Effects of Sweeteners on cAMP.

<p>A. 3T3-L1 cells at Day 0 (undifferentiated), Day 2 and Day 6 of differentiation were stimulated without (control) or with sucralose (20 mM) or saccharin (20 mM) for 30 min at 37°C, and the cellular cAMP contents were measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’. Results are shown as the mean ± SE (n = 3)., P<0.05, P<0.01 (vs. control). B. 3T3-L1 cells at Day 6 of differentiation were transfected with 30 μg of the expression plasmid encoding Epac1-camps cDNA by electroporation, seeded on a 35 mm glass bottom dish and cultured for 24 hours before the measurement of the cytosolic cAMP concentrations ([cAMP]_c). At the time point indicated with the arrow, cells were stimulated with 20 mM of sucralose. The [cAMP]_c was shown as the reciprocal of the emission ratio of EYFP/ECFP (i.e. ECFP/EYFP). C. HEK293 cells were transfected with pcDNA3.1 vector (20 μg) alone (mock) or with the vector containing mouse T1R3 cDNA (20 μg) by electroporation and seeded on a 12-well culture plate. After incubation for 24 hours, cells were stimulated without (control) or with sucralose (20 mM) or saccharin (20 mM) for 30 minutes at 37°C and the cellular contents of cAMP were measured as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054500#s2" target="_blank">Materials and Methods</a>’. Results are shown as the mean ± SE (n = 4–6)., P<0.05, P<0.01 (vs. control). D. HEK293 cells transfected by electroporation with pcDNA3.1 vector (mock), T1R2 (20 μg), T1R3 (20 μg), or both T1R2 and T1R3 (10 μg, each) were seeded on a 35 mm glass bottom dish, cultured for 24 hours and stimulated with 20 mM of sucralose at the time point indicated with the arrow. The [cAMP]_c were monitored as described in B.