2R43/hTAS2R44 agonists directly thereafter they reported unaltered bitterness of these compounds (Figure 3a). Similarly, subjects with declined bitter responses after prolonged tasting of any of the three hTAS2R43/hTAS2R44 agonists as first stimulus reported normal bitterness of salicin when given as the second stimulus (Figure 3b). When, however, subjects with decreased bitter responses to any of the hTAS2R43/hTAS2R44 agonists given as first stimuls were subsequently given another hTAS2R43/hTAS2R44 agonist as second stimulus, they showed largely diminished responses also to the second treatment (Figure 3b). Adaptation to taste responses was reversible. These results indicate that subjects adapted to the bitter taste of all compounds. Adaptation was apparently receptor-specific as the bitterness of such compounds that activate different receptors was unaltered, whereas the bitterness of all compounds that activate the same receptor was diminished. The data further suggest that aristolochic acid and the sulfonyl amide sweeteners signal through the same mechanisms and that salicin signals through a different mechanism. Human TAS2R43 and hTAS2R44 were identified by in situ hybridization in taste receptor cells of human circumvallate papillae (not shown). As the TAS2R receptors are assumed to occur in the same set of taste receptor cells and use the same intracellular signaling cascade, we conclude that in vivo hTAS2R16 mediates the salicin response and hTAS2R43 and hTAS2R44 mediate the bitterness of aristolochic acid and sulfonyl amide sweeteners. Recently, individual differences were seen among 65 subjects in the bitter responses to saccharin and acesulfame K , which were not correlated to propylthiouracil tasting. Although this observation has not been followed up so far, it is interesting to note here that intense genetic variability has been observed for the TAS2Rs (22). The best-studied example is the TAS2R38 gene. This has been identified to determine the ability to taste phenylthiocarbamide and other thioamides (23, 24). Single nucleotide polymorphisms (SNPs) are present at three positions in the hTAS2R38 gene specifying five haplotypes, referred to as P A V , PVI, A A V , A A I and A V I depending on the amino acids present in the three positions (24). These haplotypes give rise to receptor variants that, when expressed in cell lines, differ in their responses to various thioamides . Whereas



236 the P A V variant is a very sensitive receptor for these compounds, the A V I variant is totally insensitive and the other variants are of intermediate sensitivities (23). Moreover, receptor sensitivity measured in vitro correctly predicted the sensitivity of subjects for tasting phenylthiocarbamide (23). Thus, in this case, a clear correlation of genotype and phenotypical tasting a particular bitter compound is seen. It has also been shown that the two known hTAS2R16 variants 172N and 172K differ in their sensitivities for various βglucopyranosides and that these differences have behavioral consequences for humans (25, 26). SNPs also occur in the hTAS2R43 and hTAS2R44 genes, namely 6 and 11, respectively (22). Although the functional consequences of this variability have not been addressed so far, a genetic basis for differences in saccharin and acesulfame Κ tasting appears likely.

Inhibition of the sweet taste receptor hTASlR2/hTASlR3 by saccharin and acesulfame Κ As a high potency sweetener saccharin is commonly employed to sweeten food and beverages at low concentrations. In marked contrast, high concentrations of saccharin taste mostly bitter and show reduced sweetness (27). This resulted from the property of saccharin to reduce its own sweetness and the sweetness of other compounds at high concentrations. When such high concentrations of saccharin are rinsed from the mouth bitterness declines while an intense sweet sensation is initiated, the sweet "water-taste". A detailed examination of these effects revealed that induction of sweet "water-taste" by a compound is associated with its ability to inhibit sweet taste (27). It was also ruled out that cognitive suppression o f sweetness by bitterness or adaptation account for the observed effects. To elucidate whether these effects were mediated by intrinsic properties of the human sweet taste receptor we characterized the hTAS2R2/hTAS2R3 dimer in HEK293T-Gal6Gust44 cells by monitoring calcium levels in response to bath application of sweet tasting compounds (27). Saccharin and acesulfame Κ showed bell shaped concentration-response relations at h T A S l R 2 / h T A S l R 3 (Figure 4a). Both compounds exerted maximal responses at a concentration of ~3 m M , while higher concentrations caused diminished responses. A t 60 m M , the highest concentration tested, responses declined by - 7 5 % indicating that high concentrations of the sulfonyl amide sweeteners cause receptor inhibition. This effect was receptor specific as the same concentrations o f another sweetener, cyclamate, elicited normal, sigmoid dose-response relations with no inhibition of hTAS 1 R2/hTAS 1R3 (Figure 4a). Moreover, also the bitter taste receptor hTAS2R44 elicited normal calcium responses when stimulated with high concentrations of the sulfonly amides lacking any sign of inhibition (Figure



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Figure 4. Functional properties of the sweet receptor hTAS!R2/hTASlR3 and the bitter receptor WTAS2R44. a, Dose-response curves of the effect of saccharin (open circles), acesulfame Κ (triangles) or cyclamate (filled circles) on cells expressing the human sweet taste receptor, b, Dose-response curves of the effect of saccharin (circles), or acesulfame Κ (triangles) on cells expressing the human bitter receptor KTAS2R44. c, Calcium responses of cells expressing the indicated sweet taste receptors that have been challenged (arrowheads) with 10 or 60 mM saccharin (sac). Scale bars, horizontal, I min; vertical, 2000 light units, d, Calcium responses of cells expressing hTAS!R2/hTASlR3 to 1 mM stevioside (stev), 5 mMarspartame (asp), 1 mM neohesperdin dihydrochalcone (neo), 5 mM acesulfame Κ or 5 mM cyclamate (eye) with (white bars) or without (black bars) 60 mM saccharin. (Reproduced with permission from reference 27. Copyright 2006 authors.)



238 4b). Apparently, the inhibitory effects of the sulfonyl amides was specific for the human sweet taste receptor as the rat rTaslr2/rTaslr3 mediated normal calcium signals at high concentrations (Figure 4c). However, a chimeric sweet taste receptor composed of rat Taslr2 and a subunit comprising the rat N-terminal extracellular domain of rTaslr3 fused to the heptahelical domain of human h T A S l R 3 was also inhibited by high concentrations of saccharin (Figure 4c). In addition saccharin inhibited the calcium responses elicited by the other sweet tasting compounds, stevioside, aspartame, neohesperidin dihydrochalcone, acesulfame K , and cyclamate (Figure 4d). Together, these results clearly show that the inhibitory cellular response to high concentrations of saccharin or acesulfame Κ was mediated specifically by the human h T A S l R 2 / h T A S l R 3 . Moreover, they reveal that the inhibitory effect was mediated through the heptahelical region of h T A S l R 3 . To mimick the induction of sweet "water-taste"in the cellular assays we washed off high concentrations of saccharin or acesulfame Κ with buffer from HEK293T-Gal6Gust44 cells expressing hTAS 1 R2/hTAS 1R3 during calcium imaging (27). While administration of 50 m M saccharin or 60 m M acesulfame Κ to the cells per se elicited no or only a small signal, the wash-off induced robust calcium resoponses. Similar results were also obtained when the sweet inhibitor lactisole was washed off the cells. The specificity of the wash out effect was verified by the observation that it is not seen in mock-transfected cells or in cells expressing the hTAS2R44 bitter taste receptor. The actions of saccharin or acesulfame Κ on the h T A S l R 2 / h T A S l R 3 dimer are best explained by assuming an allosteric receptor model with two binding sites for the sulfonyl amide sweeteners. A t low concentrations the compounds bind preferentially to a high-affinity agonist binding site causing receptor activation. At high concentration they also bind to the low-affinity allosteric site leading to receptor inhibition. Thus, the properties of hTAS 1 R2/hTAS 1R3 offer a molecular basis for the dimished sweet taste perception in subjects at high concentrations of sulfonyl amide sweeteners. In our model sweet "water-taste" is elicited by the preferential removal of these sweeteners from the allosteric site.

Conclusions We have investigated the interactions of the sulfonyl amide sweeteners saccharin and acesulfame Κ with taste receptors. Our results demonstrate that the two compounds activate the sweet taste receptor h T A S l R 2 / h T A S l R 3 and the bitter taste receptors hTAS2R43 and hTAS2R44 in an in vitro receptor assay at overlapping concentration ranges. Our results also show that both compounds at higher concentrations occupy an allosteric inhibitory site of h T A S l R 2 / hTAS!R3. The interactions of sulfonyl amide sweeteners with their cognate


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taste receptors likely account for their increased bitterness at the expense of sweetness at higher concentrations thereby producing off tastes impacting on the sensory properties of saccharin and acesulfame K . Detailed understanding of the interactions between tastants and their receptors may help designing taste-active compounds with improved sensory properties.

Acknowledgement The authors thank the German Science Foundation for support (DFG, Mel024/-l/2).

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Saccharin: Artificial Sweetener, Bitter Tastant, and Sweet Taste Inhibitor

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