Blends of Non-caloric Sweeteners Saccharin and Cyclamate Show Reduced Off-Taste due to TAS2R Bitter Receptor Inhibition  Maik Behrens, Kristina Blank, Wolfgang Meyerhof  Cell Chemical Biology  Volume 24, Issue 10, Pages 1199-1204.e2 (October 2017) DOI: 10.1016/j.chembiol.2017.08.004 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 1199-1204. e2DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Screening of Human TAS2Rs with Saccharin and Cyclamate Inhibition of Saccharin Responses (A) HEK293T-Gα16gust44 cells were transfected with 25 human TAS2Rs (R#) or empty vector controls (co). Some wells were left untransfected (u). After transfection, cells were subjected to calcium imaging analyses using an automated plate reader. Ten millimolar (dark gray rows) and 3.0 mM (light gray rows) sodium saccharin as well as C1-buffer controls (white rows) were applied to the cells and fluorescence changes were monitored. Asterisks indicate calcium responses of cells transfected with the receptors TAS2R31 and TAS2R43 (lower panel) upon stimulation. A second application of 0.1 μM somatostatin-14 stimulating endogenous somatostatin receptors served as vitality control. (B and C) TAS2R31 and TAS2R43 expressing FLP-In T-REX 293-Gα16gust44 after induction with tetracycline were stimulated with 3.0 and 10.0 mM sodium saccharin in the presence of different concentrations of sodium cyclamate. Non-induced cells served as negative controls. (D) TAS2R14 stimulated with 1 μM aristolochic acid and challenged with increasing concentrations of sodium cyclamate served as a specificity control. y axes, changes in fluorescence (ΔF/F) upon substance application (mean ± SD); logarithmically scaled x axes, sodium cyclamate concentration in mM. Experiments (n = 5) were performed in triplicate. *Significant (p < 0.05) signal reduction as determined by Student's t test. Cell Chemical Biology 2017 24, 1199-1204.e2DOI: (10.1016/j.chembiol.2017.08.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Mode of Inhibition of Saccharin Responses by Cyclamate and Comparison of Chemical Structures of TAS2R31 and TAS2R43 Ligands (A) FLP-In T-REX 293-Gα16gust44 cells expressing TAS2R31 and TAS2R43 cDNAs. Cells were challenged by co-application of sodium cyclamate and increasing concentrations of sodium saccharin (logarithmically scaled x axis, in mM). Calcium-dependent fluorescence changes (y axis, % activation, mean ± SD) were monitored with an automated plate reader. Experiments (n = 5) were performed in triplicate. Inserts: Schild regression analyses; the slope values are highlighted. (B) Depicted are the antagonists cyclamate and 4-(2,2,3-trimethylcyclopentyl)butanoic acid together with two agonists, the non-caloric sweetener saccharin and the purely bitter compound aristolochic acid. Cell Chemical Biology 2017 24, 1199-1204.e2DOI: (10.1016/j.chembiol.2017.08.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Saccharin Inhibits the Cyclamate-Responsive Receptor TAS2R1 FLP-In T-REX 293-Gα16gust44 cells transiently transfected with TAS2R1 cDNA were stimulated with 30 mM sodium cyclamate (circles, solid line) in the presence of different concentrations of sodium saccharin. Cells transfected with an empty expression vector (triangles, dashed line) served as a negative control (logarithmically scaled x axis in mM). Changes in fluorescence (ΔF/F) upon substance application were monitored (mean ± SD). Experiments (n = 5) were performed in triplicate. *Significant (p < 0.05) signal reduction determined by Student's t test. Cell Chemical Biology 2017 24, 1199-1204.e2DOI: (10.1016/j.chembiol.2017.08.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Saccharin-Cyclamate Blends Do Not Elicit Supra-additive Sweet Taste Receptor Signals HEK293 cells expressing the sweet taste receptor subunits TAS1R2 and TAS2R3 were subjected to calcium imaging experiments. Cells were challenged with increasing concentrations of sodium saccharin (EC50 = 0.043 ± 0.005 mM), sodium cyclamate (EC50 = 0.69 ± 0.05 mM), or a mixture of both sweeteners (EC50 = 0.28 ± 0.01 mM cyclamate/0.028 ± 0.001 mM saccharin). An additive curve was calculated from the ΔF/F values obtained for sodium saccharin and sodium cyclamate (EC50 = 0.56 ± 0.05 mM cyclamate/0.056 ± 0.005 mM saccharin). x axis, logarithmically scaled compound concentrations in mM; y axis, relative changes in fluorescence (ΔF/F) as mean ± SD. Cell Chemical Biology 2017 24, 1199-1204.e2DOI: (10.1016/j.chembiol.2017.08.004) Copyright © 2017 Elsevier Ltd Terms and Conditions